双硫仑对口腔鳞癌细胞上皮-间充质转化的影响及机制探讨

Effect of disulfiram on epithelial-mesenchymal transformation of oral squamous cell carcinoma cells

目的:基于Smad4突变状态探讨双硫仑对口腔鳞癌(oral squamous cell carcinoma,OSCC)细胞上皮-间充质转化(epithelial-mesenchymal transformation,EMT)的影响。方法:将2017年6月-2018年6月在安徽医科大学附属口腔医院进行肿瘤切除术的46例OSCC患者手术切除的癌组织和癌旁组织切片纳入研究,同时购置人舌鳞状细胞癌细胞系SCC-25和CAL-27。采用免疫组织化学染色观察组织中Smad4的表达情况,Western免疫印迹测定细胞中Smad4的表达情况,分析双硫仑对TGF-β1诱导的细胞EMT迁移、侵袭、形态学以及p38、JNK和ERK表达的影响。采用SPSS 20.0软件包对数据进行统计分析。结果:癌组织中Smad4阳性表达率显著低于癌旁组织(P<0.05)。双硫仑浓度为5、10、20μmol/L时,SCC-25和CAL-27细胞存活率与对照组相比无显著差异(P>0.05),双硫仑浓度≥30μmol/L后,SCC-25和CAL-27细胞存活率显著小于对照组(P<0.05)。经TGF-β1处理后,SCC-25和CAL-27细胞形态由上皮样转变为间质样,上皮性钙黏附蛋白(E-cadherin)表达显著降低,波形蛋白(Vimentin)和神经性钙黏附蛋白(Snail)表达显著升高,迁移能力显著增强(P<0.05)。经双硫仑+TGF-β1处理后,随着双硫仑浓度上升,SCC-25和CAL-27细胞形态改变逐渐减小,E-cadherin蛋白表达逐渐升高,Vimentin和Snail蛋白表达逐渐降低,迁移能力逐渐减弱(P<0.05)。TGF-β1刺激后5 min后,SCC-25和CAL-27细胞中p-ERK水平逐渐上升,在15 min时达到最大值,随后逐渐降低(P<0.05)。经20μmol/L双硫仑+TGF-β1处理后,SCC-25和CAL-27细胞中p-ERK水平随作用时间延长而逐渐下降(P<0.05)。结论:双硫仑可通过阻断MAPK信号通路中ERK磷酸化,达到抑制Smad4突变与Smad4非突变OSCC细胞EMT作用。

PURPOSE: To investigate the effect of disulfiram on epithelial-mesenchymal transformation(EMT) in oral squamous cell carcinoma(OSCC) cells based on Smad4 mutation. METHODS: The cancer tissues and adjacent normal tissues of 46 patients with OSCC who underwent tumor resection in the Affiliated Stomatological Hospital of Anhui Medical University from June 2017 to June 2018 were included in the study. Immunohistochemical staining was used to observe the expression of Smad4 in tissues, Western blot was used to determine the expression of Smad4 in tumor cells.The effects of disulfiram on TGF-β1-induced cell EMT migration, invasion, morphology and expression of p38, JNK and ERK were analyzed. SPSS 20.0 software package was used to analyze the data. RESULTS: The positive expression rate of Smad4 in cancer tissues was significantly lower than that in adjacent normal tissues(P<0.05). The survival rates of human tongue cancer SCC-25 and CAL-27 cells were not significantly different from those of the control group at disulfiram concentrations of 5, 10, and 20 μmol/L(P>0.05). The survival rate of human tongue cancer SCC-25 and CAL-27 cells was significantly lower than that of the control group at disulfiram concentrations ≥30 μmol/L(P<0.05). After treatment with TGF-β1, the morphology of SCC-25 and CAL-27 cells changed from epithelial to mesenchymal. E-cadherin expression was significantly reduced, Vimentin and Snail expression were significantly increased, and migration and invasion were enhanced(P<0.05). After disulfiram + TGF-β1 treatment, as the concentration of disulfiram increased, the morphological changes of SCC-25 and CAL-27 cells gradually decreased, the expression of E-cadherin protein gradually increased, the expression of Vimentin and Snail protein gradually decreased, and migration ability gradually weakened(P<0.05). After 5 minutes of TGF-β1 stimulation, p-ERK levels in SCC-25 and CAL-27 cells gradually increased, reached a maximum at 15 minutes, and then gradually decreased(P<0.05). After 20 μmol/L disulfiram + TGF-β1 treatment, p-ERK levels in SCC-25 and CAL-27 cells gradually decreased with the increase of the treatment time(P<0.05). CONCLUSIONS:Disulfiram can inhibit EMT of Smad4 mutant and Smad4 non-mutated OSCC cells by blocking ERK phosphorylation in the MAPK signaling pathway.