Norcantharidin triggers cell death and DNA damage through S-phase arrest             and ROS-modulated apoptotic pathways in TSGH 8301 human urinary bladder carcinoma             cells.
Norcantharidin (NCTD) is one of the ingredients of blister beetles which have been used in Chinese medicine for a long time. The purpose of this study was to investigate the inhibitory effects of NCTD on TSGH 8301 human bladder cancer cells in vitro and the mechanisms through which it exerts its anticancer action. Cell morphological analysis was performed using a phase-contrast microscope. The percentage of viable cells, cell cycle distribution, sub-G1 phase (apoptosis), reactive oxygen species (ROS) production and the levels of mitochondrial membrane potential (∆Ψ(m)) were analyzed by flow cytometry. DNA condensation and damage were determined by DAPI staining and comet assay. Apoptosis-associated protein level changes in TSGH 8301 cells following exposure to NCTD were examined, measured and determined by western blotting. Analysis of protein translocation was conducted by immunostaining and confocal laser microscopy. The results indicated that NCTD promoted cytotoxic effects, including the induction of cell morphological changes and the decrease in the percentage of viability, the induction of S-phase arrest as well as sub-G1 phase (apoptosis) in TSGH 8301 cells. The activities of caspase-3 and -9 were upregulated following NCTD treatment. Western blotting indicated that NCTD upregulated Fas, FasL, Bax, Bid, cytochrome c, caspase-3, -8 and -9 that led to the induction of apoptosis through the Fas extrinsic pathway. Furthermore, NCTD induced AIF and Endo G that were released from mitochondria to induce apoptosis through the mitochondrial-independent pathway. NCTD upregulated ROS production, downregulated ∆Ψ(m) and ERK, JNK, p38 protein kinases in TSGH 8301 cells. These findings suggest that NCTD triggers apoptosis in TSGH 8301 human bladder cancer cells via the Fas receptor, activation of the caspse-8, -9 and -3, mitochondrial-dependent and -independent pathways. NCTD may be useful for developing new therapeutic regimens for the treatment of bladder cancer.
School of Pharmacy, China Medical University, Taichung 404, Taiwan,                     R.O.C.
Chien-Chih
CC
Yu
Fang-Yu
FY
Ko
Chun-Shu
CS
Yu
Chin-Chung
CC
Lin
Yi-Ping
YP
Huang
Jai-Sing
JS
Yang
Jing-Pin
JP
Lin
Jing-Gung
JG
Chung
D017209:Apoptosis / Q000187:drug effects*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D053148:Caspase 3 / Q000096:biosynthesis; D053453:Caspase 9 / Q000096:biosynthesis; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004249:DNA Damage; D053222:Fas Ligand Protein / Q000096:biosynthesis; D006801:Humans; D008516:Medicine, Chinese Traditional; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015854:Up-Regulation; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D019014:fas Receptor / Q000096:biosynthesis
D019086:Bridged Bicyclo Compounds, Heterocyclic; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; C069741:norcantharidin; D053148:Caspase 3; D053453:Caspase 9