间歇给药联合ERK1/2-MITF通路激活剂抑制耐药黑色素瘤细胞增殖的研究

目的 探讨ERK1/2通路在耐药黑色素瘤药物成瘾性中的作用,并在此基础上,探究间歇性给药联合ERK1/2-MITF通路激活剂对耐药黑色素瘤细胞增殖的影响。方法 体外培养黑色素瘤药物敏感株（A375和M14细胞）和对威罗菲尼耐受的细胞株（A375/R0.5、A375/R2.0、M14/R0.5和M14/R2.0）,用MEK抑制剂U0126抑制ERK1/2激活,用Western-blot检测ERK1/2等蛋白的表达,用克隆形成实验观察细胞的增殖能力。利用活性分子TBHQ激活ERK1/2通路,利用ML329抑制MITF,二者均可上调ERK1/2-MITF通路活性,导致下游MITF抑制。采用耐药细胞A375/R2.0观察“撤除威罗菲尼”联合“TBHQ或ML329”对细胞增殖的影响。结果 克隆形成实验显示,在撤除威罗菲尼后,耐药细胞增殖减慢,如在撤药同时,用MEK抑制剂U0126抑制ERK1/2活化,可逆转撤药导致的增殖抑制,证明ERK1/2活化是耐药细胞药物成瘾性的关键机制。相反,如在撤除威罗菲尼同时,添加ERK1/2激活剂TBHQ或MITF抑制剂ML329,以进一步激活ERK1/2-MITF通路,结果发现,二者均可进一步抑制耐药细胞的增殖,提示撤药和ERK1/2-MITF通路活化产生了协同抑瘤效应。结论 本研究从新的角度证明了ERK1/2通路在耐药黑色素瘤细胞药物成瘾性中的作用,并提示间歇性给药联合ERK1/2-MITF通路激活剂可更有效地抑制耐药黑色素瘤增殖,有望为防治黑色素瘤耐药提供新思路。

The combined inhibitory effect of intermittent therapy plus ERK1/2-MITF pathway activation on the proliferation of drug-resistant melanoma

Objective Drug-resistant melanoma cells are generally addicted to targeted drugs. This study is intended to investigate the role of ERK1/2 pathway in drug addiction using drug-resistant melanoma cells and to further explore the effect of intermittent administration combined with ERK1/2-MITF pathway activator on the proliferation of drug-resistant melanoma cells. Methods Drug-sensitive melanoma cell lines (A375 and M14 cells) and vemurafenib-resistant cell lines (A375/R0.5, A375/R2.0, M14/R0.5 and M14/R2.0) were cultured in vitro. MEK inhibitor U0126 was applied to inhibit ERK1/2 activation. The expression of ERK1/2 and other proteins was detected by Western blot, and the proliferation ability of the cells was observed by clonal formation experiment. Besides, the molecular activator TBHQ was taken to activate ERK1/2 pathway and ML329 was brought to inhibit MITF, both of which up-regulated the ERK1/2-MITF pathway and resulted in downstream MITF inhibition. The effect of "vemurafenib withdrawal" combined with "TBHQ or ML329" on the proliferation of drug-resistant cells was observed by A375/R2.0. Results Clone formation experiments showed that the proliferation of drug-resistant cells was slowed down after vemurafenib removal. If the activation of ERK1/2 was inhibited by U0126 during drug withdrawal, the inhibition of cell proliferation caused by drug removal could be reversed, indicating that ERK1/2 activation was the key mechanism of drug addiction in drug-resistant cells. On the contrary, if ERK1/2 activator TBHQ or MITF inhibitor ML329 were added at the moment of drug clearance to further activate ERK1/2-MITF pathway, it was found that both of them could further suppress the proliferation of drug-resistant cells, suggesting that drug withdrawal and ERK1/2-MITF pathway activation produced a synergistic antitumor effect. Conclusion This research prove the role of ERK1/2 pathway in drug addiction of drug-resistant melanoma cells from a new point of view, and suggest that the intermittent administration combined with ERK1/2-MITF pathway activator can more effectively inhibit the proliferation of drug-resistant melanoma, which is expected to provide a new idea for the prevention and treatment of drug resistance of melanoma.