DOI: 10.31038/CST.2017244

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.

DOI: 10.31038/CST.2017243

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.
• IARC (2003) World cancer report 2003. Lyon, International Agency for Research on Cancer.
• 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,
• Mallik RM and Panigrahi N (1998) Study of Domestic and Commercial Use of, including Marketing of NTFPs. SCANDIA CONSULT NATURA, Sweden.
• Gbile ZO, Ola-Adams BA and Soladoye MS (1981) Endangered species of the Nigerian flora. Niger J For 8: 14-20
• Oguntola AB, Soladoye MO, Ugbogu OA, Fasola TR (1996) A review of endangered tree species of cross river state and environs. Proceedings of the Workshop on Rain Forest of South Eastern Nigeria and South Western Cameroon Calabar, Nigeria 120-125.
• Adekunle AA (2001) Ethnobotanical studies of some medicinal plants from Lagos State, Nigeria. Nigerian Journal of Botany.
• Adodo A (2004) Nature and Power: A Christian Approach to Herbal Medicine. Benedictine Pub Nigeria 289p
• Adodo A (2005) New Frontiers in African Medicine.Pax Herbal clinic Nig. Ltd. Ewu Edo State
• Adodo A (2003) The Healing Radiance of the Soul: A Guide to Holistic Healing. Agelex Publications
• FAO (1987) Forest Products Yearbook, 1987.

DOI: 10.31038/CST.2017233

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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DOI: 10.31038/CST.2017242

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

DOI: 10.31038/CST.2017241

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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• 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]
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DOI: 10.31038/CST.2017232

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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DOI: 10.31038/CST.2017225

Abstract

The recent discoveries of the existence of natural anticancer agents either from plants, such as Aloe, Myrrh and Magnolia, or from the human body, namely the pineal hormones, allowed the possibility to elaborate new therapeutic natural combinations as a link between the commonly used palliative and curative cancer therapies, which would have not considered in a separate manner. The present study was carried out to evaluate the influence of the spiritual status on the efficacy of a natural anticancer combination containing pineal anticancer hormones in association with Aloe, Myrrh and Magnolia extracts in a group of 70 untreatable metastatic solid tumor patients with life expectancy less than 1 year. The spiritual sensitivity was evaluated by an appropriate faith test for patients affected by an untreatable disease. The percentages of both objective tumor regressions and disease control obtained in patients with high faith score were significantly higher with respect to those found in patients with low faith score. On the same way, the 3- year percent of survival achieved in patients with high faith score was significantly longer than that found in the other group. This study would suggest the efficacy of an antitumor therapeutic strategies with natural anticancer agents also in metastatic cancer patients form whom no other standard antitumor treatment was available, with a greater efficacy in the presence of a real status of spiritual faith.

Keywords

spirituality, cancer disease, psychoneuroimmunology

Introduction

Being cancer a biological war between a human host and an apparently unconscious tumor mass, it is obvious that the prognosis of the neoplastic diseases may depend on both tumor characteristics and the psychobiological identity of cancer patients. Tumor characteristics regard histology, disease extension, biological grading and eventual genetic mutations of cancer cells. At the other side, the individual identity of the single cancer patients involves their consciousness status, psychological behaviour, life style, but also and mainly their endocrine, neuroendocrine and immune status in addition to their clinical conditions [1]. Until some years ago, the human diseases were considered to be due to organicistic or psychosomatic reasons. On the contrary, with the progressive advances in the area of Psychoneuroendocrinoimmunology (PNEI), it was understood that the psychospiritual status of patients may influence the biological body not only through the nervous system, but also through complex nervous, neuroendocrine and endocrine interactions with the immune cells, which after their activation may interact with the endocrine and nervous systems by releasing immunomodulating proteins, the so-called cytokines, which are also able to exert neuroendocrine effects by realising complex feed-back circuits between neuroendocrine and immune systems [2].

As far as the psychological and spiritual point of view is concerned, must be remarked that until few years ago and yet up to now by most researchers, the spirituality has been simply considered only as a part of the psychological status of humans, and only recently some preliminary clinical investigations have suggested that the spirituality is a different condition from both psychology and religion [3]. As far as the relation between psychology and spirituality is concerned, it is possible to affirm that the Psychology represents the analysis of the emotional life, which has its energetic matrix in the sexuality, whereas the Spirituality regards the reality of the different consciousness states. At the other side, the relation between Religion and Spirituality, according to a definition previously reported in the literature [4], the Spirituality is the research of the ultimate meaning of life, while Religion is only a set of beliefs and ritual practices within a well defined religious institution, then it would simply represents one of the possible ways to realize own self spirituality, even though more widely followed with respect to an individual manner to live and feel the spiritual dimension. Then, the individual spirituality may be realized through the same religion or other mysticai experiences, and it Is not a simpie set of emotions, but it constitutes a status of consciousness. Moreover, in agreement with PNEI discoveries [5], both emotions and consciousness states require a well defined psychoneuroendocrine mediation. Then, from a clinical point of view, the two major problems concern the identification of adequate methods to clinically investigate not only the religious profile of patients, but also their spiritual sensitivity, as well as of possible eventual blood biochemical parameters able to reflect the psychological and spiritual status of patients and its influence on the clinical course of the neoplastic disease. However, most studies carried out up to now have been generally limited to the investigation of the influence of the personal religion rather than the real status of cancer patients. In any case, even though limited to the investigation of the influence of religion on the prognosis of cancer, preliminary clinical results seem to suggest that the religious support may allow an increase in the survival time of advanced cancer patients and to improve their clinical status, even though through still unknown mechanisms [3, 4]. The recent advances in PNEI knowledgements, by demonstrating that the immune responses in vivo are physiologically under a psychoneuroendocrine modulatory control [6,7], which represents the biochemical mediation of the spiritual and psychological status of the patients, may allow the hypothesis that the spiritual status may influence the clinical course of the neoplastic disease and the efficacy of the different antitumor therapies by stimulating the immune system and piloting it in an antitumor way through the activation of well-defined psychoneuroendocrine circuits [8]. Moreover, it has to be considered that until about 20 year ago, almost all scientific investigations in the oncological area were limited to the identification of possible carcinogens in the nature, either endogenous molecules, such as estrogens and androgens, or exogenous substances, capable of inducing the malignant transformation. On the contrary, more recent researches have demonstrated the existence of several antitumor plants containing well characterized anticancer molecules, in particular aloe hemodin from Aloe [9], guggulsterone from Myrrh [10] and honokiol from Magnolia [11], as well as more surprisingly the evidence of anticancer endogenous molecules, which would be responsible for the natural immunobiological resistance against cancer onset and growth, in particular some indole hormones released by the pineal gland, namely melatonin (MLT) [12] and 5-methoxytryptamine (5-MTT) [13], and the great group of beta-carbolines [14], which are mainly produced by pineal gland itself. All those natural anticancer agents has no important toxicity. Therefore, the existence of both endogenous and exogenous anticancer agents with a complete lack of biological toxicity but with well known antitumor properties, would justify their empioyment in the medical Oncology in an attempt to realize a link between the simple palliative and the curative therapies of cancer, since several anticancer natural agents, according to the PNEI knowledgements, may deserve both palliative and antitumor effects on cancer progression at least in terms of survival time. The present study was performed to investigate the influence of the spiritual status of consciousness on the antitumor efficacy of a psychoneuroendocrine regimen with antitumor pineal hormones in association with the most investigated anticancer plants in a group of metastatic solid tumor patients, for whom there is no other standard effective therapy of their tumor, by evaluating the spiritual status through a previously described clinical test to explore the spiritual faith in patients affected by an untreatable disease [15].

Materials and Methods

The study included 70 untreatable metastatic solid tumor patients. Eligibility criteria were, as follows: histologically proven metastatic solid neoplasm, measurable lesions, no availability of standard antitumor therapies because of progression on previous chemotherapies, age or low performance status (PS), and life expectancy less than 1 year. Patients affected by metastatic breast cancer or prostate carcinoma were excluded from the study, because of the availability for those tumors of well tolerated hormonal therapies also by the standard medical Oncology. The faith test for patients affected by an untreatable disease employed in the study was performed by the observation of the clinicians in an attempt to exclude possible unconscious mental manipulations in their answers by the patients, and it consisted of the analysis of five major criteria [15], by assigning 20 points to each single criterion, with a maximum score of 100 points and by defining the presence of a real status of spiritual faith for a minimal score of at least 60 points or more. The five criteria were, as follows: 1) complete self-consciousness by the patients of the severity of their diagnosis and prognosis in terns of life expectancy: the absence of an adequate knowledge of the severe prognosis would transform the faith in a simple illusion; 2) lack of excessive anxiety: the anxiety would represent the opposite mental condition with respect to a real spiritual faith; 3) lack of an exaggerated attribution of value by the patients to the professional capacities of the single clinicians, being their disease as considered as untreatable on the basis of the standard medical therapies;4) lack of an excessive analytic tendency by the patients to understand the chemical mechanisms involved in the efficacy of treatments instead of their significance in terms of reactivation of an effective biological natural anticancer resistance; 5) perception of own neoplastic disease not only as a personal problem, despite pain and other intolerable symptoms, but also as an individual manifestation of a general universal suffering involving all humans. The clinical characteristics of patients are reported in Table 1. Lung cancer, pancreatic adenocarcinoma and colorectal cancer were the neoplasms most frequent in our patients. The PNEI strategy of cancer cure consisted of the oral administration of the two most investigated anticancer pineal hormones, MLT and 5-MTT, in association with a phyto-therapeutic regimen consisting of the administration of extracts of the most investigated antitumor plants, including Aloe arborescens, Myrrh and Magnolia. MLT was given at 100 mg/day during the dark period of the day, while 5-MTT was administered at 5 mg in the early afternoon. Magnolia cortex, with a honokiol content of at least 50%, was given at 500 mg twice/day. Finally, Aloe and Myrrh were given at a dose of 10 ml twice/day of a mixture of 60% Aloe and 40% Myrrh. Patients with brain metastases also received Boswellia at 1000 mg/day in the morning, because of its anti-oedema effect. The clinical response was assessed by the WHO criteria by repeating the radiological examinations at 3-month intervals. Data were statisticaliy analyzed by the chi-square test. The survival curves were calculated by the Kaplan-Meyer method and statisticaliy analyzed by the log-rank test.

Table 1. Clinical characteristics of 70 untreatable metastatic solid tumor patients.

Results

The clinical response achieved in our patients is reported in Table 2. A complete response (CR) was obtained in 2/70 (3%) patients, who were affected the former by gastric cancer and the latter by lung adenocarcinoma. A partial response (PR) was achieved in other 9 patients (colon cancer: 2; melanoma: 2; lung cancer:1; pancreatic cancer:1; endometrial adenocarcinoma:1; biadder cancer:1; biliary tract carcinoma: 1). Then, an objective tumor regression was observed in 11/70 (16%) patients. A stable disease (SD) was found in other 41 patients. Therefore, a disease control (CR + PR + SD) was obtained in 52/70 (74%) patients, whereas the remaining 18 patients (26%) had a progressive disease (PD). A faith score of at least 60 points was found in 51/70 (73%) patients. By considering faith score in relation to the other individuai variables, no significant differences between males and females was observed in the percent of values of at least 60 points (28/37 (76%) vs 22/33 (67%). On the same way, no difference in the percent of high faith score occurred in relation to the three most frequent neoplasms (lung: 12/18 (67%); colon: 9/13 (69%); pancreas: 9/14 (64%)). Moreover, more surprisingly there was no significant difference in the percent of faith score of at least 60 between patients who followed a specific religion and those who had no religion or no defined religion (22/29 (76%) vs 29/41 (71%). Finally, by considering the clinical response in relation to the faith score, the percent of objective tumor regressions (CR+PR) achieved in patients with faith score of 60 or more was significantly higher with respect to that found in patients with values iess than 60(11/51(19%) vs 1/19 (5%), P<0.05). On the same way, the percent of DC (CR+ PR+SD) achieved in patients with high faith score was significantly higher than that observed in those with low faith score (44/51(86%) vs 8/19 (42%), P< 0.01). Table 3 shows the clinical response in relation to the differeni values of faith score. A progressive increase in the percent of DC occurred concomitantly with the increase in faith score values. Finally, the 3-year survival curves observed in our patients are illustrated in Figure 1. The percentage of 3-year survival reached by patients with faith score of at least 60 was significantiy higher than that found in patients with low faith score (P<0.05).

Table 2. Clinical response (WHO criteria) in 70 untreatable cancer patients in relation to their faith score

+ CR: complete response; PR: partial response; SD: stable disease; DC (CR + PR + SD): disease control; PD: progressive disease
* P< 0.05 vs low faith score; ** P< 0.01 vs low faith score

Table 3. Clinical response (WHO criteria) in 70 untreatable cancer patients in relation to the different values of faith score

Discussion

This study, carried out in a considerable number of untreatable metastatic cancer patients, would suggest that a neuroendocrine approach with endogenous anticancer molecules, such as the antitumor pineal hormones, and natural antitumor plants, may counteract cancer growth also in patients, who had been considered as untreatable according to the standard anticancer treatments. Moreover, the study shows that the efficacy of therapy is higher in cancer patients with a true spiritual faith, al least in the untreatable ones, even though it cannot be excluded that the reduced therapeutic efficacy observed in patients with low faith score may be simply due to an interruption or a discontinuation of therapy. In any case, even though we are only at the beginning of the possibility to understand the psychochemical mechanisms responsible for mediating the influence of the spiritual faith on the clinical course of the neoplastic diseases, the recent advances in PNEI knowledgements have demonstrated the possibility to modulate the immune system, including the anticancer immunity, by acting on its psychoneuroendocrine regulation [2, 16]. Then, in agreement with the PNEI discoveries, showing a stimulatory effect of both pleasure and spiritual sensitivity and an inhibitory one of stress and depression on the anticancer immunity, it is probable that the increased efficacy of cancer therapies with natural antitumor agents and the prolonged survival time achieved in patients with evidence of spiritual faith may mainly be due to an improvement in the potency of the immune reaction against cancer dissemination [17-19]. Moreover, the study would show that the presence of a real spiritual faith is relatively independent from the adhesion to a specific well defined religion, then it would represent an individual variable rather than to depend on external behaviours, such as the religious practices, by confirming the observations of previous authors, who had considered religion and spirituality as different human conditions [3, 4]. In more detail, since the anticancer action of the pineal hormones and of most antitumor piants is due to both antiproliferative and immunomodulating effects [20], at present, according to the PNEI discoveries, it is possible at present to identify two major functional psychoneuroendocrine systems involved in the mediation of the influence of emotions and spirituality on the anticancer immunity, consisting of the former brain opioid system-pituitary adrenal gland, which is related to stress, pain, anxiety and depression and which plays an inhibitory effect on the anticancer immunity by stimulating T regulatory (T reg) and inhibiting T helper-1 (TH1) lymphocyte functions [21], and the latter brain cannabinergic-mirror neuron-pineal gland functional axis, which on the contrary is related to both pleasure perce1ption and spiritual sensitivity, and which enhances the anticancer immunity by stimulating TH1 and inhibiting T reg activities [22-24]. In any case, both systems would be essential for the survival of the biological species, since the opioid system-pituitary-adrenal gland functional axis would play a fundamental role in the adoptive mechanisms to the environmental and social conditions, while at the other side the cannabinergic system-mirror neuron systems-pineal gland axis would be in relation to the both biological and mind evolution , as suggested by the appearance of cannabinoid receptors in a subsequent time with respect to that of the opioid ones [22], as well as by the evidence of the fundamental role of mirror neurons in the processes of imitation, learning, language, memory and self-consciousness [23] and of the involvement of pineal molecules, such as the beta-carbolines, in mind expansion [25]. If successive studies will confirm the possibility to prolong the survival time and improve the clinical status of metastatic cancer patients, for whom no other standard therapy may be available, by the administration of natural endogenous and exogenous anticancer molecules, the application of the faith score could allow to predict the probability of efficacy of natural treatments themselves, as well as for the commonly used anticancer therapies in relation to the different tumor histotypes and disease extensions.

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