Abstract

Nothing makes sense except considering evolution, by which understanding how life originates it is important to be able to decipher the mysteries that lead to the abnormal behavior of the cancer cell. So far, the emergence of life is explained in religious terms, but in scientific terms, it was not possible. The various existing theories had gaps that could not be resolved. The discovery of unsuspected capacity of melanin transform light into chemical energy, such as chlorophyll in plants; It is a watershed in biology, it turns out that melanin is the missing link in the chain of events that leads to the emergence of life. Now the Outlook is different, we can advance faster in the study and treatment of diseases that currently constitute a scourge to mankind, such as cancer. We are on the threshold of a new era in biology and in medicine.

Introduction

The unsuspected bio-energetic role of melanin has been described previously [1]. Electromagnetic radiation, melanin and water are substances that do not present any data that allow us to think that they have some thought as an evolution process. You could say that its main physical and chemical characteristics are the same since the beginning of time.

What we call evolution, can realize to the biochemical scaffolding that nature has patiently developed over eons of years, to increasingly optimize the properties of light, melanin and water, which have not changed a whit.

Eukaryote cell is becoming more sophisticated, biochemical gear is increasingly complex, and what is most striking, the power supply remains the same since the beginning of time. This is: the first living entities, had similar amount of available chemical energy derived from the melanin that human beings today.

It is something that has not changed despite the passage of time. What, has changed continuously over time, is the number of molecules, organelles, enzymes, etc., have been bolted together to make increasingly more sophisticated, increasing the complexity of the cell. And the term sophisticated, applied to any system; means that it contains many parts.

Continues to call me the attention that the power source is still, on the one hand, the sunlight mainly, on the other hand, melanin, which has not changed in the slightest despite the course of millions of years; and so on the perfect substrate which is water.

It is interesting that one of the elements, melanin; is completely dark and we can’t see through it, either with the naked eye or with instruments, and to this we attribute the fact that its formula is not known. But, on the other hand; water is completely transparent, we observe it with the naked eye, and yet we don’t understand as the hydrogen and oxygen atoms that compose it are organized, due to diverse factors, i.e. because its arrangement is dynamic and not uniform.

The nature of light, we neither speak, don’t even understand if it is a wave or a particle, or both. So, from three natural phenomena which we know little or nothing, life originated. Melanin is the missing link between the different theories that try to explain how emerged those we named life.

Light, Melanin, and Water

The physical and chemical properties of light, melanin and water whether we understand them or not, they possess characteristics that seem to be immutable, they inner dynamic had not change in the slightest despite the passage of time. And it seems paradoxical that the union of these quite stable elements generates the very dynamic phenomenon that we call life; which is characterized by constant changes, in all times, everywhere.

Charles Darwin said that life originated in warm water, with low oxygen levels, and without any other form of present life. And it is all spent out there, because the amniotic fluid is warm water with low oxygen levels and without any other way of life.

The evolution of the human eukaryote cell, requiring billions of years of evolution, based on primitive cells, is repeated in each pregnancy, and reducing days. Thus, the initial cells during pregnancy are primitive cells, whose structure is not as complex as mature or specialized eukaryote cell.

But despite differences that may be, at least in appearance; different cell lines retain the same fundamental process of generation and distribution of energy from the melanin.

In eukaryotes normal cells, of any cell line; organelles are very similar, even in size; which reflected a surprisingly uniform pattern, which cannot be a result of chance. And that consistency comes from the origin of life, as both light, melanin, and water, are very dynamic elements but do not change, do not seem to evolve in the least; It seems that they reached perfection and do not need to go further.

What if it evolves, and does so constantly, all the time, is what we call life. And one of the features of the evolution, is the increasingly complex management, in many respects, from the chains of carbon mainly from photosynthesis-derived glucose, this is: a homogenously arrangement of carbon, oxygen, and hydrogen atoms, with C_nH_2nO_n as general formula, and whose most soluble example is glucose. While more advanced is a cell in the evolution, more efficient is the way in which atoms and carbon chains entwine each other forming increasingly intricate and complicated processes and structures, but in the end, reflects an increasingly efficient use of the energy that emanates from the melanin in the form of molecular hydrogen and high-energy electrons. A kind of energy astonishing accurate.

The proliferative cell

It is a deep-rooted dogma that normal differentiated cells, rely primarily on mitochondrial oxidative phosphorylation to generate energy needed to impel cellular processes.

So, it is not surprising that the metabolism of embryonic cells present findings consistent with the Warburg effect [2], or aerobic glycolysis. Which is an inefficient way to generate adenosine 5´-triphosphate (ATP), allowing to incorporate nutrient into biomass, rather than efficient and too much complicated, ATP production.

However, mitochondria and ATP biological functions are regulation of temperature and phosphate levels, but no energy production. Therefore, ATP biological function is regarding temperature control and phosphate levels, compounds that are chemically unstable but thermodynamically stables.

Thereby, embryonic cells require generating biomass so quickly, and gradually more and more complicated processes appear inside these cells, reflected, i.e.; by the increasing mitochondria number, whose function is the temperature control as much as possible, due to the chemical reactions inside normal differentiated cells are astonishing accurate, and temperature is no exception.

By other hand, uncontrolled proliferation is due to the cells that turn back in evolution, being the explanation due to the generation and distribution of melanin´s energy has been affected chronically.

Cancer cells are not generally controlled by normal regulatory mechanisms [3]. Thereby, the characteristic disordered tumor growth is dependent of difficult-to-understand mixture of local factors. A good example are the processes involved in angiogenesis and vascular remodeling implied in the vascularization of malignant tumors.

Total spectrum of morphogenic and molecular events required to form a neovascular network are way beyond our abstraction capacity, because casualness is a significant factor. And worst, those events are significantly different of normal vasculogenesis.

But in the light of the bio-energetic unsuspected role of melanin, we can reconsider such processes initiating the analysis as follows: the blood cannot carry energy, and on the other hand, the amount of chemical energy available in a cancer cell, is significantly lower than in a normal cell, therefore we have a series of events that reflect a step backwards in the evolution of the diseased cell and their chemical and anatomical, physical characteristics indicate that address.

While more evolved it is a cell, its operation is farther from random, and vice versa.

The intricacy of the processes that make up a specialized cell, they make these increasingly more and more precise, and one of the purposes of evolution is gradually reducing the interference of random in intracellular biochemical processes. Since any alteration in its sequence, temporality, or spatial location, would have devastating effects on perfectly than a specialized eukaryote cell represents.

Anti-vascular therapy of cancer

It has been over 30 years since Judah Folkman hypothesized that tumor growth is angiogenesis dependent [4]. But after years of clinical trials based in Dr. Folkman theory, results have been disappointing. Inhibition of VEGF, the main molecular mediator in capillary sprouting has been insufficient to halt tumor progression permanently in many cancer types.

The reasons can be diverse. i.e. the existence of multiple vascularization mechanisms and additional growth factor pathways. In our experience, the main cause of this failed therapeutic focus is that blood cannot carry on energy, just cell´s building blocks as glucose, aminoacids, lipids, etc., and CO₂.

Cancer cells

Most solid tumors display distinct aneuploid karyotypes (abnormal chromosomal numbers) and frequently miss-segregate whole chromosomes in a phenomenon called chromosomal instability (CIN). CIN positively correlates with poor patient prognosis, indicating that reduced mitotic fidelity contributes to cancer progression by increasing genetic diversity among tumor cells [5].

Mechanisms leading to the loss of mitotic fidelity in CIN are not known. A common mitotic defect in tumor cells with CIN is the persistence of erroneous attachments of chromosomes to spindle microtubules (merotely).

In normal diploid cells erroneous attachments arise spontaneously and are efficiently corrected to preserve genomic stability. Paradoxically, kinetochore microtubule attachments in cancer cells with CIN are more stable than those in normal diploid cells, thereby, accounting for the persistence of mal-attachments into anaphase, causing chromosome miss-segregation. Seems that cancer cells have a diminished capacity to correct erroneous kinetochore-microtubules attachments; a dysfunction with a widespread occurrence of CIN in tumors.

The mechanisms involved in ploidy protection and genomic integrity are highly complex, not understood, and astonishingly accurate. In example, initiation of a new round of DNA replications should be restricted until after completion of the previous mitosis. Thereby DNA replication depends of biochemical pathways that occur exactly in time, space, location, amount; etc., in an amazing way; but are largely unknown.

The failures in these poor-understood regulatory mechanisms are generalized. Therefore, the plausible explanation is energy. Other theories proposed in the scientific literature, trying to explain abnormalities observed in cancer cells, has been proved elusive, given the inherent complexity of specialized eukaryotic cell after four billion yeas of evolution.

However, we are in front of a new era in the molecular biology of cancer cells; thanks to unsuspected bio-energetic role of melanin. And the explanation seems as quite simple: a specialized eukaryotic cell whose levels of generation and distribution of energy (from melanin) are impaired by contaminated water, polluted air, pesticides, herbicides, fertilizers, transition metals, heavy metals, addictive drugs, solvents of diverse types, industrial waste, stress, etc., tends to turn back in evolution. Simply because the complicated biochemical cell scaffolding that gives vital and highly-ordered support to the poorly understood specialized eukaryotic cell, requires astonishingly accurate amounts of energy.

Insofar as the available quantities of energy inside the cell to reduce enough, for example, in time and form, the very complicated biochemical pathways start to collapse in a disorderly way. Apparently, keeping only the most basic functions; or at least the first that appeared during the evolution, which gives us an idea that cell not is being able to keep relatively recent occurrence mechanisms.

In humans, aneuploidy is linked to pathological defects such as development abnormalities, mental retardation, or cancer, but the underlying mechanisms remain elusive. There are many types of aneuploidy whose origin remains unknown. A common response independent of the type of aneuploidy can be a novel target for cancer therapy.

Genomic stability requires that genetic material must be equally distributed between the two daughter cells during mitosis. An apparently straightforward process, by far beyond our abstraction capacity. By other side, an aberrant number of chromosomes, or aneuploidy, has been recognized as a feature of human malignancies for over a century, but until today without compelling evidence of causality.

Molecular logic underlying proper and aberrant chromosome segregation are extremely complex. It is undoubtedly that chromosome instability is detrimental for the fitness and survival of normal cells, being also the hallmark of cancer cells. Paradoxically, cells with an elevated proliferative potential, are also highly aneuploid.

Theoretically, Aneuploidy is a direct consequence of chromosome segregation errors in mitosis, while structural aberrations are caused by improperly repaired DNA breaks, but also exists a cross-talk between them. The long-lasting efforts to selectively inhibit proliferation of tumor cells has been infructuous.

Toxins against microtubules exert anti-tumor effects in some patients, but it is unclear its action´s mechanism. Apparently, these compounds acts interfering with microtubule dynamics during mitosis activates the spindle checkpoint, causing a prolonged mitotic arrest. Thereby, cells either then undergo death in mitosis or slippage; returning to interphase without dividing; but it is unclear what dictates the balance between these two fates.

Therapeutic inhibition of major mitotic kinases and kinesins has given a modest clinical activity at best.

Thereby, the maintenance of chromosomal stability involves the whole cell, not only genes. So, generation and distribution of energy from melanin has a fundamental role in cell biology (Figure 1).

Figure 1. Diagram of the hitherto unknown intrinsic capacity of melanin to transform visible and invisible light into chemical energy, dissociating the water molecule, as chlorophyll in plants.

The energy that melanin provides incessantly, night and day, in a quite consistently form, both in and outside the cell, explains finally, the origin of life.

In chlorophyll, the water dissociation is irreversible; but in melanin is reversible. The process is as follows:

2H₂O → 2H₂ + O₂ →2H₂O + 4e^–

The liquid water is sublimated by melanin into its gaseous components molecular Hydrogen (H₂) and Oxygen (O₂) in astonishing and accurate way, this is: always the product of the water dissociation is molecular hydrogen and molecular oxygen. Thereby, it is a quite precise process even in a diversity of environmental conditions, because melanin acts also as attemperator of surrounding sudden changes, for instance, a significant increase or decrease in the amount of light; keeping the output of H₂ and O₂ between narrow ranges.

The biochemical process of cell (and body) is amazing accurate, otherwise life is not possible; but this astonishing precise start since the very first step of life that is the energy production. Evolution can be interpreted as the increasing and gradual organization of the distribution of energy that is more and more exquisite, efficient; and precise as time pass by. Recall that is the same energy since the beginning of time, both in nature and amount.

The specialized eukaryotic cell does not generate more energy from melanin, because melanin energy generation has a top, a guarded proportion; unless more melanin is added to the system. Instead it is much more efficient in the distribution and thereby using of H₂ and e- that the melanin dissociation and re-formation of water produces constant and exactly during night and day.

Eukaryote cell, mature, specialized; It is a whole that works perfectly in every one of its parts, starting from the generation and distribution of energy; which has been patiently optimizing over millions of years of evolution. You could say that nothing or almost nothing of what happens inside a specialized cell is the result of chance.

In any system, not only in biology; energy, defined as everything that produces a change; It is the most sensitive segment as an alteration in this area produces widespread failures, such as that seen in cancer cells.

Hence how difficult that has been to develop a successful therapy in cancer cells, it seems virtually impossible, because when designing strategies that act on a certain part of the cell, for example microtubules; the cell simply changes its behavior, which is now largely influenced by random, totally opposed to a normal cell; and quickly becomes resistant to instituted treatment.

And the explanation is that simply the imbalance now is expressed differently, but the actual background of the problem doesn´t change, and the proof is that proliferation does not diminish. Many of the deviations observed in a cancer cell should be taken care at the same time, so cell tends to function normally, as it has millions of years, millions of times.

Experiments in this regard have shown that partial reparation of the altered intracellular processes is not the solution, it is necessary to address the entire e ideally at the same time.

This is not currently possible, except to increase the generation and distribution of energy that comes from the melanin, and since all the biochemical processes that occur inside the eukaryote cell depend entirely on the H₂ and e^–, to normalize these levels, all processes tend to operate normally, because ultimately, they are chemical reactions quite exact, that may not happen or inadequately occurs if the power level is not suitable.

QIAPI 1^® and Cell Proliferation

The following Tables (1-4) show results of experiments carried out in cultured human cells. The results are part of the development of medication implemented in our laboratory, and which is in phase of patent in several countries, which has already been granted in several of them.

Table 1. WI-38 (human diploid cell line from normal embryonic (3 months gestation) lung tissue)

Table 2. A549 (human lung adenocarcinoma epithelial cell line)

Table 3. HS 683 (human neuronal glioma cell line)

Table 4. Inhibitory concentrations of QIAPI 1^®.

These results present them as a proof of concept, since the mechanism of action of QIAPI 1^® is intensifying the dissociation and reformed the water, by the melanin molecule. (Tables 1-4)

The similarity in the results, although cultures of different cell lines, support our theory that acting on the generation and distribution of energy, cells, regardless of human classifications, they tend to recover complex order that is required so that their behavior is normal, proper; as millions of years, has done millions of times.

QIAPI 1^® is a new therapeutic agent [6] that gradually will appear in the market, as legal requirements should be fitted.

Conclusion

The apparent chaos that reigns in efforts to decipher the bases of the anomalous behavior of cancer cells, appears to take a totally different direction when we do aside dogma that the main source of cell energy is glucose, then the discovery of unsuspected intrinsic property of melanin make visible and invisible light into chemical energy, such as chlorophyll in plants; through the dissociation of the water molecule, it is a watershed in cell biology.

We have a new view in the study of the biology of cancer, and while we move faster in that sense, the benefit that can give every day to numerous patients affected by the disease will be higher.

Acknowledgement: This work was supported by Human Photosynthesis® Research Center.

References

• Solis-Herrera, A. Arias-Esparza, MC. Solis-Arias, RI. Solis-Arias, PE. Solis Arias, Martha P (2010) The unexpected capacity of melanin to dissociate the water molecule, fills the the gap between the life before and after ATP. Biomed Res 21: 224-227.
• Vander Heiden, Matthew Cantley, Lewis, Thompson, Craig B (2009) Understanding the Warburg effect: The Metabolic Requirements of Cell Proliferation. Science 324: 1029-1033.
• Döme, Balázs Hendrix, Mary JC, Paku, Sändor Tóvári, József, Timár, Jozsef (2013) Biological perspectives. Alternative Vascularization Mechanisms in Cancer. Pathology and Therapeutic implications. Am J Path.
• Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285: 1182-1186. [crossref]
• Compton, Duane (2013) Mechanism of chromosomal instability in human tumor cells. Geisel School of Medicine at Dartmouth, Hanover, USA.
• Solis-Herrera, A Ashraf, Ghulam M, Arias-Esparza, MC Solis-Arias, et al. (2015) Biological Activities of QIAPI 1 as Melanin Precursor and its therapeutic effects in Wistar Rats exposed to Arsenic Poisoning. CNS Agen in Med Chem 2: 99-109.

Commentary

The “biogenea pharmaceuticals” being the first biotech company in Southeast Europe, has the task to systematically promote the Science of Pharmaceutical Biotechnology, including the production of advanced therapy medicinal products ATMPs as defined by the European Medicines/Agency EMEA. On behalf of “biogenea pharmaceuticals” and its scientific board we are pleased to announce our innovative biotechnological services Nanostemogenea™ for the collection, processing, cryopreservation and therapeutic use of our advanced biotechnology applications utilizing next generation biocompatible complexes of stem, immune cells and nanoparticles in the field of magnetic guided regenerative medicine. Our GENEA cells ™ are totally safe and obtained from different sources of the human body for autologous cell therapy purposes in Renaissance medicine.

Biogenea Pharmaceuticals™ is the first Inter-Balkan Pharmaceutical Biotechnology Company since leading the way since 2005 in Red Biotechnology applications, in Cryobiology and in Autologous Cellular Therapy of Degenerative Diseases (cardiological diseases, neurological conditions and metabolic disorders).

Biogenea Pharmaceuticals focuses

• On the collection, processing, cryopreservation and cGMP (according to Good Manufacturing Practice) production -for solely autologous use – of cellular therapeutical solutions from blood (bone marrow, peripheral blood, cord blood) or blood compounds for human use.
• In collaboration with Regenetech on stem cell expansion technologies, which were created in the research laboratories of NASA (National Aeronautics and Space Administration).
• On the cGMP production of advanced medicinal products (1394/2007/ΕC) for solely autologous use from skin, dental pulp, cord tissue). (In preclinical-research phase: 2008-2009).
• On certified genetic analyses in collaboration with International Referral Centers.
• On copyright protection according to the American and/or European Copyright Agency. The “biogeneapharmaceuticals” is the only European bank that has been recognized by the European Medicines Agency EMEA as a pharmaceutical company and has the possibility of cryopreservation of hematopoietic stem cells and conducting clinical trials (EMEA/Qualification of an enterprise as an SME – GrigoriadisBros – Biogenea- Cellgenea Ltd, with registration number: EMA/SME/084/10).

In “biogenea pharmaceuticals” taking advantage of the unique properties of super paramagnetic nanoparticles as this high magnetic moment and susceptibility but also the existence yperparagnitikis behavioural development multitude biological applications agglomerate formation with these petidika molecular and different kinds STEM CELL for autologous use in the Renaissance medicine.

Biogenea Pharmaceuticals Ltd combines excellent trained scientific personnel with the most modern techniques, concerning cell expansion, that are used today in the field of biotechnology and have been developed by NASA. Thus our company is able to verge into the demanding field of clinical trials concerning stem cell treatments. One can understand the big potential of these stem cells to play a role in Regenerative Medicine by looking at the amount of clinical trials all over the world that use stem cells as a way to treat an increasing number of diseases in a supportive manner.

The “biogenea pharmaceuticals” provides the following services:

• Cardiogenea™: Autologous intracoronary, intraarterial or intracardiac injection of autologous blood, bone marrow and heart stem cells derive active cardiopoeitic spheres coated with superparamagneticnanoparticles for the restoration of myocardial infarction.
• Dendrigenea™: Autologous adjuvant immune hybridomatic therapy for cancer patients using advanced complexes of superparamagnetic iron oxide nanoparticles coated mature dendritic and dendritic tumor cell fusions as a cancer ‘cell vaccine’.
• Cartigenea™: Autologous and exvivo expanded chondrogenic cells coated with super paramagnetic nanoparticles for their intended use in Cartilage Defects.

Biogenea Pharmaceuticals Ltd is in the process of developing novel therapies for the effective treatment of several diseases. These therapies make use of Stem Cells (ADSCs) and a special class of nanoparticles, referred to as Super paramagnetic Iron Oxide Nanoparticles (SPIONs). SPIONs have recently attracted the interest of the scientific community, due to several attributes, such as their paramagnetism and their potential us in a number of therapeutic approaches. SPIONs are small synthetic γ-Fe2O3 (maghemite), Fe3O4 (magnetite) or α-Fe2O3 (hermatite) particles with a core ranging from 10 nm to 100 nm in diameter. In addition, mixed oxides of iron with transition metal ions such as copper, cobalt, nickel, and manganese, are known to exhibit superparamagneticproperties and also fall into the category of SPIONs. However, magnetite and maghemite nanoparticles are the most widely used SPIONs in various biomedical applications. SPIONs have an organic or inorganic coating so that they can be tolerated by cells and tissues.

Our Personalized cancer therapy is determined by the needs and specificities of a particular oncology patient to provide the optimum desired therapeutic effect with minimal toxicity, strengthening the immune system of cancer patients with the use TARGETED complex dendritic cells and super-paramagnetic nanoparticles in simultaneous increase the concentration of magnetic particles into the tumor.

Our technique serves the philosophy of personalized cancer treatment based on tumor pluripotent cells (cancer stem cells), consisting of four specialized areas in the prevention, diagnosis, treatment and rehabilitation of cancer patients while capitalizing on the therapeutic effects of magnetic hyperthermia the implementation of magnetic field for the local heating of the cancer cells with a view to the disaster.

Preventive Cryopreservation Insurance of Primordial Cells & Therapeutic Applications

• Continuous update and prompt training of interested Donors and Clinicians of Public and Private Health Centers and Hospitals, about the progenitor cells applications.
• Immediate availability of the stored samples 365 days a year, 7 days a week, 24 hours a day.
• Complete attunement with the specifications set by the European Accreditation Organization for cellular therapy (FACT/JACIE/ NETCORD), the American Association of Blood Banks (AABB) and the relevant Greek.
• Presidential Decretal 2004/23 for the tissue/cell banks, with the use of cGMP methods.
• Control of Plasticity of progenitor cells based on the detailed Validation Master Plan of the Standard Operating Procedures (SOPs) regarding their hematopoietic origin (Methocult).
• Fully Automated Viability Control of the cryopreserved progenitor cells with automatic luminometric device, which increases the reproducibility and the accuracy of the results in contrast to the common laboratory techniques for detecting dead cells microscopically (Trypan Blue staining).
• Validated Molecular Diagnostics service provision (detection of HIV1/2, HBV, HCV, CMV, Syphillis, Toxoplasma) applying Real- Time PCR technology (Roche LightCycler).
• Validated ex-vivo cellular and/or tissue expansion of the recently processed cells/tissues, as well as of the cryopreserved cell/ tissues in advanced technology Bioreactors, created in the NASA research laboratories, offering unique micro-gravity conditions in alternating electromagnetic field! Cellgenea is the UNIQUE company in Greece able to expand and to use ex-vivo expanded cord blood and adult progenitor cells for therapeutic reasons and clinical trials thanks to the relevant know-how transfer from the research laboratories of NASA and Regenetech Biotechnology Company.
• Storing of progenitor cells in two-chambered bags and cryovials in complete (24 hours a day) controlled and automated liquid nitrogen tanks.
• Continuous control and cross-tracking of the cryopreserved samples with validated LIS-ERP software which ensures the ability to track down and to identify the sample during all the steps of its supply, the processing, the control, the storage and the distribution. The tracking is also used for controlling and identifying all the relevant data about the products and the materials that come in contact with these samples (2004/23/ΕΚ).
• Immediate availability and distribution of the sample inside a validated cryotank, in case of therapeutic application.
• Strict security concerning personal data, confidentiality and safety according to the Personal Data Protection Agency.
• Complete bacteriological and serological control of the samples using the automatic analyzers BacT/ALERT and Architect i1000 without any extra rate.
• Life insurance provided to all the members of the different programs of preventive cryopreservation (Cellgenea, Dentogenea, Dermigenea, Angiogenea, DΝΑgenea, Neurogenea, Cardiogenea) in cooperation with insurance companies.
• DNA & pharmacogenetic control services for personalized treatment without side effects.
• Offer of validated prenatal and DNA/RNA control for chromosomal anomalies screening in a variety of biological materials (adult peripheral blood, cord blood, tissue biopsies) in cooperation with the Technological Park of Ioannina.
• Continuous training and schooling of the scientific staff on the new

Nanuristemogenea™

Advanced biotechnology applications utilizing next generation biocompatible complexes of stem and hybridoma immune cells with nanoparticles in the field of magnetic guided regenerative medicine.

The “biogenea pharmaceuticals” being the first biotech company in Southeast Europe, has the task to systematically promote the Science of Pharmaceutical Biotechnology, including the production of advanced therapy medicinal products ATMPs as defined by the European Medicines/Agency EMEA.

On behalf of “biogenea pharmaceuticals” and its scientific board we are pleased to annpounce our innovative biotechnological services Nanostemogenea™ for the collection, processing, cryopreservation and therapeutic use of our advanced biotechnology applications utilizing next generation biocompatible complexes of stem, immune cells and nanoparticles in the field of magnetic guided regenerative medicine. Our GENEAcells™ are totally safe and obtained from different sources of the human body for autologous cell therapy purposes in Renaissance medicine.

Biogenea Pharmaceuticals™ is the first Inter-Balkan Pharmaceutical Biotechnology Company since leading the way since 2005 in Red Biotechnology applications, in Cryobiology and in Autologous Cellular Therapy of Degenerative Diseases (cardiological diseases, neurological conditions and metabolic disorders).

Biogenea Pharmaceuticals focuses

• On the collection, processing, cryopreservation and cGMP (according to Good Manufacturing Practice) production -for solely autologous use – of cellular therapeutical solutions from blood (bone marrow, peripheral blood, cord blood) or blood compounds for human use.
• In collaboration with Regenetech on stem cell expansion technologies, which were created in the research laboratories of NASA (National Aeronautics and Space Administration).
• On the cGMP production of advanced medicinal products (1394/2007/ΕC) for solely autologous use from skin, dental pulp, cord tissue). (In preclinical-research phase: 2008-2009).
• On certified genetic analyses in collaboration with International Referral Centers.
• On copyright protection according to the American and/or European Copyright Agency. The “biogeneapharmaceuticals” is the only European bank that has been recognized by the European Medicines Agency EMEA as a pharmaceutical company and has the possibility of cryopreservation of hematopoietic stem cells and conducting clinical trials (EMEA/Qualification of an enterprise as an SME – GrigoriadisBros – Biogenea- Cellgenea Ltd, with registration number: EMA/SME/084/10).
• In “biogenea pharmaceuticals” taking advantage of the unique properties of super paramagnetic nanoparticles as this high magnetic moment and susceptibility but also the existence yperparagnitikis behavioral development multitude biological applications agglomerate formation with these petidika molecular and different kinds STEM CELL for autologous use in the Renaissance medicine.
• Biogenea Pharmaceuticals Ltd combines excellent trained scientific personnel with the most modern techniques, concerning cell expansion, that are used today in the field of biotechnology and have been developed by NASA. Thus our company is able to verge into the demanding field of clinical trials concerning stem cell treatments. One can understand the big potential of these stem cells to play a role in Regenerative Medicine by looking at the amount of clinical trials all over the world that use stem cells as a way to treat an increasing number of diseases in a supportive manner.
• The “biogenea pharmaceuticals” provides the following services:
• Cardiogenea™: Autologous intracoronary, intra-arterial or intracardiac injection of autologous blood, bone marrow and heart stem cells derive active cardiopoeitic spheres coated with super paramagnetic nanoparticles for the restoration of myocardial infarction. 
• Dendrigenea™: Autologous adjuvant immune hybridomatic therapy for cancer patients using advanced complexes of super paramagnetic iron oxide nanoparticles coated mature dendritic and dendritic tumor cell fusions as a cancer ‘cell vaccine’. 
• Cartigenea™: Autologous and ex-vivo expanded chondrogenic cells coated with super paramagnetic nanoparticles for their intended use in Cartilage Defects.

Biogenea Pharmaceuticals Ltd is in the process of developing novel therapies for the effective treatment of several diseases. These therapies make use of Stem Cells (ADSCs) and a special class of nanoparticles, referred to as Super paramagnetic Iron Oxide Nanoparticles (SPIONs). SPIONs have recently attracted the interest of the scientific community, due to several attributes, such as their paramagnetism and their potential us in a number of therapeutic approaches. SPIONs are small synthetic γ-Fe2O3 (maghemite), Fe3O4 (magnetite) or α-Fe2O3 (hermatite) particles with a core ranging from 10 nm to 100 nm in diameter. In addition, mixed oxides of iron with transition metal ions such as copper, cobalt, nickel, and manganese, are known to exhibit super paramagnetic properties and also fall into the category of SPIONs. However, magnetite and maghemite nanoparticles are the most widely used SPIONs in various biomedical applications. SPIONs have an organic or inorganic coating so that they can be tolerated by cells and tissues.

Our Personalized cancer therapy is determined by the needs and specificities of a particular oncology patient to provide the optimum desired therapeutic effect with minimal toxicity, strengthening the immune system of cancer patients with the use TARGETED complex dendritic cells and super-paramagnetic nanoparticles in simultaneous increase the concentration of magnetic particles into the tumor.

Our technique serves the philosophy of personalized cancer treatment based on tumor pluripotent cells (cancer stem cells), consisting of four specialized areas in the prevention, diagnosis, treatment and rehabilitation of cancer patients while capitalizing on the therapeutic effects of magnetic hyperthermia the implementation of magnetic field for the local heating of the cancer cells with a view to the disaster.

Preventive Cryopreservation Insurance of Primordial Cells & Therapeutic Applications

• Continuous update and prompt training of interested Donors and Clinicians of Public and Private Health Centers and Hospitals, about the progenitor cells applications.
• Immediate availability of the stored samples 365 days a year, 7 days a week, 24 hours a day.
• Complete attunement with the specifications set by the European Accreditation Organization for cellular therapy (FACT/JACIE/ NETCORD), the American Association of Blood Banks (AABB) and the relevant Greek Presidential Decretal 2004/23 for the tissue/cell banks, with the use of cGMP methods.
• Control of Plasticity of progenitor cells based on the detailed Validation Master Plan of the Standard Operating Procedures (SOPs) regarding their hematopoietic origin (Methocult).
• Fully Automated Viability Control of the cryopreserved progenitor cells with automatic luminometric device, which increases the reproducibility and the accuracy of the results in contrast to the common laboratory techniques for detecting dead cells microscopically (Trypan Blue staining).
• Validated Molecular Diagnostics service provision (detection of HIV1/2, HBV, HCV, CMV, Syphillis, Toxoplasma) applying Real- Time PCR technology (Roche Light Cycler).
• Validated ex-vivo cellular and/or tissue expansion of the recently processed cells/tissues, as well as of the cryopreserved cell/tissues in advanced technology Bioreactors, created in the NASA research laboratories, offering unique micro-gravity conditions in alternating electromagnetic field! Cellgenea is the UNIQUE company in Greece able to expand and to use ex-vivo expanded cord blood and adult progenitor cells for therapeutic reasons and clinical trials thanks to the relevant know-how transfer from the research laboratories of NASA and Regenetech Biotechnology Company.
• Storing of progenitor cells in two-chambered bags and cryovials in complete (24 hours a day) controlled and automated liquid nitrogen tanks.
• Continuous control and cross-tracking of the cryopreserved samples with validated LIS-ERP software which ensures the ability to track down and to identify the sample during all the steps of its supply, the processing, the control, the storage and the distribution. The tracking is also used for controlling and identifying all the relevant data about the products and the materials that come in contact with these samples (2004/23/ΕΚ).
• Immediate availability and distribution of the sample inside a validated cryotank, in case of therapeutic application.
• Strict security concerning personal data, confidentiality and safety according to the Personal Data Protection Agency.
• Complete bacteriological and serological control of the samples using the automatic analyzers BacT/ALERT and Architect i1000 without any extra rate.
• Life insurance provided to all the members of the different programs of preventive cryopreservation (Cellgenea, Dentogenea, Dermigenea, Angiogenea, DΝΑgenea, Neurogenea, Cardiogenea) in cooperation with insurance companies.
• DNA & pharmacogenetic control services for personalized treatment without side effects.
• Offer of validated prenatal and DNA/RNA control for chromosomal anomalies screening in a variety of biological materials (adult peripheral blood, cord blood, tissue biopsies) in cooperation with the Technological Park of Ioannina.
• Continuous training and schooling of the scientific staff on the new scientific developments.

Proposal Description

Abstract

Objectives and task definition, based on the state of the art in terms of technology and knowledge. Detailed description of the problem which shall be solved, description of the beneficiary and user of the project results.

Study of the prognostic marker microRNA-Mir-216a/217 in hepatocellular carcinoma patients and development of an autologous vaccine with tumorlysate pulsed dendritic cells, genetically modified for the expression of the Mir-216a/217 and HAb18G/CD147 antigen.

Aim

The aim of the present protocol is the development of the techniques for the detection and quantification of the prognostic marker microRNA-Mir- 216a/217 in serum and liver tissue from hepatocellular carcinoma patients submitted to hepatectomy. Based on the above, an experimental (in vitro) protocol will be developed for the production and differentiation of dendritic cells derived from the peripheral blood of mononuclear cells, genetically modified for the expression of the microRNA-Mir-216a/217 and HAb18G/CD147 antigen and their maturation by the cell lysate from tumor resections of hepatocellular carcinoma patients. The ultimate goals of the present study are the early diagnosis of recurrence of hepatocellular carcinoma and the development of an autologous dendritic cell vaccine for additional immunotherapy of these patients.

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide with over 600,000 patients dying from this disease annually [1]. The age standardized incidence rates (ASR) of HCC in men in Europe, adjusted to the European Standard Population, is about 8 per 100,000, with a peak in Southern Europe of 10.5 per 100,000 (http://globocan.iarc.fr/). Liver transplantation and tumor resection have proven to be the most effective standard therapies and provide 5-year survival rates of 70% for patients within the Milan criteria, i.e., single tumour < 5 cm in size or up to three tumors < 3 cm in size [2]. These rates reach 50% with radiofrequency ablation and transarterial chemoembolization, which are the next preferred lines of therapy. However, these therapeutic procedures most often do not provide a complete cure, as half of the treated patients experience tumor recurrence within 3 years. A further major problem is that only 30–40% of patients are eligible for the above-described treatments due to the fact that HCC frequently remains undiagnosed until an advanced stage has been reached and occurs in the setting of advanced liver disease due to cirrhosis. These patients have a median survival time ranging from 3 to 16 months [3]. Thus, there is currently ongoing development and testing of alternative drug-based therapies for HCC that target tumor signalling pathways and vasculature. However, so far, the only drug that significantly prolonged survival (by nearly 3 months) in patients with advanced HCC is sorafenib, a multi-targeted tyrosine kinase inhibitor [4]. In view of these facts, new strategies are required. In this study we focus on the identification of predictive molecular factors for HCC recurrence and the development of immunotherapy methods for these patients.

Early Diagnosis of HCC Recurrence with Micro-RNA Techniques

MicroRNAs (miRNAs) are a class of small endogenously expressed non-coding RNAs. The miRNAs in blood, called circulating miRs (cmiRNAs) are potential biomarkers for detecting and monitoring cancer progression [5]. The ability of some miRNAs to function as tumor promoters (miR-30d, miR-151 and miR-210) or suppressors (miR-122, let-7g, miR-29b, miR-193b, miR-194, miR-139 and miR-124) in hepatocarcinogenesis have led to new insights into the molecular pathways involved in HCC [6]. Recently, upregulation of the miR-216a/217 cluster identified to be associated with the early HCC recurrent disease, by comparing the miRNA expression profiles of HCC liver tissue from patients with early-recurrent and non-recurrent disease [7]. In this protocol we intend to develop a technique for the detection and quantification of miR-216a/217 in the blood of HCC patients submitted to liver resection in order to identify recurrence of the disease.

Specific Description of the Proposed Solution

A sensitive and predictable method for the detection and identification of the microRNA216a/217 as the ideal biomarker for prognosis of Hepatocellular Carcinoma

Paraffin-embedded tissue analysis

Paraffin-embedded Hepatocellular Carcinoma (PE-HCC) Tissue samples of the primary tumor are obtained from HCC patients who underwent surgical treatment or biopsy at our centers.

RNA Extraction from serum samples

Total RNA was extracted from 500 μL of serum by using TRI reagent BD (Molecular Research Center). Ten sections, each 10- μm thick, were cut from each PE-HCC block. Deparaffinized tissue sections were digested by using proteinase K, and RNA was extracted by using a modified protocol of the RNAWiz Isolation kit (Applied Biosystems). The RNA was quantified and assessed for purity by using ultraviolet spectrophotometry and the Quant-iT RiboGreen RNA assay kit (Invitrogen) [8,9].

RT-qPCR assay with extracted RNA

10 ng of total RNA extracted from Hepatocellular tumor tissue, normal tissue, or serum are dissolved in 5 μL H2O (2 ng/μL) for reverse transcription with the addition of 5 μL of a reaction mixture containing 5Χ first strand buffer, 10 mmol/L deoxynucleoside-5’- triphosphate, RNasin, reverse transcriptase, and miR-specific RT primers (Exiqon). After the mixture was incubated in 37°C for 2 h, the transcribed specific cDNA was diluted 10-fold with molecular grade H2O before use as a qPCR template. Each qPCR contained 2.5 μL of diluted cDNA, 5 μL of 2ΧPerfeCTa SYBR Green FastMix for iQ (Quanta Bioscience), miR-specific, locked nucleic-acid– based forward primer targeting the specific microRNA216a/217 and universal reverse primer (Exiqon).

RT-qPCR directly in serum

This assay requireσ only a small aliquot of serum. To deactivate or solubilize proteins that might inhibit the RT-qPCR, 2.5_L of each serum sample are mixed with 2.5 μL of a preparation buffer that contained 2.5% Tween 20 (EMD Chemicals), 50 mmol/L Tris (Sigma- Aldrich), and 1 mmol/L EDTA (Sigma-Aldrich). 5 μL ofRT reagent mixture are added which contain the same RT reagents used for RT-qPCR with extracted RNA RNA, directly to 5 μL of serum in preparation buffer; a 2-h incubation at 37°C is followed by a 5-min enzyme inactivation at 95°C. The transcribed cDNA is diluted 10- fold by H2O and then centrifuged at 9000 g for 5 min to eliminate the protein precipitant. A 2.5-μL volume of the supernatant cDNA solution is used as the template for qPCR. qPCR conditions, primers and reagents, and data analysis were duplicated for those described in RT-qPCR with extracted RNA section.

Real time quantitative PCR (qPCR)

Real-time qPCRs were performed using SYBR Green PCR Master Mix and 7300 Realtime PCR System (Applied Biosystems, Foster City, CA USA). Real-time qPCRs were also used to detect miRs, as reported [10]. Sequences of miR-specific primers for cDNA synthesis and reverse primers for miR-216a were: 5′-CATGATCAGCTGGGCCAAGACACAGTTGCCAGCTG-3′ and 5′-TAATCTCAGCTGGCAA-3′. Primers for miR-217 were: 5′ CATGATCAGCTGGGCCAAGAATCCAGTCAGTT-3′ (cDNA synthesis) and 5′-TACTGCATCAGGAACT-3′ (reverse primer). miR expression levels were also confirmed by poly-adenylation of mature miR and cDNA synthesis primed by oligo-dT primer tagged with universal primer sequence (miScript System, Qiagen). cDNA was amplified using miR specific and universal primers. For miR-216a and miR-217 PCRs, the same reverse primers mentioned above and the primers provided by Qiagen were used as miR-specific primers. 5S RNA or 18S RNA served as internal control (Ambion) [7]. Genetically modified dendritic cells.

The limited improvements in clinical outcomes with dendritic cells loaded with tumor associated antigens (TAAs) led to trials with genetically modified DCs to further enhance antigen presentation and immunostimulation N. Recent studies have shown that the miR- 216A/217 cluster is consistently and significantly up-regulated in HCC tissue samples and in cell lines associated with early tumor recurrence, poor disease free survival and an epithelial mesenchymal transition (EMT) phenotype [7-31]. Stable over-expression of miR-216a/217- induced EMT, increased the stem like cell population, migration and metastatic ability of epithelial HCC cells. PTEN and SMAD7 are subsequently identified as two functional targets of miR-216a/217, and both PTEN and SMAD7 are down regulated in HCC. Ectopic Expression of PTEN or SMAD7 partially rescued miR216a/217 mediated EMT phenotype, cell migration and stem-like properties in HCC cells. Previously was shown that SMAD7 is a TGF-β1 antagonist. Recently, it has been also shown that Epithelial–mesenchymal transition (EMT) induced by transforming growth factor-β (TGF-β) is implicated in hepatocarcinogenesis and hepatocellular carcinoma (HCC) metastasis [32]. On the other hand, HAb18G/CD147, which belongs to the CD147 family, is an HCC-associated antigen that has a crucial role in tumor invasion and metastasis. Upregulation of HAb18G/CD147 is induced by TGF-β coupled promoting the idea that CD147 may be a potential target for the treatment and prevention of HCC. Other studies have also shown that overexpression of miR216a/217 act a positive feedback regulator for the TGF-b pathway and the canonical way involved in the activation of the PI3K/Akt/ Mtor in HCC cells [33,34].

These facts led us to the second proposal of our protocol:

Experimental (in vitro) protocol for the production and differentiation of dendritic cells derived from the peripheral blood fraction of mononuclear cells, genetically modified for the expression of the microRNA-Mir-216a/217 and HAb18G/CD147 antigen and their maturation by the cell lysate from tumor resections of hepatocellular carcinoma patients

1. MS2 VLP-based delivery of microRNA-216a/217 as a potential delivery approach for the production of clinical grade genetically modified monocyte derived dendritic cells for the immunization of patients suffered from Hepatocellular Carcinoma

Recent studies suggest that microRNA-216a/217 microRNA (miR-216a/217) plays an essential role in immunoregulation and may be involved in the pathogenesis of Hepatocellular Carcinoma. Therefore, it is of interest to investigate the potential therapeutic application of miR-216a/217-induced dendritic cells in Hepatocellular Carcinoma, a concept that has not been thoroughly investigated thus far. Virus-like particles (VLPs) are a type of recombinant nanoparticle enveloped by certain proteins derived from the outer coat of a virus. Herein, we describe a novel miRNA-delivery approach via bacteriophage MS2 VLPs and investigate the therapeutic effects of miR–216a/217-induced dendritic cells against patients suffering from Hepatocellular Carcinoma.

2. An Innovative Methodology for the miR216a/217 MicroRNA Delivery in peripheral blood monocyte derived antigen presenting dendritic cells via magnetic nanoparticles

Peripheral Blood Monocyte derived dendritic cells show promising potential in the vaccination of HCC patients. Recently, gene silencing strategies using microRNAs (miR) emerged with the aim to expand the therapeutic potential of genetically modified dendritic cells. However, researchers are still searching for effective miR delivery methods for clinical applications. Therefore, we aimed to develop a technique to efficiently deliver miR216a/217 microRNA into patient’s dendritic cells with the help of a magnetic non-viral vector based on cationic polymer polyethylenimine (PEI) bound to iron oxide magnetic nanoparticles (MNP), whose uptake efficiency and cytotoxicity will be determined by flow cytometry. The Present Proposal is directed in different magnetic complex compositions and determined. Additionally, the present proposal is focusing on the monitoring of the release, processing and functionality of delivered miR216a/217 microRNA with confocal laser scanning microscopy, real-time PCR and live cell imaging, respectively. On this basis, we will focus on the established parameters for construction of magnetic non-viral vectors with optimized uptake efficiency (~75%) and moderate cytotoxicity in patients peripheral blood monocyte derived dendritic cells. Furthermore, we aim to introduce the magnetic non-viral vector based on cationic polymer polyethylenimine (PEI) transfection as a long term beneficial strategy for the successful genetic modification of the HCC-TAAs presenting dendritic cells against Hepatocellular Carcerous cells for future in vivo applications.

Generation of peripheral blood monocyte-derived miR-216a/217 induced DCs

Monocyte-derived DCs from Hepatocellular Carcinoma Patients (obtained with following informed consent and approved by our institutional review board) are generated. In brief, peripheral blood mononuclear cells (PBMCs) are prepared from whole blood by Ficoll density-gradient centrifugation. The PBMCs are suspended in tissue culture flask in RPMI 1640 supplemented with 1% heat inactivated autologous serum for 60 minutes at 37°C to allow for adherence. The nonadherent cells are removed and the adherent cells are cultured overnight. To generate immature miR-216a/217 induced DCs (DCs), the nonadherent and loosely adherent cells are collected the following day and placed in RPMI 1640 medium containing 1% heat-inactivated autologous serum, 1000 U/ml recombinant human GM-CSF (Becton Dickinson, Bedford, MA, USA), and 500 U/ml recombinant human IL-4 (Becton Dickinson) for 6 days. In order to assess the effects of HCCsp on miR-216a/217 induced DCs generation, we are focusing on the creation of four types of miR-216a/217 induced preparation: 1) miR-216a/217 induced DCs; 2) miR-216a/217 induced s generated in the presence of HCCsp during the entire culture period (DCs/sp); 3) miR-216a/217 induced DCs exposed to 0.1 KE/ml (0.1 KE equals of 0.01 mg of dried streptococci) penicillin-inactivated Streptococcus pyogenes (OK-432) (Chugai Pharmaceutical) for 3 days (OK- miR- 216a/217 induced DCs) as described previously; 4) OK- miR-216a/217 induced s generated in the presence of HCCsp during the entire culture period (OK-DCs/sp). Four types of miR-216a/217 induced are generated in the presence of equal amounts of GM-CSF and IL-4 during the entire culture.

To generate monocyte-derived miR-216a/217 induced DCs for vaccination, PBMCs derived from the HCC patient are freshly isolated (obtained with following informed consent and approved by our institutional review board). Autologous miR-216a/217 induced s are generated in RPMI 1640 medium containing 1% heat-inactivated autologous serum, 1000 U/ml recombinant human GM-CSF, 500 U/ml recombinant human IL-4, and 10 ng/ml recombinant TNF-α (Becton Dickinson) [30]. On day 6 of culture, DCs harvested from the nonadherent and loosely adherent cells are used for fusion. The firmly adherent monocytes are harvested and used as an autologous target for the CTL assays.

HCC Patient selection

The clinical trial protocol will be approved by the Institutional Review Boards of our University Hospitals. Patients will be informed of the investigative nature of this study, and written consent in accordance with institutional regulations is obtained prior to study entry. Eligibility criteria included HCC patients submitted to liver resection or biopsy and transarterial chemoembolization (TACE) with radiological diagnosis of primary HCC by computed tomography (CT), classified in stage II and III according to the tumor-node-metastasis (TNM) classification; age over 20 years/both male and female; Eastern Cooperative Oncology Group scale 0–1; and indicators of acceptable hematological (hemoglobin ≥8.5 g/dl, white blood cells ≥2,000/mm3, platelet ≥50,000/mm3), hepatic (Child Pugh score ≤7, alanine aminotransferase, aspartate aminotransferase ≤5x upper normal limit) and renal (creatinine ≤1.5 mg/dl) function. Important exclusion criteria consisted of organ transplantation; a medical history of autoimmune disease, immunodeficiency, or autoimmune disease that might be aggravated by immunotherapy; not exceeding 2 weeks after antibiotic treatment needed due to a serious infectious disease; seropositivity for human immunodeficiency virus antigen; use of immunosuppressive drug such as cyclosporin A and azathioprine; any cardiopulmonary disability judged by the investigator; a medical history of psychological disease or epilepsy; and evidence of another active malignant neoplasm.

Autologous DC generation

DCs are generated from blood monocytes, as reported previously, with modifications. White blood cells obtained from the HCC patients through leukapheresis. DCs are prepared in a GMP-compliant facility at our hospitals. Peripheral blood mononuclear cells (PBMCs) are separated from WBC by Ficoll-Paque™ PLUS (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation. PBMCs are stored in a liquid nitrogen tank until necessary for DC generation. PBMCs thawed, ished with Hanks’ Balanced Salt Solutions, resuspended in RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with autologous heat-inactivated plasma, and then incubated in CellSTACK Culture Chambers (Corning, Corning, NY, USA). After 0.5–1 h incubation at 37°C in a 5% CO2 incubator, non-adherent cells are removed by gentle ishes.

The adherent monocytes are cultured in X-VIVO15 (Cambrex, East Rutherford, NJ, USA) supplemented with 100 ng/ml of granulocyte macrophage-colony stimulating factor (GMP grade: LG Life Science, Seoul, Korea) and 300 ng/ml of interleukin (IL)-4 (JW CreaGene Inc., Seongnam, Korea) for 5 days. On day 5, nonattached immature DCs are harvested and pulsed with CTP-fused human AFP, MAGE-1 and GPC-3 recombinant proteins at a final concentration of 5 μg/ml each. Antigen-pulsed dendritic cells are matured in the presence of cytokine cocktail, IL-6 (Peprotech, Rocky Hill, NJ, USA), IL-1β (Peprotech), tumor necrosis factor (TNF)-α (Peprotech), prostaglandin E2 (PGE2) (Sigma Chemical Co., St. Louis, MO, USA), interferon (IFN)-γ (LG Life Science), OK432 (Chugai Pharmaceutical Co., Tokyo, Japan), and poly I: C (Sigma) for 1 or 2 days depending on surface phenotypes and cell population. On day 6–7, the DCs are harvested, ished, and resuspended in 1.2 ml of cryopreserving solution containing 5% dimethyl sulfoxide (Bioniche Pharma USA, Lake Forest, IL, USA). Finally fully equipped DCs are packed into a sterile glass vial (4×107 cells/vial), sealed with a snap-cap, and stored at an ultralow freezer for >12 h.

Quality control of dendritic cell vaccine

Safety test

For safety, endotoxin, germ-free and mycoplasma-free tests are performed according to the KFDA-approved JW CreaGene standard and test guidelines. Endotoxin is evaluated using gel-clot techniques. The endotoxin of the product should be less than 10 EU/ml per 1.2- ml vial. Mycoplasma test is performed by both direct culture and PCR methods using e-Myco™ Mycoplasma PCR detection kit (Intron Biotechnology, Seongnam, Korea), which contains primer sets specifically designed to detect major contaminants of Mycoplasma in cell cultures such as M. arginini, M. faucium, M. fermentans,M. hyorhinis, M. orale, and A. laidlawii as well as other broad spectrum of mycoplasma.

Cell size and granularity

During the differentiation from monocytes to miR-216a/217 induced dendritic cells, cell size and granularity increase. Based on these principles, the cell size and granularity of each miR-216a/217 induced DC vaccine are assessed by flow cytometric analysis. PBMCs are used for gating control.

Phenotypic analysis

The phenotype of miR-216a/217 induced DC vaccine is determined by flow cytometry using a FACSCalibur™ flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The following monoclonal antibodies are used: i) fluorescein isothiocyanate-conjugated mouse antihuman IgG2a isotype control; ii) phycoerythrin-conjugated mouse antihuman IgG1 isotype control; iii) anti-CD14, anti-CD19, anti-CD40, anti-CD80, anti-D86, anti-HLA-ABC, and anti-HLA-DR (BD Pharmingen, San Diego, CA, USA).

Viability

The viability of miR-216a/217 induced DC vaccine is assessed by propidium iodide (PI) staining. PI (BD Pharmingen) is added to a sample and kept in the dark at room temperature for 20 min. Cell viability is examined by flow cytometry using a FACSCalibur™ (Becton Dickinson). Viability is represented as 100-[(PI+ of sample)−(PI+ of control)] (%).

Lymphocyte proliferation assay

One vial from each miR-216a/217 induced DC vaccine lot is used to test of T cell stimulation capacity according to the standard lymphocyte proliferation assay. T cells are isolated from cryopreserved PBMC using nylon wool column (Polysciences, Warrington, PA, USA). Purified T cells (1×105) are cultured with serially diluted DC vaccine (starting from 1×104 cells to 0.33×103 cells) at 37°C for 5 days. T cell proliferation is assessed by 3-(4,5-di-methylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, yellow tetrazole: MTT) assay following manufacturer’s protocol (CellTiter 96 Non-Radioactive proliferation assay kit; Promega, Madison, WI, USA). R2 represent the standard curve of MTT assay for the validation of a data set.

Cytokine production assay

Either culture supernatant of each antigen-pulsed miR-216a/217 induced DC or co-cultured medium of T cells/ miR-216a/217 induced DC at the ratio of 10: 1 is collected and stored at −80°C until this assay. The concentration of IL-12p70, IL-10, IFN-γ, and IL-4 is measured with corresponding human immunoassay kits (BD OptEIA™ kit, BD Pharmingen) based on the manufacturer’s instruction. Each experiment is performed 3 times and the result is described as the mean ± standard deviation.

Treatment protocol of Hepatocellular Carcinoma Patients

The screening evaluation shall be performed in 3 weeks before the start of dendritic cell-based immunotherapy and consisted of the following: complete history, thorough physical examination, chest X-ray, electrocardiogram, urine analysis, hematological and immunological parameters, serum chemistry, tumor markers [AFP and protein induced by vitamin K absence or antagonists-II (PIVKA-II)], ultrasonography and abdominal CT scan. Eligible HCC patients underwent liver resection or TACE and biopsy, 2 weeks before the start of the vaccination. PBMC collection by leukapheresis is performed 1 week before the first planned vaccination. Tumor antigen-pulsed miR-216A/217 Professional Antigen Presenting DCs are injected intravenously in 20 mL of phosphate-buffered saline over 10 minutes on day 12. Patients were observed for 2 hours after each vaccination to assess any immediate complications. During the first cycle, 6 vaccinations are administered at biweekly intervals. Medical history and standard blood tests and urine analysis are performed at each vaccination. Vital signs are monitored during and after each injection. Response evaluation is performed 4 weeks after fourth vaccination (10 weeks after first vaccination). Two further vaccinations are administered at biweekly intervals, and final response evaluation is performed at 18 weeks after first vaccination. Tumor markers, including qRT-PCR miR-216A/217, and serological tests for autoantibodies, including anti-nuclear antibody, are evaluated every 4 weeks.

Clinical response and toxicity assessment

Clinical responses to vaccination are evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Complete response is defined as disappearance of all target lesions. Partial response is defined as 30% decrease in the sum of the longest diameter of target lesions. Progressive disease is 20% increase in the sum of the longest diameter of target lesions. Stable disease is defined as small changes that do not meet above criteria. Toxities are classified according to the National Cancer Institute Common Toxicity Criteria.

Description of the German and Greek partners from higher education institutions, research establishments and commercial companies who are involved in the project (key competencies, capabilities, infrastructure, etc).

The Division of Transplantation, Department of Surgery, Aristotle University of Thessaloniki Medical School is the leading center in Greece, performing liver transplantation and liver surgery in patients with hepatocellular carcinoma and has the ability to perform clinical studies on the prognosis and therapy on this subject.

The Department of Surgery, Essen University Hospital, is a leading center worldwide in liver transplantation and liver surgery in patients with hepatocellular carcinoma and has performed many multi institutional clinical studies on the prognosis and therapy on this subject.

Justify the scope of the collaboration between the partners of both countries and the mutual benefit provided through this collaboration. Overview of previous contacts in and collaborations with the partner country.

Immunotherapy of HCC

Immunotherapy aims to provide a more efficient and selective targeting of tumor cells by inducing or boosting the existing tumor-specific immune response. The rationale for immunotherapy for HCC is based on several lines of evidence of a protective role the immune system, e.g., by controlling tumor growth. First, HCC patients with an intratumoral accumulation of lymphocytes had a superior 5-year survival rate and a prolonged recurrence-free survival after liver transplantation or resection [11,12]. A certain level of protection was especially conferred by cytotoxic CD8+ T cells [11]. Of note, these tumor infiltrating lymphocytes (TILs) were associated with an inflammatory microenvironment that was a predictor of overall patient survival, indicating a protective role of TILs in HCC [13,14]. Furthermore, a strong CD8+ T cell response against several tumor-associated antigens (TAAs) was found to coincide with improved recurrence-free survival after liver resection [15]. The important role of CD8+ T cells in HCC control is further supported by a study in mice. It was shown that interferon γ (IFNγ) produced by CD8+ TILs could be one effector mechanism for apoptosis of hepatoma cells [16]. These data imply a central role of T cells in modulating tumor progression and provide strong justification for T cell immunotherapy.

Immunotherapy approaches for HCC

A limited number of immunotherapy trials for HCC have been conducted based on several strategies, with yet modest results. Cytokines have been used to activate subsets of immune cells and/or increase the tumor immunogenicity [17,18]. Further strategies have been based on infusion of tumor infiltrating lymphocytes or activated peripheral blood lymphocytes [19-21]. Alternatively, direct delivery of genetically modified or designer T cells (dTc) into the hepatic artery has been recently proposed as a promising novel strategy and is currently evaluated in a phase I human clinical trial (ClinicalTrials. gov Identifier: NCT01373047). Indeed, the latter strategy has recently been successfully used for treatment of different cancers and several human clinical trials are currently ongoing [22].

Alternatively, considering active immunotherapy strategies (i.e. therapeutic vaccination), the number of human clinical trials published to date is extremely small. The first HCC vaccine clinical trial was conducted by Butterfield et al. based on CD8+ T cell epitopes specific for alpha fetoprotein (AFP), showing the generation of AFP-specific T cell responses in vaccinated subjects [23]. To improve the immune response, the same authors performed a subsequent phase I/II trial administering AFP epitopes presented by autologous DCs loaded ex vivo. This treatment, however, resulted only in transient CD8+ T cell responses, possibly caused by the lack of CD4+ help [24,25]. To overcome this limitation and to increase the number of tumor associated antigens (TAAs) targeted by the immune response elicited by the vaccine, few vaccine approaches based on autologous DCs pulsed ex vivo with a lysate of the autologous tumor [26] or of hepatoblastoma cell line HepG2 [27,28] have been evaluated in human clinical trials, showing limited improvements in clinical outcomes . The last clinical trial in the literature is based on a combination of low dose cyclophosphamide treatment followed by a telomerase peptide (GV1001) vaccination which did not show antitumor efficacy [29].

Prospects for the success of the proposed measures and implementation concepts describing how the project outcomes can be utilized after the funding period (utilization plan)
Dendrigenea: A manufacturing patient-specific cell therapy product

Overview and case study of dendrigenea’s cell therapy technology: Our Dendrigenea cellular therapy product is currently progressing through clinical development with the potential to address unmet medical needs affecting millions of patients suffered from cancer –tumor related diseases. Dendrigenea is an autologous cell-based therapeutic product which has already received regulatory approval and reach the market. Our primary challenge is to quickly become a Leader dendritic cell manufacturing facility of such products in sufficient volume to meet patients demand.

Biogenea Pharmaceuticals Ltd has developed a cancer-specific dendritic cell therapy technology for use in an autologous patient-specific vaccine therapy and is conducting late-stage clinical trials both in Greece and other Countries from Southeaster Europe. Dendritic cells are derived from a patient’s own peripheral blood and processed through a short four-week isolation, maturation, culture and fusion procedure under media perfusion conditions that lend potent functional anti-cancer vaccination properties to the final vaccine-cell product.

Development and clinical use of a Peripheral Monocyte Derived Cell Therapy Product (PMDCTP) such as Dendrigenea has raised unique manufacturing challenges that we have addressed through a series of patented innovative solutions, many of which could have broad application in the field of cancer immunotherapy for the production of commercial-scale dendritic cell based manufacturing Vaccines.

Peripheral Blood as a source of dendritic vaccines has key advantages for patients. In particular, autologous (PMDCTP) (treatments derived from a patient’s own monocytes) has a favorable safety and risk profile not available from allogeneic (universal donor) therapies.

Concerns over immune rejection and disease transmission are minimal. In addition, short-term culture and monocyte maturation procedures reduce the risk of tumorgenic transformation, which is possible in universal donor cell products typically generated from longer-term serially passaged cultures. Dendrigenea has demonstrated a high level of safety in more than 250 patients treated successfully for a variety of tumor-cancer related medical indications, with both local and systemic administration, showing no sign of cell-related adverse events.

Our dendritic cells are expanded from cell populations existing within patient’s own peripheral blood that are associated with natural cell immune response and tissue homeostasis as well as healing. Local transplantation of these expanded, patient-specific peripheral blood derived dendritic cells are expected to immunize patients suffering from Tumor related diseases. They are currently under clinical evaluation for a range of cancer cell treatments including Melanoma, Leiomyosarcoma, Glioma, Glioblastoma, Neuroblastoma and Ovarian, Breast Cancer.

Dendrigenea goes through of an ex-vivo, culture, maturation and cell fusion process have been developed to expand specific subpopulations of primary monocyte derived dendritic cells found within patient’s own peripheral blood, including early stem and progenitor cells, without triggering cell differentiation to other malignant tissue specific cell-lines . The result is a mixed population of dendritic cells genetic or not modified targeted directly to patient’s own cancer cells.

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Short Commentary

Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. In summary, in “biogenea pharmaceuticals ltd” we successfully generated several immune T and dendritic cell line living vaccines using conditional immortalization where the generated dendritic cell lines demonstrate the characteristic immunophenotype of primary dendritic cells to facilitate further studies on immunomodulatory properties of dendritic cells.

Genea-Pancretovimortal24585

A Mixture of two allogeneic human pancreatic derived immature dendritic cancerous immortalized cell lines stably transduced with a retroviral vector for the endogenous encoding of the CEA, Muc-1, TRICOM, CO1 7-1 A, OC 125 and B72.3, DU-PAN-2 respectively on Allogeneic human immature dendritic cell lines derived from a class I & II HLA3-A3-B44 CD34+ progenitor cell.

Numerous clinical trials have demonstrated the safety of dendritic cells vaccines, and more than 1000 patients have received dendritic cell vaccines with no serious adverse events associated with the therapy and clinical responses in one half of patients The CA19-9, CA125, DU-PAN-2, and B72.3 antigens have been shown to be expressed in many human pancreatic cancer cells, and C01 7-1A and B72.3 have being used for immunotherapy. Here, in Biogenea Pharmaceuticals Ltd we provide a method of enhancing immunity by modifying a dendritic cell (DC) in vivo or ex vivo to produce an immature immortalized dendritic cell line enhancing immunity in pancreatic cancer patients. OurGenea-Pancretovimortal24585 composition is a pancreatic cancer patient derived tumor lysate pulsed with a immortalized cell line mixture dedicated to the treatment of pancreatic cancer by the use of lentivirus of the CEA, Muc-1, TRICOM, CO1 7-1 A, OC 125 and B72.3, DU-PAN-2 antigen transfected immature transduced dendritic cell lines as an advanced semi-autologous living (DC)-vaccine. Genea-Cordimmortalun24874: Immortalized Human Cord Blood- Derived Stem Cells for the generation of conditionally immortalized universal progenitor cell lines with multiple lineage potential and Immunosuppressive Characteristics.

Cell banking of mesenchymal stem cells (SCs) from various human tissues has significantly increased the feasibility of SC-based therapies. Sources such as adipose tissue and amnion offer outstanding possibilities for allogeneic transplantation due to their high differentiation potential and their ability to modulate immune reaction. Limitations, however, concern the reduced replicative potential as a result of progressive telomere erosion, which hampers scaleable production and long-term analysis of these cells. In Biogenea Pharmaceuticals Ltd for the first time we incorporated methods for preparing multi-potential immortalized stem cells having a pre-selected expression of MHC antigens. Our Genea-Cell lines consisting of two human cord blood-derived immortalized somatic stem cell linesgenerate by ectopic expression of the catalytic subunit of human telomerase (hTERT). hTERT overexpression resulted in continuously growing SC lines that were largely unaltered concerning surface marker profile, morphology, karyotype, and immunosuppressive capacity with similar or enhanced differentiation potential for up to 87 population doublings. can be Our universal stem cell lines can used to generate histocompatible tissues/organs for transplantation. The process comprises the use of targeting vectors capable of gene knockout, insertion of site-specific recombination cassettes, and the replacement of histocompatibility alleles in the stem cell. We incorporated novel knockout vectors which are used to delete designated HLA-B44, HLA-B7, HLA-B8, HLA-B35, HLA-B52, HLA-B60, HLA-B39, and HLA-B48 HLA-DR7, HLA-DR4, HLA-DR13, HLA-DR15, HLA-DR3, HLA-DR1, HLA-DR11, HLA-DR8, HLA-DR9, HLA-DR12, HLA-DR14 and HLA-DRBL, HLA-A allele selected from the group consisting of HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A29, and HLA-A33 regions of one chromosome. Recombination cassette vectors were used to delete the same region on the second chromosome and deposit a site-specific recombination cassette which can be utilized by replacement vectors for inserting the new MHC genes on the chromosome of the engineered cell. Our advanced methodology pertains to cells, tissues, for the generation of conditionally immortalized progenitor cell lines with multiple lineage potential.

Genea-Cellgeneroglimmortal737

A Combinations of Transgenes in LV for Reprogramming Immune Precursors into two Antigen-Loaded immortalized Dendritic Cell lines for the DC-endogenous expression of the cALLa/NEP, ABCC3, GPNMB, NNMT, and SEC61γ trasnduced antigens on immortalized class I & II HLA3-A3B-44-Dendritic Cells lines (iDC) for T Cell Expansion after Stem Cell Transplantation.

Malignant brain tumors carry a poor prognosis even in the midst of surgical, radio-, and chemotherapy. With the poor prognosis of brain tumors the available therapeutic treatments, there exists a significant need for more effective therapies to treat such tumors. Our Genea- Cellgeneroglimmortal737 is based, on the discovery that vaccines based on cancer stem cell antigens are exceptionally useful for therapy of cancer. Immunization of patients with endogenous expressed of the cALLa/NEP, ABCC3, GPNMB, NNMT, and SEC61γ trasnduced dendritic cell lines pulsed with autologous tumor lysate from isolated cancer circulated stem cells provided a significant survival benefit as compared to immunization with dendrit The principle of this approach is to educate immortalized immature antigen-presenting cells, such as dendritic cells, to recognize tumor antigens by fusing them on pulsed differentiated tumor cells. Cancer stem cells were found to express major histocompatibility (MHC), indicating that they can display antigens. Our advanced cell fusions can be useful in providing antigenic compositions for treatment of cancers (e.g., neural cancers such as gliomas).

Genea-Hivotranceral437856

A cord blood derived Non-Transformed, transduced, Immortalized Human double negative il-2 depended T-tropic lymphocyte cell-line for the expression of the CD4D1D2CAR and HTLV-1 p30II oncoprotein.

Genea-Hivotranceral437856 advanced medicinal services include a composition comprising a cord blood derived Non-Transformed, transduced, non-transformed, immortalized T-lymphocyte cell-line, wherein the T lymphocytes are IL-2 dependent and interact with an extracellular matrix and supports productive infection and replication by T-tropic HIV. Our T-lymphocyte cell-line cells are polyclonal. In another aspect, the T-lymphocyte cell-line are infected with a lentivirus that expressed THE CD4D1D2CAR and an HTLV- 1 p30II oncoprotein. Our Genea-Hivotranceral437856 T cell line composition endogenously express the chimeric antigen receptor of CD4 extracellular, transmembrane domains and a CD3 zeta signaling domain where the CD4 extracellular domain binds gpl20 expressed on the surface of cells infected with HIV.

Biogenea PharmaceuticalsTM is the first Inter-Balkan Pharmaceutical Biotechnology Company since leading the way since 2005 in Red Biotechnology applications, in Cryobiology and in Autologous Cellular Therapy of Degenerative Diseases (cardiological diseases, neurological conditions and metabolic disorders).

Biogenea Pharmaceuticals focuses

• on the collection, processing, cryopreservation and cGMP (according to Good Manufacturing Practice) production -for solely autologous use – of cellular therapeutical solutions from blood (bone marrow, peripheral blood, cord blood) or blood compounds for human use.
• in collaboration with Regenetech on stem cell expansion technologies, which were created in the research laboratories of NASA (National Aeronautics and Space Administration).
• on the cGMP production of advanced medicinal products (1394/2007/ΕC) for solely autologous use from skin, dental pulp, cord tissue). (In preclinical-research phase: 2008-2009).
• on certified genetic analyses in collaboration with International Referral Centers.
• on copyright protection according to the American and/or European Copyright Agency
• Preventive Cryopreservation Insurance of Primordial Cells & Therapeutic Applications.
• Continuous update and prompt training of interested Donors and Clinicians of Public and Private Health Centers and Hospitals, about the progenitor cells applications.
• Immediate availability of the stored samples 365 days a year, 7 days a week, 24 hours a day.
• Complete attunement with the specifications set by the European Accreditation Organization for cellular therapy (FACT/JACIE/NETCORD), the American Association of Blood Banks (AABB) and the relevant Greek Presidential Decretal 2004/23 for the tissue/cell banks, with the use of cGMP methods.
• Control of Plasticity of progenitor cells based on the detailed Validation Master Plan of the Standard Operating Procedures (SOPs) regarding their hematopoietic origin (Methocult).
• Fully Automated Viability Control of the cryopreserved progenitor cells with automatic luminometric device, which increases the reproducibility and the accuracy of the results in contrast to the common laboratory techniques for detecting dead cells microscopically (Trypan Blue staining).
• Validated Molecular Diagnostics service provision (detection of HIV1/2, HBV, HCV, CMV, Syphillis, Toxoplasma) applying Real-Time PCR technology (Roche LightCycler).
• Validated ex-vivo cellular and/or tissue expansion of the recently processed cells/tissues, as well as of the cryopreserved cell/tissues in advanced technology Bioreactors, created in the NASA research laboratories, offering unique micro-gravity conditions in alternating electromagnetic field! Cellgenea is the UNIQUE company in Greece able to expand and to use ex-vivo expanded cord blood and adult progenitor cells for therapeutic reasons and clinical trials thanks to the relevant know-how transfer from the research laboratories of NASA and Regenetech Biotechnology Company.
• Storing of progenitor cells in two-chambered bags and cryovials in complete (24 hours a day) controlled and automated liquid nitrogen tanks.
• Continuous control and cross-tracking of the cryopreserved samples with validated LIS-ERP software which ensures the ability to track down and to identify the sample during all the steps of its supply, the processing, the control, the storage and the distribution. The tracking is also used for controlling and identifying all the relevant data about the products and the materials that come in contact with these samples (2004/23/ΕΚ).
• Immediate availability and distribution of the sample inside a validated cryotank, in case of therapeutic application.
• Strict security concerning personal data, confidentiality and safety according to the Personal Data Protection Agency.
• Complete bacteriological and serological control of the samples using the automatic analyzers BacT/ALERT and Architect i1000 without any extra rate.
• Life insurance provided to all the members of the different programs of preventive cryopreservation (Cellgenea, Dentogenea, Dermigenea, Angiogenea, DΝΑgenea, Neurogenea, Cardiogenea) in cooperation with insurance companies.
• DNA & pharmacogenetic control services for personalized treatment without side effects.

Abstract

Medicinal plants are used to address the twin problems of promoting sustainable livelihoods and treatment of numerous illnesses in Nigeria. The study examined the medicinal value of forest products in the treatment of cancer in South-west Nigeria. Primary data was obtained in a cross section survey of 327 respondents comprising 127 Traditional Medicine Practitioners (TMPs), 100 Orthodox Medicine Practitioners (OMPs) and 100 respondents from the General Public drawn by multistage sampling technique from the study area. Interview schedule was used in collection of data on the effectiveness of forest products in cancer treatment. The result showed that seven species were identified belonging to seven different families; Rutaceae, Asteraceae, Anarcardiaceae, Annonaceae, Meliaceae, Guttiferaceae and Leguminaceae topped the TMPs priority list. Result of economic analysis shows minimal competition in the anti-cancer forest product market and a high level of monopoly with a Gini coefficient of 0.83. The rate of return on investment was 180 .08% indicating that the TMPs were making profit. Five of the plants were tested against cancer cell lines MCF7 and Hs578T while Doxorubicin (a synthetic anticancer drug) was used as the control treatment. Three plants; Saccharum offinarum (Stem), Sucurinega virosa (Root) and Piper guineensii (Seed) produced no result; Garcinia kola (Bark) did not exhibit any anticancer effect even at a concentration of 10u1/m1 while only one plant species was effective against the cancer cell line at 1u1/m1. It is therefore concluded that forest products are effective in the treatment of cancer.

Keywords

medicinal plants; cancer; traditional medicine practitioners (TMPs); forest products and southwest Nigeria

Introduction

Medicinal plants are important for a number of reasons. A large proportion of the world’s rural population depends on these plants for their health care needs (Largo) [1]. They also provide the basic raw material for the production of traditional medicines (FAO, 1995, 2005) [2, 3]. The collection and processing of medicinal plants provide employment and income opportunities for a large number of people in rural areas (Marshall, et al.) [4]. The importance of traditional medicinal plants in conservation of biological diversity also merits attention (Okoli) [5].

WHO has been conducting studies on medicinal plants. These studies prompted the initial identification of 20000 species of medicinal plants and a more detailed investigation of a short list of 200 (WHO, 2002, WHO, 2006, Olopade, Odugbemi) [6-9] reported that a great number of these plants have their origins in the world’s tropical forests and their present use is largely rooted in traditional medicines which play a major part in maintaining the health and welfare of both rural and city dwellers in developing countries.

More than 60% of world’s total new annual cases occur in Africa, Asia and Central and South America. These regions account for 70% of the world’s cancer deaths. It is expected that annual cancer cases will rise from 14 million in 2012 to 22 million within the next two decades (IARC 2003, WHO 2008) [7,10]. Consequently, there is need to institute measures that will ensure the availability of anticancer forest products in the forest of Southwest Nigeria and ensure the sustainability of the practice of the TMPs who use forest products to treat cancer.

It has been estimated that as many as 75% to 90% of the world’s rural people rely on herbal traditional medicine as their primary health care (WHO, 2006) [11] and this is a source of income for the growers of such plants and the TMPs (USAID, 2013) [12]. African flora is potential for new compounds with pharmacological activities. Such efforts have led to the isolation of several biologically active molecules that are in various stages of development as pharmaceuticals.

The main objective of this study is to evaluate the economic and medicinal value of forest products in the treatment of cancer in southwest Nigeria, particularly Ogun State and the specific objectives are:

i. To determine the availability of medicinal plants used for the treatment of cancer in Southwest Nigeria.
ii. To determine the efficacy of some of the forest products used for the treatment of cancer in Southwest Nigeria.
iii. To investigate the stakeholders’ socioeconomic characteristics and their involvement in the usage of forest products for the treatment of cancer in Southwest Nigeria.
iv. To determine the factors that affect the income of the TMPs in the study area and the market structure of forest products used for the treatment of cancer in Southwest Nigeria.

Sampling Method, Sample Selection and Data Collection

Data sources and collection

For the purpose of data collection in this study, field trips, collection of available medicinal plant species used for the treatment of cancer, determination of their species type, oral interviews of Traditional Medicine Boards officials, administration of structured questionnaires on relevant target groups, that is, Traditional Medicine Practitioners (TMPs), Orthodox Medicine Practitioners (OMPs) and the General Public (GP) were carried out. Ethno medicinal surveys were also conducted in the study area for collection of data related to the medicinal use of forest products in the treatment of cancer in addition to the pharmacological screening of the plants to determine the level of their efficacy in the treatment of cancer and to validate the claims of the TMPs. To identify the locations with high concentration of TMPs in the Study Area, primary data were obtained through oral interviews of the officials of the Hospital Management Department of the Federal Ministry of Health, Federal college of Complementary and Alternative Medicine (FEDCAM), Abuja and the Nigeria Natural Medicine Development Agency, Lagos. Multistage sampling technique was employed. The South Western Nigeria was first stratified into six states to produce primary units namely: Ekiti, Lagos, Ogun, Ondo, Osun and Oyo. Out of these primary units, Ogun State was purposively sampled because of the high concentration of TMPs in the State (Figure 1).

Figure 1. Map of Southwest Nigeria
Inset: Lagos and Ogun States

Results

Availability of medicinal plants used for the treatment of cancer in South-Western Nigeria

Thirty eight species of Medicinal Plants were identified from the information supplied by the TMPs. (Table 1) shows the distribution of the species in relation to the source, availability status, parts of the plant used, form of the plant used, products and the species regeneration in the study area.

The life forms of these plants (Table 1) shows that the trees constituted the highest number (66%), followed by shrubs (20%), herb (11%) and rhizome (3%) In all, the family Leguminosae was dominant with 4 species. This was followed by Annonaceae, Anacardiaceae Euphorbiaceae, and Caesalpinioideae (3 species each).The existence of other plant families in (Table 3) demonstrates the rich forest diversity in Southwest Nigeria. This also shows the dynamism in ecosystem maintenance. A number of them also serve economic purposes and are consumed as food in one way or the other (Malik) [13]. Some of these include: Anacardium occidentalis, A, Mangiferaindica, Musa sapientum, Citrus medica, Vernoniaamygdalina, etc.

(Table 1) show that majority of the TMPs source their medicinal plants from free areas and rarely cultivate them. Table 1 shows that some of the plants are already scarce and species regeneration is by wilding. According to the reports by Gbile et al. [14] and Oguntala et al. [15] the Nigerian ecosystems are at greater risk of extinction if urgent attention is not given to the cultivation of medicinal plants. Table 1 shows that 90% of the TMPs use the whole plant for treatment that is, they make use of the fruits, stems, barks and leaves at the same time. Table 1 also shows that the forest products used for the treatment of cancer are multipurpose; they are used as firewood, medicine, foods, chewing sticks and animal feeds (Agerantum conyzoides).This corroborate the works of Adekunle [16].

Table 2 projects the second objective of this work, it shows that 90% of the TMPs use the green and dry forms of the forest products; afterwards they use water to soak or boil them. Also, using water the TMPs make juices from plants like Citrus medica, Morinda lucida, Vernonia amygdalina, Sida acuta and Agerantum conyzoides. Table 2 shows that 65% of the TMPs administer their medications twice daily while 23% of the TMPs adopt the thrice daily dosage. This helps to ensure frequent interactions and effective communication between the TMPs and their clients unlike the orthodox physicians. This was also reported by Adodo in 2003, 2004 and 2005 [17-19, 20]. Weekly wash is employed by 14% of the TMPs.

Inferential Statistics Results for TMPs in Southwest Nigeria

Inferential Statistics is used to further achieve objectives three and four. Table 3 is the result of the regression analysis showing the relationship between the profit of the Traditional Medicine Practitioners (TMPs) and their demographic data. Three (3) functional forms of production model including linear, semi-log and Cobb-Douglas (double-log) functions were fitted for the regression analysis. This was done to select the function which gave the result with the best fit. The estimated functions were evaluated in terms of the statistical significance of the coefficient of multiple determination (R2) as indicated by F value, the significance of the coefficients and the magnitude of the standard errors. The R2 is the coefficient of multiple determinations which measures the extent to which the variation in the dependent variable is explained by the explanatory variables. The F-value measures the goodness of fit of the model. Based on these statistical and economic criteria, Cobb-Douglas functional form was selected as the lead equation. The coefficient of multiple determination (R2) obtained for the Cobb-Douglass, that is, 0.437 shows that 43.7% of the variation in the profit of the TMPs were explained by the included explanatory variables, while the remaining 56.3% unexplained was due to the variables not included in the model which was the error term. Number of patients received, total cost of production, age of the practitioners and their years of experience are the significant factors influencing the profit of the practitioners; each of these variables has positive sign, which suggests that an increase in these variables would lead to an increase in the profit of the practitioners.

Table 4 gives the regression analysis result showing the relationship between the profit of the Traditional Medicine Practitioners (TMPs) and some selected variables other than the demographic data of the practitioners. Number of patients per year, duration of treatment, remedy shelf-life, daily application, and time of harvest are shown to have significant positive influence on the profit of the TMPs, which suggests that an increase in these variables would lead to an increase in the profit of the TMPs. However, number of people referred is shown to have a significant negative influence on the profit suggesting that the more that number of people referred by the TMPs the lesser their profits just as it would be expected.

Table 5 is the result of the t-test analysis showing comparison of some selected parameters of the Traditional Medical Practitioners (TMPs) and the Orthodox Medical Practitioners (OMPs). The result shows that there is significant difference in the number of patients recovered, number of deaths recorded, number of referral and the cost of production between the two groups of practitioners with the mean values estimated as follows: number of patients recovered – TMPs (11.92), OMPs (1.99); number of deaths recorded – TMPs (1.75), OMPs (6.61); number of referral – TMPs (3.32), OMPs (8.26) and cost of production – TMPs (N17,246.58), OMPs (N106,750.00). However, the result shows that there is no significant difference in the number of patients treated by the two groups of practitioners.

Result of the economic analysis shows minimal competition in the anti-cancer forest product market and a high level of monopoly with a Gini coefficient of 0.83 (Table 8). Net profit was N650,769.98 (Table 7).Table 7 also shows Rate of Return (280.08%) and the Rate of Return on Investment (180.08%)indicating that the TMPs are making profit.

Table 9 shows the test result against cancer cell lines Hs578T while Doxorubicin (a synthetic anticancer drug) was used as the control treatment. Garcinia kola (Bark) did not exhibit significant anticancer effect even at a concentration of 10u1/m1 while Erythropleum sauveoleons was effective against the cancer cell line at 1u1/m1.i

Table 10 shows the Test result against cancer cell lines MCF7 while Doxorubicin (a synthetic anticancer drug) was used as the control treatment. Garcinia kola (Bark) did not exhibit significant anticancer effect even at a concentration of 10u1/m1 while Erythropleum sauveoleons was effective against the cancer cell line at 1u1/m1.i

Conclusion

Forest products are effective in treatment of cancer; therefore inorder to achieve the millennium development goals on health; there is need for government to ensure the uniformity of herbal medicine practices. Factors such as, sources and identity of the plant, physical characteristics, chemical constituents, the pharmacological and biological activities of the crude drug and method of preparation, uses and storage, amongst others, need to be identified and documented. This study has justified the importance of plant species in the maintenance of ecosystem and as a source of livelihood for man.

References

• Largo M (2014) The Big, Bad Book of Botany: The World’s Most Fascinating Flora Out now from William Morrow, an imprint of HarperCollins Publishers. Slate’s animal blog.
• FAO (1995) Report of the International Expert Consultation on NTFP. Rome.
• FAO (2005) The Support Role: The Use of Forest Resources in other Production Sectors. World Bank Publication.
• Marshall E, Newton AC, Schreckenberg K (2003) Commercialisation of non-timber products: First steps in analysing the factors influencing success. International Forestry Review 5:128–137.
• Okoli RI, Aigbe O, Ohaju-Obodo JO,  Mensah JK (2007)  Medicinal herbs used for managing some common ailments among esan people of edo state, Nigeria. Pak J Nutr 6: 490-496.
• WHO (2007) Cancer control: knowledge into action: WHO guide for effective programmes: early detection.
• WHO (2008) The global burden of disease: 2004 update.
• Olapade EO (2002) The herbs for good health. The 50thAnniversary Lecture of University of Ibadan. Nature cureseries Vol 3.230p.
• Odugbemi T (2008) A Textbook of Medicinal Plants from Nigeria. University of Lagos Press, Lagos, Nigeria.
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• WHO (2006) Report on traditional medicine, my documents/ WHO traditional medicine.
• USAID (2013) Nigeria biodiversity and tropical forests 118/119 assessment. USDA Forest Service Office of International Programs,
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Commentary Article

Uterine sarcomas comprise a group of rare tumors with differing tumor biology, natural history and response to clinical treatment. Diagnosis is often made following surgery for presumed benign disease. Currently pre-operative imaging does not reliably distinguish between benign leiomyomas (LMAs) and other malignant pathology. Human uterine leiomyosarcoma (Ut-LMS) is neoplastic malignancy that typically arises in tissues of mesenchymal origin. The identification of novel molecular mechanism leading to human Ut-LMS formation and the establishment of new clinical therapies has been hampered by several critical points. Our research group earlier reported that mice with a homozygous deficiency for Proteasome beta subunit (Psmb)9/ β1i, an interferon (IFN)-γ inducible factor, spontaneously develop Ut-LMS. The use of research findings obtained from the research experiments with mouse model has been successful in increasing our knowledge and understanding of how alterations, in relevant oncogenic, tumor suppressive, and signaling pathways directly impact sarcomagenesis. The IFN-γ pathway is physiological important for control of tumor progression, and has been implicated in several malignant tumors. In this study, the experiments with human clinical materials revealed a defective expression of PSMB9/β1i in human UtLMS that was traced to the IFN-γ pathway and the specific effect of somatic mutations of Janus kinase (JAK) 1 molecule and/or promoter region on the locus cording PSMB9/β1i gene. Understanding the biological characters of human Ut-LMS may lead to identification of new diagnostic candidates or therapeutic targets against human Ut-LMS.

Uterine mesenchymal tumors have been traditionally divided into benign tumor leiomyoma (LMA) and malignant tumor, i.e. leiomyosarcomas (LMS) based on cytological atypia, mitotic activity and other criteria. Uterine LMS (Ut-LMS), which is some of the most common neoplasms of the female genital tract, is relatively rare uterine mesenchymal tumor, having an estimated annual incidence of 0.64 per 100,000 women [1]. They account for approximately one-third of uterine sarcomas, of only 53% for tumors confined to the uterus [2, 3]. Generally, patients with Ut-LMS typically present with vaginal bleeding, pain, and a pelvic mass. Gynecological tumors, e.g. breast cancer and endometrial carcinomas, are strongly promoted by female hormones, but the rate of expression of hormone receptor in human Ut-LMS is reported to vary in comparison with normal myometrime. Importantly, in case of elder patients, low expressions of hormone receptors were found to unclearly correlate with the promotion of initial disease or with the overall survival of patients with Ut-LMS.

As Ut-LMS is resistant to chemotherapy and radiotherapy, and thus surgical intervention is virtually the only means of clinical treatment for this malignant tumor, however, molecular targeting therapies against tumors have recently shown remarkable achievements [4-8]. It is noteworthy that, when adjusting for stage and mitotic count, human Ut-LMS has a significantly worse prognosis than carcinosarcoma; developing an efficient adjuvant therapy is expected to improve the prognosis of the disease [9]. A trend towards prolonged diseasefree survival is seen in patients with matrix metalloproteinase (MMP)-2-negative tumors [10]. Although typical presentations with hypercalcemia or eosinophilia have been reported, this clinical abnormality is not an initial risk factor for human Ut-LMS. To the best of our knowledge, little is known regarding the biology of human UtLMS; therefore, the risk factors that promote the initial development of human Ut-LMS and regulate their growth in vivo remain poorly understood.

The mice with a targeted disruption of proteasome beta-subunit 9 (PSMB9)/β1i, which is interferon (IFN)-γ-inducible proteasome subunit, exhibited a defect in tissue- and substratedependent physiological function of immune-proteasome, and female PSMB9/ β1i-deficient mice shown to develop Ut-LMS, with a disease prevalence of 37% by 14 months of age [11,12]. Defective expression of PSMB9/β1i is likely to be one of the risk factors for the development of human Ut-LMS, as it is in PSMB9/β1i-deficient mice [12]. Recent report shows that stable expression of PSMB9/β1i contributes to cell proliferation, which directly correlates to the progressive deterioration with increasing stage and the tumor aggressive grade. As the importance and involvement of the IFN-γ pathway for the activation of shared-promoter of PSMB9/β1i and the transporter associated with antigen processing (TAP) 1 have been established, it is demonstrated that the defective expression of PSMB9/β1i is attributable to G871E somatic mutation in the adenosine triphosphate (ATP)-binding region of JAK1 molecule in SKN cell line, which is established from patient with Ut-LMS. It is furthermore likely that the expressions of PSMB9/β1i are significantly down regulated in human Ut-LMS tissues such like human Ut-LMS cell line. Our research group demonstrates that there are serious mutational defects in the factors on the IFN-γ pathway, which is the key signal cascade for PSMB9/β1i expression and promoter region of PSMB9/β1i gene, in human Ut-LMS. The somatic mutational defects in the IFN-γ pathway may induce the initial development of Ut-LMS. Recent advances in our understanding of the biological characters of Ut-LMS have concentrated on the impaired IFN-γ pathway. It is clear that somatic mutations in key regulatory genes alter the behavior of cells and can potentially lead to the unregulated growth seen in malignant tumor. Therefore, continued improvement of our knowledge of the molecular biology of Ut-LMS may ultimately lead to novel therapies and improved outcome.

The effects of IFN-γ on expression of PSMB9/β1i was examined using five cell lines [13]. Expressions of PSMB9/β1i were not markedly induced by IFN-γ treatment in human Ut-LMS cell lines, although cervical epithelial adenocarcinoma cell lines and normal human uterus smooth muscle cells underwent strong induction of PSMB9/β1i following IFN-γ treatment [13]. Furthermore, the immunohistochemistry (IHC) experiments revealed a serious loss in the ability to induce expression of PSMB9/β1i in human Ut-LMS tissues in comparison with normal myometrium tissues located in same tissue sections and 4 various mesenchymal tumor types. Of 58 Ut-LMS, 50 cases were negative for PSMB9/β1i, 4 cases were focally positive, 2 cases were weakly positive, and 2 cases were positive. IHC analyses showed positivity for ki-67/MIB1 and differential expression of estrogen receptor (ER), progesterone receptor (PR), tumor protein 53 (TP53), and calponin h1. In addition, the expression level of PSMB9/β1i was also examined in the skeletal muscle metastasis from human Ut-LMS, the histological diagnosis was consistent with metastatic LMS for skeletal muscle lesions. Pathological study of surgical human samples showed presence of a mass measuring 3 cm at largest diameter in lumbar quadrate muscle without a fibrous capsule. All lymph nodes were negative. In western blotting and RTPCR experiments, PSMB9/β1i was expressed in normal myometrium, LMA, and IFN-γ-post-treated HeLa cells, but not in human Ut-LMS.

The both research experiments strongly supported the research findings obtained from IHC experiments.

Most frequently, human Ut-LMS have appeared in the uterus, retroperitoneum or extremities, and although histologically indistinguishable, they have different clinical courses and chemotherapeutic responses. The molecular basis for these differences remains unclear, in addition, physiological significance of mutational defect is reportedly associated with progression of malignant tumors. Therefore, the molecular examinations of 23 human Ut-LMS tissue regions and normal tissue regions located in the tissue sections obtained from individual patients were performed to detect somatic mutations in the IFN-γ pathway, i.e. JAK1, JAK2, signal transducer and activator of transcription 1 (STAT1) and promoter region of PSMB9/β1i gene (Figure 1). As the catalytic domains of these IFN-γ signal molecules are most likely to harbor mutations that inactivate the gene product, we focused on stretches (exons) containing the kinase domains, transcriptional activation domains and enhancer/ promoter region. Over all, nearly 43.5% (10/23) of human Ut-LMS tissues had serious mutations in the ATP binding region or kinasespecific active site of JAK1; furthermore, 43.5% (10/23) of human Ut-LMS tissues had serious mutations in transcriptional activation sites of the promoter region of PSMB9/β1i gene, which is required for transcriptional activation of PSMB9/β1i gene. No somatic mutation in essential sites, e.g. Tyr701 and Ser727, which are required for physiological function of STAT1 as transcriptional activator, was elucidated in human Ut-LMS. Nearly 21.7% (5/23) of human Ut-LMS tissues unexpectedly had somatic mutations in the intermolecular region of STAT1, which is not yet reported to be important for biological function as transcriptional activation. No somatic mutation in the ATP-binding region and kinase-active site of JAK2 molecule was detected in human Ut-LMS. MOTIF Search profiling [14] and NCBI’s Conserved Domain Database and Search Service, v2.17 analysis also revealed that somatic mutations, which were identified in the catalytic domains of these genes, resulted in impaired physiological functions of tyrosine kinases or transcriptional factor [15]. In a recent report, a comparative genomic hybridization (CGH)-based analysis of human Ut-LMS using a high resolution genome-wide array gave genome-level information about the amplified and deleted regions that may play a role in the development and progression of human Ut-LMS. Other reports showed that among the most intriguing changes in genes were losses of JAK1 (1p31-p32) and PSMB9/β1i (6p21.3) [16,17]. It has also been demonstrated that a correlation exists between the development of malignant tumors and ethnic background, so we conducted CGH experiments with tissue samples obtained from Japanese patients in order to obtain genome-level information. Our results showed that human Ut-LMS having a clear functional loss at JAK1 (1p31-p32) and PSMB9/β1i (6p21.3) also harbored one nonsense mutation and one deletion, suggesting a possible homozygous loss of function. The discovery of these mutational defects in a key signal pathway may be important in understanding the pathogenesis of human Ut-LMS.

Uterine LMS are relatively rare mesenchymal tumors, having an estimated annual incidence of 0.64 per 100 000 women. They account for approximately one-third of uterine sarcomas and 1.3% of all uterine malignancies. They are the disease with extremely poor prognosis, considering aggressive malignancies with a 5-year survival rate of only 50% for tumors confined to the uterus. At present, surgical intervention is virtually the only means of treatment for Ut-LMS [4- 8]. Although adjuvant pelvic irradiation appears to decrease the rate of local recurrence, adjuvant therapy does not appear to significantly improve survival. Furthermore, gynecological cancer, for instance breast cancer and endometrial carcinomas, are strongly promoted by female hormones, but the rate of expression of estrogen receptor and progesterone receptor is reported to vary in human Ut-LMS compared with normal myometrium. In case of elder patients, low receptor expressions were found to not correlate with the promotion of initial disease or with the overall survival of patients with Ut-LMS; however, molecular targeting therapies against tumors have recently shown remarkable achievements [18]. To improve the prognosis of human Ut-LMS, research experiments were performed to identify the key role of pro- or anti-oncogenic factors that have an important function in their pathogenesis and that could serve as molecular targets for tumor treatment. For this purpose, several research facilities conducted a microarray procedure between human UtLMS and normal myometrium and showed that several known prooncogenic factors, such as brain-specific polypeptide PEP-19 and a transmembrane tyrosine kinase receptor, c-KIT, may be associated with the pathogenesis of human Ut-LMS [19-21]. However, in terms of the sarcomagenesis of human Ut-LMS, merely comparing the expression of potential pro-oncogenic factors between normal and malignant tissues is not sufficient because the results obtained may be the consequence of malignant transformation and, therefore, not necessarily the cause. In addition, dysregulation of apoptotic cascade has also been implicated in many human malignancies. Although the significant differential expression of apoptotic and cell cycle regulators in human Ut-LMS, such as B-cell Lymphoma-2 (BCL-2), BCL-2- Associated X protein (BAX), p16 inhibits CDK4 (P16/INK4a), p21 cyclin-dependent kinase inhibitor 1 (P21/CIP1), p27 kinase inhibitor protein 1 (P27/KIP1), cellular v-KIT Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (c-KIT), mitogen-inducible gene-2 (MIG-2), MDM2, tumor protein 53 (TP53), have all been reported and compared to normal myometrium, there exists no scientific evidence to show that abnormal expression of these factors directly correlates to the initiation and promotion of human Ut-LMS. PSMB9/β1i-dificient mice were reported to be prone to the development of Ut-LMS, but not in their parental mice, C57BL/6 mice [12]. The percentage of mice with overt tumors increased with age after six months, with a cumulative prevalence of Ut-LMS in female mice of 37% by 14 months of age and no apparent plateau at this late observation time. Histopathological examinations of PSMB9/β1i-deficient uterine neoplasms revealed common characteristic abnormalities of Ut-LMS. In addition, recent research reports show the loss in the IFN-γ-inducible ability of PSMB9/β1i expressions in SKN cell line and other primary Ut-LMS cells established from patients. The histopathological experiments revealed serious loss in the ability to induce the expression of PSMB9/ β1i in human Ut-LMS tissues in comparison with normal myometrim tissues located in same tissue sections. IFN-γ treatment markedly induced the expression of PSMB9/β1i, a subunit of the proteasome, which alters the proteolytic specificity of proteasomes. Sequence analysis demonstrated that the loss of IFN- γ responsiveness in the human Ut-LMS cell line was attributable to the inadequate kinase activity due to a G781E somatic mutation in the ATP-binding region of JAK1 molecule [13]. The defect was localized to JAK1 activation, which acts upstream in the IFN-γ pathway since IFN-γ treatment could not strongly induce JAK1 kinase activity in human Ut-LMS cell lines. Genetic alterations in tyrosine kinases have previously been firmly implicated in tumorigenesis, but only a few serine/threonine kinases are known to be mutated in human malignant tumors [22-27]. For instance, mice carrying homozygous deletion of Phosphatase and tensin homolog deleted from chromosome 10 (Pten) alleles developed widespread smooth muscle cell hyperplasia and abdominal LMS [28], and JUN oncogene amplification and over-expression block adipocytic differentiation in highly aggressive sarcomas. Most frequently, LMS have appeared in the uterus, retroperitoneum or extremities, and although histologically indistinguishable, they have different clinical courses and chemotherapeutic responses. The molecular basis for these differences remains unclear, therefore, the examination of human Ut-LMS tissues (23 Ut-LMS tissue sections and normal tissue sections located in the same tissue) was performed to detect somatic mutations in the IFN-γ signal molecules. In a recent report, highresolution genome wide array comparative genomic hybrydization (CGH) analysis of human Ut-LMS cases gave gene-level information about the amplified and deleted regions that may play a role in the development and progression of human Ut-LMS. Among the most intriguing genes, whose copy number sequence was revealed by CGH, were loss of JAK1 (1p31-p32) and PSMB9/β1i (6p21.3) [16,17]. The discovery of these mutational defects in a key cell-signaling pathway may be an important development in the pathogenesis of human UtLMS. The growth of JAK1-deficient cell lines is reportedly unaffected; similarly, the cell cycle distribution pattern of freshly explanted tumor cells derived from JAK1-deficient tumors shows no response to IFN-γ signaling [29]. The growth of the original SKN cells, which had defective JAK1 activity, was unaffected by IFN-γ treatment. In contrast, the growth of JAK1-transfected SKN cells, which had strong exogenous JAK1 activity, was prevented by IFN-γ treatment. Interestingly, when PSMB9/β1i-transfected SKN cells, which have marked the expression of PSMB9/β1i, were analyzed, expression of exogenous PSMB9/β1i resulted in cell growth inhibition. Conversely, the growth of PSMB9/β1i-transfected SKN cells was unaffected by IFN-γ signal pathway. Taken together, IFN-γ response to cell growth inhibition may be attributable to the physiological significance of PSMB9/β1i.

In conclusion, it is clear that in this challenging clinical group of diseases early recognition and diagnosis of human Ut-LMS is critical in order to improve patient outcomes. The down regulation of expression of major histocompatibility complex (MHC)-related factors, including the TAP1 and PSMB9/β1i genes, is one of the biological mechanisms tumor cells use to evade host immune surveillance [30- 32]. Recently, the incidence of IFN-γ unresponsiveness in human tumors was examined in several malignant tumors, and revealed that approximately 33% of each group exhibited a reduction in IFN-γ sensitivity [33]. Nevertheless, the expression of PSMB9/β1i, rather than providing an escape from immune surveillance, seems to play an important role in the negative regulation of human Ut-LMS cell growth. Defective expression of PSMB9/β1i is likely to be one of the risk factors for the development of human Ut-LMS, as it is in the PSMB9/β1i-deficient mouse. Thus, gene therapy with PSMB9/β1i expression vectors may be a new clinical treatment for Ut-LMS that exhibits a defect in the expression of PSMB9/β1i. Because there is no effective therapy for unresectable human Ut-LMS, our results may bring us to specific molecular therapies to treat this disease [34-39].

Disclosure: The Authors report no conflicts of interest.

Acknowledgments

We sincerely appreciate the generous donation of PSMB9/β1ideficient breeding mice and technical comments by Dr. Van Kaer L, Vanderbilt University Medical Center. We thank Isamu Ishiwata for his generous gift of the Ut-LMS cell lines. This work was supported by grants from the Ministry of Education, Culture, Science and Technology, the Japan Science and Technology Agency, the Foundation for the Promotion of Cancer Research, Kanzawa Medical Research Foundation, and The Ichiro Kanehara Foundation.

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Introduction

Malignant acrospiroma (MA) – is rather rare tumor with eccrine ductal and secretory differentiation, including clear cell component. This type of tumor appears more often in elderly people (average male patient age is 51 years, female patient – 55 years). The tumor corresponds to solitary intradermal, exophytic or mixed type node 0.5-2 cm or more in diameter, hemispheric, tight-elastic texture, on wide base, covered with unchanged skin, sometimes with ulcers. Small percent of cases have clear discharge from tumor. Considering the same architecture of malignant nodular hydradenoma and benign analogue, it is difficult to find the difference between them, although undisputed manifestation of malignance is vessel and perineural invasion, lymphogenic metastasis.

The tumor is very aggressive and liable to metastasis. It is important to notice that there are no direct histological and clinical signs, predicting biological behavior of the tumor. Despite chemotherapy, patients with metastasis has fatal outcome vary rapidly. There no proved data of chemotherapy impact on malignant acrospiroma.

Description of MA clinical case

Patient, 58 years, applied to Dnipropetrovsk Multi-field Clinical hospital #4 in Jan 2017 to the Department of Oncology and Medical Radiology of Dnipropetrovsk Medical Academy with complaints to fatigue and tumor formation.

Anamnesis

Patient consider himself sick since Autumn 2016, when above mentions complaint appeared for the first time. Incisional biopsy was performed in Nov 2016, and then the patient was sent to the Department of Oncology and Medical Radiology of the Dnipropetrovsk Multiffield Clinical Hospital #4

Pathohistological conclusion as of 16 Nov 2016:

Tumor has the structure of malignant eccrine acrospiroma with ulceration.

Life history

Catarrhal diseases are 1-2 times per year. No addictions. No allergies. No hemotrasfusions, traumas and surgeries. Diabetes mellitus type II, more than 2 years. No cancer family history.

Physical examination

Formation in left parietal region, round in shape with indeterminate boundaries, 2 cm in diameter, with tissue lysis and perifocal inflammation.

Investigation data

1. Pathohistological conclusion №10291-96/16 as of 16 Nov 2016: Tumor has the structure of malignant eccrine acrospiroma with local ulceration.

2. X-ray assessment of the chest as of 11 Jan 2017: Radiological signs of metastasis in lungs.

3. Endocrinologist assessment as of 13 Jan 2017: Diabetes mellitus type II, compensated.

4. Therapeutist assessment as of 13 Jan 2017: Essential hypertension II gr. Coronary heart disease: atherosclerotic cardiosclerosis, Heart failure I.

5. CT assessment of chest, abdominal cavity and pelvis as of 17 Jan 2017: CT-signs of multiple secondary changes on lungs, increased mediastinal lymph nodes, metastasis in liver S4. Cholelithiasis. Concernments in right kidney. Mass lesion in left lobe of thyroid gland.

6. CT assessment of brain as of 17 Jan 2017: CT-signs of encephalopathy.

7. Echocardiography as of 20 Jan 2017: Satisfactory myocardial contractility, LVEF – 60%.

8. Punctate from thyroid gland as of 26 Jan 2017: Accumulation of polymorphic cells of follicular epithelial tissue, stroma elements.

9. CA results as of 20 Jan 2017: 19.05 U/mL.

10. Hematology as of 13 Jan 2017

– HB 120 g/L

– RBC 4.08 × 1012/L

– WBC 7.56 × 109/L

– SOE 28 mm/h

– lymphocytes 28.7 %</p

Hematology was done 2 days before chemotherapy and 5 days after. Patient had chemotherapy-induced leucopenia and neutropenia grade I-II CTC AE, recovered after drug administration. No chemotherapy cycles delayed.

11. Hematology as of 03 Jul 2017

– HB 134 g/L

– RBC 4.58 x 10^12/L

– WBC 5.34 x 10^9/L

– SOE 8 mm/h

– lymphocytes 28.7 %

– monocytes 6%

12. Chemistry as of 13 Jan 2017

– total bilirubin – 7.3 mmol/L

– ALT – 21 IU/L

– AST – 23 IU/L

– total protein – 55.8 g/L

– alkaline phosphatase – 74 IU/L

13. RW as of 13 Jan 2017 – negative

14. AID as of 13 Jan 2017 – negative

15. Hepatitis B and C as of 13 Jan 2017 – negative

Diagnosis

Malignant acrospiroma, T2N0M1 stage IV (mts in lungs and liver), clinical group 2

Clinical case was discussed on board of doctors. Chemotherapy was prescribed.

Received treatment:

1. Carboplatin – VISTA AUG 6

2. Docetaxel – VISTA 75 mg/m2 IV

3. Fluorouracil – VISTA 500 mg/m2 IV

Visual clinical dynamics in the course of the treatment.

Recommendation at discharge

1. Family doctor supervision

2. Control blood analysis in 7-21 days.

3. CT assessment of chest, abdominal cavity and pelvis after 3months:

Complete diagnosis at discharge

Malignant acrospiroma, T2N0M1 stage IV (mts in lungs and liver), condition after non-radical surgery, after 8 cycles of chemotherapy clinical group II.

Patient had positive dynamics after chemotherapy treatment, complete response as per RECIST 1.1. Patient had not complaints.

CT assessment of chest, abdominal cavity and pelvis with IV contrast as of 28 Jun 2017: CT-signs of stable size of thyroid gland left lobe formation, positive dynamics due to disappearance of lesions in lungs, mediastinal lymph nodes, liver lesions. No new lesions.

Patient has fully active lifestyle, and he is under family doctor and oncologist supervision [Figure 1].

Figure 1. Clinical Case of Malignant Acrospiroma

Abstract

Aim: The aim of this retrospective study the effect of body mass index on the efficiency of treatment of breast cancer, improve treatment outcomes for breast cancer by individualization of treatment measures taking into account the characteristics of the metabolism of the patient.

Keywords:

body mass index, breast cancer, obesity, overall survival

Background

The incidence of breast cancer in the world in general and in Ukraine in particular is growing. In 2015, in Ukraine the incidence reached 70.0 per 100 thousand female populations.

According to the Ministry of Health in Ukraine 26% of the female population for 2015 was overweight or obese. Obesity – a chronic metabolic character, which is the result of the interaction of the endogenous factors, environmental conditions and lifestyle. Endogenous factors could be considered a violation of the genetic and hormonal balance. The external conditions include irregular rhythm nutrition, use of substandard products. By disorders include sedentary lifestyle lifestyles.

Obesity is the first risk factor for metabolic syndrome, diabetes type II, cardiovascular disease and some forms of cancer, including breast cancer.

Since overweight is a risk factor for breast cancer, there is reason to believe that among patients with breast cancer the percentage of obese women is higher than in the population. The risk of breast cancer in postmenopausal women by 30% more than in premenopausal, women with obesity – 50%. Furthermore it was proven that obesity is associated with poor prognosis in patients with breast cancer, regardless of menopausal status. [1]

The leading role in achieving long-term results of treatment with systemic methods, such as chemotherapy or hormone therapy. The purpose of systemic therapy is the eradication of micro metastases in the case of radical surgical treatment or reduction of tumor load in case of treatment of locally advanced or metastatic cancer. The calculation of the dose of chemotherapy conducted mainly in the area of the body. [2] Thus to avoid complications associated with overdose of chemotherapy, the standard practice is to calculate the dose of 2.0 m2 patients whose body area more than this. Preparations hormonal action used in standard dosage for an adult without constitutional features. Along with this recent literature there is information that women are overweight effectiveness of systemic treatments may be lower than expected. Other data refute this information. [3]

In view of the above, the study on the impact of body mass index on the effectiveness of systemic treatment for breast cancer is an actual scientific problem and promising area of research.

Overexpression of Her-2/neu in ER-positive breast cancer cells can cause Tamoxifen to behave as an agonist and stimulate cell growth. Implicit in this mechanism for resistance is cross-talk activation between the ER and the epidermal growth factor receptor (EGFR/ Her-2/neu) pathways [3]. Treatment with various signal transduction inhibitors has been used in combination with endocrine therapy to overcome resistance, such as Gefitinib, which targets the internal tyrosine kinase domain of EGFR, and Trastuzumab, which blocks the external domain of Her-2/neu [4].

Recently, complementary and alternative medicine (CAM) is widely accepted among patients with breast cancer, which may provide several beneficial effects including reduction of therapy-associated toxicity, improvement of cancer-related symptoms, fostering of the immune system, and even direct anticancer effects [5]. Carnitine is a trimethylated amino acid, naturally synthesized in the liver, brain and kidney from protein-bound lysine and methionine. Several factors such as sex hormones and glucagon may impact on Carnitine distribution and level in tissues [6]. L-Carnitine plays an important role in cell energy metabolism through mediating the transport of long chain fatty acids across the inner mitochondrial membrane. Carnitine has a modulating effect on the function of acetylcholine excitatory neurotransmitter, glutamate excitatory amino acid,insulin growth factor-1 (IGF-1) and nitric oxide (NO). L-Carnitine may have a dual protective effect by enhancing the energy dynamics of the cell and inhibiting cell membrane hyper excitability [15], which make it an ideal nutrient for cancer prevention and treatment [7]. ex hormones, especially estrogens, have been implemented in the development of breast cancer. Breast cancer risk increases after menopause, where aromatization of androgens to estrogens in adipose tissue is the most important source of estrogen in blood and peripheral tissues [11]. Weight increase and obesity subsequent to menopause have been identified as the most important risk and negative prognostic factors for breast cancer in postmenopausal women. Obesity results in increased circulating levels of insulin and insulin-like growth factor, which by acting as mitogens for epithelial breast cells, stimulate their growth and neoplastic degeneration. Mechanisms may combine to explain the association which links together menopause, the subsequent body weight increase, and hormone-dependent breast cancer [12]. Body mass index of Letrazol-treated breast cancer patients included in the present study was positively correlated with estrogen level (E2) which is consistent. [11], who showed that the increased breast cancer risk seen in postmenopausal women with adiposity might be related to elevated sex hormone level.

Materials and Methods

The study included 754 patients with breast cancer between the ages of 30 and 77 (57.6 ± 1) years of age who were treated according to our clinic, department of oncology and medical radiology.

Dnipropetrovsk medical academy at Municipal Institution “Dnipropetrovsk City Multi-field Clinical Hospital #4”, Dnepropetrovsk state medical academy from 2005-2016. All patients were evaluated according to the following data: stage of the disease, age and BMI at the time of diagnosis, the size, histological type and metastases. IHC type, MRI methods, Bioelectrical impedance analysis, Ultrasounds analysis.

Tumor size was evaluated after measuring its maximal diameter and distributed in accordance with the International TNM-classification (7th edition, 2009). The histological type and degree of differentiation of the tumor was evaluated respectively by the National Standards of diagnostics and treatment of malignant neoplasms, reflecting the recommendations of leading international organizations. BMI is calculated by the formula: I = m×h2, where m – body weight (kg); h – height (m). According to these calculations the patients were divided in accordance with the WHO criteria into the following groups: those with a BMI 30 kg/m2 – obese. The material for the histopathological study was obtained during surgery. We examined the relative risk of relapse and death with regard to the BMI categories adjusting for eight factors known to be predictors of disease-free survival (DFS) and overall survival (OS): menopausal status, nodal status tumor size, vessel invasion, estrogen receptor (ER) status, progesterone receptor status, tumor grade and treatment regimens, ECOG.

By analyzing archival material to consider the particular response to systemic treatment of breast cancer women with deficiency of body weight, normal, high and overweight. Explore options for determining the individual characteristics of lipid metabolism of patients with breast cancer and their possible use for predicting the effectiveness of treatment. To determine the lipid metabolism will be applied anthropological research methods, bioimpedansnoho measurement, CT [13,14].

Results

In this retrospective study, among 754 patients with breast cancer, 45% were identified with excess body weight, and 31% – of various obesity degree. Patients with a BMI 30 kg/m2, 10 % more often associated with metastatic RLN, which is an indirect sign of higher metastatic potentials. Patients with normal BMI had significantly longer overall survival (OS) and disease-free survival (DFS) than patients with intermediate or obese BMI in pairwise comparisons adjusted for other factors. We found a strong correlation between obesity and lymph node involvement These observations suggest that obesity may potentiate the metastatic spread of breast tumors. Distant metastases were also found more often in obese patients in bone or visceral sites in patients <45 years of age at diagnosis. Patients with normal mass by IHC with triple negative cancer 45% and 20% with BRCA + and patients with obesity 55% that’s with IHC luminal A.B but 2 group receive L Carnitine in group with L carnitine by ECOG better and calendar Chemotherapy was as planed and less Adverse Advents than group Patients without support L Carnitine And less hematological complication.

Conclusions

In conclusion, this retrospective investigation of our patient demonstrates that BMI is an independent prognostic factor for OS in patients with breast cancer. We have supporting evidence that obese BMI represents a poor risk feature for outcome, especially in pre-/premenopausal patients, most of whom received chemotherapy without hormonal therapy.A lifestyle intervention reducing dietary fat intake, with modest influence on body weight, may improve relapsefree survival of breast cancer patients receiving conventional cancer management. Longer, ongoing nonintervention follow-up will address original protocol design plans, which requires 3 years of follow-ups after completion of recruitment. The prominent role of L–Carnitine in the present study belongs to the level of Her-2/neu, Ki67, which were significantly reduced after L-Carnitine supplementation. Thus, L-CAR as add on therapy to TAM, in addition to its ability to foster the immune system and improve the patients` fatigue and quality of life, may offer better cancer prognosis, which may be, in part, a prospective trial to overcome Chemotherapy and Letrazol resistance.

References

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• Samaddar JS, Gaddy VT, Duplantier J, Thandavan SP, Shah M, et al. (2008) A role for macroautophagy in protection against 4-hydroxytamoxifen-induced cell death and the development of antiestrogen resistance. Mol Cancer Ther 7: 2977-2987. [crossref]
• Baneshi MR, Warner P, Anderson N, Edwards J, Cooke TG, et al. (2010) Tamoxifen resistance in early breast cancer: statistical modelling of tissue markers to improve risk prediction. Br J Cancer 102: 1503-1510. [crossref]
• Johnston SR (2009) Enhancing the efficacy of hormonal agents with selected targeted agents. Clin Breast Cancer 9 Suppl 1: S28-36. [crossref]
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Abstract

Although much has been published on the domain structures of human alpha-fetoprotein (AFP), the AFP third domain (AFP-3D) has emerged as an important fragment regarding the binding, docking, and interaction sites for hydrophobic ligands, multiple receptors, ion channels, and cell cycle proteins. In keeping with previous reports, studies have shown beyond doubt that certain amino acid (AA) sequences on AFP-3D provide a docking interface for protein-to-protein interactions (complexing) for such proteins. By means of a computer software program designed to study such “in silico” interactions, certain AA sequences on AFP-3D were identified which could plausibly interact with a group of DNA damage-sensing and repair (DDSR) proteins. The DDSR proteins identified included: 1) BRCA1 and BRCA2 2) FANC1 and FANCD2 3) nibrin 4) ATM and ATR and 5) DNA-PK kinase. Following the mapping of the AFP-3D with DDSR protein interaction sites, the computer-derived AFP-AA identification sequences were examined for similarities and comparisons to previously reported ligand, receptor, channel and other protein interaction sites on AFP-3D. Literature searches revealed that the association of AFP with the DDSR proteins showed correlations not only with clinical serum AFP levels, but also with an intracytoplasmic nonsecreted form of AFP, which interacts with transcription factors, cell death (apoptosis) proteins, nuclear receptors, and enzymes (caspases). The DDSR proteins that interacted with AFP were also found to be involved with cell cycle checkpoint proteins, cyclins and their dependent kinases, and ubiquitin ligases. Finally, both the clinical and experimental reports on the AFP-3D association with DDSR proteins were consistent with the “in silico” findings of this report.

Key Words

Alpha-fetoprotein, DNA repair, BRCA proteins, chromosome instability, Fanconi anemia

Introduction

Human alpha-fetoprotein (HAFP) has a long history of clinical use as a tumor-associated biomarker, employed to detect both fetal defects during pregnancy and adult cancers.[1, 2] Moreover, much of the biochemistry of the HAFP polypeptide has been elucidated over the five decades since AFP was first discovered. HAFP is a single chain polypeptide with an average molecular mass of 69 kDa, depending on its carbohydrate micro-heterogeneity.[3, 4] The secondary structure of this oncofetal protein exhibits a triplicate domain molecular structure, configured by intramolecular loops dictated by 15 disulfide bridges culminating in a helical V- or U-shaped structure.[3] This fetal protein has been classified as a member of the albuminoid gene family, consisting of AFP, albumin, alpha-albumin, vitamin D binding protein, and the AFP-related (ARG) protein.[5] Similar to albumin, HAFP binds to a vast array of ligands, including various drugs, dyes, steroid hormones, heavy metals, flavonoids, fatty acids, and phytoestrogens.[6] Unlike albumin, AFP has proven to be a notable growth factor capable of either cellular enhancement or inhibition.[7].

HAFP is known to bind to multiple cell surface receptors and intracytoplasmic proteins. Recent reviews by the author (GJM) and others have reported the existence of at least three major groups of cell surface receptors, namely, 1) including the scavenger receptor protein family 2), the mucin glycoprotein superfamily, and 3) the chemokine receptor family of proteins.[8-10] The intracellular HAFP binding proteins encompass the a) retinoic acid receptor b) the caspases c) PI3K/AKT (protein kinase-A), d) mTOR e) GAAD153 and f) PTEN. [11, 12] During the last decade, the carboxy-terminal third domain of HAFP (AFP-3D) has been confirmed to be a major binding interface for both cell surface receptors and hydrophobic ligands. [13, 14] Furthermore, the AFP-3D has been touted as a promising agent (fragment) for the selective delivery of anti-cancer agents.[15- 17] Recombinant fragments of AFP-3D have been produced which demonstrate high purification yields, good efficiency of expression, recoverable refolding capabilities, and retention of biological activities. [18-20] In some instances, the AFP-3D recombinant fragment behaves similarly to full-length AFP, while maintaining its capabilities to bind cell surface receptors and intracytoplasmic proteins.[19]

The localization of additional protein binding and interaction sites on AFP-3D, other than the three major receptor and intracellular binding sites mentioned above, continues to be a topic of focus in the biomedical literature. The pursuit to identify additional protein binding/interaction sites on AFP-3D fragments has not abated. Activity sites of interest include receptor blockade and/or inactivation, decoy ligand binding, blunting receptor responses, selective delivery of drugs, and nucleotide agents (miRNAs) and other cargos that are transported into cancer cells or other targeted cells. Such participating cells include lymphoid/leukemic cells, monocytes, macrophages, T-cells, dendritic cells, and various bone marrow cells (stem cells). Thus, knowledge gained from such activities of AFP could conceivably make it possible to modulate, control, and monitor target site interactions and might affect, dictate, or influence signal transduction pathways.

Aims and Objectives

The aims of the present review and prospectus were to search out, identify and localize, and describe plausible sites of interaction of DNA damage-sensing and repair (DDSR) proteins on the AFP-3D fragment. To achieve these aims, computer modeling and molecular software were used to pinpoint sites of possible interaction between the AFP-3D fragment and various proteins of the DDSR protein pathways. The identified proteins and their respective AFP-3D amino acid docking sequences are discussed concerning their relevance to protein-to-protein binding interactions and possible outcomes for DNA repair. Computer modeling and analysis were also employed to compare the DNA-repair protein localized sites to the ligands, receptors, and protein interaction sites previously localized on the AFP-3D fragment. Members of the DNA-repair pathways identified by this process are addressed regarding their biological activities with other ligand and protein interaction sites on AFP-3D. Finally, prior experimental and/or clinical reports of AFP-derived peptide interactions with DNA-repair proteins are addressed in view of their present “in silico” localizations.

Computer Molecular Docking Software

The computer modeling and molecular docking interaction sites of the DDSR proteins were identified and localized by use of a proprietary computer software (Peptimer Discovery Platform) developed and generously provided by Serometrix, LLC (Pittsfield/Syracuse, NY). This software tool was described in detail in earlier publications.[8, 21, 22] Use of the software simulation of protein-to-protein interaction site localization has been repeatedly confirmed and validated by means of in vitro cell-based assays and microarray analyses including receptor binding kinetics. Previous experimental verifications of AFP- 3D interaction sites using this software simulation have included cell cycle proteins, scavenger receptors, immunodeficiency-associated proteins, chemokine receptors, selective and non-selective cation channels, and lysophospholipid and mucin receptors.

DNA Damage Sensing and Repair

Most, if not all cancer cells, have an unstable genome comprising DNA-damaged pathways. In fact, it is uncommon to find a single tumor without a genetic defect. Genomic instability arises either from losing telomeres from the end of a chromosome or from breaks in the DNA contained in the chromosome. After a cell has divided multiple times, its telomeres become critically short. Often, the cell either dies or stops growing, as in end-stage differentiation or aging. If the cell does not stop dividing (i.e., cancer), it leaves chromosomes with broken ends and DNA breaks in mid-chromosome regions. Such breaks are meant to be addressed by a DNA repair mechanism to restore the damage, but if neglected or bypassed, can lead to loss of gene function and a predisposition to cancer.

Since DNA damage can lead to cancer, the cell possesses an intrinsic repair response to DNA damage and to agents causing it. Many human cancers are related to mutations that affect proteins involved in a cellular DNA damage response. For example, DDSR protein mutations in ataxia telangiectasia-mutated (ATM) and Fanconi Anemia (FANCD2) genes [23] can be linked directly to a predisposition to both leukemias and lymphomas. Mutations in other DDSR proteins, such as p53, BRCA1, and BRCA2, can cause ovarian and breast cancers. Mutations in others, such as ATM kinase, are the root causes of chromosome instability in DNA repair disorders which lead to lymphoid and leukemic cancers. When nuclear DNA is damaged, cells rely on specific intracellular signaling pathways to halt cell division before the DNA is copied into another cell. Two such pathways are the cell cycle ATM-CHK2 (checkpoint-2) and the ATRCHK1 (checkpoint-1) pathways.

DNA Repair Proteins

1) The BRCA1 and BRCA2 Proteins

The breast cancer susceptibility genes BRCA1 and BRCA2 were the first breast cancer genes to be identified. BRCA1 and BRCA2 display autosomal inheritance, and the primary tumors are associated with female breast and ovarian cancers. Mutations in BRCA1 and BRCA2 proteins occur in 10-30% of women with germline alterations; such alterations inactivate the BRCA2 allele, while a second allele is inactivated by somatic mutations.[24, 25] Both the DDSR genes are known to participate in homologous recombination pathways and cell cycle control.[26] Interestingly, many of the characteristics of the BRCA2 protein are similar to the FANCD1 gene (see below), and BRCA1 proteins share biological effects common to both proteins. The FANCD1/BRCA2 and BRCA1 proteins interact by binding and forming multi-protein complexes with FANCN proteins, and these complexes function in the DNA repair pathways.[27] Moreover, the FANC and BRCA, RAD51, and CHEK2 proteins can work in concert as multi-protein complexes in the repair of DNA damage.

A) BRCA1. The BRCA1 gene is located on the long q-arm of chromosome 17, consisting of 1,863 amino acids, which encompasses four major domains including a 1) zinc finger (C3HC4 type) 2) nuclear localization signal 3) nuclear export signal motif and 4) BRCA1 C-terminus (BRCT) domain. There are six isoforms which are known to be associated with BRCA1. The human gene encodes a tumor suppressor protein that is responsible for repairing damaged DNA and for destroying cells when DNA cannot be repaired. BRCA1 is also involved in the repair of chromosomal damage, with a role in the repair of DNA double-stranded breaks.[28, 29] If BRCA1 is damaged by mutation and DNA damage is not properly repaired, these events may increase the risk for breast cancer. BRCA1 and BRCA2 are known as proto-oncogenes termed “breast cancer susceptibility type 1 genes” and code for proteins regulating cell growth and differentiation in cells of breast and other tissues. BRCA1 can combine with other proteins, such as tumor suppressors, DNA damage sensors, RNA polymerase-II, and histone deactylase to form large multi-subunit protein complexes. BRCA1 can also play roles not only in DNA repair, but in transcription, ubiquitination, transcriptional regulation, and other cell functions.[30, 31]

B) BRCA2. The BRCA2 gene and protein product, similar to BRCA1, are tumor suppressors referred to as caretaker genes/ proteins found in all humans and primates. The BRCA2 gene is referred to as the “breast cancer type 2 susceptibility gene,” responsible for repairing damaged DNA and chromosomal damage, and inducing cell death in cells where DNA cannot be repaired.[31] The gene is located on the long q-arm of chromosome-13 encoding a protein of 3,418 AAs. Some functions of BRCA2 and BRCA1 are interrelated, even though their molecular structures differ in size. BRCA2 binds to single-strand DNA and directly interacts with the recombinase enzyme RAD51 to stimulate strand invasion, which is a vital step of homologous recombination.[32] PALB2, a partner and localizer of BRCA2, functions synergistically with BRCA2 by linking to a piccolo protein to further promote strand invasion. [33] Like BRCA1, the BRCA2 protein can regulate the activity of other genes and play multiple roles during development.

C) FANCD2, FANC1.The Fanconi anemia (FA) genes comprise a total complementation groups of 19 genes, inherited in an autosomal recessive manner. The FANCD2 gene is located on chromosome 3p with 1,328 AAs, while FANC1 has been localized on chromosome 15q26 having 1,451 AAs. FA genes respond to DNA damage to repair corrupted DNA and protect against chromosome instability.[27] The DNA damage repaired by FA genes encompasses broken and misshapen chromosomes, broken chromatids, and triradial and quadri-radial structures. The lack of DNA repair allows mitosis to proceed with corrupted DNA and enhances damaged cell survival, thus increasing genomic instability. Several components of the FA-DNA repair pathway are the FANCD2-FANC1 heterodimer, the FANCD1-BRCA2 complex, and the BRCA2-interacting protein-1 dimer.[34] In response to DNA damage, the FA protein complexes are activated by the AT kinase and the AT-RAD3-related kinase (ATR).[35] The activated FA protein complexes function as E3 ubiquitin ligases which monoubiquitinate the FAND2/ FANC1 heterodimer. This protein complex then translocates to the chromatin fraction where it combines with other FANC proteins at damaged nuclear replication points.[36] Mutated FA complementation proteins have been linked to the DNA damage/repair at the G2/M checkpoint response during cell cycle progression. However, an absence of G2/M transition arrest can occur with unrepaired double stranded breaks bypassing the checkpoint (CHK1) inhibition at the G2 cell cycle phase.

D) Nibrin.The protein nibrin (NBN) is a 754 AA protein whose gene is located on chromosome 8q21. NBN is a cell cycle regulatory factor, associated with the repair of doublestranded breaks which pose the threat of serious damage to the genome.[37, 38] NBN is associated with the BRCA1/RAD50- containing complex and plays a role in the cellular response to DNA damage and the maintenance of chromosome integrity. [39] The NBN complex is involved not only in double-strand break repair, but in DNA recombination, maintenance of telomere integrity, cell cycle checkpoint control, and meiosis. The complex containing NBN displays single-strand nuclease activity and is involved in control of intra-S phase as well as G1 and G2 checkpoints. The NBN gene is the root cause of the Nijmegen breakage syndrome and related human disorders.

E) The DNA Kinases.The other DNA repair kinases involved in ataxia telangiectasia-mutated ATM, ABL, RAD-related kinases, DNA-PKs, and cell cycle checkpoint kinases, together with their AFP-3D locations, are discussed below and listed in Table 1 (see ref. 24). In addition, the kinase activities of other DNA, cell cycle, and checkpoint protein interactions with AFP-3D are listed in Table 2. Although modest in effect, these kinase assays demonstrate and confirm the interactions of AFP-3D with many DNA and cell cycle-related enzymes.

Table 1. The DNA-repair protein kinases involved in ataxia telangiectasia and RAD-related kinases are displayed according to their properties. The AFP amino acid sequences that interact with these kinases are shown in the right column.

Ser = serine; Thr = threonine; tyr = tyrosine;
PK = protein kinase; RAD3 = DNA helicase domain; FAT = Focal Adhesion Tyrosine Kinase
*Ataxia telangiectasia mutated (ATm) is P13/P14 kinase FAT domain Ser/Thr/tyr, checkpoint kinase AFP = alpha-fetoprotein.

Table 2. The percent of kinase enzyme activity^‡ following AFP-3D GIP peptide treatment is listed below.  The control assay was 100% and the inhibition or enhancement is listed as percent activity of the control assays performed in IC₅₀ titration curves.  Note that AFP-3D kinase inhibition is associated with Ser/Thr kinases while Tyr kinases are associated mostly with enhancements.

* Ser/Thr – Serine/thyronine kinase; Tyr – tyrosine kinase;
c-Src – a non-receptor kinase protein of the Ser/Thr or tyrosine type that phosphorylates these residues in other proteins
^‡ The kinase activity screen for AFP-3D peptides was performed via the commercial “kinase profiler” by the Upstate Biosignaling Corp., Dundee Technology Park, Dundee, United Kingdom.

Computer Analysis of AFP-3D Interaction with DNA-Repair Proteins

The third domain of AFP is known to interact with a myriad of proteins and compounds including hydrophobic ligands, receptors, and cytoplasmic binding proteins. Previous publications from the author (GJM) and others have confirmed and verified these reports (see above). These interacting agents include fatty acids, steroids (estrogens), retinoids, cation channels, cell cycle proteins, and chemokine, mucin, and scavenger receptors.[8, 9, 11, 13, 23] These interacting agents have previously been mapped to the aminoterminal, middle, and carboxy-terminal portions of the AFP-3D. [8, 13] The amino terminal portion of AFP-3D is known to interact with fatty acids, estrogens, steroids, retinoids and lysophospholipids, while the middle and carboxy-terminus portions react with scavenger, mucin, and cation channels. Lastly, the carboxy-terminal fragment displays interaction sites with cell cycle proteins, cation channels, chemokine receptors, and dimerizing proteins. Data from the present study now reveal that DNA repair proteins represent additional binding/interaction sites on the AFP-3D fragment.

The DNA repair protein interaction sites similar to previous ligands and receptors, were distributed in patterns of interspaced clustered groups throughout the AFP third domain. The BRCA1/BRCA2 sites were heavily distributed on the first half of the AFP-3D fragment from AA #420 to 500, with another cluster localized at AA #510 to 530 with outliers at AA #550 to 565 (Figure 1). Figure 1, Panel A shows that BRCA1/BRCA2 sites were localized within the hydrophobic ligand binding and lysophospholipid receptor subdomain. This site further overlaps with the Growth Inhibitory Peptide (GIP) and cell cycle protein segment, together with the anterior portion of the scavenger receptor sites. The BRCA1/BRCA2 interacting sites at AA #510 to 530 were found to be localized among the cell cycle and cation channel proteins and the mucin/chemokine receptor binding sites.

The FANC1/FANCD2 interaction sites were scantily localized at AA #430 to 460 and AA #480 to 490 in contrast, the FANC proteins were heavily distributed within the second half of the AFP-CD segment extending from AA #500 to 580 (Figure 1). As shown with the BRCA1/BRCA2 proteins, the FANC proteins localized among the hydrophobic ligand-binding areas and the GIP segment in the first half of the AFP-3D segment. However, in the second half of AFP-3D, the FANC protein sites were distributed among the scavenger, mucin, and chemokine receptors in addition to the cation channel protein binding/interaction sites.

The third DNA repair protein, nibrin, was localized to the AFP- 3D, largely in the second half of the AFP third domain from AA #500 to 530 and AA #565 to 580, with an outlier at AA #480. These regions correspond largely to the scavenger, mucin, and chemokine receptor regions of AFP-3D together with the corresponding protein interaction sites at the GIP AA segment.

Proposed Relationship of DNA Repair Proteins with the AFP-3D Hydrophobic Binding and Receptor Sites

As described above, the DNA repair proteins localization sites were found to coincide with previously identified hydrophobic ligand and receptor binding sites. Prior reports in the literature have described associations and interrelationships that exist between the hydrophobic ligands and the receptors hence, the DNA repair protein pairing localizations may be more than a mere coincidence. For example, the BRCA gene expression is known to be significantly reduced in human (MCF-7) rat mammary tumorigenesis by the supplementation of omega-3 fatty acids (docosahexaenoic acid) in the diet.[40, 41] Prior research showed that BRCA1 acts as a scaffold protein in multiple cellular functions such as transcription, DNA repair, and ubiquitination by interaction with acetyl-CoA carboxylase.[42, 43] BRCA1 is also implicated in novel signaling pathways associated with fatty acid-dependent breast cancer proliferation when associated with supplemented diet fatty acids and ERK1/2, p53-p21 WAF1/CIP1, MAPK, p27 KIP1, and NF-KappaB proteins.[44] The N-3 and N-6 polyunsaturated fatty acids are reported to have differential effects on gene expression of BRCA1 and BRCA2 in human breast cancer cell lines (MCF-7, MDA-MB-231).[45, 46] In contrast, BRCA1 and BRCA2 had no relationships with scavenger receptors and chemokine receptors, as shown in Figure 1 (panels A & B). Moreover, present findings support the association reported in the literature of BRCA1/ BRCA2 DNA-repair proteins with the cell cycle proteins (Figure 1, panels A & B). It was found that the cell cycle proteins were coincident with DNA repair proteins in the localization sites of AA #480 to 490 and AA #510 to 580. Prior studies support this relationship, showing a cyclin-D induced gene amplification and hypermethylation together with CdK12 inhibition in human breast cancer BRCA positive patients.[47-50] In light of the dual localization of mucin receptors and BRCA1/BRCA2 (AA #510 to 530), DNA repair proteins have been studied to determine whether pre- and postoperative CA125 levels are associated with BRCA mutation carriers in ovarian cancer screenings.[51, 52]

Regarding FANC protein localization with hydrophobic ligand and receptor interaction sites, no associations were found relating DNA repair to either fatty acids, mucin receptors, lysophospholipids or cation channels however, DNA repair interaction sites were observed at AA #481 to 504, a known GIP and chemokine receptor area (Figure 1, panel C). Indeed, one study demonstrated a link between chemokine CXCR5 receptors and FANCA-modulated neddylation pathways involved in membrane targeting and cell mobility.[53] Regarding the nibrin protein interaction sites on AFP-CD, NBN sites were largely localized to the second half of the 3D fragment. Nibrin was found to be localized with the ataxia telangiectasia-mutated (ATM) protein, checkpoint kinase-2, and the RAD-related protein (see Table 1, panels A & D), as well as the BRCA1/BRCA2 proteins all of which contribute to breast cancer susceptibility.[54-56] These protein complexes are involved in the dysfunction of specific DNA double-strand break-repair signaling pathways. Other reports of putative ATM in vitro interaction targets include nibrin, RAD17, PTS, and ATM itself.[57]

Relationship of AFP to DNA Damage and Repair Disorders

The correlation of AFP to DNA damage/repair and chromosome instability disorders is well documented, in part because AFP is a biomarker for both immunodeficiency diseases and anemia disorders.[23, 27] Elevated AFP serum levels have been reported in immunodeficiency disorders such as ataxia telangiectasia (AT) and ataxia ocular apraxia (AOA2). The AOA2 disorder displays aberrant DNA repair proteins, ATM mutated in AT and senataxin in AOA, and ATR in AT and RAD3-related disorders.[58, 59] AFP intracellular levels have also been correlated with intracytoplasmic levels of GADDI53 (growth arrest and DNA damage-inducible gene I53) in vascular smooth muscle cell death.[60]

AT is a chromosomal instability disorder caused by an autosomal recessive gene. AT is characterized by increased cell radio-sensitivity and multiple chromosomal aberrations in the DNA of immune cells these include gaps, breaks, dicentrics, and multiple-radial configurations. Most patients (90%) with AT display high serum AFP levels, which can range from 30 to 400 ng/mL.[61-63] ATM interaction sites on AFP-3D were presently localized at AA #429 to 485, AA #500 to 506, and AA #560 to 580 (Figure 1). Patients with AT also exhibit aberrant cell checkpoint proteins that allow continuation through the cell cycle, despite DNA breaks that require repair before the next replication stage occurs. As a consequence, AT patients show a propensity to develop cancer later in life.

Once cloned, the ATM protein was found to be a kinase that shares sequence homology with RAD-3, a kinase that regulates passage (via checkpoints) through the cell cycle after DNA damage has occurred. ATM is also involved with the PI3-kinase signal transduction pathway. [64] The RAD-3 kinase has been cloned and named the AT-RAD3- related (ATR) kinase. The ATR kinase was presently localized on AFP- 3D in two clusters, one at AA #426 to 444 and the other at AA #500 to 539. The former cluster lies directly within the hydrophobic ligand binding region, the cation channel, and the lysophospholipid receptor interaction sites. The latter site was localized among the scavenger, mucin, and chemokine receptor and cell cycle interaction sites. It is of interest that the latter site coincides with cell cycle-associated checkpoint proteins during cell cycle progression.[65, 66] Non-mutated AT/ATR protein kinases sense the presence of double stranded DNA damage and are known to mediate an appropriate repair response. Lastly, a phosphoinositol kinase-3 (PI3-kinase) that associates with the ATM/ATR protein complex, termed DNA-PKCS (Table 1), is a required kinase associated with DNA repair of non-homologous end joining, whose absence results in chromosomal aberrations.[67] The DNA-PKCS interaction sites were localized on AFP-3D at AA #420 to 481, coinciding with hydrophobic binding and the cation channel sites, as well as cell-cycle associated and lysophospholipid receptor interaction sites (Table 1, Figure 1, panel A).

Fanconi’s Anemia (FA) is another DNA-damage/repair disorder associated with both chromosome instability and elevated serum AFP levels both in early infancy and adults. FA represents a progressive, autosomal recessive disorder that exhibits DNA damage, chromosomal breaks, bone marrow failure, and a predisposition to malignancies. [68] Cells from FA patients further display a delay and/or arrest in the G2-to-mitotic transition phase of the cell cycle. As discussed earlier, the FANC proteins represent a complementation group made up of multiple different proteins. However, the present study only addresses FANC1 and FAND2. The origin and source of elevated AFP in FA is presently unknown, since liver dysfunction abnormalities and disease (cancer) are not involved with FA. The author.[27] has suggested that the origin of AFP synthesis and production may lie in the existence of three stem-progenitor cell types present in adult bone marrow namely, fetal hepatic stem/progenitor cells and intrinsic hematopoietic stem/ progenitor cells (HSPC). A third stem bone marrow cell termed the “mesenchymal stem cell” is capable of migrating to the liver and differentiating into hepatocyte-like stem cells following hepatic failure, regeneration, and liver transplantation. Interestingly, the classical hepatic oval cell population surrounding bile ducts are the actual cells that secrete AFP and express the immature stem cell markers CD34 and CD45. Thus, small to moderate amounts of AFP production/secretion could occur in acutely anemic bone marrow with no detectable liver damage, dysfunction, or disease in the FA patient.

Concluding Remarks

It is well-established in the literature that the AFP-3D houses subdomain interaction (interface) sites for a myriad of ligands, receptors, cation channels, cell cycle proteins most recently, DNA damage/repair proteins have been added to this list.[8, 9, 13] These third domain protein interaction sites were first detected by computer analysis, and then verified in cell-based assays, microarray analysis, in vitro cell cultures, and in vivo animal (xenograft) models. For example, an RNA global microarray analysis using AFP-3D derived peptides (see GIP sequence, Figure 1) demonstrated that DNA repair proteins do indeed react with the AFP-GIP amino acid sequences #464-496.[14] The microarray analysis showed that the GIP AA sequences downregulated the mRNA of FANCD2 and up-regulated BRCA1 and RAD54c (Table 2). In addition, histone-1-H4g (DNA-repair) and checkpoint suppressor-1 were greatly downregulated, while multiple DNA repair proteins were modestly upregulated in proteins such as BRCA1 ring domain and RAD5/c (Table 3). Hence, published data confirms that AFP AA sequences on AFP-3D can interact and regulate the RNA of DNA repair proteins in conjunction with cell cycle progression proteins. It is conceivable that the different ligands, proteins, channels, and receptors could react simultaneously in combination with, or in competition with, direct or adjacent interaction sites.

Table 3. Global RNA microarray data following AFP-derived peptide treatment:  Transcripts displaying 1.0 or larger log fold (log base 2.0) decrease for genes associated with cell division and proliferation processes, ubiquitization, and DNA repair proteins obtained from human MCF-7 breast cancer cells in vitro.*

* Expression of 716 transcripts was significantly altered in MCF-7 cells after 8 days of treatment with GIP as compared to treatment with the scrambled peptide.  Four-hundred thirty RNAs were down-regulated, while 286 RNAs were upregulated.
** Real time PCR.  Collaborative data was provided by Kathleen Arcaro, University of Massachusetts, Amherst, MA (14).

The interaction sites described in this report obviously have links to other proximal and/or distal sites along the AFP third domain fragment. As discussed above, literature-based reports have documented that DNA-repair proteins do in fact interact with cell cycle checkpoint proteins to arrest cell cycle progression.[69] Furthermore, BRCA1/BRCA2 can act as scaffold proteins in the ubiquitinization of cell cycle proteins through a proteasomal pathway [70] As shown above, BRCA1/BRCA2 were found to be associated with fatty acid-dependent breast cancer growth.[40-43] Prior reports have further demonstrated that computer-derived cation channel interaction sites were localized at hydrophobic ligand as well as lysophospholipid receptor binding sites.[71] Thus, it is plausible that the clustered localization of channel and cell cycle proteins with DNA repair proteins could be physiologically relevant.

In the present report, evidence was presented that several mutated proteins of the DNA-damage/repair pathways are associated with cancer susceptibility, tumorigenesis, and enhancement of tumor progression, most notably in breast and ovarian cancer, but in other cancers as well. The BRCA and FA-related protein mutations leading to anemia subjects these patients to develop tumors later in life.[72] A significant connection of AFP to FA-mutated DNA repair proteins lies in the elevations of serum AFP in such anemic patients. There are further correlations with breast cancer and its associated BRCA1/ BRCA2 mutated proteins. Sarcione et al. has reported that a circulating bound form of serum AFP, as opposed to free circulating AFP, exists in some female breast cancer patients. This bound form of AFP could be experimentally released by high KCl solutions and measured by immunological assays.[73] Although the bound entity is not known, an IgM molecule has been similarly reported to complex with serum AFP as a bound form.[73] It has also been reported that an intracytoplasmic non-secreted form of AFP is present in normal cells, as well as cancer cells. The non-secreted cytoplasmic AFP (cAFP) form has been shown to participate in kinase regulation, transcription, apoptosis, nuclear hormone binding, transnuclear passage, and regulation of nuclear gene expression.[74, 75] One mechanism of this AFP interaction in cytoplasmic protein activities involves the heterodimerization of AFP with proteins such as cytoplasmic caspases and retinoic acid nuclear receptors.[76] (Figure 1, panel A). Thus, these observations support the contention that cAFP reacts with intracytoplasmic proteins such as the BRCA1/BRCA2, FANC1/FANCD2, nibrin, ATM, and ATR, as suggested by the present report. Furthermore, the reported observations of interaction of AFP-3D with cell cycle proteins, together with the DNA-repair proteins association with cell cycle checkpoints, allows for speculation that AFP could mask, interfere, enhance, or interpose itself into the DNA-repair process of the cell cycle checkpoint regulation pathway. The RNA microarray analyses (Table 3) are consistent with this supposition. The above studies beg the question of whether cAFP (by means of the third domain) is a prime regulatory factor in the overall scheme of DNA repair during cell cycle progression.

Acknowledgements: The author thanks Kathleen Arcaro, University of Massachusetts, Amherst, MA for providing data on the RNA microarray analysis. The author also wishes to thank Mr. Andrew Bentley (Wadsworth Center Photography and Medical Illustration Department) for his expertise in producing the figures and graphic art illustrations and Ms. Tracy Godfrey for her typing, corrections, revisions, and processing of this manuscript.

Competing interests: The author declares that he has no competing interests.

Funding information: The author has no such involvement

Abbreviations

AA – amino acid
AFP – alpha-fetoprotein 3D – third domain
DNA – deoxyribonucleic acid
DDSR – DNA damage-sensing and repair
PI3K – phosphoinositol kinase
mTOR – mechanistic target of rapamycin
GAAD153 – growth arrest and DNA damage-inducible-protein-153
PTEN – phosphatase and tensin homolog
ATM – ataxia telangiectasia-mutated
FA – Fanconi’s anemia
CHK – checkpoint
BRCA – breast cancer
RAD (ATR) – AT-related repair of DNA
NBN – nibrin
ABL – Abelson leukemia oncogene
GIP – growth inhibitory peptide
CdK-cyclin dependent kinase
PTS – 6-pyruvoyltetrahydropterin synthase
AOA – ataxia ocular apraxia
PKC – protein kinase- C.

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