非受体酪氨酸激酶抑制剂CYT387对多发性骨髓瘤细胞增殖、凋亡影响及作用机制

Effects of janus kinase inhibitor CYT387 on proliferation and apoptosis of multiple myeloma cells and mechanism of action

目的探讨非受体酪氨酸激酶(JAK)抑制剂CYT387对多发性骨髓瘤(MM)细胞增殖、凋亡的作用及其相关信号通路的关系。方法选取4株MM细胞株(RPMI-8226、LP-1、U266、XG-7)及北京积水潭医院自2012年3月至2015年3月收治的26例MM患者为研究对象。对26例MM患者的骨髓及肿瘤组织进行免疫组化磷酸化JAK2(p-JAK2)表达检测。采用蛋白免疫印迹法检测4株MM细胞株中JAK2的磷酸化水平。采用蛋白免疫印迹法对XG-7细胞株用不同浓度CYT387处理后的磷酸化信号转导及磷酸化STAT3(p-STAT3)进行检测。采用不同浓度CYT387处理4株MM细胞株和JAK2不同磷酸化水平的MM原代肿瘤细胞后,进行细胞活力检测。蛋白免疫印迹法检测相同浓度CYT387处理后LP-1细胞株在不同时间点B细胞淋巴瘤/白血病2(BCL2)、B细胞淋巴瘤-特大型(BCL-xL)、半胱氨酸蛋白酶3(caspase-3)、聚腺苷二磷酸核糖聚合酶(PARP)的表达状况。结果26例MM肿瘤组织中,13例(50%)呈p-JAK2阳性表达。4株细胞株全部p-JAK2阳性表达。随着CYT387浓度增加,XG-7和LP-1细胞株的p-STAT3的表达显著降低(P<0.05)。外源白细胞介素6(IL-6)的刺激可整体提高p-STAT3的表达,但并不能改变CTY387呈浓度依赖性显著降低p-STAT3表达的趋势(P<0.05)。4株MM细胞株和3例MM原代肿瘤细胞,随着CYT387浓度增加细胞活力均显著降低(P<0.05),p-JAK2阳性的原代MM细胞更为显著(P<0.05)。用相同浓度的CYT387处理LP-1细胞株后,检测caspase-3表达处于高水平,而BCL-xL、BCL2、PARP表达水平逐渐下降,具有时间依赖性(P<0.05)。结论CYT387可通过阻断MM细胞内IL-6/JAK/STAT信号通路抑制肿瘤增殖,同时可以引发凋亡相关蛋白活化诱导MM细胞凋亡。

Objective To investigate the effects of janus kinase(JAK)inhibitor CYT387 on the proliferation and apoptosis of multiple myeloma(MM)cells and the relationship of related signaling pathways.Methods Four MM cell lines(RPMI-8226,LP-1,U266,XG-7)and 26 MM patients admitted to Beijing Jishuitan Hospital from March 2012 to March 2015 were selected as the research subjects.The expression of phosphorylated-JAK2(p-JAK2)was detected in bone marrow and tumor tissues of 26 MM patients.The phosphorylation of JAK2 in 4 MM cell lines was detected by western blotting.The phosphorylation signal transduction and phosphorylated-STAT3(p-STAT3)of XG-7 cell lines treated with different concentrations of CYT387 were detected by western blotting.Four MM cell lines and MM primary tumor cells with different levels of JAK2 phosphorylation were treated with different concentrations of CYT387 to detect cell viability.The expression levels of B-cell lymphoma/leukemia 2(BCL2),B-cell lymphoma-extra large(Bcl-XL),cysteine aspartic acid specific protease-3(caspase-3)and poly ADP-ribose polymerase(PARP)in LP-1 cell lines treated with CYT387 at different time points were detected by western blotting.Results Of the 26 MM tumors,13(50%)showed positive expression of p-JAK2.All 4 cell lines were positive for p-JAK2 expression.With the increase of CYT387 concentration,the expression of p-STAT3 in XG-7 and LP-1 cell lines was significantly decreased(P<0.05).Exogenous interleukin-6(IL-6)stimulation could improve the expression of p-STAT3,but did not change the trend of CTY387 significantly decreasing the expression of p-STAT3 in a concentration-dependent manner(P<0.05).The cell viability of 4 MM cell lines and 3 MM primary tumor cells was significantly decreased with the increase of CYT387 concentration(P<0.05),and the p-JAK2-positive primary MM cells were more significant(P<0.05).After LP-1 cell line was treated with the same concentration of CYT387,the expression of caspase-3 was detected at a high level,while the expression levels of Bcl-XL,BCL2 and PARP decreased gradually in a time-dependent manner(P<0.05).Conclusion CYT387 inhibits tumor proliferation by blocking the IL-6/JAK/STAT signaling pathway in MM cells and induces apoptosis by activating apoptosis-related proteins.