3-O-(3-甲氧基肉桂酰)-甘草次酸酯对HepG2细胞体外抑制作用研究

Inhibition of 3-O-(3-methoxy cinnamoyl)-glycyrrhetinic acid ester against HepG2 cells in vitro

目的探讨3-O-(3-甲氧基肉桂酰)-甘草次酸酯对Hep G2细胞体外抑制作用及其作用机制。方法采用MTT法考察药物对肝癌细胞Hep G2、宫颈癌细胞He La、结肠癌细胞HT-29、心肌细胞H9C2、犬肾上皮细胞MDCK和血管内皮细胞HY926的细胞毒性,利用细胞染色法观察不同浓度3-O-(3-甲氧基肉桂酰)-甘草次酸酯对Hep G2细胞形态的影响,流式细胞术评价该化合物诱导Hep G2细胞的凋亡。结果 3-O-(3-甲氧基肉桂酰)-甘草次酸酯具有较好的抑制Hep G2细胞增殖活性作用,其半数有效抑制浓度(IC50)为8.28μmol/L,明显强于甘草次酸的活性(IC50>50μmol/L)。3-O-(3-甲氧基肉桂酰)-甘草次酸酯及其合成原料甘草次酸、3-甲氧基肉桂酸对非肿瘤细胞MDCK、HY926、H9C2细胞毒性较弱(IC50>24μmol/L)。3-O-(3-甲氧基肉桂酰)-甘草次酸酯浓度在12.5μmol/L时,对MDCK、HY926、H9C2细胞抑制率分别为17.05%、16.08%、4.66%,与对Hep G2细胞的抑制效果相比差异明显。不同浓度3-O-(3-甲氧基肉桂酰)-甘草次酸酯对Hep G2细胞Giemsa染色、H33342染色的细胞形态有明显差异。随着3-O-(3-甲氧基肉桂酰)-甘草次酸酯浓度的升高,早期凋亡率逐渐增加,而晚期凋亡率无明显变化,提示3-O-(3-甲氧基肉桂酰)-甘草次酸酯可以有效地诱导细胞早期凋亡,并呈现一定的量效关系。结论 3-O-(3-甲氧基肉桂酰)-甘草次酸酯具有良好的抗肿瘤作用,对Hep G2细胞抑制效果最好,对非癌细胞MDCK、HY926、H9C2没有明显抑制效果,该作用主要通过诱导Hep G2细胞凋亡,且呈现一定的量效关系。

Objective To study the inhibitory effect of 3-O-（3-methoxy cinnamoyl）-glycyrrhetinic acid ester against Hep G2 cells in vitro and explore its mechanism. Methods Cytotoxicities of 3-O-（3-methoxy cinnamoyl）-glycyrrhetinic acid ester against three tumour cells lines（Hep G2, He La, HT-29） and three normal cell lines（H9C2, MDCK, and HY926） were detected by MTT assay. Effects of various concentration of 3-O-（3-methoxy cinnamoyl）-glycyrrhetinic acid ester on morphological changes of Hep G2 cells were investigated by Giemsa staining and H3334 staining. Apoptosis of Hep G2 cells induced by the compound was evaluated by flow cytometry. Results 3-O-（3-Methoxy cinnamoyl）-glycyrrhetinic acid ester had better inhibition of Hep G2 cell proliferation activity with IC50 of 8.28 μmol/L than that（IC50〉 50 μmol/L） of glycyrrhetinic acid. 3-O-（3-Methoxy cinnamoyl）-glycyrrhetinic acid ester and its precursor drugs glycyrrhetinic acid and（3-methoxy） cinnamoyl acid had weak cytotoxicities against MDCK, HY926, and H9C2 cells. The inhibitory rates of 3-O-（3-methoxy cinnamoyl）-glycyrrhetinic acid ester at concentration of 12.5 μmol/L against MDCK, HY926, and H9C2 cells were 17.05%, 16.08%, and 4.66%, which had significant differences compared with the inhibitory effects on Hep G2 cells. There were obvious differences on morphological changes of Hep G2 cells at various concentration of 3-O-（3-methoxy cinnamoyl）-glycyrrhetinic acid ester stained by Giemsa staining and H3334 staining methods. With 3-O-（3-methoxy cinnamoyl）-glycyrrhetinic acid ester concentration increased, the early apoptosis rate increased gradually, but had no obvious change rate of late apoptosis, and the results showed that the drug effectively induced Hep G2 cell apoptosis, with dose-dependent relationship. Conclusion 3-O-（3-Methoxy cinnamoyl）-glycyrrhetinic acid ester displays good anti-tumor effect, which has a considerable inhibition against Hep G2 but low toxicity effect on normal cells MDCK, HY926, and H9C2. Its mechanism may be related to apoptosis of Hep G2 cells, and has a dose-response relationship.