Mini-oral session: Fri 2/15 4:00 pm Oncology IX: Apoptosis, immunology, and angiogenesis| Volume 144, ISSUE 2, P389, February 2008

QS308. HSP70 Inhibition Sensitizes to TRAIL Induced Apoptotic Cell Death in Pancreatic Cancer Cells

      Pancreatic cancer is highly resistant to current conventional therapeutic options because it expresses different mutations that increase early invasiveness, metastasis and resistance to apoptosis after chemo- or radiotherapy. We have previously shown that pancreatic cancer cell lines overexpress Heat Shock Protein 70 (HSP70) and that HSP70 inhibition either with siRNA or triptolide, a novel inhibitor of HSP70 expression induces apoptosis in pancreatic cancer cells. Furthermore, we have also shown that triptolide reduces tumor growth rate as well as the loco-regional spread of tumors in an orthotopic model of pancreatic cancer. Also, our data suggests that inhibition of HSP70 expression releases cytochrome c from the mitochondria, thus activating the intrinsic apoptotic pathway. Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) is emerging as a therapeutic option in some tumors. Previous studies have suggested that TRAIL can activate both apoptotic pathways, but most of its effects are exerted through the activation of the extrinsic pathway. TRAIL increases apoptosis in tumor cells without affecting normal cells, however pancreatic cancer cells are known to be highly resistant to TRAIL. The purpose of this study was to evaluate whether or not inhibition of HSP70 expression sensitizes pancreatic cancer cells to TRAIL induced cell death. Further, we are interested to know whether or not TRAIL influences the level of HSP70 expression in pancreatic cancer cells. Methods: Pancreatic cancer cells (Panc-1 and MiaPaCa 2) were treated with different concentrations of triptolide (25-200 nM), TRAIL (1.25-20 ng/ml), or a combination of both. The effect on cell viability and early apoptosis were assessed by CCK8 and analysis of annexin V staining by flow cytometry respectively. HSP70 expression was analyzed by western blotting. Results: Both triptolide and TRAIL, used separately, reduce the viability of cells in MiaPaCa-2 and Panc-1 cells (Table 1). Remarkably, there is a significant synergistic effect when both drugs are combined. The percentage of cells undergoing apoptosis also increases when both drugs are combined in both cell lines (Table 1). Both triptolide and TRAIL reduce the expression ofHSP70 protein as measured by western blotting. Conclusion: TRAIL and triptolide induce cell death and apoptosis in pancreatic cancer cells and act synergistically when used together. This has great implication for the treatment of pancreatic cancer. In this study, we have shown for the first time that TRAIL inhibits expression of HSP70, a phenomenon hitherto unknown. The elucidation of the mechanism by which TRAIL inhibits the expression ofHSP70 will be of great clinical significance.
      TABLE 1Effect of Trail and Triptolide in the Cell Viability and Apoptosis in Pancreatic Cancer Cells
      Drug Cell Death (% of Control) Apoptosis (% of Cells)
      MiaPaCa-2 PANC-1 MiaPaCa-2 PANC-1
      Triptolide 50 nM 29 ± 6% 22 ± 4% 16.6 ± 5.5% 18.6 ± 4.9%
      TRAIL 1.25 ng/ml 9 ± 9% 1 ± 7% 11.2 ± 2.3% 15 ± 2.8%
      Triptolide 50 nM + TRAIL 1.25 ng/ml 65 ± 4% 51 ± 10% 39 ± 6.7% 66 ± 10.8%
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