Combination of FIP-gts and Chloroquine Induces Cell Death via Autophagy and Caspase-independent Pathway in Cisplatin-resistant Urothelial Cancer Cells

• Main Authors: Cai-Chun Ciou, 邱彩醇
• Other Authors: You-Cheng Hseu
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/73480218249741556676

碩士 === 中山醫學大學 === 醫學研究所 === 103 === Urothelial carcinoma (UC) is the most common cancer in Taiwan men. The average age at the time of diagnosis is over 60. Surgery of transurethral resection using cytoscope is considered to be an option only when the disease is in its early stages. Cisplatin-based chemotherapy is highly effective for metastatic urothelial carcinoma. However, the progression free survival is short and the median overall survival ranges from only 8 to 14 months in metastatic UC patients. Effective salvage therapy in patients in whom cisplatin based regimens fail was lacking until the introduction of new active cytotoxic agents, such as paclitaxel, in recent years. Although the treatment have approximately 50% response, the median overall survival is poor. In our previous study, FIP-gts, a fungal immunomodulatory protein, was cloned from Ganoderma tsugae and purified. FIP-gts possesses antitumor activity against solid tumors and inhibits telomerase activity. In additional, FIP-gts and Bafilomycin A1 combined treatment was found to lead to enhancement of apoptosis along with inhibition of autophagy in parental, NTUB1 and cisplatin-resistant, N/P cells. The purpose of this study was to examine the effects of FIP-gts and chloroquine co-treatment on inhibiting urothelial cancer cell viability. Using CCK-8 assay and Western blot, chloroquine increased FIP-gts-induced cytotoxicity and autophagosome accumulation in parental and cisplatin-resistant urothelial cancer cell lines. On flow cytometry, chloroquine enhanced FIP-gts-mediated sub-G1 accumulation, annexin V positive signal and mitochondrial membrane potential loss. Combination treatment with FIP-gts and chloroquine did not elicit the cleavage of caspase-3/7. Furthermore, z-VAD-fmk, a pan-caspase inhibitor, did not reverse the FIP-gts and chloroquine-suppressed cell viability. This suggest that FIP-gts and chloroquine induced caspase-independent cell death. The capabilities of FIP-gts and chloroquine to induce cytotoxicity and sub-G1 phase accumulation were abolished in autophagy-defective cells (LC3 shRNA). Using H2DCFDA staining and flow cytometry, FIP-gts and chloroquine did not induce ROS production. N-acetyl cysteine, a ROS scavenger, inhibited the cytotoxicity and LC3-II accumulation in FIP-gts and chloroquine-treated N/P cells. Necrostatin-1, a necroptosis inhibitor, did not reverse the FIP-gts and chloroquine-suppressed cell viability. We conclude that Chloroquine enhanced FIP-gts-induced autophagy dependent caspase-independent cell death via abundant autophagosome accumulation.