亚砷酸钠联合粉防己碱对乳腺癌MDA-MB-231细胞的增殖抑制作用及机制研究
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摘要
目的:探讨亚砷酸钠联合粉防己碱对乳腺癌MDA-MB-231细胞的增殖抑制作用。方法:MTT法检测细胞活力,Annexin V-FITC/PI双染法检测细胞凋亡,Hoechst33258染色观察细胞染色质固缩情况,碘化丙啶PI单染法检测细胞周期,Western blot检测p-GSK3β、GSK3β及凋亡相关蛋白PARP、bcl-2的表达。结果:不同浓度的亚砷酸钠或粉防己碱单用均可抑制MDA-MB-231细胞增殖(P <0. 01);与单独用药相比,两药联合使用可显著增强细胞增殖抑制效果(P <0. 000 1)。两药联用可显著诱导MDA-MB-231细胞凋亡(P <0. 01),细胞凋亡率呈现浓度依赖性递增,并出现细胞核染色质固缩。联合用药浓度为亚砷酸钠10μmol/L+粉防己碱4μg/ml时,S期细胞所占百分比显著增高(P <0. 05);联合用药浓度为亚砷酸钠15μmol/L+粉防己碱4. 5μg/ml时,G_2/M期细胞所占百分比显著增高(P <0. 001)。联合用药后,凋亡相关蛋白PARP的表达显著上调(P <0. 000 1); bcl-2的表达显著下调(P <0. 05); p-GSK3β、GSK3β蛋白表达均显著上调(P <0. 000 1)。结论:亚砷酸钠联合粉防己碱对乳腺癌MDA-MB-231细胞具有增殖抑制作用,其机制与诱导细胞周期S期、G_2/M期阻滞、GSK3β蛋白水平上调及促进细胞凋亡有关,促凋亡作用与bcl-2蛋白水平下调、PARP蛋白水平上调有关。
Objective: To investigate the effects of sodium arsenite combined with tetrandrine on proliferation inhibition in human breast cancer cell line MDA-MB-231. Methods: Cell proliferation was measured using MTT assay.Cell apoptosis was examined by Annexin V-FITC/PI double staining analysis. Chromatin condensation was observed by Hoechst33258 staining. Cell cycle was examined by PI staining. The expression of p-GSK3β,GSK3β and apoptosis-related proteins PARP,bcl-2 were detected by Western blot. Results: Both alone sodium arsenite and alone tetrandrine could inhibit the proliferation of MDA-MB-231 cells( P < 0. 01). Compared with each drug alone,the combination could enhance the inhibition rate( P < 0. 000 1). Apoptosis was induced significantly by the combination. Chromatin condensation was observed,and the apoptosis rate was dose-dependent. After treatment with NaAsO_2 10 μmol/L + tetrandrine 4 μg/ml for 48 h,the rate of S phase was increased significantly( P < 0. 05). After treatment with NaAsO_2 15 μmol/L + tetrandrine 4. 5 μg/ml for 48 h,the rate of G_2/M phase was increased significantly( P <0. 001). After treatment with the combination,the expression levels of PARP,p-GSK3β,GSK3β were up-regulated( P < 0. 000 1),while bcl-2 was down-regulated( P < 0. 05). Conclusion: Sodium arsenite combined with tetrandrine could inhibit the proliferation of MDA-MB-231 cells. The mechanism was related to cell cycle S phase or G_2/M phase arrest,the up-regulation of GSK3β protein,and apoptosis induction.
Objective: To investigate the effects of sodium arsenite combined with tetrandrine on proliferation inhibition in human breast cancer cell line MDA-MB-231. Methods: Cell proliferation was measured using MTT assay.Cell apoptosis was examined by Annexin V-FITC/PI double staining analysis. Chromatin condensation was observed by Hoechst33258 staining. Cell cycle was examined by PI staining. The expression of p-GSK3β,GSK3β and apoptosis-related proteins PARP,bcl-2 were detected by Western blot. Results: Both alone sodium arsenite and alone tetrandrine could inhibit the proliferation of MDA-MB-231 cells( P < 0. 01). Compared with each drug alone,the combination could enhance the inhibition rate( P < 0. 000 1). Apoptosis was induced significantly by the combination. Chromatin condensation was observed,and the apoptosis rate was dose-dependent. After treatment with NaAsO_2 10 μmol/L + tetrandrine 4 μg/ml for 48 h,the rate of S phase was increased significantly( P < 0. 05). After treatment with NaAsO_2 15 μmol/L + tetrandrine 4. 5 μg/ml for 48 h,the rate of G_2/M phase was increased significantly( P <0. 001). After treatment with the combination,the expression levels of PARP,p-GSK3β,GSK3β were up-regulated( P < 0. 000 1),while bcl-2 was down-regulated( P < 0. 05). Conclusion: Sodium arsenite combined with tetrandrine could inhibit the proliferation of MDA-MB-231 cells. The mechanism was related to cell cycle S phase or G_2/M phase arrest,the up-regulation of GSK3β protein,and apoptosis induction.
引文
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[3]Li Y,Wu J,Zheng YJ,et al.Effects of sodium arsenite on skinkeratinocytes related gene expression[J].J Xinjiang Med Univ,2016,39(2):164-168.[李煜,吴军,郑玉建,等.亚砷酸钠对皮肤细胞角化相关基因表达的影响[J].新疆医科大学学报,2016,39(2):164-168.]
[4]Zhang TJ,Guo RX,Li X,et al.Tetrandrine cardioprotection in ischemia-reperfusion(I/R)injury via JAK3/STAT3/Hexokinase II[J].Eur J Pharmacol,2017,813:153-160.
[5]Wu XL,Li JX,Li ZD,et al.Protective effect of tetrandrine on sodium taurocholate-induced severe acute pancreatitis[J].Evid Based Complementary and Alternat Med,2015,2015:129103.
[6]Liu T,Liu X,Li W.Tetrandrine,a Chinese plant-derived alkaloid,is a potential candidate for cancer chemotherapy[J].Oncotarget,2016,7(26):40800-40815.
[7]Hong-Duck Um.Bcl-2 family proteins as regulators of cancer cell invasion and metastasis:A review focusing on mitochondrial respiration and reactive oxygen species[J].Oncotarget,2016,7(5):5193-5203.
[8]Senkevitch E,Li W,Hixon JA,et al.Inhibiting Janus Kinase 1 and BCL-2 to treat T cell acute lymphoblastic leukemia with IL7-Rαmutations[J].Oncotarget,2018,9(32):22605-22617.
[9]Syed O Ali,Farhaan A Khan,Miguel A Galindo-Campos,et al.Understanding specific functions of PARP-2:New lessons for cancer therapy[J].Am J Cancer Res,2016,6(9):1842-1863.
[10]Natalia Bailon-Moscoso,Gabriela Cevallos-Solorzano,Juan Carlos Romero-Benavides,et al.Natural compounds as modulators of cell cycle arrest:Application for anticancer chemotherapies[J].Curr Genomics,2017,18(2):106-131.
[11]S Phukan,VS Babu,A Kannoji,et al.GSK3β:Role in therapeutic landscape and development of modulators[J].Br J Pharmacol,2010,160(1):1-19.
[12]Wu F,Chen J,Fan LM.Analysis of the effect of rutin on GSK-3βand TNF-αexpression in lung cancer[J].Exp Ther Med,2017,14(1):127-130.