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目的:探讨丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPKs)信号转导通路在胰岛素促人乳腺癌细胞MCF-7增殖中的作用机制。方法:通过MTT比色法观察不同浓度(0、25、50、100、200 nmol/L)胰岛素促MCF-7细胞的增殖效应,及用JNK、ERK1/2通路特异性抑制剂(SP600125、PD98059)阻断MAPKs信号通路对胰岛素促MCF-7细胞增殖效应的影响;Western blot检测胰岛素干预对磷酸化JNK、磷酸化ERK1/2蛋白表达水平的影响以及JNK、ERK1/2、JAK2特异性抑制剂对上述蛋白表达的影响。结果:各浓度胰岛素均能不同程度促进人乳腺癌细胞MCF-7增殖,25 nmol/L胰岛素的促增殖效应最强(P<0.01);分别用SP600125、PD98059、AG490阻断JNK、ERK1/2、JAK/STAT信号通路均可不同程度抑制胰岛素促MCF-7细胞的增殖效应;胰岛素可诱导MAPKs信号通路中靶蛋白的磷酸化激活,Western blot结果显示,100 nmol/L胰岛素作用于MCF-7细胞5 min后即可磷酸化激活JNK途径,且该活化状态可持续1hr,而ERK1/2蛋白在胰岛素作用30 min后才被磷酸化激活;10μmol/L PD98059(ERK通路抑制剂)可显著抑制200 nmol/L胰岛素作用30 min对ERK1/2的磷酸化激活,而20μmol/LSP600125(JNK通路抑制剂)以及20μmol/L AG490(JAK/STAT通路抑制剂)对ERK1/2磷酸化激活的抑制作用则不明显。结论:胰岛素可促进人乳腺癌细胞MCF-7增殖,该增殖效应可能与其激活MAPKs信号通路及下游其他信号转导有关,上述通路的激活可能促进乳腺癌的发生发展。
AIM: To investigate the mechanism of mitogen activated protein kinase (MAPKs) signal transduction pathway in the proliferation of human breast cancer cell line MCF-7. Methods: The proliferation of MCF-7 cells induced by different concentrations of insulin (0, 25, 50, 100 and 200 nmol / L) was observed by MTT colorimetric assay and the effects of JNK, ERK1 / 2 pathway specific inhibitor (SP600125, PD98059 ) Blocked the effects of MAPKs signal pathway on the proliferation of insulin-stimulated MCF-7 cells; Western blot was used to detect the effect of insulin on the expression of phosphorylated JNK and phosphorylated ERK1 / 2 protein and JNK, ERK1 / 2 and JAK2 specific inhibitors Effects on the above protein expression. RESULTS: Insulin at various concentrations promoted the proliferation of human breast cancer cell MCF-7 to varying degrees, and the effect of 25 nmol / L insulin was the strongest (P <0.01). The effects of JNK and ERK1 / 2 were blocked by SP600125, PD98059 and AG490 respectively , JAK / STAT signaling pathway can inhibit the proliferation of insulin-induced MCF-7 cells to varying degrees; insulin can induce phosphorylation of target protein in MAPKs signaling pathway, Western blot results showed that 100 nmol / L insulin on MCF-7 After 5 min, the JNK pathway was activated by phosphorylation, and this activation state lasted for 1 hr. However, ERK1 / 2 protein was activated by phosphorylation only 30 min after insulin treatment; 10 μmol / L PD98059 (ERK pathway inhibitor) The phosphorylation of ERK1 / 2 was induced by 200 nmol / L insulin for 30 min, while the inhibition of ERK1 / 2 phosphorylation by 20 μmol / LSP600125 (JNK pathway inhibitor) and 20 μmol / L AG490 (JAK / STAT pathway inhibitor) It is not obvious. CONCLUSION: Insulin can promote the proliferation of human breast cancer cell line MCF-7, which may be related to its activation of MAPKs signal pathway and other downstream signal transduction. The activation of these pathways may promote the development of breast cancer.