目的:膀胱癌(BC)主要采用以顺铂为基础的化疗,但是顺铂耐药的发展往往导致BC复发。本研究的重点是评估藤黄酸(GA)在减轻BC细胞顺铂耐药,以及对所涉及的潜在机制的分析。
方法:将顺铂以各种浓度梯度给予人膀胱移行细胞癌细胞(T24)以诱导顺铂耐药(T24-DDP)细胞。设置几个实验组:T24组,T24-DDP组,T24-DDP+DDP组,T24-DDP+GA组,T24-DDP+DDP+GA组,T24-DDP+DDP+GA+miR-NC组,和T24-DDP+DDP+GA+miR-205-5p抑制剂组。细胞计数试剂盒-8(CCK-8)测定,Transwell迁移试验,和划痕试验分别用于评估细胞增殖,入侵,和移民。进行蛋白质印迹分析以检测E-cadherin的蛋白表达,ZEB1Vimentin,N-钙黏着蛋白,LRP,MRP,和细胞中的P-gp,而miR-205-5p的相对表达水平通过qRT-PCR测定。
结果:与T24-DDP组相比,T24-DDP+GA组细胞对顺铂的敏感性增强.此外,如CCK-8测定所示,GA提高了T24-DDP细胞对顺铂的敏感性,增强了顺铂的作用,并对入侵产生抑制作用,扩散,以及T24-DDP细胞的迁移。通过蛋白质印迹分析,表明GA显著抑制N-cadherin的表达,E-cadherin,还有Vimentin,以及顺铂耐药蛋白MRP,P-GP,和BC细胞中的LRP。此外,进一步的实验表明,GA促进miR-205-5p表达,同时抑制细胞内ZEB1表达。
结论:GA通过miR-205-5p/ZEB1轴介导的上皮-间质转化途径减轻BC细胞顺铂耐药。
OBJECTIVE: Bladder cancer (BC) is primarily treated with cisplatin-based chemotherapy, but the development of cisplatin resistance often leads to BC recurrence. This study is focused on assessing the potential of gambogic acid (GA) in mitigating BC cells\' cisplatin resistance, along with an analysis of the underlying mechanism involved.
METHODS: Cisplatin was administered to human bladder transitional cell carcinoma cells (T24) at various concentration gradients to induce cisplatin-resistant (T24-DDP) cells. Several experimental groups were set: T24 group, T24-DDP group, T24-DDP+DDP group, T24-DDP+GA group, T24-DDP+DDP+GA group, T24-DDP+DDP+GA+miR-NC group, and T24-DDP+DDP+GA+miR-205-5p inhibitor group. The cell counting kit-8 (CCK-8) assay, Transwell migration assay, and scratch assay were respectively carried out for assessment of cell proliferation, invasion, and migration. Western blot analysis was conducted for detection of the protein expression of E-cadherin, ZEB1, Vimentin, N-cadherin, LRP, MRP, and P-gp in the cells, while the relative expression level of miR-205-5p was determined by qRT-PCR.
RESULTS: In comparison with the T24-DDP group, cells in the T24-DDP+GA group showed enhanced sensitivity to cisplatin. Furthermore, as indicated by CCK-8 assay, GA improved T24-DDP cells\' sensitivity to cisplatin, potentiated the effects of cisplatin, and exerted an inhibitory effect on the invasion, proliferation, as well as migration of T24-DDP cells. Through Western blot analysis, GA was revealed to significantly inhibit the expression of N-cadherin, E-cadherin, and Vimentin, as well as that of cisplatin-resistant proteins MRP, P-gp, and LRP in BC cells. In addition, shown by further experiments, GA promoted miR-205-5p expression and simultaneously inhibited ZEB1 expression within the cells.
CONCLUSIONS: GA alleviates BC cells\' cisplatin resistance through the epithelial-mesenchymal transition pathway mediated by the miR-205-5p/ZEB1 axis.