布托啡诺抑制Notch1信号通路对肺癌细胞紫杉醇敏感性的影响

Effect of butorphanol on Taxol sensitivity of lung cancer cells by inhibiting Notch1 signaling pathway

目的研究布托啡诺对肺癌细胞紫杉醇敏感性的影响及其机制。方法将紫杉醇耐药A549/Taxol细胞株分为单独布托啡诺处理组、布托啡诺联合紫杉醇处理组和Jagged1/FC干预组,其中单独布托啡诺处理组接受不同浓度的布托啡诺(0、1、2、4、8和16μmol/L)处理24 h,布托啡诺联合紫杉醇处理组接受布托啡诺(8μmol/L)联合紫杉醇(5 nmol/L)处理24 h,Jagged1/FC干预组接受布托啡诺(8μmol/L)联合紫杉醇(5 nmol/L)以及Jagged1/FC(500μg/L)处理24 h。使用MTT法检测细胞增殖,利用Transwell法评估细胞增殖能力,采用Western blot法检测细胞侵袭及细胞增殖侵袭相关蛋白(CyclinD1、p21、MMP2和MMP9)以及Notch信号通路中关键蛋白(Notch1、Jagged1和Hes5)的表达量。结果与空白对照组相比,布托啡诺不影响A549/Taxol细胞活力(P>0.05)。与紫杉醇处理组相比,布托啡诺和紫杉醇联合组的A549/Taxol细胞活力明显降低(P<0.05)。相较于单纯紫杉醇处理组,紫杉醇联合布托啡诺处理组的细胞侵袭数目明显降低(P<0.05)、细胞增殖迁移相关(CyclinD1、MMP2和MMP9)蛋白表达降低(P<0.05)、Notch信号通路中关键蛋白(Notch1、Jagged1和Hes5)的表达水平明显降低(P<0.05),但是p21蛋白表达水平升高(P<0.05)。使用Jagged1/FC激活紫杉醇联合布托啡诺处理组细胞中的Notch信号通路后,A549/Taxol细胞活力升高、细胞增殖能力增加,并且CyclinD1、MMP2和MMP9的蛋白表达水平升高而p21蛋白表达降低,上述差异均有统计学意义(P<0.05)。结论布托啡诺可抑制紫杉醇耐药细胞的中Notch1信号通路的过度活化,并由此增加化疗耐药细胞系对紫杉醇的敏感性。

Objective To study the effect of butorphanol on the sensitivity of lung cancer cells to paclitaxel and its mechanism.Methods Paclitaxel-resistant A549/Taxol cell lines were divided into butorphanol treated group,butorphanol combined paclitaxel treated group,and Jagged1/FC intervention group.The butorphanol treated group was treated with different concentrations of butorphinol(0,1,2,4,8,16μmol/L)for 24 h,the butorphanol combined with paclitaxel treated group was treated with butorphanol(8μmol/L)combined with paclitaxel(5 nmol/L)for 24 h,the Jagged1/FC intervention group was treated with butorphanol(8μmol/L)combined with paclitaxel(5 nmol/L)and Jagged1/FC(500μg/L)for 24 h.MTT method was used to detect cell proliferation.Transwell method was carried out to evaluate cell proliferation ability.Western blot was applied to detect the expression levels of cell invasion and cell proliferation,invasionrelated proteins(CyclinD1,p21,MMP2,MMP9)and key proteins in the Notch signaling pathway(Notch1,Jagged1,Hes5).Results Compared with the blank group,butorphanol did not affect the viability of A549/Taxol cells(P>0.05).Compared with the paclitaxel treated group,the A549/Taxol cell viability of the butorphanol and paclitaxel combined group was significantly reduced(P<0.05).Compared with the paclitaxel treated group,the number of cell invasion in the paclitaxel combined with butorphanol treated group was significantly reduced(P<0.05).In addition,the cells in paclitaxel combined with butorphanol treated group showed declined expression level of cell proliferation and migration-related proteins(CyclinD1,MMP2 and MMP9)and key proteins in Notch signaling pathway(Notch1,Jagged1,Hes5)(P<0.05).However,the expression level of p21 protein increased(P<0.05).After using Jagged1/FC to activate the Notch signaling pathway in the cells treated with paclitaxel combined with butorphanol,A549/Taxol cell viability and cell proliferation increased,and the protein expression levels of CyclinD1,MMP2 and MMP9 increased,while the expression of p21 protein increased(P<0.05).Conclusion Butorphanol can inhibit the overactivation of Notch1 signaling pathway in paclitaxel-resistant cells,and thereby increase the sensitivity of chemotherapy-resistant cell lines to paclitaxel.