阿霉素联合TRAIL对人肝癌细胞增殖与TRAIL受体表达的影响

目的 探讨阿霉素联合肿瘤坏死因子相关凋亡诱导配体（TRAIL）对肝癌细胞HepG2和SMMC-7721增殖及TRAIL受体表达的影响。方法 采用MTT法分别检测阿霉素、TRAIL及低浓度阿霉素联合TRAIL处理HepG2和SMMC-7721细胞的生长抑制率；RT-PCR和Western blotting分别检测阿霉素作用前后HepG2和SMMC-7721细胞TRAIL受体DR4、DR5的mRNA和蛋白表达水平。结果 3种浓度阿霉素（0.86、8.6、86μmol／L）作用24h后，对HepG2和SMMC-7721细胞的生长抑制率分别为（8.84±0.44）％和（8.67±1.22）％、（24.12±1.11）％和（25.39±2.26）％、（64.55±4.05）％和（66.2±3.74）％，呈浓度依赖性，不同浓度组之间差异均有统计学意义（P＜0.05）。4种浓度TRAIL（10、100、500、1000ng／ml）作用24h后，对HepG2和SMMC 7721细胞的生长抑制率分别为（5.83±0.25）％和（5.66±0.56）％、（9.60±1.38）％和（8.96±1.13）％、（11.87±1.43）％和（12.11±1.84）％、（15.12±3.84）％和（16.16±1.41）％，其他3种浓度分别与10ng／ml TRAIL比较，差异均有统计学意义（P＜0.01）。低浓度阿霉素联合TRAIL作用于HepG2和SMMC-7721细胞，低浓度阿霉素能够增加HepG2和SMMC-7721细胞对TRAIL治疗的敏感性，并且随着时间的延长和TRAIL浓度的增加，细胞抑制率逐渐增加，呈时间 效应和剂量 效应关系；无论在mRNA还是蛋白水平，阿霉素处理后HepG2细胞死亡受体DR4、DR5的表达水平较未处理组显著增加；在SMMC-7721细胞，阿霉素处理后，DR5的表达水平显著增加，而DR4的表达水平与阿霉素未处理组相似。结论 低浓度阿霉素能够增加肝癌细胞对TRAIL治疗的敏感性，其机制可能是阿霉素增加死亡受体特别是DR5的表达水平，从而诱导细胞凋亡增加，TRAIL在肝癌治疗方面存在潜在的临床应用价值。

Effect of doxorubicin combined TRAIL on hepatocellular carcinoma cells and TRAIL receptors

Objective To investigate the effect of doxorubicin and TRAIL on proliferation and the expression of TRAIL receptors in human hepatocellular carcinoma cells HepG2 and SMMC-7721.Methods Growth inhibition rate was examined respectively by MTT assay under treatment with TRAIL or doxorubicin or in combination.The change of TRAIL receptors（DR4/DR5） in mRNA and protein levels was quantified by RT-PCR and Western blotting analysis respectively.Results The growth inhibiting rates of HepG2 and SMMC-7721 cells treated by 0.86,8.6 and 86μmol/L doxorubicin after 24h were（8.84±0.44）% and（8.67±1.22）%,（24.12±1.11）% and（25.39±2.26）%,and（64.55±4.05）% and（66.2±3.74）%,which were in a concentration dependent manner.There were obvious differences between different groups treated with doxorubicin in inhibiting rates（P〈0.05）.The growth inhibiting rates of HepG2 and SMMC-7721 cells treated by 10,100,500,1000ng/ml TRAIL after 24h were（5.83±0.25）% and（5.66±0.56）%,（9.60±1.38）% and（8.96±1.13）%,（11.87±1.43）% and（12.11±1.84）%,and（15.12±3.84）% and（16.16±1.41）%.There were obvious differences between 10ng/ml group and other concentration groups treated with TRAIL（P0.01）.Low-concentration doxorubicin combined with TRAIL treating HepG2 and SMMC-7721 cells could enhance the sensibility of those cells to TRAIL.With time expansion and concentration increase,cell growth inhibiting rates rised with time and concentration dependent manner.The increase of TRAIL death receptors（DR4/DR5） in mRNA and protein levels was detected in HepG2 cells treated with doxorubicin,however,significant increased DR5 expression was observed in SMMC-7721 cells but not in DR4.Conclusion Low-concentration doxorubicin in combination with TRAIL presents synergistic effect,and the possible mechanism lies in doxorubicin may enhance TRAIL-induced apoptosis through up-regulating death receptors,especially DR5,indicating the potential of TRAIL to treat hepatocellular carcinoma.