头颈部鳞状细胞癌瞬时受体电位M7通道蛋白与肿瘤细胞增生的关系

Transient receptor potential melastain 7 channel protein in human head and neck carcinoma cells and role in cell proliferation

目的探测头颈鳞状细胞癌（简称鳞癌）细胞中可能存在的离子通道蛋白瞬时受体电位通道M7（transient receptor potential melastatin 7，TRPM7），了解离子通道与肿瘤细胞生长和增生的关系。方法运用免疫组化方法对人类头颈鳞癌SCC-25细胞株进行TRPM7抗体检测，结合被检测到的离子通道蛋白，运用非特异性离子通道阻断剂钆离子（Gd^3＋）和2-氨基乙氧基苯硼酸（2-aminoethoxydiphenyl borate，2-APB）阻断离子通道，并运用特异性TRPM7-siRNA抑制通道的表达，以了解该通道与细胞生长和增生的关系。细胞数量的评估选用乳酸脱氢酶实验。结果所有被检测的SCC-25细胞均对1：100稀释比例的TRPM7抗体呈阳性反应，阴性对照组未能检测到免疫反应。非特异性离子通道阻断剂Gd^3＋对SCC-25细胞生长具有明显的抑制作用，10μmol／L Gd^3＋（n=16）及100μmol／LGd^3＋（n=16）均可显著抑制SCC-25细胞的生长，与不加药的空白对照组比较差异有统计学意义（t值分别为4．1414和6．2661，P值分别为0．0256和0．0082）。离子通道阻断剂2-APB对SCC-25细胞生长的抑制作用十分强大，100μmol／L2-APB（n=16）几乎使SCC-25细胞生长完全停止，与不加药的空白对照组比较差异有统计学意义（t=13．4493，P=0．0008）。特异性TRPM7-siRNA能明显抑制SCC-25细胞的生长，在TRPM7-siRNA的终浓度为30nmol／L时，细胞乳酸脱氢酶释放实验结果显示TRPM7-siRNA能显著降低SCC-25细胞的生长速率（t=4．3446，P=0．0002，n=32），而阴性对照siRNA则不影响细胞的生长。结论人类头颈鳞癌SCC-25细胞株中存在TRPM7通道；运用离子通道阻断剂或运用特异性TRPM7-siRNA抑制该通道的表达均能明显抑制肿瘤细胞的生长；2-APB对人类头颈鳞癌细胞生长的强大抑制作用表明TRPM7通道蛋白将来可能成为头颈鳞癌治疗的重要靶点之一。

Objective To detect the presence of ion channel protein and its role in cell growth and proliferation in human head and neck squamous carcinoma cells （SCC）. Methods Human head and neck squamous carcinoma SCC-25 cell line was tasted with transient receptor potential melastatin 7 （ TRPM7 ） antibody using the method of immunocytochemistry. The role of TRPM7 in cell growth and proliferation was evaluated through its blockade by ion channel blockers and specific siRNA using lactate dehydrogenase （LDH） assay technique. Results Clear immunoreactivity against TRPM7 was detected in almost all SCC-25 cells tested, whereas no immunoreactivity was observed in negative control. The inhibitory effect of Gd^3＋, a non-specific ion channel blocker, on cell growth and proliferation was potent. Addition of 10 μmol/L Gd^3＋ （n = 16） and 100 μmol/L Gd^3＋ （n = 16） in the culture medium significantly inhibited the growth of SCC-25 cells, as compared with control cells growing in normal medium （ t was 4. 1414 and 6. 2661, P was 0. 0256 and 0. 0082 respectively ）. However, the effect of 2-APB was striking. Cell proliferation was almost totally suppressed in the presence of 100 μmol/L 2-APB（ t = 13.4493 ,P = 0. 0008, n = 16 ）compared with cells growing in normal medium. Suppression of TRPM7 expression by siRNA also significantly inhibited the growth and proliferation of these cells （t = 4. 3446, P = 0. 0002, n = 32, compared with nontransfected cells） ,whereas cells transfected with negative control siRNA showed no difference in cell proliferation compared with nontransfected cells. Conclusions All of those results strongly suggest the existence of TRPM7 channel in human head and neck squamous carcinoma cells. Ion channel blockers serve as a potent inhibitor of SCC-25 cell proliferation. The striking inhibitory effect of 2-APB on cell growth and proliferation may promise clinical workers an inspiring remedy for fighting against carcinoma.