Mode of action of a human cancer cell active toxin (Parasporin-3) from Bacillus thuringiensis

Bacillus thuringiensis (Bt) crystal (Cry) proteins, used for decades as insecticidal toxins worldwide, are well known to be toxic to certain insects, but not to mammals. The three domain Cry toxins represent the biggest group with pore formation as a widely accepted model for insect killing. A novel group of Cry proteins has been identified known as parasporins. They do not show insecticidal or hemolytic activity, but exert a strong cytocidal effect against some human cancer cells. The preferential activity of parasporins has potential for anticancer drug design but at the same time the knowledge that some Bt toxins are able to kill mammalian cells may raise concerns about the use of Bt-based pesticides in the future. Out of 19 parasporins Parasporin-3 (PS-3) most closely resembles the commercially used insecticidal toxins and is toxic to a narrow range of human cancer cell lines. In this study the effect of recombinant PS-3 on the human hepatic cancer cell line HepG2 was investigated to elucidate its mode of action. Results are consistent with PS-3 being a pore forming toxin. The toxin induced: pore formation in artificial and biological membranes, irreparable membrane damage, cell swelling, rapid decrease in ATP levels and drop in metabolic activity. The toxin did not induce activation of caspases or oxidative stress. In response to the toxin, cells activated p38 MAPK, a conserved signalling pathway induced in host cells by pore forming toxins. PS-3 induced very stable lesions and p38 MAPK did not facilitate cell recovery. Identification of a proteinous receptor was unsuccessful, but the toxin interaction with the membrane was prevented by EGTA facilitated chelation of membrane associated cations, suggesting the existence of a cation dependent receptor.