重组ProTα诱导肝癌细胞自噬的作用及机制

The effects and mechanisms of recombinant ProTαinduced autophagy in hepatoma cells

目的通过体内外实验探讨ProTα对肝癌细胞自噬和增殖的影响及机制。方法以人肝癌细胞株HepG2为研究对象,给予不同剂量的ProTα和干扰素-γ(IFN-γ)处理,CCK-8法检测ProTα和IFN-γ对HepG2细胞增殖的影响;Western blot法检测HepG2自噬相关LC3B蛋白表达;取C57BL/6雄性小鼠50只,左侧腋下注射H22细胞株复制H22肝癌实体瘤模型,筛选肿瘤直径>2 mm的小鼠40只,随机数表法分为四组,每组各10只,分别为对照组、ProTα组、奥沙利铂组、ProTα+奥沙利铂组。干预组小鼠奥沙利铂腹腔注射2周,对照组小鼠给予等体积生理盐水注射。2周后处死小鼠,取出肿瘤组织,测定小鼠体重、瘤质量并计算抑瘤率;Western blot法检测肿瘤组织中自噬相关LC3B、P62蛋白及p-mTOR蛋白表达水平。结果CCK-8检测显示,与对照组比较,不同浓度的ProTα对HepG2细胞存活率比较,差异无统计学意义(P>0.05);使用100 ng/ml IFN-γ处理HepG2细胞24、48 h的存活率均明显降低,分别为61.03%,52.16%,抑制作用明显,差异有统计学意义(P<0.01);自噬标记物LC3B蛋白水平显著增加,与对照组比较,差异有统计学意义(P<0.01)。动物实验表明,ProTα可明显降低H22实体瘤小鼠的瘤质量系数,抑瘤率达34.7%;ProTα+奥沙利铂组抑瘤率明显高于ProTα组(P<0.05);与对照组及奥沙利铂组比较,ProTα和ProTα+奥沙利铂组胸腺指数较高(P<0.05);与对照组比较,ProTα组、奥沙利铂组和ProTα+奥沙利铂组瘤组织中自噬相关LC3B蛋白的表达显著上调,P62及p-mTOR蛋白表达下调,差异均有统计学意义(P<0.05)。结论ProTα可以诱导H22荷瘤小鼠肝癌细胞的自噬,主要通过调节免疫细胞释放干扰素IFN-γ等细胞因子并下调p-mTOR表达诱导自噬发挥抗肿瘤作用;并且ProTα可以提高奥沙利铂的抗癌疗效。

Objective To investigate the effects and m echanisms of prothymos inα(P roTα)on autophagy and proliferation in hepatoma cells through in vivo and in vitro experiments.Methods Human hepatoma cell line HepG2 was selected as the study subject and was treated with different doses of ProTαand interferon-γ(IFN-γ).The effect of ProTαand IFN-γwere detected on the proliferation of HepG2 cells by CCK-8 method,and HepG2 autophagy-associated light chain 3b(LC3B)protein expression was detected by Western blot method.50 C57BL/6 male mice were injected with H22 cell line under the left axillary to establish and replicate the H22 hepatoma solid tumor models.40 mice with tumor diameter greater than 2 mm were screened and divided into 4 groups with 10 mice in each group by random number table method,namely the control group,ProTαgroup,oxaliplatin group and ProTα+oxaliplatin group.The mice in the experimental groups were injected with oxaliplatin intraperitoneally for 2 weeks.The mice in the control group were injected with equal volume of normal saline.2 weeks later,the mice were executed and the tumor tissues were removed,and the body mass and tumor mass were measured and the tumor inhibition rate was calculated.In addition,the expression levels of autophagy-associated LC3B,P62 and p-mTOR proteins in the tumor tissues were measured by Western-blot method.Results CCK-8 assay showed no significant differences between the experimental groups and the control group in the survival rate of HepG2 cells with different concentrations of ProTα(P>0.05).The survival rate of HepG2 cells treated with 100 ng/ml IFN-γfor 24 h and 48 h were significantly reduced to 61.03%and 52.16%,respectively,suggesting obvious inhibition effect,with statistically significant differences(P<0.01).The level of autophagy marker LC3B protein was significantly increased in the experimental groups,with statistically significant difference from the control group(P<0.01).Animal experiments showed that ProTαsignificantly reduced the tumor mass coefficient of mice with H22 solid tumors,and the tumor inhibition rate reached 34.7%,and the tumor inhibition rate of ProTα+oxaliplatin group was significantly higher than that of the ProTαgroup(P<0.05).The thymus indexes in the ProTαgroup and the ProTα+oxaliplatin group were higher than those in the control group and the oxaliplatin group(P<0.05).The expressions of autophagy-associated LC3B protein were significantly upregulated while the expressions of P62 and p-mTOR prot eins were downregulated in the tumor t issues of the Pro Tα,oxaliplatin and Pro Tα+oxaliplatin groups,with statistically significant differences from the control group(P<0.05).Conclusion ProTαmainly by regulating the release of IFN-γand other cytokines from immune cells and down-regulating p-mTOR expression to induce autophagy in hepatoma cells of H22-bearing mice,thus exerting an anti-tumor effect.Besides,ProTαcan improve the anti-cance r efficacy of oxaliplatin.