Bengalin initiates autophagic cell death through ERK–MAPK pathway following suppression of apoptosis in human leukemic U937 cells.

Abstract: Aims: The aim of this study was to assess the autophagy inducing ability of the scorpion venom toxin Bengalin in human leukemic U937 cells. The same toxin was previously shown to induce apoptosis in human leukemic cells. Main methods: Bengalin was purified from Indian black scorpion (Heterometrus bengalensis) venom by ion exchange chromatography and HPLC. In human leukemic U937 cells, Bengalin associated MAPK (mitogen activated protein kinase) pathway was determined by western blotting. Downstream to MAPK, the Bengalin induced apoptosis-mediator caspase-3 was blocked by chemical inhibitor and reconfirmed by siRNA mediated gene knockdown. Subsequent to caspase-3 blocking, the autophagic response was evaluated by quantification of acidic vesicle organelles formation and modulations of Atg's, Beclin-1, LC3-1 and LC3-II expression by western blotting. Key findings: In U937 cells, Bengalin increased ERK1/2 expression to bring about cell death. However in subsequent caspase-3 blocked conditions, Bengalin downregulated p-Akt, p-mTOR and decreased apoptosis. It had also increased the percentage of acidic vesicle organelles positive cells. Bengalin could induce autophagic response by augmenting Beclin-1, Atg12, Atg7, Atg5 and Atg3 in U937 cells. Moreover a time dependant reciprocal relation was observed between LC3-I and LC3-II expression upon Bengalin treatment. The decrease in LC3-II was inhibited in the presence of lysozomal enzyme blockers thereby suggesting lysosome involvement in the autophagic response. Significance: We have for the first time demonstrated that scorpion venom-component could induce an alternate cell death pathway other than apoptosis in the form of autophagy in human leukemic U937 cells. [Copyright &y& Elsevier]