白蛋白结合型紫杉醇对肺鳞癌H520细胞株PD-L1、ERK、p-ERK的调控作用观察

Regulatory effects of albumin-bound paclitaxel on PD-L1,ERK,and p-ERK in lung squamous carcino⁃ma H520 cells

目的探讨白蛋白结合型紫杉醇(ab-PTX)对肺鳞癌H520细胞株的程序性死亡蛋白1配体(PD-L1)表达的影响及其可能的作用机制。方法(1)本研究采用的ab-PTX浓度及作用时间的确定:用0.001、0.01、0.1、1、10μmol/L ab-PTX培养H520细胞株24、36、48 h,计算细胞增殖抑制率,最终选择细胞增殖抑制作用最显著的48 h及其对应的ab-PTX IC50值(1.221μmol/L)进行后续试验。(2)给予不同浓度ab-PTX培养后H520细胞株PD-L1表达观察:培养基中加入0、0.001、0.01、0.1、1、10μmol/L ab-PTX体外培养肺鳞癌H520细胞株48 h(基于(1)中确定的ab-PTX作用时间),采用流式细胞术测算PD-L1阳性细胞率,采用qRT-PCR法检测PD-L1 mRNA。(3)单独或联合给予ab-PTX、细胞外调节蛋白激酶(ERK)抑制剂培养后ERK、p-ERK的表达观察:将H520细胞株分为Control组、abPTX组、PD98059(ERK抑制剂)]组、TPA(ERK激活剂)组、ab-PTX联合PD98059组、ab-PTX联合TPA组,ab-PTX组加入1.221μmol/L ab-PTX(基于(1)中确定的ab-PTX浓度);PD98059组加入50μmol/L PD98059;TPA组加入50nmol/LTPA;ab-PTX+PD98059组加入1.221μmol/Lab-PTX和50μmol/LPD98059;ab-PTX+TPA组加入1.221μmol/L ab-PTX和50 nmol/L TPA,培养48 h,流式细胞术测算PD-L1阳性细胞率,Western blotting法检测ERK、p-ERK。结果(1)不同浓度ab-PTX培养后H520细胞株PD-L1表达:采用0、0.001、0.01、0.1、1、10μmol/L ab-PTX培养的H520细胞株PD-L1阳性细胞率分别为33.9%±1.1%、39.5%±2.7%、52.6%±3.3%、60.3%±3.4%、69.9%±2.3%、77.8%±2.2%,PD-L1 mRNA相对表达量分别为1.06±0.07、1.65±0.22、2.12±0.16、3.25±0.11、3.79±0.14、4.45±0.28,随着ab-PTX浓度的增加,PD-L1阳性细胞率、PD-L1 mRNA相对表达量逐渐上升(P均<0.05)。(2)单独或联合给予ab-PTX、ERK抑制剂培养后ERK、p-ERK的表达及PD-L1阳性细胞率:与Control组相比,ab-PTX组p-ERK相对表达量升高,PD98059组p-ERK相对表达量降低,TPA组p-ERK相对表达量升高;ab-PTX联合PD98059组较ab-PTX组p-ERK相对表达量降低,较PD98059组p-ERK相对表达量升高;ab-PTX联合TPA组较ab-PTX组及TPA组p-ERK相对表达量升高(P均<0.05)。与Control组相比,ab-PTX组PD-L1阳性细胞率升高;与ab-PTX组比较,ab-PTX联合PD98059组PD-L1阳性细胞率降低;与PD98059组比较,ab-PTX联合PD98059组PD-L1阳性细胞率升高;与ab-PTX组及TPA组比较,ab-PTX联合TPA组PD-L1阳性细胞率升高(P均<0.05)。结论ab-PTX可上调H520细胞株PD-L1表达,一定范围内呈剂量依赖性;ab-PTX上调PD-L1表达的作用机制可能部分与ERK信号通路激活有关。

Objective To investigate the effect of albumin-bound paclitaxel(ab-PTX)on programmed death ligand 1(PD-L1)expression in lung squamous cell carcinoma H520 cells and its possible mechanism of action.Methods 1 Determination of ab-PTX concentration and duration of effect used in this study:H520 cells were cultured with 0.001,0.01,0.1,1,and 10μmol/L ab-PTX for 24,36,and 48 h.The cell proliferation inhibition rate was calculated,and fi⁃nally,the 48 h with the most significant cell proliferation inhibition and its corresponding ab-PTX half maximal inhibitory concentration(IC50)value(1.221μmol/L)were selected for subsequent experiments.2 Observation of PD-L1 expression in H520 cells after administration of different concentrations of ab-PTX in culture:lung squamous carcinoma H520 cells were cultured in vitro with 0,0.001,0.01,0.1,1,and 10μmol/L ab-PTX in culture medium for 48 h(based on the du⁃ration of ab-PTX action determined in 1),and PD-L1 positive cell rate was measured by flow cytometry and PD-L1 mRNA was detected by qRT-PCR.3 Observation of extracellular-regulated protein kinase(ERK)and p-ERK expression after ad⁃ministration of ab-PTX,ERK inhibitor alone or in combination in culture:the H520 cells were divided into the Control group,ab-PTX group,PD98059(ERK inhibitor)group,TPA(ERK activator)group,ab-PTX combined with PD98059 group,ab-PTX combined with TPA group;cells in the ab-PTX group were added with 1.221μmol/L ab-PTX(based on the ab-PTX concentration determined in 1);in the PD98059 group,50μmol/L PD98059 was added;in the TPA group,50 nmol/L TPA was added;in the ab-PTX combined with PD98059 group,1.221μmol/L ab-PTX,and 50μmol/L PD98059 were added;in the ab-PTX combined with TPA group,1.221μmol/L ab-PTX and 50 nmol/L TPA were added,and cells were cultured for 48 h.The PD-L1 positive cell rate was measured by flow cytometry,and ERK and p-ERK were detected by Western blotting.Results 1 PD-L1 expression in H520 cells after different concentrations of ab-PTX cul⁃ture:The PD-L1 positive cell rates of H520 cell line cultured with 0,0.001,0.01,0.1,1,and 10μmol/L ab-PTX were 33.9%±1.1%,39.5%±2.7%,52.6%±3.3%,60.3%±3.4%,69.9%±2.3%,and 77.8%±2.2%,respectively,and the relative expression levels of PD-L1 mRNA were 1.06±0.07,1.65±0.22,2.12±0.16,3.25±0.11,3.79±0.14,and 4.45±0.28,respectively;with the increase of ab-PTX concentration,the PD-L1 positive cell rate and the PD-L1 mRNA relative expression gradually increased(all P<0.05).2 Expression of ERK,p-ERK,and PD-L1 positive cell rate after the administration of ab-PTX,ERK inhibitor alone or in combination in culture:Compared with the Control group,the relative expression of p-ERK increased in the ab-PTX group,decreased in the PD98059 group and increased in the TPA group;the relative expression of p-ERK decreased in the ab-PTX combined with PD98059 group compared with the ab-PTX group and increased in the PD98059 group compared with the ab-PTX group;the relative expression of p-ERK was elevated in the ab-PTX combined with TPA group compared with the ab-PTX group and the TPA group(all P<0.05).Compared with the Control group,PD-L1 positive cell rate increased in the ab-PTX group;compared with the ab-PTX group,the PD-L1 posi⁃tive cell rate in the ab-PTX combined with PD98059 group decreased;compared with the PD98059 group,PD-L1 positive cell rate in the ab-PTX combined with PD98059 group increased;compared with the ab-PTX group and the TPA group,PD-L1 positive cell rate in the ab-PTX combined with TPA group increased(all P<0.05).Conclusion Ab-PTX can up-regulate PD-L1 expression in a dose-dependent manner within a certain range.The mechanism of action of ab-PTX up-reg⁃ulating PD-L1 expression may be partly related to the activation of ERK signaling pathway.