nm23-H1通过下调PKC信号通路抑制肺癌细胞侵袭

nm23-H1 gene inhibits lung cancer cell invasion through down-regulation of PKC signal pathway

目的探讨nm23-H1基因调控肺癌细胞PKC信号通路抑制肺癌侵袭和转移的分子机制。方法应用Western-blot、Boyden-Chamber、MTT法和激光扫描共聚焦显微镜技术分别检测nm23- H1基因转染前后以及PKC抑制剂Calphostin C处理前后人高转移大细胞肺癌细胞株L9981中胞膜、胞浆PKC的分布变化;体外侵袭力和增殖力的变化;以及细胞中PKC激活转位变化和Ca2+浓度的变化。结果 (1)L9981和L9981-pLXSN细胞中PKCα、PKCβⅡ蛋白在胞膜的表达量明显较L9981- nm23-H1增多(P<0．001),表明其处于活化状态的PKC明显增加;(2)PKCα、PKCβⅡ在L9981及 L9981-pLXSN细胞中主要位于胞核、核周和质膜,明显处于激活转位状态;在L9981-nm23-H1细胞中则主要位于胞浆内,处于无活性状态,前二者胞浆中Ca2+荧光表达强度显著高于后者(P<0．001); (3)L9981-nm23-H1体外增殖力、侵袭力显著低于L9981和L9981-pLXSN(P<0．001),但后二者间比较差异无统计学意义(P>0．05);(4)抑制剂处理后,L9981和L9981-pLXSN细胞中PKCα、PKCβⅡ在胞膜的蛋白表达量和胞浆中的Ca2+荧光表达强度均较处理前明显下降(P<0．001);3株细胞体外增殖力、侵袭力均较处理前明显下降(P<0．001)。结论nm23-H1基因可能通过PKC信号通路来发挥其肿瘤转移抑制作用,细胞内钙离子可能参与了这一过程。

Objective To study the molecular mechanisms of nm23-H1 for regulating PKC signal pathway before and after transfection with nm23-H1 gene. Methods Using Western-blot,Boyden-chamber, MTT and laser scanning confocal microscopy （ LSCM ） techniques to detect the distribution of PKC in cytosol and plasma membrane, changes of invasion and proliferation activity, PKC translocation status and changes of intracellular Ca^2＋ concentration among different human pulmonary carcinoma cells with transfected or untransfected nm23-Hl gene, and changes of the three cell lines after treatment with Calphostin C, a PKC inhibitor. Results （1） The expression of PKCα, PKCβ Ⅱ on L9981 and L9981-pLXSN cell membrane, which was in activated status, was remarkably higher than those in L9981-nm23-H1 cell line （P 〈0.001 ）. The expression of PKCα. PKCβ Ⅱ in cytoslol in L9981 and L9981-pLXSN cell lines, which was in inactivated status, was lower than those in L9981-nm23-H1 cell line （P 〈0.001 ）. It means that the PKC signal pathway was activated in L9981 and L9981-pLXSN cell lines. （2） PKCα and PKCβ Ⅱ mainly located in nuclei and perinuclear area in L9981 and L9981-pLXSN cells, which were in active status, and the Ca^2＋ concentration in these cells was obviously higher than that in L9981-nm23-H1 cell line （P 〈 0. 01 ）. In L9981-nm23-H1 cell line, which was transfected with nm23-H1 gene, PKCα and PKCβ Ⅱ mainly located in soluble cytosolic section, in an inactive status. （3） The invasion and proliferation ability of L9981 and L9981 -pLXSN lung cancer cells was higher than that of L9981 -nm23-H1 cell line （ P 〈 0. 001 ）. There was no statistically significant difference beteween L9981 and L9981-pLXSN cell lines（ P 〉 0. 05 ）. （4） After treated with PKC inhibitor Calphstin C, the expression of PKC and PKCβ Ⅱ in membrane in L9981 and L9981-pLXSN cell lines was down-regulated （P〈0.001）.PKCα and PKCβ Ⅱ were mainly located in cytosolie area, mainly in an inactive status,and the Ca^2＋ concentration was found to be decreased in all the three cell lines.The invasion and proliferation ability of the three lung cancer cell lines were lbviously decreasing （P〈0.001）.However,the invasion and proliferation ability of L9981-nm23-H1 lung cancer cell line was still lower than that of L9981 and L9981-pLXSN cell lines（P〉0.05）.Conclusion The results of this study suggest that nm23-H1 gene might inbibit the invasion and metastasis of lung cancer cells by down-regulating PKC signaling pathway.The Ca^＋ in cells might be involved in this process.