S. Modi1, B. Giri1, V. Sethi1, B. Garg1, J. George1, S. Banerjee1, V. Dudeja1, A. K. Saluja1 1Sylvester Cancer Center,Surgery,Miami, FL, USA
Introduction: Pancreatic cancer is an aggressive malignancy. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to cause cancer cell death in multiple cancers with minimal toxicity to un-transformed cells. Unfortunately, pancreatic cancer (PDAC) is resistant to TRAIL. We have previously shown that triptolide (TPL), a diterpene triepoxide isolated from a Chinese herb and its water soluble pro-drug Minnelide, is effective against pancreatic cancer. Minnelide is currently undergoing Phase-I trials against advanced GI malignancies. The aim of the current study is to evaluate the anti-tumor efficacy of lowered doses of Minnelide in combination with TRAIL in multiple models including an immunocompetent and stroma rich mouse model of pancreatic cancer.
Methods: The effect of TRAIL (0-40ng/ml), low dose of TPL (50nM), or combination of TRAIL and TPL on the viability (WST-8 assay) and apoptosis (cleaved caspase-3 and cleaved PARP levels using flow cytometry) of human pancreatic cancer cell lines (S2-VP10) and human pancreatic stellate cells (hPSCs) was measured. To evaluate efficacy of combination therapy in animal models of pancreatic cancer, subcutaneous xenograft model in athymic nude mice, subcutaneous syngeneic model in C57BL/6J wild type mice and a novel tumor implantation model in which 3 mm3 pieces of KPC tumors were implanted in pancreas of C57BL/6J wild type mice. The animals were randomized into four groups and treated with Minnelide (0.21 mg/kg/day ip), TRAIL (20mg/kg 3Xweek ip) or combination of the two for 4-6 weeks. At the end of 4-6 weeks, tumors were harvested, and tissues were used for various experiments. The animal doses of Minnelide correspond to the clinically relevant and well tolerated doses from the Phase-I trial.
Results:Combination of TRAIL 1.25ng/ml and TPL 50nM decreased the viability of S2VP10 (viability, % of Control, 25±4.6% after 24h of treatment) and significantly increased cleaved caspase-3 and PARP levels suggesting the activation of apoptotic cell death, whereas TRAIL alone did not influence viability of these cancer cells (viability, % of Control, 96±8.3% at 24h). TRAIL alone caused a downregulation of DR5 receptors while addition of TPL abrogated this effect. In the xenograft SC model, Minnelide and TRAIL alone did not have any effect on tumor size while combination of the two resulted in marked decrease in tumor growth. In the tumor implantation model, combination of low doses of Minnelide and TRAIL markedly inhibited tumor burden when compared to Minnelide or TRAIL alone. Tumor weights (gm, mean±SEM) after 3 week of Rx: Saline-1.63±0.1; Minnelide-1.15±0.02; TRAIL-1.54±0.04; Minnelide+TRAIL-0.38±0.03. TUNEL staining showed significantly increased apoptosis in the combination arm.
Conclusion:Minnelide synergizes with TRAIL therapy and reverses the TRAIL resistance in multiple models of pancreatic cancer including immunocompetent stroma rich model.