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

Te-Fu Tsai ^1#, Ji-Fan Lin^2#, Shan-Che Yang², Ya-Ming Teng², Hung-En Chen¹, Yi-Chia Lin¹, Kuang-Yu Chou¹, and Thomas I-Sheng Hwang^1,3,4,5

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.

 

Running title: Quercetin induces autophagy in human bladder cancer cells.

Keywords: Apoptosis, autophagy, bladder cancer cells, quercetin.