Abstract 5417: A novel curcumin derivative DiFiD prevents pancreatic cancer growth in vivo by suppressing Notch-1

Abstract

Abstract Background and Aims: Dysregulated Notch signaling plays an important role in the progression of pancreatic cancer. Previous studies have demonstrated that the dietary preventive agent curcumin modulates Notch activity. We have developed a novel curcumin analog DiFiD and determined that it inhibits pancreatic cancer cell growth by affecting proteins that regulate Notch signaling. Methods. Five pancreatic adenocarcinoma cell lines (AsPC-1, MiaPaCa-2, PanC-1, BxPC-3 and Pan02) were used in the study. Cell proliferation was measured for hexosaminidase activity and colony formation assay. Apoptosis was determined by measuring caspase 3/7 activity and western blot analysis for caspase 3. Cell cycle analysis was performed by propidium iodide staining and flow cytometry. Real Time PCR and western blot analyses were used to measure mRNA and protein levels. For in vivo studies, nude mouse xenografts were developed with Pan02 mouse pancreatic tumor cells. Immunohistochemistry was performed for CD31 (surrogate for angiogenesis), COX-2, VEGF and Notch-1. Results. DiFiD treatment resulted in a dose- and time-dependent inhibition of proliferation and in colony formation in all 5 pancreatic cell lines, but not that of normal mouse embryo fibroblasts. Cell cycle analyses demonstrated that DiFiD induced G2/M cell cycle arrest, which was followed by the induction of apoptosis as evidenced by the increased number of cells in the sub-G1 DNA fraction and activated caspase-3. Western blot analyses demonstrated that DiFiD down-regulated the expression of cell cycle related proteins cyclin A, D1 and E, while upregulating p21Waf1. Western blot analyses demonstrated that DiFiD inhibited Notch-1 activation as evidenced by a downregulation in the intracellular domain (NICD) and its downstream targets Hes-1 and Hey1. Expression of the Notch ligand Jagged-1 was also reduced. We further determined that the inhibition of Notch-1 signaling is due to a downregulation in Presenilin-1 and Nicastrin, critical componenst of the gamma-secretase complex. DiFiD also down regulated the expression of COX-2, IL-8 and VEGF. In vivo, nude mice harboring pancreatic cancer cell xenografts showed significant reduction in tumor growth when administered the compound intraperitoneally every day for 21 d. Microvessel density, based on CD31 staining was also significantly lower in DiFiD-treated tumors when compared to in controls. Real Time PCR and immunohistochemistry analyses also demonstrated significant inhibition of COX-2, VEGF, Notch-1, and cyclin D1 expression in the tumors. Conclusion. Together, these data suggest that DiFiD is a novel potent inhibitor of pancreatic tumor growth that targets the Notch-1 activating gamma-secretase complex proteins Presenilin-1 and Nicastrin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5417. doi:10.1158/1538-7445.AM2011-5417