槲皮素(Quercetin)在膀胱癌細胞中誘發保護型細胞自噬之機制
蔡德甫1#、林致凡2#、楊尚哲2、鄧雅旻2、陳宏恩1、林宜佳1、仇光宇1、黃一勝1,2,3,4
1新光醫院外科部泌尿科、2新光醫院中央研究室、3台北醫學大學醫學院、4輔仁大學醫學院
Quercetin induces protective autophagy in human bladder cancer cells
Te-Fu Tsai1#, Ji-Fan Lin2#, Shan-Che Yang2, Ya-Ming Teng2,Hung-En Chen1, Yi-Chia Lin1,
Kuang-Yu Chou1, and Thomas I-Sheng Hwang1,3,4,5
Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital1, Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital2, Department of Urology, Taipei Medical University3, Division of Urology, School of Medicine, Fu-Jen Catholic University4, Taipei, Taiwan.
# These authors contributed equally to this work.
 
Background:Quercetin exists in flavonoids of a variety of plants, sharing antioxidant and anticancer properties, and has been demonstrated to induce apoptosis in different cancers. Previous studies demonstrated that cancer cells with mutant p53 are resistant to quercetin treatment. We therefore are interested in weather this resistant is related to autophagy induction in p53-mutated bladder cancer (BC) cells.
Materials and Methods: Human BC cell lines, including 5637 and T24 (with mutant p53) and immortalized uroepithelial cell line (SV-Huc-1, with wild-type p53) were used in this study. Cell viability in quercetin-treated cells was detected by WST-1 reagent. Induction of autophagy was monitored by (1) the processing of LC3-II autophagic marker, and the clearance of p62 cargo protein, and (2) the accumulation of LC3 puncta in the cytosol. The induction of apoptosis was determined by (1) detecting the caspase 3/7 activity, (2) the cleavage of caspase-3 and PARP, and (3) the increased level of DNA fragmentation. The mitochondria membrane potential was monitored by JC-1 staining followed by flow cytometry. The release of cytochrome C was detected in the cytosol protein of protein extracted from quercetin-treated cells. To investigate the signaling pathways involved in quercetin-induced autophagy, the expression level of phosphorylated AMPK, mTOR, ERK and p38 were detected by Western blot. Chloroquine (CQ), bafilomycin A1 (Baf A1) and shRNAs against ATG7/12 were utilized as autophagy inhibitors to investigate the role of autophagy induced by quercetin in BC cells.
Results:Quercetin treatment was found to be significant effective in inhibiting proliferation of both 5637 and T24 cell in a concentration-dependent manner after 72h of incubation. Quercetin induced BC cell death through apoptosis judging by the increased level of caspase 3/7 activity, DNA fragmentation, disruption of mitochondria membrane potential, release of cytochrome C, and cleavage of caspase 3 and PARP. We found that autophagy was induced in quercetin-treated cells as early as 12h post-treatment. Pretreatment of autophagy inhibitor strong augmented apoptosis in 5637 and T24 cells, indicating that quercetin induced protective autophagy. By screening the signaling pathway activated in quercetin-treated cells, we found that mTOR inhibition and the activation of BECN1 were responsible for the autophagy induction.
Conclusions:Our results demonstrated that quercetin induced protective autophagy in human BC cells with mutant p53. Coordinate inhibition of autophagy potentiated anticancer activity of quercetin against BC.
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    台灣泌尿科醫學會
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    2017-06-01 11:24:17
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    2017-06-01 11:34:30
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