Inhibition of grade dependent autophagy in urothelial carcinoma increases cell death under nutritional limiting condition and potentiates the cytotoxicity of chemotherapeutic agent.
OBJECTIVE
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.


METHODS
The study was performed in tumor tissue from patients with low and high grade urothelial carcinoma, in normal urothelial tissue and in the 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.


RESULTS
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 3 dependent cell death. Combined treatment with cisplatin and an autophagy inhibitor resulted in greater cell death than cisplatin treatment alone.


CONCLUSIONS
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.
Department of Urology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Rani
R
Ojha
Department of Urology, Post Graduate Institute of Medical Education and Research, Chandigarh, India. Electronic address: shrawanksingh2002@yahoo.com.
Shrawan K
SK
Singh
Department of Biophysics, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Shalmoli
S
Bhattacharyya
Department of Translational and Regenerative Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Rakesh Singh
RS
Dhanda
Department of Translational and Regenerative Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Aruna
A
Rakha
Department of Urology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Arup K
AK
Mandal
Department of Translational and Regenerative Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
Vivekanand
V
Jha
D000970:Antineoplastic Agents / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects*; D002295:Carcinoma, Transitional Cell; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival; D019008:Drug Resistance, Neoplasm; D006801:Humans; D015398:Signal Transduction; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy / Q000473:pathology*; D019459:Urothelium / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*
D000970:Antineoplastic Agents
apoptosis; autophagy; carcinoma; urinary tract; urothelium