塞来昔布联合紫杉醇诱导乳腺癌细胞凋亡的协同作用及其机制

doi:10.13481/j.1671-587x.20160306

摘要/Abstract

摘要：

目的: 观察塞来昔布联合紫杉醇对乳腺癌MCF-7细胞凋亡的影响,探讨环氧化酶2(Cox-2)抑制剂与化疗药物协同作用的可能机制。方法: 将MCF-7细胞分为空白对照组(不含药物作用的培养基)、塞来昔布组(含不同浓度塞来昔布的培养基)、紫杉醇组(含不同浓度紫杉醇的培养基)和塞来昔布联合紫杉醇组(含不同浓度塞来昔布和不同浓度紫杉醇的培养基)。Hoechst 33258染色观察各组MCF-7细胞凋亡形态;MTT法检测各组MCF-7细胞存活率;流式细胞术检测各组MCF-7细胞周期分布和细胞凋亡率;Western blotting法检测各组MCF-7细胞中B淋巴细胞瘤2(bcl-2)、细胞外调节蛋白激酶2(ERK2)和磷酸化细胞外调节蛋白激酶(p-ERK)蛋白的表达量。结果: Hoechst 33258染色,与空白对照组比较,塞来昔布组和紫杉醇组MCF-7凋亡细胞数增多,塞来昔布联合紫杉醇组MCF-7凋亡细胞最多,并随塞来昔布浓度增加而增加。MTT法检测,塞来昔布组和紫杉醇组MCF-7细胞存活率均随药物作用时间的延长而降低;而在相同时间段内,50mg·L^-1塞来昔布与30μg·L^-1紫杉醇联合应用组MCF-7细胞存活率低于单独使用两药时的细胞存活率(P<0.05)。流式细胞术检测,塞来昔布组细胞阻滞于G₀/G₁期,紫杉醇组细胞阻滞于G₂/M期;与空白对照组比较,塞来昔布联合紫杉醇组细胞周期分布改变最明显,在G₀/G₁期及G₂/M期的细胞比例均有明显升高,S期细胞比例下降。AnnexinⅤ/PI双染,药物刺激细胞6h后,塞来昔布组和紫杉醇组细胞早期凋亡率均高于空白对照组(P<0.05),塞来昔布联合紫杉醇组细胞早期凋亡率高于其他组(P<0.05)。Western blotting法检测,与空白对照组比较,塞来昔布组、紫杉醇组和塞来昔布联合紫杉醇组MCF-7细胞中bcl-2蛋白和p-ERK蛋白表达量均下调,塞来昔布联合紫杉醇组下调最明显;而ERK2蛋白表达量组间比较差异无统计学意义(P>0.05)。结论: 塞来昔布和紫杉醇在促乳腺癌MCF-7细胞凋亡方面有协同作用,其机制可能与下调bcl-2和p-ERK蛋白表达水平有关。

关键词: 塞来昔布, 紫杉醇, 乳腺肿瘤, MCF-7细胞, B淋巴细胞瘤2

Abstract:

Objective: To investigate the effect of celecoxib combined with taxol on the apoptosis of breast cancer MCF-7 cells,and to explore the possible mechanisms of the synergistic effect of Cox-2 inhibitors combined with chemotherapy drugs. Methods: The MCF-7 cells were divided into blank control group(treated with normal medium),celecoxib group(treated with different concentrations of celecoxib),taxol group(treated with different concentrations of taxol),and celecoxib combined with taxol group(combination group,treated with celecoxib combined with taxol).Hoechst 33258 staining was used to observe the morphology of apoptotic cells;MTT assay was adopted to detect the survival rate of MCF-7 cells;flow cytometry with Annexin Ⅴ FITC/PI double staining was used to detect the distribution of cell cycle and the apoptotic rate of MCF-7 cells;Western blotting method was taken to detect the protein expressions of b-cell lymphoma-2(bcl-2),extracellular regulated protein kinase2(ERK2) and phosphorylase-extracellular regulated protein kinase(p-ERK) in the MCF-7 cells. Results: The Hoechst 33258 staining results showed that compared with blank control group,the number of apoptotic cells in both celecoxib group and taxol group were increased,and the number of apoptotic cells in combination group was the highest,and the number of apoptotic cells was increased with the increasing of celecoxib concentration.The MTT assay results showed that the survival rates of MCF-7 cells in celecoxib group and taxol group were decreased with the prolongation of treatment time;during the same period,the MCF-7 cells treated with the combination of 50 mg·L^-1 celecoxib and 30 μg·L^-1 taxol had a much lower survival rate compared with celecoxib or taxol used alone(P<0.05).The flow cytometry results showed that the MCF-7 cells in celecoxib group were arrested at G₀/G₁ phase,and the MCF-7 cells in taxol group were arrested at G₂/M phase;compared with blank control group,the distribution of cell cycle in combination group changed dramatically,such as the ratio of MCF-7 cells arrested at G₀/G₁ phase and G₂/M phase were increased,and the ratio of MCF-7 cells arrested at in S phase was decreased.The Annexin Ⅴ FITC/PI double staining results showed that 6 h after drug treatment,the apoptotic rates of MCF-7 cells in celecoxib group and taxol group were higher than that in blank control group(P<0.05),and the apototic rate of cells in combination group was the highest(P<0.05).The Western blotting results showed that compared with blank control group,the expression levels of bcl-2 and p-ERK proteins in MCF-7 cells in celecoxib group, taxol group and combination group were all decreased,especially in combination group;but there were no significant differences in the ERK2 protein expression levels between various groups(P>0.05). Conclusion: Celecoxib and taxol show a synergistic effect on inducing the apoptosis of MCF-7 cells and its mechanism may be related to the down-regulation of the expressions of bcl-2 and p-ERK proteins.

Key words: celecoxib, taxol, breast carcinoma, MCF-7 cell, b-cell lymphoma-2

中图分类号:

R737.9

周贺, 刘军, 柳雅玲, 王丽, 李开济, 门秀丽. 塞来昔布联合紫杉醇诱导乳腺癌细胞凋亡的协同作用及其机制[J]. 吉林大学学报(医学版), 2016, 42(03): 446-451.

ZHOU He, LIU Jun, LIU Yaling, WANG Li, LI Kaiji, MEN XiuLi. Synergistic effect of celecoxib combined with taxol on apoptosis of breast cancer cells and its mechanism[J]. Journal of Jilin University Medicine Edition, 2016, 42(03): 446-451.

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