摘要
目的探讨鬼臼毒素衍生物（DPPC）抑制人非小细胞肺癌A549细胞的增殖作用,阐述DPPC对A549细胞周期阻滞以及DNA损伤的相关分子机制。方法体外培养A549细胞,噻唑蓝（MTT）实验观察DPPC不同浓度或不同时间处理A549细胞后肿瘤细胞的增殖情况;倒置显微镜下观察0.5μmol/L DPPC处理A549细胞后的形态学变化;细胞流式仪检测不同浓度DPPC处理A549细胞后细胞周期的变化;蛋白免疫印迹（Western blot）实验检测周期调控蛋白cyclin B1、cdc2（p34）和p-cdc2以及DNA损伤因子γ-H2AX的表达。并与依托泊苷和未予任何药物处理的A549细胞做对照。结果 MTT实验显示,DPPC不同浓度（0.001~10μmol/L）处理A549细胞48 h后或不同时间（12~72 h）同浓度（0.1μmol/L）处理A549细胞后,能够明显抑制肿瘤细胞的增殖,并呈时间和剂量依赖性;倒置显微镜下见DPPC处理后的A549细胞呈现整体萎缩,出现细胞碎片,细胞膜边缘出现小气泡等形态改变;细胞流式实验表明,DPPC能够显著影响A549细胞周期,使其多数阻滞在G2/M期;Westen blot实验结果显示,DPPC能够促进cyclin B1和cdc2（p34）的表达,同时抑制p-cdc2的表达,并使组蛋白H2AX的磷酸化增加。结论 DPPC能够显著抑制人非小细胞肺癌A549细胞的增殖,使细胞周期阻滞在G2/M期,可能与其上调周期调节蛋白cyclin B1、抑制cdc2（p34）磷酸化、诱导细胞DNA损伤有关;此外,DPPC体外表现出优越的抗肿瘤细胞增殖作用。
Objective To discuss the effect of DPPC on anti-proliferation in human non-small cell lung cancer A549 cells, and to investigate the cell cycle arrest and DNA damaged molecular mechanism by DPPC. Methods The A549 cells were cultured in vitro, and morphologic changes of tumor cells were observed using MTT （ thiazolyl blue） as-say after A549 cells were treated with different doses and times. Morphologic changes were observed under inverted mi-croscope after A549 cells were treated with 0. 5μmol/L DPPC. The changes of cell cycle were detected using flow cytom-etry after A549 cells were treated with different does DPPC. Regulatory protein expressions of cyclinB1, cdc2 （p34） and DNA damaged γ-H2AX factor in cell cycle were detected using Western blot test, and the expressions were compared with those by Etoposide treatment and control group （ without treatment） . Results MTT assay showed that tumor cells proliferation obviously inhibited after A549 cells were treated with different concentrations （0. 001-10 μmol/L） for 48 h and same concentration （0. 1 μmol/L） for different times （12-72 h） DPPC, and it had a dose-and time-dependent. In-verted microscope showed that the A549 cells were total atrophy with cell debris, and morphologic changes such as little gas vacuole was found in limbic plasmalemma. The flow cytometry showed that DPPC obviously affected A549 cell cycle to make the blocking cell cycle in G2/M phase. Western blot showed that DPPC could promote cyclinB1 and cdc2 （p34） expressions and inhibit p-cdc2 expression, and enhanced H2AX phosphorylation. Conclusion DPPC may obviously in-hibit proliferation of non-small-cell lung carcinoma cells, block cell cycle in G2/M phase, which possibly relate with up-regulation expression of regulatory protein CyclinB1, phosphorylation inhibition of cdc2（p34） and induction of DNA dam-age. In addition, DPPC shows superior effect of anti-tumor cell proliferation in vitro.
出处
《解放军医药杂志》
CAS
2015年第9期7-12,共6页
Medical & Pharmaceutical Journal of Chinese People’s Liberation Army
基金
国家自然科学基金资助项目(81372177)
全军医药卫生科研基金课题资助项目(CLZLZJB04)
