Luo Yong, He Da-lin, Jiang Yong-guang, Ning Liang, Shen Shu-lin, Zhao Jia-hui, Cui Xin-hao
Department of Urology, Affiliated Beijing Anzhen Hospital of Capital Medical University, Beijing 100035, China.
Zhonghua Yi Xue Za Zhi. 2010 Apr 27;90(16):1131-6.
Epithelial-mesenchymal transition (EMT) is an important process in tumor development. Several studies suggest that the beta-catenin signal pathway may play an important role in EMT. However, there is no direct evidence showing that this pathway actually determines the EMT induced by exogenous signal. Our previous study has successfully proved that over-expression of HIF-1alpha could induce EMT in LNCaP cells, but not in PC-3. So the present study was intended to indicate that the signal of HIF-1alpha for inducing prostate cancer cell to undergo EMT might pass through the beta-catenin pathway.
Firstly, we analyzed the expression of HIF-1alpha and its target proteins in LNCaP/HIF1alpha and PC-3/HIF1alpha by Western blot. And then EMT-associated proteins were detected by Western blot. Furthermore the potency of invasiveness and proliferation of several cell lines were evaluated by transwell and MTT assay. Lastly the expressions of beta-catenin and GSK-3beta in these cells were analyzed by Western blot and RT-PCR.
HIF-1alpha, Glut-1 and VEGF were highly expressed in LNCaP/HIF1alpha and PC-3/HIF1alpha. And PC-3, LNCaP and PC-3/HIF1alpha were EMT-negative cell lines whereas LNCaP/HIF1alpha and IA8 had undergone EMT process. LNCaP/HIF1alpha, IA8 and PC-3/HIF1alpha exhibited a much stronger potency of invasiveness and proliferation than those of PC-3 and LNCaP. The protein levels of total GSK-3beta and phospho-GSK-3beta in LNCaP/HIF1alpha, IA8 and PC-3/HIF1alpha cells significantly decreased; however, the relative ratios of p-GSK3beta/t-GSK3beta increased. Consistently, beta-catenin protein expression increased in LNCaP/HIF1alpha and IA8 cells, but not in PC-3/HIF1alpha; RT-PCR confirmed these results, except for an enhanced transcription activity of beta-catenin mRNA in PC-3/HIF1alpha.
The activation of the beta-catenin signaling pathway correlates with the characteristic of EMT and potency of invasiveness and proliferation. And it may be one critical factor directly controlling the process of EMT induced by HIF-1alpha in prostate cancer.
上皮-间质转化（EMT）是肿瘤发展过程中的一个重要过程。多项研究表明，β-连环蛋白信号通路可能在EMT中发挥重要作用。然而，尚无直接证据表明该通路实际上决定了外源性信号诱导的EMT。我们之前的研究已成功证明，HIF-1α的过表达可在LNCaP细胞中诱导EMT，但在PC-3细胞中则不能。因此，本研究旨在表明HIF-1α诱导前列腺癌细胞发生EMT的信号可能通过β-连环蛋白通路传递。
首先，我们通过蛋白质免疫印迹法分析LNCaP/HIF1α和PC-3/HIF1α中HIF-1α及其靶蛋白的表达。然后通过蛋白质免疫印迹法检测EMT相关蛋白。此外，通过Transwell实验和MTT法评估几种细胞系的侵袭和增殖能力。最后，通过蛋白质免疫印迹法和逆转录-聚合酶链反应（RT-PCR）分析这些细胞中β-连环蛋白和糖原合成酶激酶-3β（GSK-3β）的表达。
HIF-1α、葡萄糖转运蛋白-1（Glut-1）和血管内皮生长因子（VEGF）在LNCaP/HIF1α和PC-3/HIF1α中高表达。PC-3、LNCaP和PC-3/HIF1α是EMT阴性细胞系，而LNCaP/HIF1α和IA8经历了EMT过程。LNCaP/HIF1α、IA8和PC-3/HIF1α表现出比PC-3和LNCaP更强的侵袭和增殖能力。LNCaP/HIF1α、IA8和PC-3/HIF1α细胞中总GSK-3β和磷酸化GSK-3β的蛋白水平显著降低；然而，p-GSK3β/t-GSK3β的相对比值增加。一致地，β-连环蛋白蛋白表达在LNCaP/HIF1α和IA8细胞中增加，但在PC-3/HIF1α中未增加；RT-PCR证实了这些结果，但PC-3/HIF1α中β-连环蛋白mRNA的转录活性增强。
β-连环蛋白信号通路的激活与EMT特征以及侵袭和增殖能力相关。它可能是直接控制前列腺癌中HIF-1α诱导的EMT过程的一个关键因素。
