Nie Qiang, Zhou Qing-hua, Zhu Wen, Liu Lun-xu, Fu Jun-ke, Li Ding-biao, Li Yin, Che Guo-wei
Cancer Center, Guangdong Provincial People's Hospital, Guangzhou 510080, China.
Zhonghua Zhong Liu Za Zhi. 2006 May;28(5):334-6.
To study the molecular mechanisms of nm23-H1 for regulating PKC signal pathway before and after transfection with nm23-H1 gene.
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-H1 gene, and changes of the three cell lines after treatment with Calphostin C, a PKC inhibitor.
(1) The expression of PKCalpha, PKCbeta 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 PKCalpha, PKCbeta II in cytosol 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) PKCalpha and PKCbeta 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, PKCalpha and PKCbeta 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 between L9981 and L9981-pLXSN cell lines (P > 0.05). (4) After treated with PKC inhibitor Calphstin C, the expression of PKC and PKCbeta II in membrane in L9981 and L9981-pLXSN cell lines was down-regulated (P < 0.001), PKCalpha and PKCbeta II were mainly located in cytosolic 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 obviously 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 lung cancer cell lines (P < 0.001). There was also no significant difference between L9981 and L9981-pLXSN cell lines (P > 0.05).
The results of this study suggest that nm23-H1 gene might inhibit the invasion and metastasis of lung cancer cells by down-regulating PKC signaling pathway. The Ca(2+) in cells might be involved in this process.
研究nm23-H1基因转染前后调控PKC信号通路的分子机制。
采用蛋白质免疫印迹法（Western-blot）、小室侵袭实验（Boyden-chamber）、MTT法及激光扫描共聚焦显微镜（LSCM）技术，检测转染或未转染nm23-H1基因的不同人肺癌细胞中PKC在胞浆和质膜的分布、侵袭与增殖活性变化、PKC转位状态及细胞内Ca(2+)浓度变化，以及用PKC抑制剂Calphostin C处理后三种细胞系的变化。
（1）L9981和L9981-pLXSN细胞膜上处于激活状态的PKCalpha、PKCbeta II表达明显高于L9981-nm23-H1细胞系（P<0.001）。L9981和L9981-pLXSN细胞系胞浆中处于失活状态的PKCalpha、PKCbeta II表达低于L9981-nm23-H1细胞系（P<0.001）。这表明L9981和L9981-pLXSN细胞系中PKC信号通路被激活。（2）处于激活状态的L9981和L9981-pLXSN细胞中，PKCalpha和PKCbeta II主要位于细胞核及核周区域，这些细胞中的Ca(2+)浓度明显高于L9981-nm23-H1细胞系（P<0.01）。在转染了nm23-H1基因的L9981-nm23-H1细胞系中，PKCalpha和PKCbeta II主要位于可溶性胞浆部分，处于失活状态。（3）L9981和L9981-pLXSN肺癌细胞的侵袭和增殖能力高于L9981-nm23-H1细胞系（P<0.001）。L9981和L9981-pLXSN细胞系之间无统计学差异（P>0.05）。（4）用PKC抑制剂Calphstin C处理后，L9981和L9981-pLXSN细胞系膜上PKC和PKCbeta II的表达下调（P<0.001），PKCalpha和PKCbeta II主要位于胞浆区域，主要处于失活状态，且发现三种细胞系中的Ca(2+)浓度均降低。三种肺癌细胞系的侵袭和增殖能力明显下降（P<0.001）。然而，L9981-nm23-H1肺癌细胞系的侵袭和增殖能力仍低于L9981和L9981-pLXSN肺癌细胞系（P<0.001）。L9981和L9981-pLXSN细胞系之间也无显著差异（P>0.05）。
本研究结果提示，nm23-H1基因可能通过下调PKC信号通路抑制肺癌细胞的侵袭和转移。细胞内的Ca(2+)可能参与了这一过程。
