小蘗紅鹼與白藜蘆醇透過誘導GSTM2來抑制膀胱上皮癌細胞之癌特性

周主民¹、沈正煌¹、洪晨泰²、劉佩俞²、劉怡文²

¹嘉義基督教醫院泌尿外科; ²國立嘉義大學微生物免疫與生物藥學系

Suppression of Tumor Characteristics of Urothelial Carcinoma Cells by Berberrubine and Resveratrol by GSTM2 Induction

Chu-Min Chou¹, Cheng-Huang Shen¹, Chen-Tai Hong ², Pei-Yu Liu² and Yi-Wen Liu²

¹Department of Urology, Chiayi Christian Hospital,Chiayi, Taiwan; ²Department of Microbiology, Immunology and Biopharmaceuticals, National Chiayi University, Chiayi, Taiwan

Purpose:

Glutathione S-transferases (GSTs) belong to phase 2 detoxification enzymes which are important in metabolism and/or detoxification of a life. Our previous investigations demonstrated that GSTM5 plays an anti-cancer role in urothelial carcinoma cells, but it is not easy to induce GSTM5 expression because GSTM5 gene is heavy methylated. Therefore, in the present study, we further investigate whether GSTM2 inhibits the growth and metastasis of bladder cancer cells and is induced by phytochemicals.

Materials and Methods:

The metastasis and invasion assays were performed using 24-well Transwell chambers with an 8 µm pore membrane. Luciferase assay was performed using the Dual-Luciferase Reporter Assay System. Western blot and real-time PCR were used for protein and mRNA analysis.

Results:

GSTM2 gene expression is lower in high stages of bladder urothelial carcinoma than in stage 1 and normal bladder tissue. GSTM2 overexpression retards growth, invasion and migration of bladder cancer cells. Analysis of GSTM2 promoter activity shows that one SP1 site located at -48 to -40 bp is important for GSTM2 gene expression in BFTC 905 cells. An SP1 inhibitor, mithramycin A, inhibits GSTM2 promoter activity and protein expression. SP1 overexpression also increases GSTM2 expression in BFTC 905 and 5637 cells. Eight potential phytochemicals were analyzed for GSTM2 promoter activation, and results indicated that baicalein, berberrubine, chalcone, curcumin, resveratrol, and wogonin can increase promoter activity. In endogenous GSTM2 expression, berberrubine and resveratrol activated GSTM2 mRNA and protein expression the most. Berberrubine and resveratrol also increased SP1 protein expression as one of the mechanisms for GSTM2 gene expression. A DNA methylation inhibitor, 5-aza-deoxycytidine, can decrease GSTM2 gene methylation level and then increase its gene expression; 50 μM berberrubine also decreased the GSTM2 gene methylation level, providing another mechanism for activating GSTM2 gene expression.

Conclusion:

Berberrubine and resveratrol activate GSTM2 expression which inhibits cell proliferation, migration, and invasion of bladder cancer cells. The GSTM2 expression mechanism is via SP1 activation, and the effect of berberrubine is also via DNA CpG demethylation.