大黄素抑制人肝癌裸鼠移植瘤生长机制探讨

Effects of Emodin on apoptosis and expression of AIF and EndoG in transplanted tumor of hepatocarcinoma cell in nude mice

目的研究大黄素对人肝癌细胞株SMMC-7721裸鼠移植瘤细胞凋亡及凋亡诱导因子(apoptosis inducing factor,AIF)、核酸内切酶G(endonuclease G,Endo G)表达的影响,探讨大黄素体内抗癌机制。方法复制裸鼠人肝癌移植瘤模型,带瘤模型裸鼠腹腔注射大黄素。实验随机分为对照组(生理盐水+1%DMSO)、大黄素低剂量组〔25mg/(kg·d)〕、中剂量组〔50mg/(kg·d)〕和高剂量组〔100mg/(kg·d)〕,每组各8只。观察裸鼠一般生长情况和肿瘤体积变化,计算肿瘤生长抑制率以及肿瘤生长延迟时间。治疗结束次日处死裸鼠,取病变组织后,TUNEL检测肿瘤组织的凋亡指数,免疫组化检测各组AIF和EndoG表达情况。结果 32只裸鼠均成功建立模型。试验过程中裸鼠一般情况正常,实验结束后各组之间裸鼠体质量差异无统计学意义,P=0.79。治疗结束后,与对照组相比,大黄素低、中、高剂量组均有抑制肿瘤生长作用,大黄素各剂量组的肿瘤生长抑制率呈剂量效应正相关。低剂量组肿瘤生长抑制率为(37.2±1.09)%,中剂量组为(57.7±1.15)%,高剂量组为(72.3±1.21)%,高剂量组显著高于中剂量组,P<0.01;中剂量组显著高于低剂量组,P=0.019。低剂量组肿瘤生长延迟时间为(1.9±0.6)d,中剂量组为(3.4±0.9)d,高剂量组为(4.6±1.1)d。TUNEL结果显示,对照组肿瘤细胞凋亡指数为(8.23±1.65)%,低剂量组为(34.38±8.73)%,中剂量组为(48.14±9.57)%,高剂量组为(68.71±10.56)%。低剂量组较对照组明显升高,P=0.003;中剂量组较低剂量组明显升高,P=0.023;高剂量组较中剂量组明显升高,P=0.009。免疫组化结果显示,随着大黄素剂量的不断增加,肿瘤组织中AIF和EndoG蛋白表达逐渐增强,低剂量组高于对照组,P值均为0.001;中剂量组高于低剂量组,P值分别为0.015和0.004;高剂量组高于中剂量组,P值分别为0.021和0.013。免疫荧光结果显示,不同剂量大黄素组肿瘤组织中AIF及EndoG的表达位置发生了明显变化,出现由胞质向胞核移位的现象;对照组肿瘤组织中AIF、EndoG的表达位置变化不明显。结论大黄素可以明显抑制人肝癌SMMC-7721细胞在裸鼠体内的生长;经AIF和EndoG介导的非Caspase依赖线粒体通路诱导细胞凋亡,是大黄素抑制人肝癌SMMC-7721细胞移植瘤生长的机制之一。

OBJECTIVE To study the effects of emodin on apoptosis and protein of apoptosis inducing factor （AIF） and Endonuclease G（Endo G）in hepatocellular cancer in vivo, and to investigate the anti-cancer mechanism of emodin. METHODS The subcutaneously transplanted tumor model of human SMMC-7721 cells in nude mice was established. The nude mice with tumor were randomly divided into four groups： the control group, the low-dose emodin group [25 mg/（kg ~ d）], the moderate-dose emodin group [50 mg/（kg ~ d）] and the high-dose emodin group [100 mg/（kg ~ d）]. the drug was administered respectively for 14 days. Thereafter,tumor volumes was measured,and tumor rates was calculated. Apoptosis was deter mined by TUNEL assay and the expression of AIF and EndoG proteins were examined by immunohistochemistry. RESULTS Compared to the normal saline control group,all dose Emodin treated groups inhibited the growth of transplanted tumor inordinately. The apoptosis rate in high-dose Emodin-treated group （72. 3 ± 1.21）% was significantly higher than that in moderate-（57. 7±1.15）%（P=0. 01）,and that in moderate- was significantly higher than in low-（37. 2±1.09）%（P=0. 019）, As the effects of increasing doses of Emodin, the apoptosis rate increased. And the tumor growth delay time of the low- ,moderate-and high- dose Emodiwtreated group was （1.9±0. 6） ,（3.4±0. 9） and （4.6±1.1） d. The result of TUNEL test showed that compared with the control group＇s apoptosis index （8.23± 1.65） %, the low-dose Emodin-treated group＇s apoptosis index （34. 38 ± 8. 73）% was higher（P=0. 003）, and the moderate-dose group （48. 14 ± 9. 57）% was higher than the low- （P=0. 023）, the high-dose group （68. 71±10. 56）% was the highest（P=0. 009）. The result of immunohistoehemical showed that after being treated with different dose of emodin, the expression of AIF, Endo-G protein in tumor tissue increased with the increasing doses of Emodira the low-dose group＇s was higher than the control group＇s（P〈0. 01）. the moderate-dose group＇s was higher than the low-dose group＇s （P= 0. 015,P=0.004） the high-dose group＇s was higher than the moderate-dose group＇s （P=0. 021,P=0. 013）. Immunofluorescence demonstrated that the location of AIF and EndoG in the tumor changed obviously in different dose Emodin-treated group. The effect occurred in a dose-dependent manner. But these changes was not significant in control group. CONCLUSION Via the AIF, EndoG mediated Caspase-independent mitochondrial pathway may be one of the mechianism of emodin-induced apoptosis in transplanted tumor of human hepatoma SMMC-7721 cells.