抗MetFab-DOX对人肝癌细胞的多柔比星耐药作用

MetFab-DOX reduces doxorubicin-resistance of HepG2/DOX human hepatocellular carcinoma cells

目的抗MetFab-DOX能抑制肝细胞肝癌HepG2的增殖,而关于抗MetFab-DOX对多柔比星(DOX)耐药肝癌细胞的作用研究较少。文章构建DOX耐药细胞HepG2/DOX并探讨抗MetFab-DOX对该细胞耐药性的影响。方法使用大剂量间歇诱导法构建DOX耐药肝癌细胞模型HepG2/DOX;细胞分为对照组(不加药物)、DOX组(5μg/m L)、抗MetFab-DOX组(含有DOX浓度5μg/m L),采用CCK8法、流式细胞凋亡检测、细胞免疫荧光、划痕愈合实验以及Transwell侵袭实验检测抗MetFab-DOX对HepG2/DOX增殖、凋亡、药物内化以及生物学功能的影响;构建HepG2/DOX荷瘤裸鼠模型并检测抗MetFabDOX处理后对肿瘤体积和形态的影响。结果耐药细胞株HepG2/DOX细胞对DOX以及抗MetFab-DOX的敏感性均明显降低,HepG2细胞与HepG2/DOX细胞的IC50差异均有统计学意义(P<0.05)。不同药物处理24h后,抗MetFab-DOX组细胞凋亡率高于DOX组(19.87%vs 8.09%,P<0.05);当药物作用48 h时,抗MetFab-DOX组细胞凋亡率亦高于DOX组(41.27%vs16.15%,P<0.01)。抗MetFab-DOX组以及DOX组在30 min细胞内化情况没有明显的差异,而在60 min以及120 min时抗MetFab-DOX组DOX荧光强度高于DOX组。抗MetFab-DOX组24 h细胞划痕愈合率(14.46%)低于DOX组(16.80%)和空白对照组(19.88%),差异有统计学意义(P<0.05)。不同药物处理48 h后,与空白对照组细胞划痕愈合率(56.43%)和DOX组(36.96%)相比,抗MetFab-DOX处理后显著下降(22.60%),差异有统计学意义(P<0.01)。与对照组单个视野平均穿膜细胞数(1043.52个)比较,DOX组(880.51个)和抗MetFab-DOX组(646.18个)明显减少(P<0.05),且抗MetFab-DOX组明显低于DOX组(P<0.05)。药物处理第40天时,抗MetFab-DOX组抑瘤率明显高于DOX组(64.00%vs 35.27%,P<0.05)。对照组中移植瘤组织细胞排列不规则,增殖期肿瘤大小不一,数目占比大。DOX组与对照组相比,细胞增生略有下降。在抗MetFabDOX组中,肿瘤细胞进一步出现明显的皱缩变小,肿瘤细胞数量减少。结论抗MetFab-DOX能有效降低肝细胞肝癌对DOX的耐药性,其机制可能与抗MetFab-DOX能够增加药物细胞中的积聚,诱导细胞凋亡相关。

Objective MetFab-DOX can inhibit the proliferation of hepatocellular carcinoma HepG2 cells,but few researches have been conducted on the effect of MetFab-DOX on doxorubicin-resistant HepG2 cells.This study aimed to constructed doxorubicin-resistant HepG2 cell lines and explored the effect of MetFab-DOX on their drug resistance.Methods Using high-dose intermittent induction,we constructed the doxorubicin-resistant hepatocellular carcinoma cell model HepG2/DOX and divided the cells into a blank control,a DOX(5μg/mL),and an MetFab-DOX group(containing 5μg/mL doxorubicin).After treatment,we detected the effects of MetFab-DOX on the proliferation,apoptosis,internalization and biological function of the HepG2/DOX cells by CCK8 assay,FCM,cell immunofluorescence,wound healing assay and Transwell invasion assay,respectively.We also established a tumor-bearing model in the nude mouse and examined the effects of MetFab-DOX on the volume and morphology of the tumor.Results The drug resistance index of the HepG2/DOX cells treated with DOX and MetFab-DOX was markedly reduced,with statistically significant difference between the HepG2 and HepG2/DOX cells(P<0.05).After 24 hours of treatment,the cell apoptosis rate was remarkably higher in the MetFab-DOX than in the DOX group(19.87%vs 8.09%,P<0.05),and so was it at 48 hours(41.27%vs 16.15%,P<0.01).The internalization in the cells showed no statistically significant difference between the MetFab-DOX and DOX groups at 30 minutes,while the fluorescence intensity of doxorubicin was markedly higher in the former than in the latter group at 60 and 120 minutes.The cell scratch healing rate was lower in the MetFab-DOX than in the DOX and blank control groups at 24 hours(14.46%vs 16.80%and 19.88%,P<0.05),but higher in the former than in the latter two groups at 48 hours(22.60%vs 36.96%and 56.43%,P<0.01).The number of the membrane-penetrating cells per visual field was significantly decreased in the MetFab-DOX and DOX groups as compared with that in the blank control(646.18 and 880.51 vs 1043.52,P<0.05),and even lower in the MetFab-DOX than in the DOX group(P<0.05).After 40 days of treatment,the tumor inhibition rate was remarkably higher in the MetFab-DOX than in the DOX group(64%vs 35.27%,P<0.05).In the blank control group,the transplanted tumor cells were irregularly arranged and proliferative tumors varied in volume and constituted a larger proportion.The proliferation of the cells was slightly reduced in the DOX group as compared with that in the control.In the MetFab-DOX group,the tumor cells showed a significant shrinkage and a decreased number.Conclusion MetFab-DOX can effectively reduce the doxorubicin-resistance of hepatocellular carcinoma,and the underlying mechanism may be associated with its abilities of increasing the accumulation in drug-induced cells and inducing cell apoptosis.