Immunomodulatory proteins FIP-gts and chloroquine induce caspase-independent cell death via autophagy for resensitizing cisplatin-resistant urothelial cancer cells.
BACKGROUND
Chloroquine, a lysosomal inhibitor, is used for malaria, rheumatoid arthritis, and lupus erythematosus therapy. In our previous study, FIP-gts, an immunomodulatory protein from Ganoderma tsugae, inhibited cell viability in lung cancer cells and urothelial cancer cells. Urothelial carcinoma is the most common type of bladder cancer. Cisplatin resistance is an important issue in urothelial carcinoma therapy.


OBJECTIVE
The aim of this study is to investigate the effect of combination treatment with FIP-gts and chloroquine on cytotoxicity to resensitize the cisplatin-resistant cells.


METHODS
FIP-gts and chloroquine cytotoxicity were determined by evaluating CCK-8 assay. Cell death pathways, ROS and cell cycle arrested were analysed through flow cytometry and Western blot. ShRNA targeting to autophagy-related genes were tested to evaluate their autophagic cell death for resistant urothelial cells.


RESULTS
Using CCK-8 assay, chloroquine increased FIP-gts-induced cytotoxicity 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. Caspase-3/PARP cascade and z-VAD-fmk were performed to prove that FIP-gts and chloroquine induced caspase-independent cell death. 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. To elucidate the role of autophagy in caspase-independent cell death by FIP-gts and chloroquine, LC3 shRNA were used to inhibit autophagy in N/P cells. The capabilities of FIP-gts and chloroquine to induce cytotoxicity and sub-G1 phase accumulation were abolished in autophagy-defective cells. This is the first study to reveal the novel function of FIP-gts in triggering caspase-independent cell death in cisplatin-resistant urothelial cancer cells.


CONCLUSIONS
Chloroquine enhanced FIP-gts-induced autophagy dependent caspase-independent cell death via abundant autophagosome accumulation. Combination treatment with FIP-gts and chloroquine may provide a new strategy for urothelial cancer therapy.
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan; Inflammation Research & Drug Development Center, Changhua Christian Hospital, Changhua, Taiwan.
I-Lun
IL
Hsin
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan; Department of Urology, Chung-Shan Medical University Hospital, Taichung, Taiwan; School of Medicine, Chung-Shan Medical University, Taichung, Taiwan.
Shao-Chuan
SC
Wang
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan; Division of Urology, Department of Surgery, Taichung Veterans General Hospital, Taichung, Taiwan.
Jian-Ri
JR
Li
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan.
Tsai-Chun
TC
Ciou
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan.
Chih-Hsien
CH
Wu
Inflammation Research & Drug Development Center, Changhua Christian Hospital, Changhua, Taiwan; Graduate Institute of Acupuncture Science, China Medical University, Taichung, Taiwan; Department of Neurology, Changhua Christian Hospital, Changhua, Taiwan.
Hung-Ming
HM
Wu
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan; Department of Urology, Chung-Shan Medical University Hospital, Taichung, Taiwan; School of Medicine, Chung-Shan Medical University, Taichung, Taiwan. Electronic address: jlko1007@gmail.com.
Jiunn-Liang
JL
Ko
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002738:Chloroquine / Q000008:administration & dosage / Q000494:pharmacology*; D002945:Cisplatin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D005656:Fungal Proteins / Q000008:administration & dosage / Q000494:pharmacology*; D032902:Ganoderma / Q000737:chemistry; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D014571:Urologic Neoplasms / Q000188:drug therapy* / Q000473:pathology
D000590:Amino Acid Chloromethyl Ketones; D005656:Fungal Proteins; D007155:Immunologic Factors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D002738:Chloroquine; D020169:Caspases; D002945:Cisplatin
Abundant autophagosome accumulation stress; Autophagy; Caspase-independent cell death; Chloroquine; Cisplatin-resistant urothelial cells; FIP-gts