Differential effects of p38 MAP kinase inhibitors SB203580 and SB202190 on growth and migration of human MDA-MB-231 cancer cell line
Differential effects of p38 MAP kinase inhibitors SB203580 and SB202190 on growth and migration of human MDA-MB-231 cancer cell line
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DOI：
10.1007/s10616-017-0079-2
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发表时间：
2017-08-01
期刊：
影响因子：
2.2
通讯作者：
Boyaci, Ihsan
中科院分区：
文献类型：
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作者：
Duzgun, Sukru Aydin;Yerlikaya, Azmi;Boyaci, Ihsan
p38 丝裂原激活蛋白激酶 (MAPK) 属于 MAPK 超家族，可磷酸化靶蛋白的丝氨酸和/或苏氨酸残基。 p38 MAPK 的激活导致细胞生长、分化、炎症、存活或凋亡。在本研究中，我们测试了两种高度特异性和有效的p38 MAPK抑制剂（即SB203580和SB202190）对人乳腺癌细胞系MDA-MB-231的作用，以进一步阐明p38 MAPK对细胞增殖和/或细胞迁移/转移的有争议的作用。测定SB203580的IC50值为85.1μM，而SB202190的IC50值为46.6μM，这表明SB202190比SB203580稍微更有效。为了验证每种抑制剂对细胞增殖和细胞毒性的影响，用不同剂量的SB203580和SB202190处理细胞，并使用iCELLigence系统进行检查。与经DMSO处理的对照细胞长达96小时相比，1μM和5μM的两种抑制剂对细胞增殖没有显着影响。另一方面，SB203580和SB202190在50μM浓度下均显着阻止细胞增殖。SB202190再次比SB203580更有效。之后，我们使用伤口实验测试了每种抑制剂对细胞迁移的影响。 SB203580和SB202190在50μM浓度下均以时间依赖性方式显着降低细胞迁移。然而，有趣的是，观察到低且无细胞毒性剂量的5μM SB203580和SB202190在处理48小时时也确实引起显着的细胞迁移抑制，证实了p38 MAPK通路在细胞迁移中具有关键作用的事实。迁移/转移。然后，我们使用 iCELLigence 系统测试了每种 p38 MAPK 抑制剂在 3 小时的治疗期间是否对细胞粘附有影响。仅 50 μM 浓度的 SB202190 即可降低细胞粘附约 1.5 小时（p < 0.001）；这段时间之后，50μM SB202190处理的细胞中的细胞粘附恢复到对照细胞的水平。为了确定 p38 MAPK 抑制剂的生长和细胞迁移抑制作用的机制，随后通过 PathScan (R) 细胞内信号阵列试剂盒分析各种蛋白质和酶的激活/失活。低浓度（1或5μM）SB202190和SB203580不会改变ERK1/2磷酸化水平；而高浓度（50 μM）的两种抑制剂会导致 ERK1/2 磷酸化显着降低。此外，确定在这些实验条件下，两种 p38 MAPK 抑制剂都会导致 MDA-MB-231 中突变体 p53 的 Ser15 磷酸化显着增加；而SB202190比SB203580更有效。
p38 mitogen-activated protein kinase (MAPK) belongs to the MAPK superfamily, phosphorylating serine and/or threonine residues of the target proteins. The activation of p38 MAPK leads to cell growth, differentiation, inflammation, survival or apoptosis. In this study, we tested the effect of two highly specific and potent inhibitors of p38 MAPK (namely, SB203580 and SB202190) on human breast cancer cell line MDA-MB-231 to elucidate the controversial role of p38 MAPK on cell proliferation and/or cell migration/metastasis further. It was determined that the IC50 value of SB203580 was 85.1 mu M, while that of SB202190 was 46.6 mu M, suggesting that SB202190 is slightly more effective than SB203580. To verify the effect of each inhibitor on cell proliferation and cytotoxicity, the cells were treated with various doses of SB203580 and SB202190 and examined using iCELLigence system. No significant effect of 1 and 5 mu M of both inhibitors were seen on cell proliferation as compared to the DMSO-treated control cells for up to 96 h. On the other hand, both SB203580 and SB202190 significantly prevented cell proliferation at a concentration of 50 mu M. SB202190 was again more effective than SB203580. Afterwards, we tested the effect of each inhibitor on cell migration using wound assay. Both SB203580 and SB202190 significantly reduced cell migration in a time-dependent manner at a concentration of 50 mu M. However, interestingly it was observed that a low and noncytotoxic dose of 5 mu M of SB203580 and SB202190 also did cause significant cell migration inhibition at 48 h of the treatment, corroborating the fact that p38 MAPK pathway has a critical role in cell migration/metastasis. Then, we tested whether each p38 MAPK inhibitor has any effect on cell adhesion during a treatment period of 3 h using iCELLigence system. A concentration of only 50 mu M of SB202190 reduced cell adhesion for about 1.5 h (p < 0.001); after that period of time, cell adhesion in 50 mu M SB202190-treated cells returned to the level of the control cells. To determine the mechanism of growth and cell migration inhibitory effects of p38 MAPK inhibitors, the activation/inactivation of various proteins and enzymes was subsequently analyzed by PathScan (R) Intracellular Signaling Array kit. The ERK1/2 phosphorylation level was not modified by low concentrations (1 or 5 mu M) of SB202190 and SB203580; while a high concentration (50 mu M) of both inhibitors caused significant reductions in the ERK1/2 phosphorylation. In addition, it was determined that both p38 MAPK inhibitors caused significant increases on the Ser15 phosphorylation of mutant p53 in MDA-MB-231 under these experimental conditions; while SB202190 was more potent than SB203580.