目的: 膀胱癌由于其较高的复发率和有限的治疗选择,一直是临床治疗中的重大挑战。尽管索拉非尼在肝癌治疗中表现出良好的临床效果,但其长期使用常伴有不良反应。而二甲双胍在抗肿瘤治疗中展示出一定潜力,但其较高剂量的使用可能引发副作用。本研究基于中间体衍生化策略,设计并合成了新型双胍衍生物N-1-己基-N-5-（4-三氟甲氧基）氯胍（6C）,旨在评估其单独使用及与索拉非尼联合使用对膀胱癌细胞增殖的抑制作用及其潜在机制。方法: 首先,采用MTT法检测6C与索拉非尼联合应用及单独处理对膀胱癌细胞增殖的影响。通过集落形成实验进一步评估两种药物单独或联合处理对膀胱癌细胞增殖的抑制效果。其次,使用Western blotting技术检测6C单用以及与索拉非尼联合应用对表皮生长因子受体（epidermal growth factor receptor,EGFR）及自噬相关标志蛋白LC3-II和Beclin1表达的影响。最后,Co-IP检测6C处理后EGFR与Belin1的结合情况。结果: 单独使用6C或索拉非尼均能显著抑制膀胱癌细胞的增殖。6C与索拉非尼的联合治疗展现出显著的协同作用,相较于单药治疗,联合用药在抑制膀胱癌细胞的增殖方面具有更强的效果。机制研究显示,6C与索拉非尼联合使用能够显著降低EGFR蛋白的表达,并上调自噬标志蛋白LC3-II和Beclin1的水平;同时6C处理后,EGFR与Belin1的结合明显减少,诱导膀胱癌细胞自噬。结论: 6C与索拉非尼的联合应用通过降低EGFR表达并诱导自噬,显著抑制膀胱癌细胞的增殖,表明该联合治疗策略为膀胱癌的治疗提供新的治疗思路和临床应用前景。
Abstract
Objective Bladder cancer presents a significantly clinical challenge due to its high recurrence rate and the limited therapeutic options. Although Sorafenib has shown the promising clinical efficacy in liver cancer, its long-term use is often associated with adverse effect. Additionally, Metformin has demonstrated the potential in cancer therapy; however, its use with high doses may lead to side effect. This study is based on an intermediate derivatization strategy to design and synthesize a novel biguanide derivative, namely 6C, with the aim to evaluate its effect, as a monotherapy and in combination with Sorafenib, on bladder cancer cell proliferation and investigate its underlying mechanisms. Methods Firstly, the impact of 6C and Sorafenib alone on bladder cancer cell proliferation was assessed using the MTT assay. Subsequently, the effect of these drugs, both individually and in combination, on bladder cancer cell proliferation were further evaluated by colony formation assay. Finally, Western blotting was employed to examine the expression of epidermal growth factor receptor (EGFR) and autophagy-related proteins LC3-II and Beclin1 in bladder cancer cells by the treatment with 6C and Sorafenib combination therapy. Results Both 6C and Sorafenib alone significantly inhibited the proliferation of bladder cancer cells. The combination of 6C and Sorafenib exhibited a marked synergistic effect, demonstrating more potent inhibition of cell proliferation compared to either drug used alone. Mechanistic investigations revealed that the combination treatment significantly decreased the expression level of EGFR and upregulated the levels of autophagy markers LC3-II and Beclin1, thereby inducing autophagy of these cells. Conclusion The combination of 6C and Sorafenib decreases EGFR expression and induces autophagy, which inhibits bladder cancer cell proliferation. This study suggests that the combination therapy using 6C and Sorafenib may offer a novel therapeutic strategy with the promising application for bladder cancer treatment.
关键词
膀胱癌 /
双胍衍生物 /
索拉非尼 /
自噬 /
联合治疗
Key words
bladder cancer /
biguanide derivative /
sorafenib /
autophagy /
combination therapy
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基金
湖南师范大学交叉学科研究团队,生殖健康与转化医学研究团队（No.2023JC101）
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