Antibody response against neem leaf preparation recognizes carcinoembryonic antigen.

An immune serum generated in Swiss mice against an aqueous preparation from neem leaf was reactive with carcinoembryonic antigen (CEA) and a peptide sequence derived from it. Using ELISA, we have demonstrated that CEA reactive antibody titer (chiefly IgG2a) was significantly decreased after absorption of the immune sera with CEA. Neem leaf preparation (NLP) generated immune sera was also reactive with CEA in immunoblotting and CEA reactive component in the NLP sera can be immunoprecipitated. Identical recognition of CEA expressed on human colorectal cancer specimens, by anti-CEA monoclonal antibody and NLP sera was documented by immunohistochemistry. NLP generated sera could also react with NLP in ELISA and this reactivity was decreased after absorption of the NLP with anti-CEA antibody. Estimation of protein in NLP revealed the presence of it, at least 10% of the total dry weight. In addition, existence of flavone and quercetin was also evidenced from LC-MS analysis. Further studies are needed to identify the antigenic component may have some homology with CEA molecule. This unique property of neem may be utilized for the immunotherapy of CEA positive tumors.

Curcumin-induced apoptosis in human leukemia cell HL-60 is associated with inhibition of telomerase activity.

Curcumin (diferuloylmethane), a natural cancer chemopreventive compound, has been tested for its action in acute myeloblastic leukemia cell line HL-60. The results clearly show that curcumin induces apoptosis in these cells as evidenced by the release of cytochrome c from mitochondria to the cytosol and increase in the DNA content in sub G1 region as observed in FACS analysis. Apoptosis is apparently mediated by up-regulation of apoptotic gene bax and simultaneous down-regulation of anti-apoptotic gene bcl-2 followed by activation of caspases 3 and 8 and degradation of PARP. Telomerase, a reverse transcriptase, has been found to be activated in more than 80% of human cancers and, therefore, can be considered as a potential marker for tumorigenesis. Certain natural compounds have the potential of inhibiting telomerase activity leading to suppression of cell viability and induction of apoptosis. The present study shows that curcumin-induced apoptosis coincides with the inhibition of telomerase activity in a dose dependent manner.

A Mechanistic Approach for Modulation of Arsenic Toxicity in Human Lymphocytes by Curcumin, an Active Constituent of Medicinal Herb Curcuma longa Linn.

Chronic exposure of humans to high concentrations of arsenic in drinking water is associated with skin lesions, peripheral vascular disease, hypertension, blackfoot disease and a high risk of cancer. Arsenic induces single strand breaks, DNA-protein crosslinks and apurinic sites in DNA, which are prerequisites for induction of cancer. Amelioration of such damages with natural compounds could be an effective strategy to combat arsenic toxicity. Curcumin is the active ingredient of turmeric, a common household spice, which is a rich source of polyphenols and this compound has been extensively studied as a chemopreventive agent against many types of cancer. The present study investigates whether curcumin could counteract the DNA damage caused by arsenic as assessed by single cell gel electrophoresis (SCGE) using peripheral blood lymphocytes, from healthy donors. It was observed that DNA damage induced by arsenic could be efficiently reduced by curcumin and the effect was more pronounced when lymphocytes were pre-incubated with curcumin prior to arsenic insult. Arsenic caused DNA damage by generation of reactive oxygen species (ROS) and enhancement of lipid peroxidation levels. Curcumin counteracted the damage by quenching ROS, decreasing the level of lipid peroxidation and increasing the level of phase II detoxification enzymes like catalase, superoxide dismutase and glutathione peroxidase. Curcumin also enhanced the DNA repair activity against arsenic induced damage. The expression of polymerase, a repair enzyme, was found to be highly elevated when arsenite induced damaged cells were allowed to repair in presence of curcumin. Results indicate that curcumin has significant role in confronting the deleterious effect caused by arsenic, which could be an economic mode of arsenic mitigation among rural population in West Bengal, India.

In vitro mitigation of arsenic toxicity by tea polyphenols in human lymphocytes.

The groundwater arsenicals have brought dreadful misery for the people residing in the endemic regions of West Bengal, India. Arsenic-related anomalies include arsenicosis, hyperkera-tosis, gastric complications, liver fibrosis, peripheral neuropathy, and cancer. Some of these diseases have been frequently associated with overproduction of reactive oxygen species that cause DNA damage and improper functioning of body's antioxidant defense mechanism. Natural polyphenols present in tea serve as excellent antioxidants. In the present study, an attempt has been made to elucidate the role of representative polyphenols and extracts of green and black tea in modulating sodium arsenite (As III)-induced DNA damage in normal human lymphocytes. Comet assay was used to detect the DNA damage. Arsenic-induced oxidative stress was measured with generation of reactive oxygen species, lipid peroxidation, and activity of some antioxidant enzymes. Expression of some repair enzymes such as poly(ADP-ribose) polymerase and DNA polymerase beta was measured to assess the effect of tea on DNA repair. Tea afforded efficient reduction of As III-induced DNA damage in human lymphocytes. Tea also quenched the excessive production of reactive oxygen species by arsenic, reduced the elevated levels of lipid peroxidation, and increased the activity of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase. Furthermore, tea enhanced recovery of DNA damage, which was indicative of repair as confirmed by unscheduled DNA synthesis and pronounced expression of DNA repair enzyme poly(ADP-ribose) polymerase. It is speculated that the antioxidant potential and repair-inducing capacity of tea might help in combating the severe genotoxic effects induced by arsenic in the human population.

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.

Induction of apoptosis in human leukemia cells by black tea and its polyphenol theaflavin.

Treatment of human leukemic cell lines HL-60 and K-562 with extracts of green and black tea and their polyphenols epigallocatechin gallate and theaflavins, respectively, showed a dose dependent inhibition of growth as a result of cytotoxicity and suppression of cell proliferation. Based on the IC50 values obtained from cytotoxicity data it was clearly evident that black tea was as efficient as green tea. Analysis of polyphenol contents of tea extracts revealed that not only epigallocatechin gallate, which is a predominant polyphenol of green tea, but also theaflavin that is abundantly present in black tea affords significant chemotherapeutic action by imparting cytotoxicity to human leukemic cells. Electrophoretic analysis of fragmented DNA from treated cells displayed characteristic ladder pattern. Flow cytometric analysis revealed the dose dependent increase in sub-G1 peak. These criteria confirmed that cytotoxic activity of green and black tea was due to induction of apoptosis. Such induction was found to be mediated through activation of caspases 3 and 8, particularly caspase 3 and by altering apoptosis related genes as evident by down-regulation of Bcl-2 and up-regulation of Bax proteins.

Modulation of arsenic induced cytotoxicity by tea.

Arsenic, a naturally ocurring chemical element, is considered hazardous to human health. Inorganic arsenic compounds were found to induce cytotoxicity in Chinese hamster V-79 cells in culture. The arsenite form was more toxic than arsenate. Extracts of green and two varieties of black tea, as well as their principal polyphenols, (-)-epigallocatechingallate and theaflavin, efficiently counteracted the cytotoxic effects of arsenic compounds. On the basis of the amount of tea extract that afforded 50% protection to the cells from arsenic induced cytotoxicity, black tea was found to be as effective as green tea. The protective effect was attributable to the contents of not only (-)-epigallocatechingallate but also of theaflavin, the latter being a predominant polyphenol present in black tea.