Objective: To investigate the effect of platycodin D on the radiosensitivity of human hepatoma cell lines HepG2 and SMMC-7721 and related mechanisms of action. Methods: MTT assay was used to analyze the effect of different concentrations of platycodin D with different treatment times on cell viability. The cells were pretreated with 5 μg/ml platycodin D for 24 hours followed by X-ray irradiation at different radiation doses. Colony-forming assay was used to measure the radiosensitizing effect of platycodin D on cells. The quasi-threshold dose (Dq), mean lethal dose (Do), extrapolation number (N), sensitizer enhancement ratio (SER), and survival fraction (SF) at different radiation doses were calculated, and the multi-target single-hit model was used to fit the cell survival curve according to the formula SF = l-(l-e(-D/D0))N. Flow cytometry was used to investigate the distribution of cell cycle, and Western blotting was used to measure the changes in the protein expression of phosphorylated phosphatidylinositol 3'-kinase (pPI3K), phosphorylated protein kinase (pAkt), nuclear factor-κB (NF-κB), and phosphorylated nuclear factor inhibiting protein (pIκBα). A one-way analysis of variance, the t-test, or the least significant difference test was used for statistical analysis based on the type of data. Results: Platycodin D reduced the viability of HepG2 and SMMC-7721 cells in a dose-dependent manner; the IC50 value for HepG2 cells was 24.2 ± 0.61 μg/ml at 24 hours and 7.68 ± 0.46 μg/ml at 48 hours, and that for SMMC-7721 cells was 23.8 ± 0.57 μg/ml at 24 hours and 8.63 ± 0.86 μg/mL at 48 hours. After the combined treatment with platycodin D and irradiation, there were significant reductions in Dq (P = 0.002), Do (P = 0.002), and N value (P = 0.003), the survival curve markedly shifted to the left, and SER was 1.347 ± 0.04 in HepG2 cells and 1.418 ± 0.05 in SMMC-7721 cells. In addition, platycodin D significantly inhibited the increase in the proportion of cells in G2/M phase, the increases in the protein expression of pPI3k (P = 0.002), pAkt (P = 0.003), and NF-κB (P = 0.002), and the reduction in the protein expression of pIκBα (P = 0.003). Conclusion: Platycodin D can increase the radiosensitivity of HepG2 or SMMC-7721 cells, possibly by enhancing the growth inhibition effect of irradiation and inhibiting the activation of the PI3k/Akt and NF-κB pathways.
目的： 研究桔梗皂苷D对人肝癌细胞株HepG2与SMMC-7721细胞放射敏感性的作用，并探讨其相关作用机制。 方法： 四甲基偶氮唑盐法检测不同浓度不同处理时间桔梗皂苷D对细胞存活率的影响；预先给予细胞5 μg/ml桔梗皂苷D处理24 h，经过不同照射剂量的X射线照射，细胞克隆形成实验检测桔梗皂苷D对细胞的辐射增敏作用，计算准域剂量（Dq），平均致死剂量（D0），外推数（N），放射增敏比SER及不同照射剂量作用下的存活分数（SF），并依据多靶单击型模型公式SF = l-（l-e-(D/D0)）N拟合细胞存活曲线；流式细胞仪检测细胞周期的分布；Western blot检测细胞中磷酸化磷脂酰肌醇激酶（pPI3k）、磷酸化蛋白激酶（pAkt）、核因子（NF）-κB与磷酸化核因子抑制蛋白（pIκBα）的蛋白表达变化。据资料不同分别单因素方差分析（ANOVA）、t检验或LSD检验进行统计学分析。 结果： 桔梗皂苷D降低两种细胞存活率，且该抑制作用呈时间与剂量依赖性，HepG2细胞24 h的IC50为（24.2±0.61）μg/ml，48 h的IC50为（7.68±0.46） μg/ml；SMMC-7721细胞24 h的IC50为（23.8±0.57） μg/ml，48 h的IC50为（8.63±0.86）μg/ml；桔梗皂苷D联合放射处理后，Dq（P = 0.002）、D0（P = 0.002）和N值（P = 0.003）均显著降低，细胞存活曲线明显左移，放射增敏比SER分别为1.347±0.04（HepG2）与1.418±0.05（SMMC-7721）；此外，桔梗皂苷D显著降低放射引起的两种细胞周期G(2)/M期比例的上升，以及pPI3k（P = 0.002）、pAkt（P = 0.003）与NF-κB（P = 0.002）蛋白表达的增加，pIκBα（P = 0.003）蛋白的表达降低。 结论： 桔梗皂苷D增加人肝癌细胞株HepG2与SMMC-7721细胞的放射敏感性，其机制可能与桔梗皂苷D增强发射线对细胞生长抑制作用、抑制PI3k/Akt及NF-κB通路的激活有关。.
Keywords: Carcinoma, hepatocelluar; Hepatocarcinoma cells; NF-κB; PI3k/Akt; Platycodin D; Radio-sensitivity.