Jiang Yong-Guang, Luo Yong, He Da-lin, Li Xiang, Zhang Lin-lin, Peng Tao, Li Ming-Chuan, Lin Yun-Hua
Department of Urology, Affiliated Beijing Anzhen Hospital of Capital Medical University, Anzhenli Street, Chaoyang District, Beijing, China.
Int J Urol. 2007 Nov;14(11):1034-9. doi: 10.1111/j.1442-2042.2007.01866.x.
Epithelial-mesenchymal transition (EMT) is an important process in tumor development, and several studies suggest that the Wnt/beta-catenin signal pathway may play an important role in EMT. However, there is no direct evidence showing that the Wnt/beta-catenin pathway actually determines the EMT induced by an exogenous signal. Our previous research has successfully proved that overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) could induce EMT in LNCaP cells, but not in PC-3. The present study aims to determine whether the signal of HIF-1alpha for inducing prostate cancer cells to undergo EMT might possibly pass through the Wnt/beta-catenin pathway.
Epithelial-mesenchymal transition associated proteins were detected in several human prostate carcinoma cell lines by Western blot, and then we distinguished the EMT positive cell lines from the EMT negative cell lines. Furthermore, we evaluated the possible correlation between potency of invasiveness and proliferation among these cell lines with different characteristics of EMT using Matrigel transwell and thiazolyl blue tetrazolium bromide (MTT) assays. Finally, the different expression of some critical proteins and genes in Wnt/beta-catenin signaling pathway were analyzed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) in these cells with different characteristics of EMT.
Among several prostate cancer cell lines, PC-3, LNCaP and PC-3/HIF-1alpha are EMT negative cell lines, whereas LNCaP/HIF-1alpha and IA8 have undergone the EMT process. EMT positive cells (LNCaP/HIF-1alpha and IA8) exhibit much stronger potency of invasiveness and proliferation than those of PC-3 and LNCaP, which belong to EMT negative cells. Interestingly, although PC-3/HIF-1alpha had not completed the EMT process, it still displayed stronger potency of invasion and proliferation, resembling EMT positive cells. The protein expression level of total glycogensynthase kinase 3beta (GSK-3beta) and phospho-GSK-3beta in LNCaP/HIF-1alpha, IA8 and PC-3/HIF-1alpha cells significantly decreased; however, the relative ratios of p-GSK3beta/t-GSK3beta in LNCaP/HIF-1alpha, IA8 and PC-3/HIF-1alpha cells were significantly higher than PC-3 and LNCaP. Consistently, beta-catenin protein expression increased in LNCaP/HIF-1alpha and IA8 cells, but not in PC-3/HIF-1alpha; RT-PCR confirmed these results, except for the enhanced transcription activity of beta-catenin mRNA in PC-3/HIF-1alpha.
Our data suggests that activation of the Wnt/beta-catenin signaling pathway correlates with the characteristic of EMT and potency of invasiveness and proliferation. This may be the critical factor that directly controls the process of EMT induced by HIF-1alpha in prostate cancer cells.
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Wnt/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Wnt/β-连环蛋白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缺氧诱导因子-1α（HIF-1α）的过表达可诱导LNCaP细胞发生EMT，但不能诱导PC-3细胞发生EMT。本研究旨在确定HIF-1α诱导前列腺癌细胞发生EMT的信号是否可能通过Wnt/β-连环蛋白通路传递。
通过蛋白质免疫印迹法检测几种人前列腺癌细胞系中上皮-间质转化相关蛋白，然后区分EMT阳性细胞系和EMT阴性细胞系。此外，我们使用基质胶Transwell和噻唑蓝四氮唑溴盐（MTT）试验评估这些具有不同EMT特征的细胞系之间侵袭力和增殖能力的可能相关性。最后，通过蛋白质免疫印迹法和逆转录-聚合酶链反应（RT-PCR）分析这些具有不同EMT特征的细胞中Wnt/β-连环蛋白信号通路中一些关键蛋白和基因的表达差异。
在几种前列腺癌细胞系中，PC-3、LNCaP和PC-3/HIF-1α是EMT阴性细胞系，而LNCaP/HIF-1α和IA8经历了EMT过程。EMT阳性细胞（LNCaP/HIF-1α和IA8）的侵袭力和增殖能力比属于EMT阴性细胞的PC-3和LNCaP强得多。有趣的是，尽管PC-3/HIF-1α尚未完成EMT过程，但它仍表现出较强的侵袭和增殖能力，类似于EMT阳性细胞。LNCaP/HIF-1α、IA8和PC-3/HIF-1α细胞中总糖原合酶激酶3β（GSK-3β）和磷酸化GSK-3β的蛋白表达水平显著降低；然而，LNCaP/HIF-1α、IA8和PC-3/HIF-1α细胞中p-GSK3β/t-GSK3β的相对比值显著高于PC-3和LNCaP。一致地，LNCaP/HIF-1α和IA8细胞中β-连环蛋白蛋白表达增加，但PC-3/HIF-1α细胞中未增加；RT-PCR证实了这些结果，但PC-3/HIF-1α中β-连环蛋白mRNA的转录活性增强除外。
我们的数据表明Wnt/β-连环蛋白信号通路的激活与EMT特征以及侵袭力和增殖能力相关。这可能是直接控制前列腺癌细胞中HIF-1α诱导的EMT过程的关键因素。
