HSP70 INHIBITS APOPTOSIS IN PANCREATIC CANCER CELLS BY ATTENUATING INTRACELLULAR CALCIUM

Abstract

Pancreatic cancer cells are resistant to apoptosis. We have previously shown that heat shock protein 70 (HSP70) levels are increased in pancreatic cancer cells and that its inhibition by either HSP70 siRNA, Triptolide (a diterpene triepoxide) or Quercetin (a bioflavonoid) induces apoptosis. Since increased cytosolic calcium can result in apoptosis, our group's interest has been to evaluate the role of calcium (Ca2+) in apoptosis induced by inhibition of HSP70 expression. Our initial studies have shown that thermal stress induced overexpression of HSP70 in normal pancreatic duct cells attenuated the Ca2+ response to PAR-2 activating peptide. We hypothesize that HSP70 overexpression in pancreatic cancer cells prevents apoptosis by attenuation of intracellular Ca2+ concentrations. Methods: HSP70 expression was inhibited by siRNA specific for HSP70, Triptolide (200nM) or Quercetin (100 μM) and intracellular Ca2+ levels were evaluated by flow cytometry. Human pancreatic cancer cells (MiaPaCa-2 or PANC-1) were pretreated with the intracellular Ca2+ chelator BAPTA-AM (5μM) or DMSO alone and then treated with Triptolide or Quercetin for 24h. Cell viability was assessed by WST-8 assay and Annexin V was analyzed as a marker of apoptosis by flow cytometry. Results: Inhibiton of HSP70 by siRNA, Triptolide or Quercetin significantly increased intracellular Ca2+ levels (Table 1). Both triptolide and Quercetin significantly decreased the viability of pancreatic cancer cells by inhibiting the expression of HSP70 (Table 2). However, pre-treatment of both PANC-1 and MiaPaCa-2 cells with BAPTA-AM prevented the decrease in the viability of these cells induced by inhibition of HSP70 expression. Pre-treatment with BAPTA-AM decreased apoptosis induced by HSP70 inhibition in MiaPaCa-2 and PANC-1 cells as evidenced by decreased Annexin-V staining.TABLE 1: Intracellular Calcium Levels (% of Control, Mean (SE)TABLE 2: Viability and Annexin V (% of Control, Mean ± SE)Conclusion: Intracellular Ca2+ plays an important role in apoptosis induced by HSP70 inhibition in pancreatic cancer cells.