MMP-9抑制剂对人口腔鳞状细胞癌细胞系CAL27生物学行为影响机制的研究

Study on the mechanism of MMP-9 inhibitor on biological behavior of human oral squamous cell carcinoma cell line CAL27

目的:探究基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)抑制剂(BB-94)对人口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞系CAL27恶性生物学行为的影响及可能的作用机制。方法:用含不同浓度(0、0.125、0.25、0.5、1.0μg/mL)BB-94培养液培养对数生长期CAL27细胞48 h,明胶酶谱实验分析细胞培养上清液中MMP-9的活性;细胞计数试剂盒-8(cell counting kit‐8,CCK-8)实验检测细胞存活率;流式细胞术、伤口愈合实验及Transwell实验检测CAL27细胞凋亡、迁移及侵袭情况;实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)技术检测磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,PKB,又称Akt)mRNA表达水平;蛋白质印迹法(Western blotting)检测PI3K、磷酸化蛋白激酶B(phosphorylated proteinkinaseB,又称p-Akt)、MMP-9、波形蛋白(vimentin,Vim)、神经钙黏素(N-cadherin,N-cad)、上皮钙黏素(E-cadherin,E-cad)表达情况。结果:CAL27细胞MMP-9相对活性、存活率、划痕愈合率、细胞侵袭数、PI3K和Akt mRNA相对表达水平及PI3K、p-Akt、Vim、N-cad、MMP-9蛋白相对表达水平均随BB-94作用浓度增大而降低(P<0.05),CAL27细胞凋亡率、E-cad蛋白相对表达水平均随BB-94作用浓度增大而升高(P<0.05)。结论:MMP-9抑制剂可抑制人口腔鳞状细胞癌细胞系CAL27恶性生物学行为,其作用机制可能与阻止PI3K/Akt信号通路激活及上皮间质转化(epithelial mesenchymal transition,EMT)进展有关。

Objective:To explore the potential of BB-94,a matrix metalloproteinase-9(MMP-9)inhibitor,in alleviating malignant biological behavior of human oral squamous cell carcinoma(OSCC)cell line CAL27 and its possible mechanism.Methods:CAL27 cells in logarithmic growth phase were cultured in BB-94 culture medium with different concentrations(0,0.125,0.25,0.5,1.0µg/mL)for 48 hours;the activity of MMP-9 in cell culture supernatant was analyzed by gelatin zymogram;the cell survival rate was detected by cell counting kit-8(CCK-8)method;the apoptosis,migration,and invasion of CAL27 cells were detected by flow cytometry,wound healing test and Transwell test;real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(Akt);the expressions of PI3K,phosphorylated protein kinase B(p-Akt),MMP-9,vimentin(Vim),N-cadherin(N-cad)and E-cadherin(E-cad)proteins were detected by Western blotting.Results:The relative activity of MMP-9,the survival rate of CAL27 cells,the wound healing rate,the number of cell invasion,the relative expression level of PI3K and Akt mRNA and the relative expression level of PI3K,p-Akt,Vim,N-cad and MMP-9 protein decreased with the increase in the concentration of BB-94(P<0.05).The apoptosis rate of CAL27 cells and the relative expression level of E-cad protein increased as the concentration of BB-94 went up(P<0.05).Conclusion:These results indicate that MMP-9 inhibitor could inhibit the malignant biological behavior of human OSCC cell line CAL27,and its mechanism may be related to preventing the activation of PI3K/Akt signaling pathway and the progress of epithelial mesenchymal transition(EMT).