Cytotoxic Effects and Intracellular Localization of Bin Toxin from Lysinibacillus sphaericus in Human Liver Cancer Cell Line.

Simab Kanwal,Panadda Boonserm,Monrudee Srisaisup,Shalini Abeysinghe

doi:10.3390/toxins13040288

Abstract

Binary toxin (Bin toxin), BinA and BinB, produced by Lysinibacillus sphaericus has been used as a mosquito-control agent due to its high toxicity against the mosquito larvae. The crystal structures of Bin toxin and non-insecticidal but cytotoxic parasporin-2 toxin share some common structural features with those of the aerolysin-like toxin family, thus suggesting a common mechanism of pore formation of these toxins. Here we explored the possible cytotoxicity of Bin proteins (BinA, BinB and BinA + BinB) against Hs68 and HepG2 cell lines. The cytotoxicity of Bin proteins was evaluated using the trypan blue exclusion assay, MTT assay, morphological analysis and LDH efflux assay. The intracellular localization of Bin toxin in HepG2 cells was assessed by confocal laser scanning microscope. HepG2 cells treated with BinA and BinB (50 µg/mL) showed modified cell morphological features and reduced cell viability. Bin toxin showed no toxicity against Hs68 cells. The EC50 values against HepG2 at 24 h were 24 ng/mL for PS2 and 46.56 and 39.72 µg/mL for BinA and BinB, respectively. The induction of apoptosis in treated HepG2 cells was confirmed by upregulation of caspase levels. The results indicated that BinB mediates the translocation of BinA in HepG2 cells and subsequently associates with mitochondria. The study supports the possible development of Bin toxin as either an anticancer agent or a selective delivery vehicle of anticancer agents to target mitochondria of human cancer cells in the future.

Highlights

Introduction published maps and institutional affilBinary toxin (Bin toxin) is produced as crystalline inclusions during the sporulation stage of Lysinibacillus sphaericus
Culex larvae were fed with fluorescently labeled BinA and BinB, and the dissected larvae were visualized by using a confocal laser scanning microscope
To explore the cytotoxic effects of Bin toxin on human cancer cell lines compared with PS2, HepG2 cell line was used as a model system in this study

Summary

Introduction

Introduction published maps and institutional affilBinary toxin (Bin toxin) is produced as crystalline inclusions during the sporulation stage of Lysinibacillus sphaericus (hereafter L. sphaericus). Bin toxin contains two crystal proteins, namely BinA (Tpp1Aa2) and BinB (Tpp2Aa2) with the sizes of 42 and 51 kDa, respectively, and both of them act together to exert their toxicity against Culex and Anopheles mosquito larvae [1]. Both BinA and BinB share little amino acid sequence homology with those from other bacterial toxins (such as insecticidal toxins), but they share 25% sequence identity and 40% similarity among themselves [2]. Culex larvae were fed with fluorescently labeled BinA and BinB, and the dissected larvae were visualized by using a confocal laser scanning microscope.

Objectives

Methods

Results