Theses (PHD)

Bacillus thuringiensis (Bt) parasporal proteins with anti-cancer activity have gained interest due to its selectivity towards cancer cells. This study determines the efficacy and mechanism of action of parasporal proteins harvested from a Malaysian Bt strain (Bt 18) against three leukaemic cell lines (CEM-SS, CCRF-SB and CCRF-HSB-2) compared to normal T-lymphocytes. Results from the present study shows that Bt 18 parasporal proteins consist of two subunits of polypeptide (68 kDa and 28 kDa) which co-elute during ion-exchange chromatography. The N-terminal sequence of Bt 18 parasporal proteins shows that 68-kDa polypeptide has high similarity with Cry25Aa and Cry24Aa, parasporal proteins found in a mosquitocidal isolate from Malaysia, Bt serovar jegathesan. However, the 68-kDa polypeptide of Bt 18 parasporal protein is non-insecticidal. The alignment results of N-terminal sequence of 68-kDa Bt 18 parasporal protein with various parasporin groups showed that it could be a new group of parasporins. The solubilised and activated parasporal proteins of Bt 18 exhibited moderate anti-cancer potential by lowering the percentage cell viability for CEM-SS, CCRF-SB and CCRF-HSB-2 cells, whilst being non-cytotoxic to normal human T lymphocytes. Interestingly further purification of the parasporal proteins from Bt 18 lowers the concentration of 28 kDa subunit and changes in inhibition selectivity for the three cell lines was observed. Phosphatidylserine externalisation, active caspase 3 and TUNEL assays detect and confirm apoptotic activity in the leukaemic cells treated with Bt 18 parasporal proteins. Western blot analysis suggests that Bt 18 binds to Glyceraldehyde 3-phosphate dehyrogenase (GAPDH) in all three cell lines. The present study suggests that Bt 18 parasporal proteins caused cell cycle arrest at S-phase and induce apoptosis that leads to the reduction of percentage cell viability, although more than one cellular pathway may be involved. When combined with the all the results obtained, the present study suggests that Bt 18 parasporal proteins would bind to cancer cells (CEM-SS, CCRF-SB and CCRF-HSB-2) on the surface membrane binding protein GAPDH that may lead to S-phase cell cycle arrest, initiating apoptosis and reducing cell viability.