人参皂苷Rg3对膀胱癌细胞血管生成相关蛋白的表达及癌细胞生长和转移的影响

Effect of Ginsenosides Rg3 on Bladder Cancer Angiogenesis Associated Protein Expression and Cancer Cell Growth and Metastasis

目的探讨人参皂苷Rg3对表皮生长因子受体酪氨酸蛋白激酶(EGFR-TPK)和DNA拓扑异构酶I(DNA TOP I)的抑制作用,并阐明其对膀胱癌细胞增殖、侵袭和血管生成的抑制作用机制。方法不同浓度人参皂苷Rg3加入到EGFR-TPK、DNA TOP I和人膀胱癌细胞株S637中,用ELISA法研究人参皂苷Rg3对EGFR-TPK和DNA TOP I的抑制作用;溴化3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑(MTT)法测定人参皂苷Rg3对人膀胱癌细胞株S637细胞和人正常膀胱上皮细胞(SV-HUC-1)生长的抑制作用;Transwell法检测人参皂苷Rg3对S637细胞侵袭的抑制作用;酶标仪法检测人参皂苷Rg3对细胞凋亡蛋白Caspase-3活性的影响;Western blot法检测人参皂苷Rg3存在下,人膀胱癌细胞株S637中还氧化酶-2(COX-2)、血管内皮生长因子(VEGF)、缺氧诱导因子1α(HIF-1α)、小窝蛋白-1(CAV-1)及干细胞标志物(SOX-2)的表达。结果人参皂苷Rg3对EGFR-TPK和DNA TOP I(半抑制率IC_(50)分别为35.32±3.12和8.32±1.21μmol·L^(-1))、S637细胞生长(半抑制率IC_(50)为35.11±2.32μmol·L^(-1))都具有良好的抑制能力,但对于SV-HUC-1细胞,人参皂苷Rg3则表现了较低的毒性;人参皂苷Rg3与空白对照组比较能够诱导肿瘤细胞穿过滤膜的数量减少(P<0.01);与空白对照组比较,人参皂苷Rg3能显著提高S637细胞中Caspase 3活性(P<0.05);人参皂苷Rg3能够下调HIF-1α、COX-2、SOX-2及VEGF的表达,上调CAV-1的表达(P<0.05,P<0.01)。结论低毒性的人参皂苷Rg3通过多靶向的降低EGFR-TPK、DNA TOP I活性和HIF-1α、COX-2、SOX-2及VEGF的表达,上调CAV-1的表达和激活Caspase 3活性的方式,对膀胱肿瘤细胞的侵袭、增殖和血管生成产生抑制作用。

Objective To study the inhibitory effect of ginsengoside Rg3 on endothelial growth factor receptor-tyrosine protein kinase（EGFR- TPK） and DNA topoisomerase I（DNA TOP I）, and to explore its mechanism in inhibiting the proliferation and metastasis of bladder cancer cells and angiogenesis. Methods EGFR- TPK,DNA TOP I and human bladder cancer cell line S637 were separately co- cultured with various concentrations of ginsengoside Rg3.The inhibitory effect of ginsengoside Rg3 on the activity of EGFR- TPK and DNA TOP I was detected by using the ELISA method, and the inhibition of ginsengoside Rg3 on the growth of S637 and normal bladder epithelial cells SV- HUC- 1 was examined with the MTT method. Transwell method was used for detecting the inhibition of ginsengoside Rg3 on bladder cancer cell line S637,and ELISA reader was applied for the detection of apoptotic protein Caspase- 3activity. Western blot was used for detecting the expression of angiogenesis related protein cyclooxygenase 2（COX- 2）,vascular endothelial growth factor（VEGF）, hypoxia- inducible factor 1α（HIF- 1α）, caveolin 1（CAV- 1）,and sex determining region Y- box 2（SOX- 2） of S637 tumor cells treated with ginsengoside Rg3. Results Ginsengoside Rg3 showed obvious inhibition on EGFR- TPK,DNA TOP I and S637 growth,the half inhibition rate（IC50）being 35.32±3.12, 8.32 ±1.21, 35.11 ±2.32 μmol·L^-1, respectively. However, ginsengoside Rg3 showed low toxicity on SVHUC- 1. Compared with the blank group, ginsengoside Rg3 reduced the number of cancer cells passing through the artificial basement membrane,and increased Caspase 3 activity of S637,the difference being significan（P〈0.05）.Ginsengoside Rg3 could also down- regulate the expression of HIF- 1α,COX- 2,SOX- 2 and VEGF,and up- regulate CAV- 1 expression in S637 cells. Conclusion Low- toxicity ginsengoside Rg3 has inhibitory effect on proliferation and metastasis of bladder cancer cells and angiogenesis through decreasing the activities of EGFR- TPK and DNA TOP I,reducing the expression of HIF- 1α,COX- 2,SOX- 2 and VEGF,up- regulating CAV- 1 expression,and promoting Caspase 3 activities.