中华眼镜蛇毒膜毒素-12对膀胱癌细胞侵袭、转移的抑制作用及机制

Inhibitory effects of cobra venom membrane toxin 12 on invasion and metastasis of bladder cancer cells

目的观察眼镜蛇毒膜毒素12(MT-12)对膀胱癌T24、RT4细胞侵袭、转移能力的影响,探讨MT-12抑制膀胱癌细胞侵袭、转移的机制。方法取T24、RT4细胞,设置空白对照组(Ctrl组)、溶媒对照组(NC组)、0. 25μg/m L组、0. 50μg/m L MT-12组,分别应用PBS、0. 1%DMSO、0. 25μg/m L MT-12、0. 50μg/m L MT-12处理细胞24h,采用细胞划痕实验测算细胞迁移抑制率以评价细胞迁移能力,采用细胞黏附实验测算细胞黏附率以评价细胞黏附能力。设置空白对照组(Ctrl组)、MT-12组、促瘤剂佛波酯(PMA)组、PMA+MT-12组,分别应用PBS、0. 50μg/m L MT-12、100 nmol/L PMA、0. 50μg/m L MT-12+100 nmol/L PMA干预细胞24 h,采用凝胶酶谱分析法检测细胞基质金属蛋白酶(MMP)-2、MMP-9酶活性含量,采用RT-PCR法检测细胞侵袭转移相关因子细胞间黏附因子-1(ICAM-1)、血管黏附因子-1(VCAM-1)、血管生成因子(VEGF) mRNA表达,采用ELISA法检测细胞VEGF蛋白表达。结果 T24和RT4两株细胞中,0. 25μg/m L MT-12组、0. 50μg/m L MT-12组细胞迁移抑制率、细胞黏附率均低于NC组(P均<0. 05)。T24和RT4两株细胞中,MT-12组MMP-9酶活性均低于Ctrl组(P均<0. 05),PMA组MMP-9酶活性均高于Ctrl组,PMA+MT-12组MMP-9酶活性均低于PMA组(P均<0. 05); MT-12组ICAM-1及VEGF mRNA表达均低于Ctrl组(P均<0. 05),PMA+MT-12组ICAM-1、VEGF mRNA表达均低于PMA组(P均<0. 05); PMA组VEGF蛋白表达均高于Ctrl组,PMA+MT-12组VEGF蛋白表达均低于PMA组(P <0. 05)。结论MT-12可有效抑制膀胱癌细胞T24、RT4的迁移及转移能力,其分子机制与抑制MMP-9、VEGF、ICAM-1表达有关。

Objective To observe the effects of cobra venom membrane toxin12(MT-12)on the invasion and metastasis of bladder cancer cell RT4and T24,and to investigate the mechanism of MT-12inhibiting the invasion and metastasis of tumor cells.Methods T24and RT4cells cultured in vitro were divided into four group:the control group(Ctrl group),solvent control group(NC group),0.25μg/mL group,and 0.50μg/mL group,which were separately cultured in the media with PBS,0.1% DMSO,0.25μg/mL MT-12,and 0.50μg/mL MT-12 for 24h.The cell migration ability and cell migration inhibition rate were tested by cell scratch test,and the cell adhesion ability and cell adhesion inhibition rate were tested by the cell adhesion experiment.Then T24and RT4cells were divided into four group:the control group(Ctrl group),MT-12group,tumor-promoter PMA group,and MT-12+PMA group,which were separately cultured in the media with PBS,0.50μg/mL MT-12,100nmol/L PMA,and0.50μg/mL MT-12+100nmol/L PMA for 24h.The content of matrix metalloproteinases(MMP-2)and MMP-9active enzyme was detected by gel enzyme spectrum analysis.The mRNA expression of intercellular adhesion factor-1(ICAM-1),vascular adhesion factor-1(VCAM-1),and angiogenesis factor(VEGF)was detected by reverse transcription-polymerase chain reaction(rt-PCR),and the VEGF protein expression was detected by enzyme-linked immunosorbent assay(ELISA).Results In both RT4and T24 cells,the cell migration inhibition rate and cell adhesion rate of the 0.25μg/mL MT-12 group and 0.50μg/mL MT-12 group were lower than those in the NC group(both P<0.05).In both RT4 and T24 cells,MMP-9 enzyme activity in the MT-12 group was lower than that in the Ctrl group(P<0.05),MMP-9enzyme activity in the PMA group was higher than that in the Ctrl group,and MMP-9 enzyme activity in the MT-12+PMA group was lower than that in the PMA group(P<0.05).The mRNA expression levels of ICAM-1and VEGF in the MT-12 group were lower than those in the Ctrl group(both P<0.05),and the mRNA expression levels of ICAM-1and VEGF in the PMA+MT12 group were lower than those in the PMA group(both P<0.05).VEGF protein expression in the PMA group was higher than that in the Ctrl group,and VEGF protein expression in the MT-12+PMA group was lower than that in the PMA group(all P<0.05).Conclusion Mt-12 can effectively inhibit the migration and adhesion of bladder cancer cells T24 and RT4,and its molecular mechanism is related to the inhibition of MMP-9,VEGF,and ICAM-1.