白藜芦醇诱导人乳腺癌MDA-MB231细胞凋亡及自噬
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摘要
目的探讨白藜芦醇(resveratrol,Res)诱导人乳腺癌MDA-MB231细胞凋亡及自噬蛋白表达的作用与机制。方法以人乳腺癌MDA-MB231细胞为研究对象,MTT法检测Res(0、1、5、10、20、50、100、200 mg·L~(-1))抑制MDA-MB231细胞增殖情况; Res(0、5、20、60 mg·L~(-1))处理MDA-MB231细胞,划痕实验检测细胞迁移能力,Annexin V/PI双染法检测细胞早期凋亡率,免疫荧光法检测细胞LC3Ⅰ/Ⅱ的表达;Res(0、20、60 mg·L~(-1))处理MDA-MB231细胞,蛋白免疫印迹法检测细胞中自噬相关蛋白p62及Beclin-1的表达。结果Res在体外可明显抑制MDA-MB231细胞增殖,且呈浓度依赖关系。随着Res浓度的增高,乳腺癌细胞迁移能力较对照组分别下降了(8. 7±1. 1)%、(16. 5±2. 9)%和(32. 2±3. 6)%。流式细胞仪检测结果表明,早期凋亡细胞数增加;激光共聚焦检查显示,细胞质中LC3Ⅰ/Ⅱ绿色荧光增加;蛋白免疫印迹法检测出MDA-MB231细胞p62蛋白表达下降,Beclin-1蛋白表达升高。结论 Res可抑制人乳腺癌MDA-MB231细胞增殖,其机制可能与Res诱导细胞凋亡和自噬有关。
Aim To explore the induced effects of resveratrol( Res) on apoptosis and autophagy protein expression in human breast cancer MDA-MB231 cells and the mechanisms. Methods MDA-MB231 cells were cultured in vitro. MTT was adopted to detect cell proliferation of MDA-MB231 cells inhibited by Res( 0,1,5,10,20,50,100,200 mg · L~(-1)). MDA-MB231 cells were treated by Res with different concentrations( 0,5,20,60 mg · L~(-1)). The migration ability of cells was detected by scratch test. The early apoptotic rate was detected by Annexin V/PI double staining.The expression of cell LC3 Ⅰ/Ⅱ was detected by immunofluorescence method. MDA-MB231 cells were treated by Res with different concentrations( 0,20,60 mg·L~(-1)). And the autophagy related to p62 protein and Beclin 1 expression was detected by Western blot.Results Res had significantly inhibitory effects on MDA-MB231 cell proliferation in a concentration-dependent manner. With the increase of Res concentration in MDA-MB231 cells, compared with control group, cell migration ability decreased( 8. 7 ±1. 1) %,( 16. 5 ± 2. 9) % and( 32. 2 ± 3. 6) %. The early apoptotic cells increased according to the results of flow cytometry and the laser confocal examination showed that LC3 Ⅰ/Ⅱ green fluorescence increased.Western blot detected that p62 protein expression decreased and Beclin 1 protein expression increased in the cytoplasm of MDA-MB231. Conclusions Res can inhibit the proliferation of human breast cancer MDAMB231 cells in vitro and its mechanism may be related to cell apoptosis and autophagy induced by Res.
Aim To explore the induced effects of resveratrol( Res) on apoptosis and autophagy protein expression in human breast cancer MDA-MB231 cells and the mechanisms. Methods MDA-MB231 cells were cultured in vitro. MTT was adopted to detect cell proliferation of MDA-MB231 cells inhibited by Res( 0,1,5,10,20,50,100,200 mg · L~(-1)). MDA-MB231 cells were treated by Res with different concentrations( 0,5,20,60 mg · L~(-1)). The migration ability of cells was detected by scratch test. The early apoptotic rate was detected by Annexin V/PI double staining.The expression of cell LC3 Ⅰ/Ⅱ was detected by immunofluorescence method. MDA-MB231 cells were treated by Res with different concentrations( 0,20,60 mg·L~(-1)). And the autophagy related to p62 protein and Beclin 1 expression was detected by Western blot.Results Res had significantly inhibitory effects on MDA-MB231 cell proliferation in a concentration-dependent manner. With the increase of Res concentration in MDA-MB231 cells, compared with control group, cell migration ability decreased( 8. 7 ±1. 1) %,( 16. 5 ± 2. 9) % and( 32. 2 ± 3. 6) %. The early apoptotic cells increased according to the results of flow cytometry and the laser confocal examination showed that LC3 Ⅰ/Ⅱ green fluorescence increased.Western blot detected that p62 protein expression decreased and Beclin 1 protein expression increased in the cytoplasm of MDA-MB231. Conclusions Res can inhibit the proliferation of human breast cancer MDAMB231 cells in vitro and its mechanism may be related to cell apoptosis and autophagy induced by Res.
引文
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[3]Bai D P,Zhang X F,Zhang G L,et al.Zinc oxide nanoparticles induce apoptosis and autophagy in human ovarian cancer cells[J].Int J Nanomedicine,2017,12:6521-35.
[4]王坦,张艳群,曾永联,等.白藜芦醇抑制人肝癌SMMC-7721细胞增殖并降低m TOR蛋白磷酸化水平[J].中国药理学通报,2017,33(9):1309-14.[4]Wang T,Zhang Y Q,Zeng Y L,et al.Effect of resveratrol on proliferation of liver cancer SMMC-7721 cells and lowering levels of m TOR protein phosphorylation[J].Chin Pharma Bull,2017,33(9):1309-14.
[5]Rasheduzzaman M,Jeong J K,Park S Y.Resveratrol sensitizes lung cancer cell to TRAIL by p53 independent and suppression of Akt/NF-κB signaling[J].Life Sci,2018,208:208-20.
[6]Alayev A,Salamon R S,Schwartz N S,et al.Combination of rapamycin and resveratrol for treatment of bladder cancer[J].J Cell Physiol,2017,232(2):436-46.
[7]Dermani F K,Saidijam M,Amini R,et al.Resveratrol inhibits proliferation,invasion,and epithelial-mesenchymal transition by increasing miR-200c expression in HCT-116 colorectal cancer cells[J].J Cell Biochem,2017,118(6):1547-55.
[8]Zhao H G,Zhou S L,Lin Y Y,et al.Autophagy plays a protective role against apoptosis?induced by toxicarioside N via the Akt/m TOR pathway in human gastric cancer SGC-7901 cells[J].Arch Pharm Res,2018,41(10):986-94.
[9]De Urbina J J O,San-Miguel B,Vidal-Casariego A,et al.Effects of oral glutamine on inflammatory and autophagy responses in cancer patients treated with abdominal radiotherapy:a pilot randomized trial[J].Int J Med Sci,2017,14(11):1065-71.
[10]He J P,Hou P P,Chen Q T,et al.Flightless-I blocks p62-mediated recognition of LC3 to impede selective autophagy and promote breast cancer progression[J].Cancer Res,2018,78(17):4853-64.