CDK4/6抑制剂哌柏西利对人黑素瘤细胞增殖、周期和凋亡的影响  

Effects of CDK4/6 inhibitor palbociclib on proliferation,cell cycle and apoptosis of human melanoma cells

作　　者：邓龙 徐敏 付蕾 杜冠群 王利 DENG Long;XU Min;FU Lei;DU Guanqun;WANG Li(Department of Xianghongqi Outpatient,Jingxi Medical District,Chinese People’s Liberation Army General Hospital,Beijing 100091,China)

机构地区：[1]中国人民解放军总医院京西医疗区厢红旗门诊部,北京100091

出　　处：《肿瘤》2020年第8期521-530,共10页Tumor

摘　　要：目的:探讨细胞周期蛋白依赖性激酶4/6(cyclin-dependent kinases 4/6,CDK4/6)抑制剂哌柏西利对人恶性黑素瘤SK-MEL-5和A375细胞增殖、细胞周期及凋亡的影响。方法:体外培养人黑素瘤A2058、SK-MEL-5和A375细胞,以耐药A2058细胞作为阴性对照。用不同浓度的哌柏西利处理细胞24和48 h后,采用CellTiter-Glo?发光法检测哌柏西利对A2058、SK-MEL-5和A375细胞增殖的影响。碘化丙啶(propidium iodide,PI)染色后检测哌柏西利对A2058、SK-MEL-5和A375细胞周期的影响,蛋白质印迹法检测哌柏西利对SK-MEL-5和A375细胞中细胞周期相关蛋白[CDK4、CDK6、细胞周期蛋白D1(cyclin D1)、视网膜母细胞瘤蛋白(retinoblastoma protein,Rb)和磷酸化Rb(phospho-Rb,p-Rb)]表达的影响。AnnexinⅤ-FITC/PI双染法检测哌柏西利对A2058、SK-MEL-5和A375细胞凋亡的影响。采用NOD/SCID小鼠建立A2058、SK-MEL-5和A375细胞的皮下移植瘤模型,将小鼠分为2组[哌柏西利(80 mg/kg)处理组和乳酸钠缓冲液处理组],通过体内实验验证哌柏西利的安全性和疗效。结果:与阴性对照A2058细胞相比,哌柏西利对SK-MEL-5和A375细胞的增殖具有明显抑制作用(P值均<0.001)。哌柏西利能够引起SK-MEL-5和A375细胞中G1期细胞所占比例增高(P值均<0.05);蛋白质印迹法检测发现哌柏西利能够使SK-MEL-5和A375细胞中细胞周期G1/S期检查点关键蛋白CDK4、CDK6、cyclin D1以及p-Rb的表达水平明显下调(P值均<0.001)。哌柏西利对A2058、SK-MEL-5和A375细胞均未见明显的促凋亡作用(P值均>0.05)。在荷瘤小鼠模型中,哌柏西利安全性高,对小鼠移植瘤的生长具有明显的抑制作用[对SK-MEL-5细胞移植瘤的抑制率为(64.6±9.8)%,对A375细胞移植瘤的抑制率为(76.3±6.6)%](P值均<0.001)。结论:哌柏西利对黑素瘤SK-MEL-5和A375细胞的增殖具有明显抑制作用,可引起细胞周期G1期阻滞。Objective:Investigate the effects of cyclin-dependent kinases 4/6(CDK4/6)inhibitor palbociclib on proliferation,cell cycle and apoptosis of human malignant melanoma SK-MEL-5 and A375 cells.Methods:The melanoma A2058,SK-MEL-5 and A375 cells were cultured in vitro,A2058 cells were used as the negative control cells.After treatment with different concentrations palbociclib for 24 and 48 h,the proliferation of melanoma cells were detected by CellTiter-Glo?Luminescent Cell Viability Assay.The cell cycle of A2058,SK-MEL-5 and A375 cells after treatment with palbociclib was detected by FCM method[propidium iodide(PI)staining],and then the expression level of cell cycle-related proteins[CDK4,CDK6,cyclin D1,retinoblastoma protein(Rb)and phospho-Rb(p-Rb)]in SK-MEL-5 and A375 cells were detected by Western blotting.The effect of palbociclib on apoptosis rate of A2058,SK-MEL-5 and A375 cells was detected by FCM method(AnnexinⅤ-FITC/PI staining).Subcutaneous transplantation tumor models of A2058,SK-MEL-5 and A375 cells in NOD/SCID mice were established,the mice were divided into two groups:palbociclib(80 mg/kg)treatment group and sodium lactate buffer treatment group,safety and efficacy of palbociclib were detected in vivo experiments.Results:Compared with the negative control A2058 cells,palbociclib could significantly inhibit the proliferation of SK-MEL-5 and A375 cells(both P<0.001).FCM assay showed that palbociclib could increase the proportion of cells in G1 phase in SK-MEL-5 and A375 cells(both P<0.05).Western blotting showed that the expression levels of CDK4,CDK6,cyclin D1 and p-Rb proteins were significantly down-regulated in SK-MEL-5 and A375 cells(all P<0.001).Palbociclib had no significant effect on the apoptosis of A2058,SK-MEL-5 and A375 cells(all P>0.05).In the tumor-bearing mouse models,palbociclib was safe and had a significant inhibitory effect on tumor growth[the xenograft tumor growth inhibition rate of SK-MEL-5 and A375 cells were(64.6±9.8)%and(76.3±6.6)%,respectively](both P<0.001).Conclusion:Palboci

关 键 词：黑素瘤 实验性 动物实验 哌柏西利 细胞增殖 细胞周期检测点 

分 类 号：R739.5[医药卫生—肿瘤]