Antitumor effect of matrine in human hepatoma G2 cells by inducing apoptosis and autophagy
Antitumor effect of matrine in human hepatoma G2 cells by inducing apoptosis and autophagy
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DOI：
10.3748/wjg.v16.i34.4281
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发表时间：
2010-09-14
影响因子：
4.3
通讯作者：
Ren, Zhi-Jian
中科院分区：
文献类型：
--
作者：
Zhang, Jun-Qiang;Li, Yu-Min;Ren, Zhi-Jian
目的：为研究苦参碱对人肝癌细胞株HepG 2的抗肿瘤作用及其分子机制，采用3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazoliumbromide（MU）法检测细胞活力。流式细胞仪检测苦参碱对细胞周期的影响。Annexin-V-FITC/PI双染法检测细胞凋亡。倒置相差显微镜下观察细胞形态学变化。透射电镜观察苦参碱作用后细胞的超微结构变化。单丹酰尸胺（MDC）染色用于检测自噬。评价自噬是否被自噬抑制剂3-甲基腺嘌呤（3-MA）阻断。结果：苦参碱可明显抑制HepG 2细胞增殖，并呈剂量和时间依赖性，诱导HepG 2细胞周期阻滞于G1期，并诱导HepG 2细胞凋亡。未用苦参碱处理的HepG 2细胞总凋亡率为0.14%。苦参碱浓度为0.5、1.0和2.0 mg/mL时，HepG 2细胞凋亡率分别为28.91%、34.36%和38.80%。在倒置相差显微镜下观察到明显的形态学变化。苦参碱处理后的HepG 2细胞胞浆内出现大量大小不等的空泡。随着苦参碱浓度的增加，细胞质空泡化程度逐渐加重。电镜观察显示苦参碱作用后HepG 2细胞内形成大量自噬空泡。当应用特异性自噬抑制剂3-MA时，自噬空泡的数量大大减少。MDC染色显示苦参碱处理组HepG 2细胞内MDC标记的荧光密度高于对照组，MDC标记的颗粒数量多于对照组。当苦参碱处理前加入3-MA时，在3-MA和苦参碱联合处理组中观察到较少的自噬空泡，表明当苦参碱诱导肝癌G2细胞死亡时，自噬和凋亡都被激活。实时荧光定量RT-PCR结果显示，苦参碱处理组凋亡相关分子Bax基因和自噬关键基因Beclin 1的表达水平均高于对照组，提示Beclin 1参与了苦参碱诱导的自噬，苦参碱的促凋亡机制可能与上调Bax基因表达有关。苦参碱对HepG 2细胞具有较强的抗肿瘤活性，有望成为治疗肝癌的新的有效药物。(C)2010年百世登。All rights reserved.
AIM: To study the antitumor effect of matrine in human hepatoma G2 (HepG2) cells and its molecular mechanism involved in antineoplastic activities.METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MU) assay was used to detect viability of HepG2 cells. The effect of matrine on cell cycle was detected by flow cytometry. Annexin-V-FITC/PI double staining assay was used to detect cellular apoptosis. Cellular morphological changes were observed under an inverted phase contrast microscope. Transmission electron microscopy was performed to further examine ultrastructural structure of the cells treated with matrine. Monodansylcadaverine (MDC) staining was used to detect autophagy. Whether autophagy is blocked by 3-methyladenine (3-MA), an autophagy inhibitor, was evaluated. Expression levels of Bax and Beclin 1 in HepG2 cells were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR).RESULTS: Matrine significantly inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner, and induced G1-phase cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner. The total apoptosis rate was 0.14% for HepG2 cells not treated with matrine. In contrast, the apoptosis rate was 28.91%, 34.36% and 38.80%, respectively, for HepG2 cells treated with matrine at the concentration of 0.5, 1.0 and 2.0 mg/mL. The remarkable morphological changes were observed under an inverted phase contrast microscope. Abundant cytoplasmic vacuoles with varying sizes were observed in HepG2 cells treated with matrine. Furthermore, vacuolization in cytoplasm progressively became larger and denser when the concentration of matrine was increased. Electron microscopy demonstrated formation of abundant autophagic vacuoles in HepG2 cells after matrine treatment. When the specific autophagic inhibitor, 3-MA, was applied, the number of autophagic vacuoles greatly decreased. MDC staining showed that the fluorescent density was higher and the number of MDC-labeled particles in HepG2 cells was greater in matrine treatment group than in control group. Fewer autophagic vacuoles were observed in the combined 3-MA and matrine treatment group when 3-MA was added before matrine treatment, indicating that both autophagy and apoptosis are activated when matrine-induced death of hepatoma G2 cells occurs. Real-time quantitative RT-PCR revealed that the expression levels of Bax gene, an apoptosis-related molecule, and Beclin 1 gene which plays a key role in autophagy were higher in matrine treatment group than in control group, indicating that Beclin 1 is involved in matrine-induced autophagy and the pro-apoptotic mechanism of matrine may be related to its upregulation of Bax expression.CONCLUSION: Matrine has potent antitumor activities in HepG2 cells and may be used as a novel effective reagent in treatment of hepatocellular carcinoma. (C) 2010 Baishideng. All rights reserved.