蛋白酶体抑制剂MG132逆转子宫颈癌细胞HCE1多细胞球体对顺铂耐药的研究

Reversion of resistance to cisplatin induced by MG132 in cervical cancer cell HCE1 multicellular spheroid

目的 构建宫颈鳞癌细胞系HCE1的多细胞球体模型,研究蛋白酶体抑制剂MG132对其顺铂天然耐药的影响,探讨MG132是否可以逆转HCE1多细胞球体的天然耐药及其可能的机制.方法 （1）采用液体重叠法和旋转法建立HCE1多细胞球体模型;（2）用MG132、顺铂分别干预HCE1单层细胞及多细胞球体,分为MG132组、顺铂组、MG132＋顺铂组、对照组,锥虫蓝拒染法检测细胞抑制率,流式细胞仪检测细胞周期及凋亡率;（3）用蛋白印迹法和免疫组化方法分别检测HCE1单层细胞及药物干预前后多细胞球体中核因子kB（NF-kB）和B细胞淋巴瘤/白血病2蛋白（bcl-2）的表达.结果 （1）成功建立了HCE1多细胞球体模型.（2）HCE1单层细胞和多细胞球体的细胞抑制率：MG132组分别为（11.67±2.34）%和（10.78±1.17）%,两者比较,差异无统计学意义（P〉0.05）;MG132＋顺铂组分别为（92.67±2.52）%和（91.33±2.18）%,两者比较,差异也无统计学意义（P〉0.05）;顺铂组分别为（45.01±7.44）%和（9.45±5.98）%,两者比较,差异有统计学意义（P〈0.05）.（3）HCE1单层细胞和多细胞球体的凋亡率：MG132组两者凋亡率分别为8.14%和5.97%,MG132＋顺铂组两者凋亡率分别为99.01%和95.22%;顺铂组两者凋亡率分别为33.61%和0.88%.（4）NF-kB的相对表达量和bcl-2的高表达率：HCE1多细胞球体对照组分别为0.67、60%,顺铂组分别为0.85、83%,MG132组分别为0.39、20%,MG132＋顺铂组分别为0.47、33%.结论 （1）HCE1多细胞球体模型对顺铂可产生天然耐药,是体外研究宫颈癌对顺铂耐药的良好模型.（2）MG132对HCE1多细胞球体有抑制增殖、诱导凋亡的作用,可部分逆转HCE1多细胞球体对顺铂的天然耐药性,其机制可能与NF-kB、bcl-2表达下调有关.

Objective To investigate the effect of the ubiquitin-proteasome pathway inhibitor MG132 on the natural resistance to cisplatin in the human cervical cancer line （HCE1） muhicellular spheroid （HCE1/MCS） model and to probe it if MG132 could reverse the HCE1/MCS resistance to cisplatin, as well as the possible mechanism of drug resistance.Methods （1） HCE1/MCS was obtained using liquid overlay and rotating technique.（2）Four groups were established （MG132 group, cisplatin group, MG132 ＋ cisplatin group, the control group）.Cell viability were measured by trypan blue exclusion assay.Cell cycle and apoptosis were detected by flow cytometry.（3） The expression of nuclear factor （NF） kB of both HCE1 monolayer cells （HCEI/MC） and HCE1/MCS was detected by western blot, and the expression of B cell lymphoma/leukemia-2 （bcl-2） was detected by immunohistochemistry.Results （1） HCE1/MCS was established successfully.（2） Cell inhibited rate of HCE1/MC and HCE1/MCS was： in MG132 group, （11.67 ± 2.34） % vs （10.78 ± 1.17） % （P 〉 0.05） ; in MG132 ＋ cisplatin group, （92.67 ± 2.52）% vs （91.33 ±2.18）% （P〉0.05）; in cisplatin group, （45.01±7.44）% vs （9.45±5.98）% （P〈0.05）.（3）The rate of apoptosis of HCE1/MC and HCE1/MCS were： in MG132 group, 8.14% and 5.97% ; in MG132 ＋ cisplatin group, 99.01% and 95.22% ; in cisplatin group, 33.61% and 0.88%.（4）The expression level of NF-kB and the high expression rate of bcl-2 were： in HCE1/MCS of control group, 0.67 and 60% ; in HCE1/MCS of cisplatin group, 0.85 and 83% ; in HCE1/MCS of MG132 group, 0.39 and 20% ; in HCE1/MCS of MG132 ＋ cisplatin group, 0.47 and 33%.Conclusions （1） HCE1/ MCS present natural resistance to cisplatin and may become a good model for the study of cervical cancer drug resistance in vitro.（2） MG132 could induce the inhibition and apoptosis of HCE1/MCS cells and partially reverse the natural resistance of HCE1/MCS to cisplatin, of which partially reverse the natural resistance may be in relation to the down-regulation of NF-kB and bcl-2 expression.