Abstract 2497: Dual inhibition of PI3K/mTOR by PI-103 inhibits pancreatic cancer cell growth mediated via autophagy and cell cycle arrest
Authors: Xiao Dakai , Wei Hua , Feng Pei 
Published: February 2011

Abstract
Pancreatic cancer is characterized by its extremely poor prognosis and it is the 4th leading cancer death in both men and women. The limited scientific knowledge and lack of effective treatment of this cancer are critically urging to an extensive research development in the field. Emerging evidence shows that pancreatic cancer cells exhibit aberrant activation of class I phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling. To understand the molecular mechanism underlying the relationship between signaling activation and pancreatic cancer cell growth, we investigated the anti-cancer effect of a novel dual inhibitor of PI3K/mTOR, PI-103 {3-(4-(4-Morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl)phenol}, on pancreatic cancer cells. The cell proliferation experiment assessed by MTT assay showed that PI-103 (0.1-10μM) effectively inhibits the growth of p53-mutant pancreatic cancer cells including Panc-1, MIA paca-2, BxPC-3 and AsPC-1 cells. The growth inhibitory effect of PI-103 was further supported by clonogenic assay showing a complete blockage of colony formation of Panc-1 cells after 7 days treatment. Our results demonstrated that PI-103 induces autophagy in Panc-1 cells characterized by a significant increase of the conversion of LC3-1 to LC3-II, which was then attenuated by an autophagy inhibitor, 3-methyladenine (3-MA), and augmented using a thiol proteinase inhibitor, E-64d, by blocking the degradation of autolysosomes. Knockdown of the expression of beclin1 with siRNA attenuated LC3 conversion and the growth inhibitory effect of PI-103 as well as colony formation, suggesting a possible linkage of autophagy and cell growth inhibition. Furthermore, cell cycle analysis and apoptotic assay with Annexin V staining showed that PI-103 induced G0/G1 arrest significantly without an effective induction of apoptosis. Western blotting revealed that PI-103 regulated the expression of cell-cycle relevant proteins, including increased p27 and decreased CDK1 and cyclin B. In addition to the effect of PI-103 on autophagy and cell cycle arrest, to our surprise, a striking appearance of cytoplasmic vacuoles ranging from 1-4μm in diameter was observed in the cells treated with PI-103 at as early as 3 hrs. The origin of the vacuoles was explored using the markers with GFP-LAMP1 for late endosome and lysosome, and DsRed-FYVE-labeling for early endosome, respectively. The results showed the large vacuoles are LAMP1-positive lysosomes or late endosomes, but not early endosomes, which is leading to future studies. Taken altogether, our data show that PI-103 is a potent compound for inhibiting pancreatic cancer cell growth, and the anti-cancer effect may operate through the induction of autophagy and cell cycle arrest.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2497.