Objective: To explore the effects of matrine on the proliferation, tumor cell stemness, β-catenin transcriptional activity and AKT/GSK3β/β-catenin signaling pathway in human hepatocellular carcinoma (HCC) HepG2 and Huh7 cells.
Methods: The proliferation and colony formation ability of HepG2 and Huh7 cells treated with 200, 400, and 800 μg/mL matrine were evaluated with MTT assay and colony formation assay, respectively. Real-time quantitative PCR was performed to detect the mRNA expressions of CD90, epithelial cell adhesion molecule (EpCAM) and CD133, and dual-luciferase assay was used to detect the transcriptional activity of β-catenin in the treated cells. The effects of matrine on the expressions of protein kinase B (AKT), P-AKT, GSK-3β, P-GSK-3β, P-β-catenin and β-catenin proteins in the Wnt/β-catenin signaling pathway were assessed using Western blotting.
Results: Matrine inhibited the proliferation of the two HCC cell lines in a time- and concentration-dependent manner. The half-inhibitory concentrations of matrine were 2369, 1565 and 909.1 μg/mL at 24, 48 and 72 h in HepG2 cells, respectively, and were 1355, 781.8, and 612.8 μg/mL in Huh7 cells, respectively. Matrine concentrationdependently suppressed colony formation of the HCC cells, producing significant inhibitory effects at 400 μg/mL P < 0.01) and 800 μg/mL P < 0.001) in HepG2 cells and at 200 μg/mL P < 0.05), 400 μg/mL P < 0.01), and 800 μg/mL P < 0.001) in Huh7 cells. Matrine at 400 and 800 μg/mL significantly inhibited the mRNA expression of CD90, EpCAM and CD133 and the transcriptional level of β-catenin in both HepG2 and Huh7 cells P < 0.05 or 0.01). Matrine at 400 and 800 μg/mL also significantly decreased the protein levels of β-catenin, P-AKT and P-GSK-3β and increased the phosphorylation level of β-catenin in both of the cell lines.
Conclusions: Matrine inhibits the proliferation, colony formation, and the expressions of tumor stem cell markers CD90, EpCAM and CD133 in both HepG2 and Huh7 cells probably by inhibiting Wnt/β-catenin signaling pathway and the transcriptional activity ofβ-catenin.
目的: 探讨苦参碱对人肝癌HepG2和Huh7细胞增殖活性、细胞干性、β-catenin转录活性以及AKT/GSK3β/β-catenin信号通路的影响。
方法: 应用MTT法检测细胞增殖活性，平板克隆形成实验检测0 g/mL、200 g/mL、400 μg/mL、800 μg/mL苦参碱对人肝癌HepG2和Huh7细胞的克隆形成能力，荧光定量PCR分析0 g/mL、200 g/mL、400 μg/mL、800 μg/mL苦参碱对人肝癌HepG2和Huh7细胞干性基因CD90、EpCAM（上皮细胞粘附分子）和CD133 mRNA的表达水平，双荧光素酶报告基因检测0 g/mL、200 g/mL、400 μg/mL、800 μg/mL苦参碱对人肝癌HepG2和Huh7细胞内β-catenin转录活性，Western blotting检测0 g/mL、400 μg/mL、800 μg/mL苦参碱对人肝癌HepG2和Huh7细胞内AKT（蛋白激酶B）、GSK-3β和β-catenin及其相应磷酸化蛋白的表达水平。
结果: MTT实验结果显示，苦参碱呈时间-浓度依赖性地抑制肝癌HepG2和Huh7细胞的增殖，处理24、48、72 h对HepG2细胞的半数抑制浓度依次为2369、1565、909.1 μg/mL，对Huh7细胞的半数抑制浓度依次为1355、781.8、612.8 μg/mL。苦参碱浓度依赖性地抑制肝癌细胞的克隆形成能力，400 μg/mL、800 μg/mL苦参碱能够显著抑制HepG2细胞的克隆形成能力（P < 0.01，P < 0.001），200 g/mL、400 μg/mL、800 μg/mL苦参碱能够显著抑制Huh7细胞的克隆形成能力（P < 0.05，P < 0.01，P < 0.001）。苦参碱能够显著肝癌细胞内CD90、EpCAM和CD133等干细胞标志物mRNA的表达，其中400 μg/mL、800 μg/mL苦参碱能够显著抑制HepG2细胞中CD90（P < 0.01，P < 0.001）、EpCAM（P < 0.05，P < 0.01）和CD133（P < 0.01，P < 0.01）mRNA的表达，400 μg/mL、800 μg/mL苦参碱能够显著抑制Huh7细胞中CD90（P < 0.01，P < 0.01）、EpCAM（P < 0.05，P < 0.01）和CD133（P < 0.01，P < 0.01）mRNA的表达。同时400 μg/mL、800 μg/mL苦参碱显著抑制HepG2细胞（P < 0.001，P < 0.001）和Huh7细胞（P < 0.01，P < 0.001）内β-catenin的转录活性。研究结果显示400 μg/mL、800 μg/mL苦参碱能够显著降低HepG2和Huh7细胞内β-catenin和磷酸化的AKT和GSK-3β蛋白水平，增加β-catenin的磷酸化水平。
结论: 苦参碱可抑制肝癌HepG2和Huh7细胞的增殖、克隆形成以及肝癌干性基因CD90、EpCAM和CD133的表达，其机制可能与抑制AKT/GSK3β/β-catenin信号通路，从而降低β-catenin的转录活性有关。
Keywords: hepatocellular carcinoma; matrine; tumor cell stemness; β-catenin.