Screening and characterization of a non-insecticidal Bacillus thuringiensis strain producing parasporal protein with selective toxicity against human colon cancer cell lines

Parasporins are a heterogenous group of Cry proteins produced by non-insecticidal Bacillus thuringiensis strains that specifically act on human cancer cells without affecting normal ones. This unique cytotoxic property has driven researchers to explore novel non-insecticidal B. thuringiensis strains possessing cancer cell-killing proteins. In this study, we isolated 65 non-clonal native isolates from 21 coastal soil samples and subsequently tested 28 strains which were non-haemolytic and non-insecticidal for their cytotoxicity against human colon cancer cell line HCT 116 and human cervical cancer cell line SiHa. Parasporal protein from a strain designated as B.t.LDC 501 showed significantly higher cytolytic activity on HCT 116 cells than on SiHa cells. The activated protein also exerted specific cell lethality against two other colon cancer cell lines, SW480 and SW620. However, it was notably non-toxic to normal cells, such as human peripheral blood leukocytes, human embryonic kidney cell line (HEK293) and human corneal epithelial cell line (HCEC) and showed only modest toxicity on a murine fibroblast cell line (NIH/3T3). The purified 20-kDa crystal protein obtained through gel filtration chromatography exhibited a markedly higher cytopathic effect than the unpurified protein. Liquid chromatography-tandem mass spectrometry analysis of the 20-kDa fragment revealed it to be an uncharacterized protein containing a tumor necrosis factor-like domain. The non-apoptotic mode of cell death, extensive membrane permeability and aminopeptidase N-dependent cytotoxicity suggests the pore-forming nature of the protein. Further characterization of the protein and the receptor will facilitate its use as a potential therapeutic drug against cancer.