脂联素对乳腺癌细胞MCF7生长抑制的影响

Induction of cell cycle arrest in human breast cancer cells by adiponectin

目的 观察脂联素对人乳腺癌MCF7细胞株生长抑制的影响及探讨其可能的作用机制.方法 RPMI 1640培养基培养MCF7细胞.应用实时定量聚合酶链反应（RT-PCR）方法检测MCF7细胞脂联素受体的表达.观察脂联素（20、40 μg/ml）作用48 h后对细胞计数、细胞周期的影响.检测脂联素对细胞磷酸化腺苷酸活化蛋白激酶α（p-AMPKα）/总腺苷酸活化蛋白激酶α（AMPKα）、磷酸化蛋白激酶B（p-Akt）/蛋白激酶B（Akt）、细胞周期素D1（cyclinD1）及细胞周期素E2（cyclinE2）表达的影响.采用方差分析进行统计学分析.结果 （1）MCF7细胞表达脂联素受体1和受体2.（2）脂联素对MCF7细胞的生长具有抑制作用,且此作用呈现为剂量依赖性.当脂联素浓度为20和40μg/ml时,MCF7的细胞计数分别为对照组的86.7％和72.9％（F=20.438,P＜0.05）.（3）脂联素可抑制MCF7细胞的增殖,使较多的细胞停留于G1/G0期（对照组为45.1％±0.7％,20 μg/ml脂联素组49.1％±1.3％,40 μg/ml脂联素组50.1％±1.1％,F=17.339,P＜0.05）,而S期细胞比例相对减少（对照组39.9％±1.2％,20μg/ml脂联素组36.4％±1.5％,40 μg/ml脂联素组36.0％±2.1％,F=9.715,P＜0.05）,此作用呈剂量依赖性.（4）脂联素作用30 min可增加MCF7细胞p-AMPKα （Thr172）表达（对照组、20、40 μg/ml脂联素组相对表达量分别为0.44±0.01、0.67±0.02、0.72±0.03,F=91.07,P＜0.05）,作用24 h可降低cyclin D1的表达（对照组、20、40 μg/ml脂联素组相对表达量分别为0.81±0.03、0.70±0.03、0.59±0.03,F=30.25,P＜0.05）,作用48 h可降低cyclin E2的表达（对照组、20、40 μg/ml脂联素组相对表达量分别为0.83±0.04、0.72±0.02、0.64±0.02,F=21.17,P＜0.05）.结论 脂联素可抑制乳腺癌细胞株MCF7的生长,其作用机制可能与AMPK激活、降低cyclin D1和cyclinE2表达有关.

Objective To investigate the inhibitory effect of adiponectin on the human breast carcinoma cell line MCF7 and to explore the potential mechanism.Methods MCF7 cells were grown in RPMI 1640.The expression of adiponectin receptors in MCF7 cells,including adiponectin receptor 1 and adiponectin receptor 2,was detected by the RT-PCR assay.The total cell count and the percentage of cells at different stages of cell cycle were measured after the exposure to human adiponectin at 20 μg/ml or 40 μg/ml for 48 hours.The expression of phospho-AMPK (THr172)/AMPK,phospho-Akt (Ser473)/Akt,cyclin D1 and cyclin E2 was determined by Western blotting.Comparisons between groups were performed using one-way ANOVA.Results (1) MCF7 cells express both adipo-R1 and adipo-R2.(2) Treatment of the cultured MCF7 cells with human adiponectin resulted in a dosage-dependent suppressive effect on the cell proliferation.At 20 μg/ml and 40 μg/ml,human adiponectin inhibited the growth of MCF7 cells by 13.3％ and 27.1％,respectively (F =20.438,P ＜ 0.05).(3) Adiponectin treatment leads to suppression of cell proliferation,which is primarily due to the significant increase of cell populations at G1/G0 phase (control group 45.1％ ±0.7％,20 μg/ml adiponectin group 49.1％ ± 1.3％,40 μg/ml adiponectin group 50.1％ ± 1.1％,F=17.339,P ＜0.05),concomitant with these changes,the percentage of MCF7 cells in S phase was decreased (control group 39.9％ ± 1.2％,20 μg/ml adiponectin group 36.4％ ± 1.5％,40 μg/mladiponectin group 36.0％ ± 2.1％,F =9.715,P ＜ O.05).(4) Adiponectin treatment markedly increases the phosphorylation (Thr172) of AMPK in MCF7 cells within 30 min.The expression of phospho-AMPK-αwas 0.44 ±0.01,0.67 ±0.02 and 0.72 ±0.03 in the control group,the 20 μg/ml adiponectin group and the 40 μg/ml adiponectin group,respectively (F =91.07,P ＜ 0.05).Prolonged exposure to adiponectin for 24 hours resulted in the expression reduction of cyclin D1.The expression of cylcin D1 was 0.81 ± 0.03,0.70 ±0.03 and 0.59 ± 0.03 in the control group,the 20 μg/ml adiponectin group and the 40 μg/ml adiponectin group,respectively (F =30.25,P ＜ 0.05).The treatment of adiponectin for 48 hours markedly decreased the expression of cyclin E2.The expression of cylcin E2 was 0.83 ± 0.04,0.72 ± 0.02 and 0.64±0.02 in the control group,the 20 μg/ml adiponectin group,and the 40 μg/ml adiponectin group,respectively (F =21.17,P ＜ 0.05).Conclusion Direct anti-proliferative effect has been shown in the MCF7 cells,which may be mediated by the activation of AMPK pathway and the reduction in the expression of cyclin D1 and cyclin E2.