Cancer Cell Cycle Arrest and Apoptosis in vitro Induced by Newly Synthesized Phenazine Derivatives
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摘要: 目的 探讨新合成吩嗪衍生物抗癌活性及其作用机制。方法 MTT法检测细胞增殖，吖啶橙/溴乙锭（AO/EB）染色荧光显微镜观察细胞形态，流式细胞仪检测细胞周期，Annexin V-FITC/7-AAD双染检测凋亡，Western blot检测p53和caspase-3蛋白的表达。结果 pn18、pn23、pc27和pc28四种化合物在体外抑制癌细胞增殖，尤其对人结直肠癌细胞HCT116作用明显，且呈时间和剂量依赖性。pc28在四种化合物中作用最强，48 h对HepG2、HCT116和A549细胞半数抑制浓度（IC50）分别为（6.62±2.69）、（10.83±1.41）和（22.39±4.31）μmol。20 μmol化合物作用于HCT116细胞24 h后，细胞密度降低，形态变圆，细胞内明亮绿色荧光与染色质浓缩相关，死亡细胞呈橙黄色和红色与细胞膜通透性增加有关；pc28诱导的细胞晚期凋亡比例由0.345%升高至19.7%。20 μmol pn18和pc27诱导G0/G1期细胞分别升高17.5%和25.0%，pn23和pc28诱导S期细胞分别升高18.4%和11.0%。pn23和pc28诱导p53表达升高，pc28也能上调并激活caspase-3。结论 四种合成的新型吩嗪衍生物在体外能有效抑制多种癌细胞增殖，其作用机制可能与诱导细胞周期阻滞和凋亡相关。Abstract: Objective To evaluate the anticancer activity of newly synthesized phenazine derivatives and investigate the possible mechanism. Methods Cell proliferation was tested by MTT assay. The morphology of HCT116 cells was observed by fluorescent microscope after acridine orange/ethidium bromide(AO/EB) dye. Cell cycle distribution was detected by flow cytometry, and cell apoptosis was examined by Annexin V-FITC/7-AAD staining. The expressions of p53 and caspase-3 proteins were detected by Western blot. Results All compounds inhibited the growth of cancer cells, especially human colorectal cancer cells HCT116, in a time- and dose-dependent manner. Compound pc28 showed the most potent antiproliferation effect on HepG2, HCT116 and A549 cells with half maximal inhibitory concentration(IC50) being (6.62±2.69), (10.83±1.41) and (22.39±4.31)μmol respectively at 48h. 20μmol compound on HCT116 cells for 24h resulted in the decrease of cell density, morphological change to round, increase of green fluorescence related to chromatin condensation, and dead cells showed orange or red color implying cell membrane permeability increase. Besides, pc28 induced apoptosis rate of HCT116 cells in advanced stage from 0.345% to 19.7%. 20μmol pn18 and pc27 induced the increase of cell number in G0/G1 phase for 17.5% and 25.0% respectively, while pn23 and pc28 induced the increase of cell number in S phase for 18.4% and 11.0%. Compound pn23 and pc28 elevated p53 protein expression. pc28 also upregulated and activated caspase-3. Conclusion The newly synthesized phenazine derivatives show effective anticancer activity in vitro, and the mechanism might be related to inducing cell cycle arrest and apoptosis.
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Key words:
- Phenazine derivatives /
- Anticancer
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