Novel Cytotoxic Proteins of Bacillus thuringiensis Isolated from Peninsular India.

Abstract

Abstract 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.