MP34-18 INHIBITION OF AUTOPHAGY ENHANCED THE CELL DEATH INDUCING EFFECT OF EVEROLIMUS (RAD001) IN HUMAN BLADDER CANCER CELLS

Abstract

INTRODUCTION AND OBJECTIVES: mTOR, a downstream protein kinase of phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, has been recognized to play a central role in controlling cancer cell growth. The PI3K/AKT/mTOR pathway promotes tumor growth and survival while suppressing autophagy. Conversely, inhibitors of the mTOR pathway such as Everolimus (RAD001), induce autophagy that promote tumor survival and thus, these agents potentially limit their own efficacy. We hypothesized that inhibition of autophagy in combination with mTOR inhibition would improve the cytotoxicity of mTOR inhibitors in bladder cancer. METHODS: The cytotoxicity of RT4 (grade I), 5637 (grade II), HT1376 (grade III) and T24 (grade III) human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (BafA1), chloroquine (CQ) or hydroxychloroquine (HCQ)) was accessed by WST-8 cell viability kit. Inhibition of mTOR pathway by RAD001 was monitored by home-made QPCR gene array and the detection of phospho-mTOR by Western blot. The autophagy status in cells was performed by the detection of microtubule-associated light chain 3 form II (LC3-II) using immunofluorescent staining and Western blot. Acidic organelle formation in treated cells was determined by acridine orange (AO) vital staining. Induced apotposis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment. RESULTS: Advanced bladder cancer cells (5637, HT1376 and T24) were more resistant to RAD001 than RT4. AO formation was detected in cells treated with RAD001 and inhibited by the addition of 3-MA or Baf A1. Autophagy flux detected by the expression of LC3-II showed RAD001 induced autophagy. However, the LC3-II formation was not inhibited by the increasing concentration of 3-MA. Co-treatment of RAD001 with autophagy inhibitors further reduced cell viability and induces apoptosis in bladder cancer cells. CONCLUSIONS: Our data suggest that coordinate inhibition of the mTOR and autophagy pathways promotes apoptosis and could be a new therapeutic paradigm for the treatment of bladder cancer.