一种双吡嗪基脲衍生物通过ERK/Slug/vimentin/BCRP信号轴逆转表阿霉素耐药的研究

Epirubicin (Epi) is a commonly used drug for intravesical instillation in the treatment of bladder cancer, but its side effects and drug resistance result in high tumor recurrence rates. In previous study, we synthesized several series of arylurea derivatives. During the in vitro anti-cancer activity screening, it was unexpectedly found that one bipyrazinyl urea derivative BPNU with low toxicity which could significantly enhance the sensitivity of resistant cell line (T24/Epi) towards epirubicin; moreover, the combination of epirubicin and BPNU could inhibit the migration of T24 cells, and down-regulate the expressions of the invasion-, migration-, and resistance-related proteins/proteases, such as p-ERK, Slug, vimentin, BCRP, and so on, which indicated that BPNU could reverse the resistance of epirubicin. Based on our previous study, this project intends to combine BPNU with epirubicin to block the invasion and migration of bladder cancer cells through effectively intervening the ERK/Slug/vimentin/BCRP cascade signaling pathway, thereby reversing the resistance of epirubicin and reducing its side effects. The project will focus on the following two aspects through evaluating the activity of the combination at the cellular, molecular, genetic, and animal levels: (1) finding the key molecular and genetic targets; (2) clarifying the reversal mechanisms of BPNU. This project will provide a new solution to overcome the resistance of epirubicin.

表阿霉素（Epirubicin，Epi）是常用的膀胱癌灌注治疗药物，但是其毒副作用大，耐药性明显。前期研究中我们合成了数个系列双芳基脲类化合物，发现一种低毒性双吡嗪基脲衍生物BPNU可显著增强耐药细胞株（T24/Epi）对表阿霉素的敏感性，且BPNU的引入能抑制膀胱癌细胞的迁移，下调侵袭迁移和耐药相关蛋白（酶）p-ERK, Slug，vimentin和BCRP等的表达，预示BPNU可逆转表阿霉素的耐药性。本项目基于已有的研究，拟将BPNU与表阿霉素两者组合使用，通过干预ERK/Slug/vimentin/BCRP信号轴，阻断癌细胞侵袭迁移和耐药相关通路的信号传导，逆转表阿霉素的耐药性，降低其毒副作用。项目将围绕如下两项主要内容展开研究：（1）寻找到两者组合抑制侵袭迁移和逆转耐药的关键分子和基因靶点；（2）阐明BPNU逆转耐药的机制。本项目将为膀胱癌治疗中克服表阿霉素耐药性提供新的解决方案。

本研究探讨了一种双吡嗪基-芳基脲衍生物BPNU逆转T24/EPI耐药，以及青蒿琥酯基-芳基脲衍生物ARS-DPU逆转T24/Reg和T24/Sor耐药的功能，阐明了其逆转耐药的分子机制。在本课题组已报道的化合物8g基础上，合成得到两个系列喹唑啉基-芳基脲衍生物和一个系列吡嗪基-芳基脲衍生物，优选出4个能有效抑制膀胱癌细胞增殖的化合物：7j，III-30，5-23和9g，其中9g对5637细胞株的IC50 < 2 μM，然后进行了分子和基因水平验证和分析，以及小动物体内药物动力学，代谢产物，以及抑瘤活性研究。实验结果：（1）≥ 8 µM的BPNU对表阿霉素即表现出逆转作用，并能抑制耐药细胞株T24/EPI的侵袭迁移。（2）ARS-DPU与瑞戈非尼（或索拉非尼）联用，能明显增敏索拉非尼（或索拉非尼），导致胞内铁代谢失衡，耐药细胞株T24/Reg和T24/Sor死亡。（3）化合物7j可触发T24细胞三种不同的死亡模式：凋亡、铁死亡和自噬。7j和9g通过调控胞内Sxc-/GPx4/ROS信号转导通路导致细胞铁死亡。（4）化合物5-23通过激活RIPK1/RIPK3/MLKL信号通路导致T24细胞发生程序性坏死。（5）化合物9g还可通过下调胞内HMGCS1、HMGCS2和HMGCR等胆固醇合成相关蛋白（酶）的表达，影响胞内胆固醇的合成，同时抑制侵袭迁移，导致5637细胞死亡。（6）化合物9g能够显著抑制5637荷瘤裸鼠肿瘤的生长，抑瘤活性明显优于阳性参照索拉非尼和厄达替尼。（7）大鼠灌胃给予化合物9g后，体内代谢产物超过40种。本研究的创新性：首次建立了细胞内亚铁离子含量的定量分析方法，采用此法分析了ARS-DPU逆转T24/Reg和T24/Sor过程中细胞内金属离子含量的变化，并对化合物III-30诱导T24细胞铁死亡时二价和三价铁离子含量进行了定量分析。已发表密切相关的SCI论文2篇，申请中国发明专利2项，其中1项已授权。后续将对化合物9g进一步结构改造，力争获得1-2个具有良好应用前景的“me-better”化合物。