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.

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.

Prevention and repair of DNA damage by selected phytochemicals as measured by single cell gel electrophoresis.

We assessed the ability of some natural products--namely, curcumin, resveratrol, indole-3-carbinol, and ellagic acid--to modify the DNA damaging ability of the alkylating carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in cultured Chinese hamster lung fibroblast cells (CH V-79). MNNG produced DNA single strand breaks in a dose- and time-dependent manner, as observed by increase in the tail moments of the comet, when the cells were subjected to alkaline single cell gel electrophoresis. When the cells were treated in the presence of each of the natural compounds, the DNA damage caused by MNNG was considerably reduced. This effect was found to be dose related. Preincubation of cells with each of these compounds individually afforded significant protection to DNA against damage caused by subsequent treatment with MNNG, indicating a true chemopreventive role of these substances. The most remarkable aspect of the present study was that all four compounds helped in the recovery of DNA damage by accelerating DNA repair efficiency in the damaged cells. This was further substantiated by the observation on unscheduled DNA synthesis. Our results suggest that these agents are chemopreventive by virtue of their ability to protect DNA as well as to induce DNA repair.

Induction of Apoptosis in Tumor Cells by Natural Phenolic Compounds.

Investigation has been conducted to delineate the action of some phenolic compounds of natural origin in four human tumor cell lines: acute myeloblastic leukemia (HL-60), chronic myelogenic leukemia (K-562), breast adenocarcinoma (MCF-7) and cervical epithelial carcinoma (HeLa). In cells grown in appropriate media the phenolics curcumin, yakuchinone B, resveratrol and capsaicin exhibited growth inhibition as assessed by trypan blue dye exclusion. It was evident from the results of the MTT reduction assay and [(3)H]thymidine incorporation into nuclear DNA that the phenolics were cytotoxic and inhibited cell proliferation. Dose response studies indicated curcumin to be most cytotoxic towards HL-60, K-562 and MCF-7 but did not show much activity in HeLa cells. On the other hand, yakuchinone B, although less active than curcumin, displayed cytotoxicity towards all four cell lines. Resveratrol was cytotoxic only in leukemic cells, while capsaicin was marginally cytotoxic. All these phenolics did not elicit any cytotoxic activity as judged by the above parameters towards lymphocytes purified from normal human blood. When cells treated with phenolics were stained with propidium iodide and examined under a fluorescent microscope, characteristic apoptotic features such as chromatin condensation and nuclear fragmentation were observed. Scoring of cells with apoptotic and non-apoptotic features showed positive correlation of apoptotic index with dose of phenolic, and fragmented DNA extracted free of genomic DNA displayed on gel electrophoresis a typical ladder pattern. These phenolics which have human exposure are known cancer chemopreventive agents and their action as inducers of apoptosis in tumor cells suggest their potential use in a strategy for cancer control.

Cancer Chemoprevention: Tea Polyphenol Induced Cellular and Molecular Responses.

Considerable evidence is now available showing that tea infusions can prevent tumor induction in experimental animals by a variety of chemical carcinogens. Such an action is mainly attributed to the polyphenolic constituets of tea. These polyphenols possess antioxidant activity and interfere with carcinogen activation, show anti-mutagenic and anti-genotoxic properties, exhibit anti-tumor promoting activity and alter certain events in signal transduction pathways. Tea polyphenols are known to exhibit cytotoxicity towards various human tumor cell lines as well as growth inhibition that is accompanied by cell cycle arrest. It has been demonstrated that the cytotoxicity result in inducton of apoptosis. Additionally, tea polyphenols are good candidates for sensitizing tumor cells leading to apoptotic death by cytotoxic drugs.