胡椒醛环丙沙星腙诱导人肝癌 SMMC-7721 细胞生长停滞和凋亡。
Piperonal ciprofloxacin hydrazone induces growth arrest and apoptosis of human hepatocarcinoma SMMC-7721 cells.
机构信息
Institute of Neurobiology, College of Nursing, He-nan University, Kaifeng, China.
出版信息
Acta Pharmacol Sin. 2012 Feb;33(2):271-8. doi: 10.1038/aps.2011.158.
AIM
To investigate the cytotoxic effects of piperonal ciprofloxacin hydrazone (QNT4), a novel antibacterial fluoroquinolone derivative, against human hepatocarcinoma SMMC-7721 cells.
METHODS
Human hepatocarcinoma cells (SMMC-7721), human breast adenocarcinoma cells (MCF-7) and human colon adenocarcinoma cells (HCT-8) were tested. The effects of QNT4 on cell proliferation were examined using MTT assay. Cell apoptosis was determined using Hoechst 33258 fluorescence staining, TUNEL assay and agarose gel electrophoresis. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. Mitochondrial membrane potential (Δψm) was measured using a high content screening imaging system. Protein expression of caspase-9, caspase-8, caspase-3, p53, Bcl-2, Bax, and cytochrome c was detected with Western blot analysis.
RESULTS
Treatment with QNT4 (0.625-10 μmol/L) potently inhibited the proliferation of the cancer cells in time- and dose-dependent manners (the IC(50) value at 24 h in SMMC-7721 cells, MCF-7 cells and HCT-8 cells was 2.956±0.024, 3.710±0.027, and 3.694±0.030 μmol/L, respectively). Treatment of SMMC-7721 cells with QNT4 (0.2146, 2.964, and 4.600 μmol/L) for 24 h dose-dependently increased the percentage of apoptotic cells, elicited characteristic DNA "ladder" bands, and decreased the mitochondrial membrane potential. QNT4 dose-dependently increased topoisomerase II-mediated DNA breaks while inhibiting DNA relegation, thus keeping the DNA in fragments. Treatment of SMMC-7721 cells with QNT4 significantly increased cytochrome c in the cytosol, and decreased cytochrome c in the mitochondrial compartment. QNT4 (3-7.39 μmol/L) significantly increased the protein expression of p53, Bax, caspase-9, caspase-3, and the cleaved activated forms of caspase-9 and caspase-3 in SMMC-7721 cells. In contrast, the expression of Bcl-2 was decreased, while caspase-8 had no significant change.
CONCLUSION
QNT4 induced the apoptosis of SMMC-7721 cells via inhibiting topoisomerase II activity and modulating mitochondrial-dependent pathways.
目的
研究新型抗菌氟喹诺酮衍生物胡椒醛环丙沙星腙（QNT4）对人肝癌 SMMC-7721 细胞的细胞毒性作用。
方法
检测人肝癌细胞（SMMC-7721）、人乳腺癌细胞（MCF-7）和人结肠腺癌细胞（HCT-8）。采用 MTT 法检测 QNT4 对细胞增殖的影响。采用 Hoechst 33258 荧光染色、TUNEL 检测和琼脂糖凝胶电泳检测细胞凋亡。采用琼脂糖凝胶电泳法，以质粒 pBR322 为底物，测定拓扑异构酶 II 活性。采用高通量筛选成像系统测定线粒体膜电位（Δψm）。采用 Western blot 分析检测 caspase-9、caspase-8、caspase-3、p53、Bcl-2、Bax 和细胞色素 c 的蛋白表达。
结果
QNT4（0.625-10 μmol/L）处理可时间和剂量依赖性地显著抑制癌细胞增殖（24 h 时 SMMC-7721 细胞、MCF-7 细胞和 HCT-8 细胞的 IC50 值分别为 2.956±0.024、3.710±0.027 和 3.694±0.030 μmol/L）。用 QNT4（0.2146、2.964 和 4.600 μmol/L）处理 SMMC-7721 细胞 24 h，可剂量依赖性地增加凋亡细胞的百分比，引发特征性 DNA“梯状”条带，并降低线粒体膜电位。QNT4 可剂量依赖性地增加拓扑异构酶 II 介导的 DNA 断裂，同时抑制 DNA 连接，从而使 DNA 保持片段化。用 QNT4 处理 SMMC-7721 细胞，细胞质中的细胞色素 c 明显增加，线粒体部分的细胞色素 c 减少。QNT4（3-7.39 μmol/L）可显著增加 SMMC-7721 细胞中 p53、Bax、caspase-9、caspase-3 和 cleaved 激活形式的 caspase-9 和 caspase-3 的蛋白表达。相反，Bcl-2 的表达减少，而 caspase-8 没有明显变化。
结论
QNT4 通过抑制拓扑异构酶 II 活性和调节线粒体依赖性途径诱导 SMMC-7721 细胞凋亡。
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