A Study On The Cycotoxic Effect Of Bacillus Thuringiensis 18 Toxin On A Leukaemic Cell Line (CEM-SS)

Abstract

Bacillus thuringiensis (Bt) has long been used in the agriculture fields as insecticides. Recent studies showed that Bt toxins exhibit cytotoxicity against human cancer cells. This study looked at the cytotoxic effect of the toxin of a locally isolated Bt, namely Bt 18, and the mechanism of cell death it induced on a leukaemic cell line, CEM-SS. Previous works demonstrated that Bt 18 toxin preferentially killed CEM-SS cells but exerted little or no cytotoxicity on other human cancer cell lines and human T lymphocytes. Bt 18 was cultured, its toxin was harvested and purified by FPLC using Resource Q® column. Cell viability assays were carried out using the standard MTT calorimetric method. The mode of cell death was determined using the Cell Death Detection ELISAPLUS kit (Roche). Morphological analysis was carried out using light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bt 18 toxin was biotinylated for homologous competitive binding study using three leukaemic cell lines, CEM-SS, CCRF-SB, CCR-HSB-2 and a totally different class of cells, MCF-7 (breast cancer cells) for the determination of the dissociation constant (Kd) for each cell line. Heterologous competitive binding study using cisplatin, doxorubicin, etoposide, methotrexate and navelbine (commercial anticancer drugs), Btj toxin and Bt 22 toxin was carried out to determine if competition for binding sites existed between Bt 18 toxin and these test compounds on CEM-SS. To localise the binding site, the biotinylated toxin was tagged with FITC-conjugated avidin and visualised under the confocal microscope. At 72 hours, Bt 18 toxin and Btj toxin significantly reduced cell viability of CEM-SS to 72.45% and 63.62% at 20μg/ml respectively (p<0.001). Normal human T lymphocytes remained relatively unharmed with cell viability of 86.66% for Bt 18 toxin at 160μg/ml (p>0.05) but was significantly reduced to 55.77% for Btj toxin at 160μg/ml (p<0.05). The mode of cell death was found to be apoptosis for Bt 18 toxin and Btj toxin using the Cell Death ELISAPLUS kit. At 72 hours, the highest percentage of apoptotic cell death was achieved at 34.32% and 41.84% for Bt 18 toxin and Btj toxin respectively (p<0.001). Light microscopy, SEM and TEM demonstrated early apoptotic features at 3, 6 and 12 hours, followed by late secondary necrotic changes at 24 and 72 hours for both toxins. Homologous competitive binding study revealed decreasing binding affinity of Bt 18 toxin for CEM-SS, CCRF-SB and CCRF-HSB-2, with Kd of 8.44nM, 14.98nM and 17.71nM respectively. Kd for MCF-7 could not be determined due to insufficient displacement of the biotinylated toxin. Heterologous competitive study showed little competition between biotinylated Bt 18 toxin and Btj toxin, Bt 22 toxin (<30%) and all five anticancer drugs (<45%). Confocal microscopy revealed binding of biotinylated Bt 18 toxin at the periphery of the cell. In conclusion, Bt 18 toxin selectively exhibited cytotoxicity on CEM-SS but not normal human T lymphocytes. The mode of cell death was apoptosis. It most likely bound to binding sites on the cell surface of CEM-SS and its mechanism of cell death most likely differed from that of Btj toxin, Bt 22 toxin and all five anticancer drugs used in this study.