Proceedings of the Indo-U.S. bilateral workshop on accelerating botanicals/biologics agent development research for cancer chemoprevention, treatment, and survival.

With the evolving evidence of the promise of botanicals/biologics for cancer chemoprevention and treatment, an Indo-U.S. collaborative Workshop focusing on "Accelerating Botanicals Agent Development Research for Cancer Chemoprevention and Treatment" was conducted at the Moffitt Cancer Center, 29­31 May 2012. Funded by the Indo-U.S. Science and Technology Forum, a joint initiative of Governments of India and the United States of America and the Moffitt Cancer Center, the overall goals of this workshop were to enhance the knowledge (agents, molecular targets, biomarkers, approaches, target populations, regulatory standards, priorities, resources) of a multinational, multidisciplinary team of researcher's to systematically accelerate the design, to conduct a successful clinical trials to evaluate botanicals/biologics for cancer chemoprevention and treatment, and to achieve efficient translation of these discoveries into the standards for clinical practice that will ultimately impact cancer morbidity and mortality. Expert panelists were drawn from a diverse group of stakeholders, representing the leadership from the National Cancer Institute's Office of Cancer Complementary and Alternative Medicine (OCCAM), NCI Experimental Therapeutics (NExT), Food and Drug Administration, national scientific leadership from India, and a distinguished group of population, basic and clinical scientists from the two countries, including leaders in bioinformatics, social sciences, and biostatisticians. At the end of the workshop, we established four Indo-U.S. working research collaborative teams focused on identifying and prioritizing agents targeting four cancers that are of priority to both countries. Presented are some of the key proceedings and future goals discussed in the proceedings of this workshop.

Curcumin protects DNA damage in a chronically arsenic-exposed population of West Bengal.

Groundwater arsenic contamination has been a health hazard for West Bengal, India. Oxidative stress to DNA is recognized as an underlying mechanism of arsenic carcinogenicity. A phytochemical, curcumin, from turmeric appears to be potent antioxidant and antimutagenic agent. DNA damage prevention with curcumin could be an effective strategy to combat arsenic toxicity. This field trial in Chakdah block of West Bengal evaluated the role of curcumin against the genotoxic effects of arsenic. DNA damage in human lymphocytes was assessed by comet assay and fluorescence-activated DNA unwinding assay. Curcumin was analyzed in blood by high performance liquid chromatography (HPLC). Arsenic induced oxidative stress and elucidation of the antagonistic role of curcumin was done by observation on reactive oxygen species (ROS) generation, lipid peroxidation and protein carbonyl. Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase, glutathioneS-transferase, glutathione peroxidase and non-enzymatic glutathione were also analyzed. The blood samples of the endemic regions showed severe DNA damage with increased levels of ROS and lipid peroxidation. The antioxidants were found with depleted activity. Three months curcumin intervention reduced the DNA damage, retarded ROS generation and lipid peroxidation and raised the level of antioxidant activity. Thus curcumin may have some protective role against the DNA damage caused by arsenic.

Tea-induced apoptosis in human leukemia K562 cells as assessed by comet formation.

Programmed cell death or apoptosis is a physiological process by which genetically damaged cells or undesired cells can be eliminated. Various morphological and molecular changes undergoing during the process of apoptosis are the formation of apoptotic blebs of the cell membrane, cell shrinkage, condensation of chromatin and the disruption of deoxyribonucleic acid (DNA) into typical fragments of multiples of 180 base pairs. These changes can be detected in a number of ways. DNA ladder formation, which is observed following gel electrophoresis technique although is widely accepted but does not reflect the DNA breakdown in individual cell and also may miss contributions from small sub-populations in a heterogeneous cell population. Alkaline comet assay as measured by single cell gel electrophoresis, on the other hand, accurately measures DNA fragmentation on a single cell level and allows analysis of subpopulation of cells. The assay was originally developed for measuring DNA damage of cells exposed to any genotoxic agent. However, the comet image generated by an apoptotic cell is different from that obtained with a cell treated for a short time with a genotoxic agent. Correlation of comet formation with various other established parameters of apoptosis is very important. The present study aims to correlate different features of apoptosis with the formation of comet tail in human leukemia K-562 cells using tea extracts. Apoptosis as measured by formation of apoptotic bodies, flow cytometric analysis, activation of caspase 3 and 8, and expressions of apoptosis related genes such as bcl-2 and bax showed high degree of correlation with comet tail moment. This indicates that comet assay can accurately reflect measure of DNA fragmentation and hence can be used to detect a cell undergoing apoptosis.

Black tea protects thymocytes in tumor-bearing animals by differential regulation of intracellular ROS in tumor cells and thymocytes.

The accumulated in vitro evidence indicates that many tumors induce T-cell apoptosis as a mechanism of inhibiting antitumor activity. This downregulation of cell-mediated immune functions occurring at the late stages of the disease may be causally related to the thymic involution, because the thymus is the major site of T-cell maturation, extensive proliferation, and differentiation. Our results showed that in Erhlich's ascites carcinoma cell (EAC)-bearing mice, the number of EAC was inversely proportional to the thymocyte count in the host's thymus, which is the primary immune organ. Further studies indicated the presence of tumor-induced thymocyte apoptosis in EAC bearers. Black tea prolonged the survival of the tumor bearer by successfully restricting tumor progression as well as protecting the thymus from tumor insult. In fact, black tea inhibited thymic apoptosis while inducing programmed cell death of EAC. Interestingly, the tea regulated the oxidant status differentially in EAC and thymocytes--i.e., it reduced the EAC-induced reactive oxygen species (ROS) generation in the thymus while activating the same in the EAC. A similar effect of black tea was obtained when thymocytes were cultured in the presence of cell-free ascitic fluid, thereby indicating that black tea could directly reduce oxidative stress, an activity independent of its tumoricidal property. As a result, the maturation block in thymocyte subpopulations in tumor bearers was ameliorated significantly in black tea-treated animals. Our results demonstrate that black tea protects thymocytes in the tumor bearer by regulating the intracellular ROS in tumor cells and thymocytes differentially, thereby strengthening its candidacy in future anticancer therapeutic regimens.

Correlation of apoptosis with comet formation induced by tea polyphenols in human leukemia cells.

Induction of apoptosis is an important approach to cancer control. Apart from morphological changes in cells, apoptosis is characterized by fragmentation of nuclear DNA. The characteristic DNA ladder formation that is observed on gel electrophoresis does not reflect the DNA breakdown in individual cells; contributions from small subpopulations are usually overlooked. On the other hand, alkaline comet assay as measured by single cell gel electrophoresis accurately measures DNA fragmentation at a single cell level. The comet assay was originally developed as a cytogenetic test to measure the genotoxicity of various chemicals. However, the comet image generated by an apoptotic cell is different from that obtained with a cell treated for a short time with a genotoxic agent. In the present study using human leukemic cells, typical apoptotic features such as morphological characteristics, FACS analysis, caspase activation, and expression of apoptosis-related genes as induced by tea polyphenols have been found to correlate with the comet tail formation. It is apparent from the high degree of correlation observed between the comet tail moment and each parameter of apoptosis that the comet assay can accurately reflect the measure of DNA fragmentation and, hence, can be used to detect a cell undergoing apoptosis.

Amelioration of sodium arsenite-induced clastogenicity by tea extracts in Chinese hamster v79 cells.

Since the early 1980s, an alarming problem of groundwater arsenic (As) contamination has devastated many districts of West Bengal in India. People drinking As-contaminated water have been suffering severe health problems such as hyperkeratosis, blackfoot disease, neuropathy, and cancer of various sites. DNA damage and genetic instability induced by the inorganic arsenicals present in water are thought to be prerequisites for the initiation of carcinogenesis. Many natural polyphenols, which are consumed through our daily diet, possess excellent cancer chemopreventive properties. Tea, a popular beverage worldwide and rich in polyphenols, has exhibited many health benefits. The present study was conducted to examine the anticlastogenic action of tea extracts (both green and black) against the As-induced chromosomal aberrations. We also evaluated the role of tea in inducing antioxidant enzymes such as superoxide dismutase and catalase to provide protection against the oxidative stress induced by As. Our results demonstrated that tea extracts, particularly Darjeeling tea extract, are effective in counteracting the clastogenicity (chromatid breaks, in particular) of the most potent form of As, sodium arsenite. The antioxidant function of tea in reducing clastogenicity may be partly due to the induction of phase II detoxification enymes, such as superoxide dismutase and catalase. Our results suggest that the use of tea may be an effective approach in combating the health crisis generated by As.

Arsenic-induced micronuclei formation in mammalian cells and its counteraction by tea.

The Gangetic plain of West Bengal, India, has been engulfed by a disastrous environmental calamity of arsenic contamination of the ground water. Chronic arsenic toxicity caused by drinking arsenic-contaminated water has been one of the worst health hazards gradually affecting nine districts of West Bengal since the early 1980s. Over and above hyperpigmentation and keratosis,weakness, burning sensation of the eyes, swelling of the legs, liver fibrosis, chronic lung disease, gangrene of the toes, neuropathy, and skin cancer are other manifestations. Induction of cancer is frequently associated with DNA damage, changes in ploidy of cells, and non-random chromosome aberrations. Counteraction of these genotoxic and cytogenetic abnormalities with natural dietary polyphenols could be a useful strategy to combat arsenic-induced DNA damage and thereby cancer. A review of the literature showed that it is the antioxidant property of tea polyphenols that affords protection against various types of cancer. The present study was conducted to investigate whether the extracts of green tea and black tea (Darjeeling and Assam) as well as their polyphenols could ameliorate this arsenic-induced genotoxicity. The normal mammalian cell culture derived from male Chinese hamster lung fibroblast cells (V79) was used as the test system to assess the genotoxicity by micronucleus assay. The results showed that both green tea and black tea extracts have equal potential in modulating the arsenic-induced genotoxicity. This effect was perhaps induced by the constituent polyphenols present in green and black tea. In addition, the repair activity of the damaged cells was enhanced when treated with these tea extracts and their polyphenols. Thus, tea and its polyphenols may have a promising role in counteracting the devastating effects of arsenic.

Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol.

Modulation of events characteristic of carcinogenesis or of cancer cells is being emphasized as a rational strategy to control cancer. Green tea polyphenol epigallocatechin gallate (EGCG) has been shown to be highly active as a cancer chemopreventive agent. Certain cellular and molecular events relevant to carcinogenesis are also modified by EGCG. The present investigation was carried out to examine the effects of EGCG on the cytogenetic change and DNA damage induced by toxicant H(2)O(2) and carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster V-79 cells in culture. Cytogenetic change as evident by the formation of micronuclei and DNA damage in the form of comet tail length during single cell gel electrophoresis was found to be significantly suppressed by EGCG in a dose dependent manner. Cells preincubated with EGCG were protected from subsequent damage by the genotoxic agents. Apoptosis, a highly organized physiological mechanism to eliminate injured or abnormal cells, is also implicated in multistage carcinogenesis. Initiated cells, cells at promotional stage or fully transformed cells can be eliminated through apoptosis. It was observed that EGCG suppressed growth and proliferation of K-562 cells derived from human chronic myelogenic leukemia. Morphological features of treated cells and characteristic DNA fragmentation revealed that the cytotoxicity was due to induction of apoptosis. This was mediated by activation of caspase 3 and caspase 8. Results show that EGCG not only protects normal cells against genotoxic hazard but also eliminate cancer cells through induction of apoptosis.