黄芩苷-金属配合物对人肝癌SMMC-7721细胞的毒性作用及其与肝癌细胞DNA相互作用性能关联分析

Relationship between toxicity of baicalin-metal complexes on human hepatoma SMMC-7721 cells and interaction of baicalin-metal complexes with hepatoma cell DNA

目的：探讨黄芩苷（BC）-金属（Ni2＋，Co2＋和 Cu2＋）配合物（BmC）与肝癌细胞 DNA 的结合能力与其细胞毒性的关联性。方法络合配位法合成 BmC 并表征其组成和结构；mTT 法、PI 单染法和 AnnexinⅤ-FITC 双染法检测 BmC 对人肝癌 SmmC-7721细胞增殖、周期和凋亡的影响，结合形态学观察探讨其对SmmC-7721细胞的毒性作用；以提取的肝癌 SmmC-7721细胞 DNA 为靶点，采用电化学循环伏安法和交流阻抗法研究 BmC 与 DNA 的相互作用，探讨二者的作用机制。结果成功制备3种新型 BmC，即 BC-铜（BC-Cu）、BC-钴（BC-Co）和 BC-镍（BC-Ni），解析获得配合物的分子式为 Na2 Ni（C21 H16 O11）2·10H2 O， Na2 Co（C21 H16 O11）2·8H2 O 和 Na2 Cu（C21 H16 O11）2·8H2 O；细胞增殖和形态学检测结果显示，BmC 6.25~100 mg·L-1作用24，48和72 h 后，能明显抑制 SmmC-7721细胞增殖，细胞毒性顺序为 BC-Cu﹥BC-Co﹥BC-Ni﹥BC，并存在明显的时间效应和浓度效应关系（P﹤0.01）；细胞周期和凋亡检测结果显示，BmC 使细胞阻滞于 G0/ G1期，阻止进入 S 期和 G2/ m 期，促使 SmmC-7721细胞发生凋亡；电化学分析显示，BmC 与肝癌 SmmC-7721细胞 DNA 通过静电作用和插入作用的混合模式形成非电活性超分子化合物，结合数 m＝2，结合常数βBC ＝2.77×106 L·mol-1，βBC-Ni ＝5.46×106 L·mol-1，βBC-Co ＝7.74×106 L·mol-1和βBC-Cu ＝1.21×107 L·mol-1，BC 与金属离子配位后与 DNA 的结合能力明显增强，强弱顺序为 BC-Cu﹥BC-Co﹥BC-Ni﹥BC。结论 BmC 通过阻滞细胞周期抑制细胞增殖，促进细胞凋亡，表现出细胞毒性作用，且 BmC 与 DNA 结合能力与其细胞毒性一致，具有关联性，表明 BmC 进入 SmmC-7721细胞后与 DNA 结合，阻滞 DNA 复制，抑制细胞增殖，促进细胞凋亡，进而表现出抗肿瘤活性。

OBJECTlVE To investigate the correlation between baicalin metal（Ni2＋,Co2＋,Cu2＋） compIexes（BmC）with their anti-tumor activity and the abiIity of BmC to bind to hepatoma cell DNA. METHODS The chelating Iigand method was used to synthesize BmC,and the composition and struc-ture of BmC were characterized. mTT,PI staining method and AnnexinⅤ-FITC double staining method were used to analyze the effect of BmC on SmmC-7721 cell proliferation,cycIe and apoptosis,and to expIore their cytotoxic effect on SmmC-7721 cells in combination with morphology. With DNA extracted from hepatoma cells as a target,cycIic voltammetry and AC impedance were used to study the interaction of BmC with DNA. The interaction mechanism between BmC and DNA was expIored. RESULTS Three new types of BmS were successfuIIy prepared. The molecuIar formuIas of compIexes were Na2 Ni（C21 H16 O11 ）2·10H2 O,Na2 Co（C21 H16 O11 ）2·8H2 O,and Na2 Cu（C21 H16 O11 ）2·8H2 O,respec-tively. Cell proliferation and morphology detection revealed that BmC 6.25-100 mg·L-1 treatment for 24, 48 and 72 h couId inhibit SmmC-7721 cell survival. BmC cytotoxicity was Iisted as follows：baicalin-cop-per（ BC-Cu）﹥ baicalin-cobalt（ BC-Co）﹥ baicalin-nickel（ BC-Ni）﹥ baicalin（ BC）,in a concentration-dependent manner（P﹤0.01）and time-dependent manner（P﹤0.01）. According to the resuIts of cell cycIe and apoptosis detection,BmC retarded the growth of cells from G0 / G1 phase into S phase or G2 / m phase whiIe inducing apoptosis of SmmC-7721 cells. The resuIts of electrochemical analysis showed that BmC and hepatoma SmmC-7721 cell DNA formed a non-electroactive supermolecuIar compound through the mixed-mode of electrostatic interaction and insertion effect. The binding parameters were obtained：the binding number m = 2,the binding constant βBC = 2.77 ×106 L·mol-1 ,βBC-Ni = 5.46 ×106 L·mol-1 ,βBC-Co =7.74×106 L·mol-1 ,and βBC-Cu =1.21×107 L·mol-1 . The abiIity of BmC to bind to DNA was signifi-cantIy enhanced by BC compIexes with metal ions,and their abiIity was Iisted as follows：BC-Cu﹥BC-Co﹥BC-Ni﹥BC. CONCLUSlON BmC shows cytotoxicity by blocking the cell cycIe,inhibiting cell proliferation and motivaing apoptosis. The abiIity of BmC to bind to DNA is consistent with its cytotoxicity. BmC,after binding to cell DNA,wiII block DNA repIication,inhibit cell proliferation,Iead to cell apoptosis,and exhibit anti-tumor activity.