天南星多糖对人肾癌细胞系GRC-1增殖及凋亡作用的影响
|
摘要
目的:探讨天南星多糖(ARPS)对人肾癌细胞系GRC-1的增殖、凋亡及Wnt/β-连接素(β-catenin)信号通路的影响。方法:采用RPMI-1640培养液常规培养获对数生长期的GRC-1细胞后,给予0,20,50,100,200 mg·L~(-1)ARPS处理,0 mg·L~(-1)ARPS组为空白组,细胞计数试剂盒(CCK-8)法检测各浓度ARPS处理24,48,72和96 h的增殖抑制率,分别采用AnnexinFITC/PI双染法和PI单染法检测各浓度处理48 h后的细胞凋亡率和细胞周期情况,蛋白质免疫印迹(Western blot)检测各浓度处理48 h后Wnt/β-catenin信号通路中β-catenin及下游靶分子原癌基因(C-myc),细胞周期素D1(Cyclin D1)蛋白水平。结果:与空白组比较,在20~200 mg·L~(-1),ARPS对GRC-1细胞的增殖有明显抑制效果,细胞增殖抑制率随ARPS作用时间的延长和质量浓度的增加而升高,总体上抑制效应呈剂量和时间依赖的方式,以上差异均有统计学意义(P<0.05),50~200 mg·L~(-1)ARPS的早期凋亡率及20~200 mg·L~(-1)ARPS的晚期凋亡率和总凋亡率均升高(P<0.05),20,50,100,200 mg·L~(-1)ARPS组的G_0期/G_1期高于空白组,S期,G2期/M期及β-catenin,C-myc和Cyclin D1蛋白水平均低于0 mg·L~(-1)ARPS(P<0.05),20~200 mg·L~(-1)各质量浓度间的以上指标的差异均有统计学意义(P<0.05)。结论:ARPS对人肾癌细胞系GRC-1的增殖有一定抑制作用,同时可诱导细胞凋亡和G_0/G_1期阻滞并可抑制Wnt/β-catenin通路激活。
Objective: To explore Arisaematis Rhizoma polysaccharide( ARPS) on the proliferation,apoptosis and Wnt/β-catenin signaling pathway of human renal cell carcinoma cell line GRC-1. Method: After entering into the logarithmic growth phase with RPMI-1640 culture fluid under routine condition,the GRC-1 cells were treated with different doses of ARPS( 0,20,50,100,200 mg·L~(-1)). The 0 mg·L~(-1)ARPS group was the blank group. The inhibition rates of proliferation at 24,48,72 and 96 h after the treatment were measured by viable cell counting kit assay( CCK-8). The apoptosis rate and cell cycle at 48 h after the treatment were detected by Annexin-FITC/PI double staining and PI staining methods via flow cytometry,respectively. Western blot was employed to detect the expression level of β-catenin in Wnt/β-catenin signaling pathway and its downstream target molecule C-myc and Cyclin D1 at 48 h after the treatment. Result: Compared with the blank group,ARPS within the range of 20-200 mg·L~(-1)presented an inhibitory effect on GRC-1 cell proliferation. The proliferation inhibition rate increased with the rise in time and concentration of ARPS in a dose-and time-dependent manner,and the above differences were statistically significant( P < 0. 05). Compared with the 0 μg·m L~(-1)ARPS,the early apoptosis rate of 50-200 mg·L~(-1)ARPS,the late apoptosis rate of 20-200 mg·L~(-1)and the total apoptosis rate were all elevated( P < 0. 05). There was a higher G_0/G_1 phase ratio than 20,50,100,200 mg·L~(-1)ARPS groups,and lower S and G2/M phase and β-catenin,C-myc and Cyclin D1 protein levels than 0 mg·L~(-1)( P < 0. 05). Indexes within the concentration range between 20-200 mg·L~(-1)were statistically significant( P < 0. 05). Conclusion: ARPS inhibited the proliferation of renal cell carcinoma cell line GRC-1,and induced cell apoptosis and G_0/G_1 arrest,with the effect of inhibiting the activation of Wnt/β-catenin signaling.
Objective: To explore Arisaematis Rhizoma polysaccharide( ARPS) on the proliferation,apoptosis and Wnt/β-catenin signaling pathway of human renal cell carcinoma cell line GRC-1. Method: After entering into the logarithmic growth phase with RPMI-1640 culture fluid under routine condition,the GRC-1 cells were treated with different doses of ARPS( 0,20,50,100,200 mg·L~(-1)). The 0 mg·L~(-1)ARPS group was the blank group. The inhibition rates of proliferation at 24,48,72 and 96 h after the treatment were measured by viable cell counting kit assay( CCK-8). The apoptosis rate and cell cycle at 48 h after the treatment were detected by Annexin-FITC/PI double staining and PI staining methods via flow cytometry,respectively. Western blot was employed to detect the expression level of β-catenin in Wnt/β-catenin signaling pathway and its downstream target molecule C-myc and Cyclin D1 at 48 h after the treatment. Result: Compared with the blank group,ARPS within the range of 20-200 mg·L~(-1)presented an inhibitory effect on GRC-1 cell proliferation. The proliferation inhibition rate increased with the rise in time and concentration of ARPS in a dose-and time-dependent manner,and the above differences were statistically significant( P < 0. 05). Compared with the 0 μg·m L~(-1)ARPS,the early apoptosis rate of 50-200 mg·L~(-1)ARPS,the late apoptosis rate of 20-200 mg·L~(-1)and the total apoptosis rate were all elevated( P < 0. 05). There was a higher G_0/G_1 phase ratio than 20,50,100,200 mg·L~(-1)ARPS groups,and lower S and G2/M phase and β-catenin,C-myc and Cyclin D1 protein levels than 0 mg·L~(-1)( P < 0. 05). Indexes within the concentration range between 20-200 mg·L~(-1)were statistically significant( P < 0. 05). Conclusion: ARPS inhibited the proliferation of renal cell carcinoma cell line GRC-1,and induced cell apoptosis and G_0/G_1 arrest,with the effect of inhibiting the activation of Wnt/β-catenin signaling.
引文
[1]Tan X,Liu Y,Hou J,et al.Targeted therapies for renal cell carcinoma in Chinese patients:focus on everolimus[J].Onco Targets Ther,2015,8:313-321.
[2]种铁,牛建强,王子明,等.苦参碱抑制人肾癌细胞系GRC-1细胞株增殖和促凋亡的实验研究[J].中西医结合学报,2006,4(4):388-391.
[3]张晓莉,赵富生,武庚,等.红花多糖对小鼠Lewis肺癌移植瘤生长及转移的影响[J].国际免疫学杂志,2010,33(6):486-489.
[4]李珊珊,金银萍,姚春林,等.人参多糖的结构与活性研究进展[J].中国中药杂志,2014,39(24):4709-4715.
[5]王景春,刘蔚,杨瑞玲,等.川芎多糖对人肝癌细胞Hep G2增殖及凋亡的影响[J].南京中医药大学学报,2014,30(5):461-464.
[6]Huang C F,Yang R S,Liu S H,et al.Evidence for improved neuropharmacological efficacy and decreased neurotoxicity in mice with traditional processing of Arisaematis Rhizoma[J].Am J Chin Med,2011,39(5):981-998.
[7]杨宗辉,尹建元,魏征人,等.天南星提取物诱导人肝癌SMMC-7721细胞凋亡及其机制的实验研究[J].中国老年学杂志,2007,27(2):142-144.
[8]Chen G,Xu J,Miao X,et al.Characterization and antitumor activities of the water-soluble polysaccharide from Arisaematis Rhizoma[J].Carbohydr Polym,2012,90(1):67-72.
[9]姜爽,李建睿,苑广信,等.天南星多糖对荷瘤小鼠的抗肿瘤活性[J].中国老年学杂志,2014,34(18):5183-5184.
[10]Kuo H S,Hsu F N,Chiang M C,et al.The role of Cdk5 in retinoic acid-induced apoptosis of cervical cancer cell line[J].Chin J Physiol,2009,52(1):23-30.
[11]赵陈琛,顾康生.PI3K/Akt/m TOR在表阿霉素抑制Jurkat细胞增殖和诱导凋亡中的作用[J].临床肿瘤学杂志,2012,17(9):780-784.
[12]Zhao B,Li L,Liu J,et al.Exposure to perfluorooctane sulfonate in utero reduces testosterone production in rat fetal Leydig cells[J].PLo S One,2014,9(1):e78888.
[13]Stewart D J,Chang D W,Ye Y,et al.Wnt signaling pathway pharmacogenetics in non-small cell lung cancer[J].Pharmacogenomics J,2014,14(6):509-22.
[14]牛志宏,丁森泰,高德轩,等.肾癌组织WISP-1表达及其临床意义的探讨[J].中华肿瘤防治杂志,2009,16(16):1238-1241.
[2]种铁,牛建强,王子明,等.苦参碱抑制人肾癌细胞系GRC-1细胞株增殖和促凋亡的实验研究[J].中西医结合学报,2006,4(4):388-391.
[3]张晓莉,赵富生,武庚,等.红花多糖对小鼠Lewis肺癌移植瘤生长及转移的影响[J].国际免疫学杂志,2010,33(6):486-489.
[4]李珊珊,金银萍,姚春林,等.人参多糖的结构与活性研究进展[J].中国中药杂志,2014,39(24):4709-4715.
[5]王景春,刘蔚,杨瑞玲,等.川芎多糖对人肝癌细胞Hep G2增殖及凋亡的影响[J].南京中医药大学学报,2014,30(5):461-464.
[6]Huang C F,Yang R S,Liu S H,et al.Evidence for improved neuropharmacological efficacy and decreased neurotoxicity in mice with traditional processing of Arisaematis Rhizoma[J].Am J Chin Med,2011,39(5):981-998.
[7]杨宗辉,尹建元,魏征人,等.天南星提取物诱导人肝癌SMMC-7721细胞凋亡及其机制的实验研究[J].中国老年学杂志,2007,27(2):142-144.
[8]Chen G,Xu J,Miao X,et al.Characterization and antitumor activities of the water-soluble polysaccharide from Arisaematis Rhizoma[J].Carbohydr Polym,2012,90(1):67-72.
[9]姜爽,李建睿,苑广信,等.天南星多糖对荷瘤小鼠的抗肿瘤活性[J].中国老年学杂志,2014,34(18):5183-5184.
[10]Kuo H S,Hsu F N,Chiang M C,et al.The role of Cdk5 in retinoic acid-induced apoptosis of cervical cancer cell line[J].Chin J Physiol,2009,52(1):23-30.
[11]赵陈琛,顾康生.PI3K/Akt/m TOR在表阿霉素抑制Jurkat细胞增殖和诱导凋亡中的作用[J].临床肿瘤学杂志,2012,17(9):780-784.
[12]Zhao B,Li L,Liu J,et al.Exposure to perfluorooctane sulfonate in utero reduces testosterone production in rat fetal Leydig cells[J].PLo S One,2014,9(1):e78888.
[13]Stewart D J,Chang D W,Ye Y,et al.Wnt signaling pathway pharmacogenetics in non-small cell lung cancer[J].Pharmacogenomics J,2014,14(6):509-22.
[14]牛志宏,丁森泰,高德轩,等.肾癌组织WISP-1表达及其临床意义的探讨[J].中华肿瘤防治杂志,2009,16(16):1238-1241.