Parasporins, a class of non-insecticidal crystal proteins of Bacillus thuringiensis (Bt) are being explored as promising anticancer agents due to their specific toxicity to cancer cells. The present study has identified 25 Bt isolates harbouring parasporin genes from Western Ghats region, the hotspot of biodiversity in India. Among these, the isolate, KAU 41 (Kerala Agricultural University isolate 41) contained non-hemolytic homogenous crystals showing specific cytotoxicity towards cancer cells. SDS-PAGE analysis of this crystal, isolated by aqueous biphasic separation, revealed a 31 kDa sized peptide. The N-terminal sequence deciphered in BLAST analysis showed homology to a hypothetical Bt protein. Upon proteolysis, a 29 kDa active peptide was generated which exhibited heterogenic cytotoxic spectrum on various cancer cells. HeLa cells were highly susceptible to this peptide with IC 50 1 lg/mL and showed characteristics of apoptosis. RT-qPCR analysis revealed the overexpression of APAF1, caspase 3 and 9 by 14.9, 8 and 7.4 fold, respectively which indicates the activation of intrinsic pathway of apoptosis. However, at higher concentrations of peptide (greater than 3 lg/mL), necrotic death was prominent. The results suggest that the 31 kDa protein from Bt isolate, KAU 41 is a parasporin that may have high therapeutic potential.
Apodytes dimidiata, belonging to the family Icacinaceae, is used for treating inflammation and various gastrointestinal ailments in Zulu traditional medicine. In the present study, significant cytotoxicity was exhibited by the methanolic extract of the A. dimidiata leaf against various cancer cell lines. The extract was purified partially through silica gel column by successive elution using various solvents of increasing polarity. Among these, the active methanolic fraction was found to be the most cytotoxic with IC50 values ranging from 0.92 to 3.95 µg/mL for Ehrlich's ascites carcinoma (a carcinoma cell line), Jurkat (human T lymphocyte cell line), and SK-BR-3 (mammary tumour cell line). The treated cells showed morphological alterations characteristic of apoptosis. Upon oral administration of active methanolic fraction at a dose of 250 mg/kg body weight, the solid tumour volume in mice was significantly reduced to 55.14% and the life span of the ascites tumour-bearing mice increased to 44.65% compared to untreated control. The active fraction with Rf value 0.56 was purified from the methanolic fraction by preparative thin-layer chromatography and was subjected to high-performance thin-layer chromatography, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and nuclear magnetic resonance analysis. The iridoid glycoside genipin was identified as the active component.
Scutellaria species of Western Ghats showed cytotoxic and antioxidant potential and the presence of baicalein. This suggests that S. colebrookiana and S. violacea could be used as alternative sources for baicalein in view of the reported scarcity of S. baicalensis.
Results indicate that S. colebrookiana and S. violacea are capable of protecting erythrocytes from oxidative damage. This cytoprotective effect of the extract is possibly by its antioxidant property.
Bacillus thuringiensis (Bt) is a versatile soil born bacterium with potential insecticidal activity over a wide range of species of agriculture pests. These unusual properties are due to the crystal (Cry) inclusion proteins largely produced during the sporulation. Recent findings indicate that some non-insecticidal crystal inclusions are selectively cytotoxic to cancer cells. The toxicity is due to the induction of membrane permeability and apoptosis in susceptible cells. Recently, we have reported the cytotoxic effect of proteins extracted from three native strains of Bacillus thuringiensis from the Western Ghats of Kerala (India). Studies have shown that these potent toxins exhibit selective cytotoxicity against some human breast cancer cells including MCF-7, MDA-MB-231, ZR-75 and SKBR3. Presently, we report the cytotoxic properties of more number of Bacillus thuringiensis isolates from Peninsular India, which are already assessed for their diversity. The cytotoxic properties of isolates were primarily analysed by the amplification of DNA using specific primers designed for cytotoxic parasporal genes. The strains showing positive result were subjected to in vitro cytotoxic analysis using cancer cell lines. The crystal proteins were harvested from 72h old cultures in sporulating medium, after confirming the lysis of the cells and release of the protein. The cytotoxic proteins were isolated by aqueous biphasic (sodium dextran sulfate/polyethylene glycol) separation method and purified by various chromatographic (sephadex/nickel coated columns) and electrophoretic procedures. The molecular weight of proteins isolated from different isolates was varying and ranged from 15 to 80 kDa. The pro-protein isolated was activated and subjected in vitro cytotoxicity assay on various normal as well as breast cancer cell lines. The protein was found to have strong cytocidal activities against tumour cells and the treated cells showed remarkable morphological alterations and apoptotic cell death. Thus, a new 14 isolates were found to be cytotoxic by the study. The study revealed that the diversity of cytotoxic Cry genes in Peninsular India is perplexingly high and specific to certain cancers. Considering these results, we suggest that the receptor-mediated action of crystal proteins against tumour cells may find a new place in the therapeutic as well as diagnostic field of cancer therapy and the bacteria and its genetic potential may be utilized as versatile tools against cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3143.
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