Inhibition of Grade Dependent Autophagy in Urothelial Carcinoma Increases Cell Death under Nutritional Limiting Condition and Potentiates the Cytotoxicity of Chemotherapeutic Agent

Abstract

We evaluated the status of autophagy in different grades of urothelial carcinoma and explored autophagy modulators as a potential adjunctive therapeutic agent for urothelial carcinoma. The study was performed in tumor tissue from patients with low and high grade urothelial carcinoma, in normal urothelial tissue andinthe T24 cell line. Autophagic vesicles and the expression of various autophagic proteins were studied in tissue samples by transmission electron microscopy and Western blot, respectively. The effect of autophagy induction and inhibition was evaluated by measuring AMPK and mTOR expression, cell viability and mitochondrial membrane potential. The therapeutic implication of autophagy was studied using cisplatin alone or combined with an autophagy inhibitor. High grade urothelial carcinoma showed a higher number of autophagic vesicles and significantly higher expression of autophagic proteins. Upon starvation cells cultured from high and low grade urothelial carcinoma demonstrated significant autophagy induction associated with AMPK activation and mTOR inhibition. AMPK inhibition decreased the autophagic response and increased cell death. Autophagy inhibition by wortmannin, 3-methyladenine and chloroquine increased mitochondrial hypopolarization as well as caspase-9 and 3dependent cell death. Combined treatment with cisplatin and an autophagy inhibitor resulted in greater cell death than cisplatin treatment alone. Autophagy is related to urothelial carcinoma grade and regulated via the AMPK pathway for tumor cell survival. Autophagy inhibition leads to cancer cell death through an intrinsic apoptotic pathway. The potential application of autophagy inhibitors as an adjunct to chemotherapy for urothelial carcinoma must be explored.