Huang Chao, Wen Bin
Institute of Pi-Wei, Guangzhou University of Chinese Medicine, Guangzhou, 510000, China.
Mol Biol Rep. 2016 Oct;43(10):1069-78. doi: 10.1007/s11033-016-4038-3. Epub 2016 Jul 11.
Transforming growth factor-β1 (TGF-β1) within tumor microenvironment has a pivotal function in cancer initiation and tumorigenesis, and hence this study was to observe the malignant transformation induced by TGF-β1 in an immortalized colon epithelial cell line NCM460 for better understanding the mechanisms of colon carcinogenesis. Immortalized colon epithelial cell line NCM460 was used as the model of this study, and was treated with different concentrations of TGF-β1 for different time. Then, immunofluorescence was performed to observe the change of phenotype hallmarks including adherent junction protein E-cadherin, cytoskeleton protein vimentin, and tight junction marker ZO-1, western blotting analysis was performed to detect the expression of the above three markers and two transcription factors (Snail and Slug) involved in the transformation by TGF-β1. In addition, chromosome instability (CHI) including analysis of DNA-ploid was detected by flow cytometry. Our results revealed significant loss or reduction of ZO-1 and E-cadherin, and robust emergence of vimentin in the cell line NCM460 after a 15-, 20-, and 25-day treatment with 10 ng/ml TGF-β1. Interestingly, 20 and 25 days after stimulation with 5 ng/ml TGF-β1, expression of E-cadherin and ZO-1 revealed a pattern roughly similar to that of 10 ng/ml TGF-β1, especially, both expressions was vanished and vimentin expression was dramatically increased at days 25 after TGF-β1 stimulation. After a stimulation with 10 ng/ml TGF-β1 for 15, 20, and 25 days, the levels of Snail and Slug expression in the cells were significantly up-regulated, compared with the cells treated with TGF-β1 inhibitor LY364947, PBS or balnk control (P < 0.01). Our results found that many abnormal mitotic patterns including lagging chromosomes and anaphase bridges in NCM460 cells were induced by TGF-β1 after its stimulation for 15, 20, and 25 days. Very few mitotic cells with treatment of PBS for 15, 20 and 25 days were non-diploid whose DNA content was greater or less than 4 N, but these cells were significantly increased after exposure to TGF-β1 for 15, 20, and 25 days, which was associated with the induction of hypo-diploid, hyper-diploid, and poly-diploid (P < 0.05).These data indicate that TGF-β1 induces a phenotypic transformation of normal colon epithelium similar to its pro-tumoral behaviors in TME, involving in alteration of chromosome stability.
肿瘤微环境中的转化生长因子-β1（TGF-β1）在癌症起始和肿瘤发生过程中具有关键作用，因此本研究旨在观察TGF-β1诱导永生化结肠上皮细胞系NCM460发生恶性转化，以更好地理解结肠癌发生的机制。将永生化结肠上皮细胞系NCM460作为本研究的模型，用不同浓度的TGF-β1处理不同时间。然后，进行免疫荧光观察包括黏附连接蛋白E-钙黏蛋白、细胞骨架蛋白波形蛋白和紧密连接标志物ZO-1在内的表型标志物的变化，进行蛋白质免疫印迹分析以检测上述三种标志物以及参与TGF-β1诱导转化的两种转录因子（Snail和Slug）的表达。此外，通过流式细胞术检测包括DNA倍体分析在内的染色体不稳定性（CHI）。我们的结果显示，用10 ng/ml TGF-β1处理15、20和25天后，NCM460细胞系中ZO-1和E-钙黏蛋白显著缺失或减少，波形蛋白大量出现。有趣的是，用5 ng/ml TGF-β1刺激20和25天后，E-钙黏蛋白和ZO-1的表达呈现出与10 ng/ml TGF-β1大致相似的模式，特别是在TGF-β1刺激25天时，两者表达均消失且波形蛋白表达显著增加。用10 ng/ml TGF-β1刺激15、20和25天后，与用TGF-β1抑制剂LY364947、PBS或空白对照处理的细胞相比，细胞中Snail和Slug的表达水平显著上调（P < 0.01）。我们的结果发现，TGF-β1刺激15、20和25天后，NCM460细胞中诱导出许多异常有丝分裂模式，包括滞后染色体和后期桥。用PBS处理15、20和25天的有丝分裂细胞中很少有非二倍体，其DNA含量大于或小于4N，但在暴露于TGF-β1 15、20和25天后，这些细胞显著增加，这与亚二倍体、超二倍体和多倍体的诱导有关（P < 0.05）。这些数据表明，TGF-β1诱导正常结肠上皮发生表型转化，类似于其在肿瘤微环境中的促肿瘤行为，涉及染色体稳定性的改变。
