02.05 Inhibition of lysosomal enzyme activity enhances antitumor effect of gemcitabine in pancreatic cancer cells.

R. Hamura1,2, Y. Shirai1,2, Y. Shimada2, N. Saito1,2, T. Horiuchi1,2, H. Sugano1,2, N. Takada1,2, T. Taniai1,2, Y. Kanegae3, T. Ohashi2, K. Yanaga1  1The Jikei University School of Medicine,Department Of Surgery,Minato-ku, TOKYO, Japan 2Research Center for Medical Science, The Jikei University School of Medicine,Division Of Gene Therapy,Minato-ku, TOKYO, Japan 3Research Center for Medical Science, The Jikei University School of Medicine,Core Research Facilities Of Basic Science (Molecular Genetics),Minato-ku, TOKYO, Japan

Introduction: Autophagy plays an important role in metabolism of anticancer agents and chemoresistance. Suppression of autophagy is expected to be a new strategy for cancer. Because autophagy depends on hydrolysis by lysosome enzymes, we hypothesized that down-regulation of lysosomal enzymes may induce autophagy dysfunction and enhance chemosensitization. In this study, we knocked down the gene of an lysosomal enzyme acid-αglucosidase (GAA), to evaluate the anti-tumor effects on pancreatic cancer.

Methods: The autophagy levels, apoptosis signals, cell viabilities and the enzyme activities of GAA were assessed using gemcitabine-resistance pancreatic cancer cell lines (PANC-1 and MIA PaCa-2) by Western blot analysis, cell viability MTT assay and GAA enzyme activity assay. These experiments were assessed by GAA knock down with siRNA.

Results: In both cell lines, the expression levels of LC3-II and LAMP2 protein were increased and p62 was deceased by gemcitabine. The expression levels of GAA protein were elevated by gemcitabine. Also, the enzyme activities of GAA were elevated by treatment of gemcitabine in a dose dependent manner (PANC1; gemcitabine (1μM)  120±8.0 % of control, p=0.034, MIA PaCa-2; gemcitabine (0.5μM); 157±7.3 % of control, p<0.01). Knockdown of GAA protein enhanced  gemcitabine-induced expression levels of apoptotic signals (Cleaved caspase-3, -8, -9 and cleaved PARP), which were superior to the effects by an autophagy inhibitor, bafilomycin A1. Moreover, antiproliferative effects were increased by suppression of GAA as compared to gemcitabine mono therapy (MIA PaCa-2; gemcitabine vs. siGAA+gemcitabine, 110 ± 6.00 vs. 89.7± 9.69% of control, p<0.01). 

Conclusion:Down-regulation of GAA enzyme activity induces autophagy dysfunction and enhances anti-tumor effect of gemcitabine on pancreatic cancer.