Bufalin抑制食管癌TE13细胞迁移机制探讨  

Mechanism of Bufalin in inhibiting the migration of TE13 esophageal cancer cells

作　　者：张玲玲[1] 韩金薇[1] 王晓琳[1] 王小玲[1] 刘月平[1] 

机构地区：[1]河北医科大学第四医院病理科,河北石家庄050011

出　　处：《中华肿瘤防治杂志》2016年第13期837-843,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金青年基金(81303271)

摘　　要：目的蟾蜍灵(Bufalin)是中医抗癌药物蟾酥的有效成分之一,但其抗肿瘤的机制并不十分清楚。本研究通过分析不同浓度Bufalin对食管癌TE13细胞株迁移距离、MEK1/2及其活化形式P-MEK1/2和基质金属蛋白酶(matrix metalloproteinase,MMP)表达的影响,初步探讨Bufalin抑制食管癌TE13细胞迁移能力的作用机制。方法采用四甲基偶氮唑蓝[3-(4,5-dimethylthiazol-yl)-2,5-diphenyl tetrazolium bromide,MTT]法测定细胞药物毒性,细胞划痕实验分别测量不同浓度(0、10、25、50和100nmol/L)Bufalin处理组食管癌TE13细胞的迁移距离。蛋白质印迹法及免疫细胞化学法检测MEK1/2和P-MEK1/2蛋白的表达。逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测MMP-2、MMP-9mRNA的表达情况。结果 MTT法结果显示,Bufalin药物最大无毒浓度(TC0)为100nmol/L。细胞划痕实验结果显示,不同浓度Bufalin处理组在划痕36h后与对照组相比迁移的距离不同,随着Bufalin浓度的增加,细胞迁移距离下降,100nmol/L迁移距离最小(F=243.163,P<0.01),Bufalin能有效抑制食管癌细胞迁移距离。蛋白质印迹法及免疫细胞化学检测结果显示,Bufalin对MEK1/2蛋白的表达无影响,但能显著抑制其活化形式P-MEK1/2的表达,并呈剂量依赖性,随Bufalin浓度的升高(0、10、25、50和100nmol/L),P-MEK1/2相对表达量逐渐下降,分别为0.710±0.006、0.676±0.010、0.656±0.010、0.599±0.020和0.521±0.021,F=76.369,P<0.01。P-MEK1/2阳性细胞数随着Bufalin浓度的升高而减少,分别为(80.330±1.905)%、(68.407±2.219)%、(67.297±1.797)%、(52.617±2.368)%和(26.97±2.912)%,F=243.348,P<0.01。RT-PCR结果显示,随着Bufalin药物浓度的升高,MMP-2mRNA相对表达量分别为0.772±0.010、0.725±0.019、0.663±0.015、0.582±0.019和0.519±0.019,F=112.990,P<0.01;MMP-9mRNA相对表达量分别为0.783±0.013、0.740±0.010、0.703±0.006、0.601±0.017和0.531±0.010,均低于对照组,F=179.417,P<0.01。结论] OBJECTIVE Bufalin is one of the active ingredients of toad venom, however, its anti-tumor mechanisms are still unclear. The objective of this study was to test the expression of MEK1/2,P-MEK1/2,MMPs and migration dis- tance in human esophageal carcinoma cell lines TEl3 by different concentrations of Bufalin, and investigate the potential mechanism of Bufalin in affacting the activation of MEK and the migration. METHODS Cell drug toxicity was measured by using MTT assay. The esophageal carcinoma TEl3 cells were divided into Bufalin 0 nmol/L group, Bufalin 10 nmol/L group, Bufalin 25 nmol/L group, Bufalin 50 nmol/L group and Bufalin 100 nmol/L group. The migratory range was measured in different groups through wound healing assay. Western Blot and immunocytochemistry were performed to de- tect the expression quantities of MEK and P MEK. The MMP-2 and MMP-9 mRNA expression levels were detected by RT-PCR. RESULTS MTT assay showed that the TC0 of Bufalin was 100 nmol/L. According to results of Wound heal ing assay： after scratched 36 h, the cell migration distance in Bufalin 10 nmol/L group, Bufalin 25 nmol/L group, Bufalin 50 nmol/L group and Bufalin 100 nmol/L group were significantly shorter than that in control group. The effect of inhibi- tion was markedly clearer in the group of using Bufalin lOOnmol/L（F=243.163, P〈0. 01）. Western Blot and immuno- cytochemistry method results showed that compared with the control group （0. 710±0. 006）, treatment with Bufalin significantly decreased the level of P-MEK1/2 protein （0. 676i0. 010, 0. 656±0. 010, 0. 599±0. 020, 0. 521±0. 021）, F=76. 369,P〈0.01. The number of positive cells which in Bufalin groups was gradually fewer than that in control group, which were （80. 330±1. 905）%, （68. 407!2. 219）% （67. 297±1. 797）%, （52. 617±2. 368）% and （26.97±2. 912）% respectively, F=243. 348,P〈0.01. RT-PCR results showed that Bufalin suppressed the nuclear translocation of MMP-2, MMP-9 in TEl3. The exepression levels of MMP-2 mRNA and MMP

关 键 词：食管肿瘤 蟾蜍灵 MEK1/2 基质金属蛋白酶2 基质金属蛋白酶9 细胞迁移 

分 类 号：R735.1[医药卫生—肿瘤]