PKCε經由Ras/Raf/ERK途徑促進人類肺癌細胞生長和轉移

Protein kinase Cε(PKCε)為特異的PKC異構物中的代表成員之一，表示其活化時不需要依賴鈣但是可以藉由phorbol esters、二醯基甘油(diacylglycerol) 以及磷脂醯絲胺酸(phosphatidylserine)活化。PKCε能夠調控許多重要的細胞的功能包含細胞增生、分化和存活。此外，PKCε也促進細胞轉移的能力以及抵擋抗癌治療的能力。在本實驗室先前研究證明過度表現PKCε可能會透過調控survivin的表現進而降低TRAIL所誘發的細胞凋亡。在本實驗中，觀察PKCε在H460人類肺癌細胞中高度表現時對於細胞生長、群落形成造成影響，而在細胞週期的部份我們觀察到，當PKCε被過度表現後會使細胞週期S期百分比上升並且使G1期百分比降低，細胞週期調控分子cyclin D、cyclin E及CDK2蛋白表現增加，而CDK4及CDK6蛋白表現下降；CDK inhibitor的部份則是p16蛋白表現有下降的情形，而p21蛋白表現則有增強的情形。在細胞貼附實驗中發現過度表現PKCε會影響細胞貼附能力，本實驗室利用傷口癒合、爬行和侵襲實驗，發現PKCε的高度表現提高細胞爬行、侵襲和傷口癒合的能力。根據文獻報告，PKCε與許多訊息傳遞路徑有關，包含了Ras/Raf/MAPK和PI-3K/Akt等路徑，而MAPK和Akt分別和調控許多不同的細胞功能有關。我們得到在H460和A549細胞株中高度表現PKCε時K-Ras、Raf-1和pERK的表現量會增加。觀察人類肺癌細胞株的蛋白表現，結果顯示PKCε藉由K-Ras/Raf-1/ERK這條路徑影響細胞功能表現。使用PKCε siRNA或K-Ras siRNA在H460 PKCε細胞會降低細胞中PKCε或K-Ras的表現，結果顯示降低PKCε或K-Ras的表現之後，細胞生長以及細胞爬行能力會降低；使用ERK抑制劑(PD98059)與H460 PKCε細胞作用降低細胞中磷酸化ERK的表現後，細胞生長以及爬行的能力降低；在動物實驗中，我們得到PKCε的高度表現會促進腫瘤轉移。綜合以上結果，在人類肺癌細胞中PKCε會藉由K-Ras/Raf-1/ERK這條路徑影響人類肺癌細胞轉移和生長的能力。

關鍵字

肺癌； PKCε ； Ras/Raf/ERK ；轉移；生長

並列摘要

Protein kinase Cε (PKCε) is a representative member of a family of novel PKC isoforms that are calcium independent, but can be activated by phorbol esters, diacylglycerol and phosphatidylserine. PKCε is capable of modulating crucial cell functions, including proliferation, differentiation and survival. Furthermore, PKCε also promotes tumor metastatic capacity and resistance to anti-cancer therapy. Our previous study demonstrates that overexpression of PKCε decreased TRAIL-induced apoptosis in H460 lung cancer cells via modulation of survivin expression. In this study, we found that overexpression of PKCε could increase cell growth and colony formation in H460 human lung cancer cells. In the cell cycle analysis, we observed that PKCε overexpression would increase the cell population in S phase and decrease cell population in G1 phase.. Expressions of cyclin D, cyclin E and CDK2 were up-regulated; however, CDK4 and CDK6 expressions were down-regulated. About CDK inhibitor, p16 expression was decreased, p21 expression was enhanced in PKCε overexpressing H460 cells. We found out when overexpression of PKCε, it would have negative influence on cell adhesion. In this study, we did the following experiments: wound healing assay, cell migration assay and cell invasion assay. We found out PKCε overexpressed lung cancer cells, it would increase cell migration, invasion and wound healing ability. Several studies suggested PKCε interacts with several signaling pathways, including the Ras/Raf/MAPK and PI-3K/Akt pathway. MAPK and Akt coordinate various extracellular signals to regulate different crucial cell functions. Our results suggest that overexpression of PKCε were associated with increased in K-Ras, Raf-1, and ERK activity in H460 and A549 cells. In this study, we demonstrated that PKCε can affect cell function via K-Ras/Raf-1/ERK pathway. Finally, we used siRNA to block PKCε or K-Ras activity in H460 PKCε cells. Knockdown of PKCε or K-Ras expression by siRNA resulted in decreased cell growth rate and migration ability. Effects of increased cell growth rate and migration ability were significantly but incompletely reversed when PKCε overexpressing cells were treated with a specific ERK inhibitor. In animal model, the increased ability of tumor migration was observed in PKCε overexpressed cells. Taken togehter, our results suggest that PKCε may increase cell growth rate and cell migration ability in human lung cancer cells through integration with K-Ras/Raf-1/ERK cascade.

並列關鍵字

Lung cancer ； PKCε ； Ras/Raf/ERK ； metastasis ； cell growth

參考文獻

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Albanese, C., Johnson, J., Watanabe, G., Eklund, N., Vu, D., Arnold, A., and Pestell, R.G. (1995). Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem 270, 23589-23597.

Aziz, M.H., Manoharan, H.T., Church, D.R., Dreckschmidt, N.E., Zhong, W., Oberley, T.D., Wilding, G., and Verma, A.K. (2007). Protein kinase Cepsilon interacts with signal transducers and activators of transcription 3 (Stat3), phosphorylates Stat3Ser727, and regulates its constitutive activation in prostate cancer. Cancer Res 67, 8828-8838.

Bae, K.M., Wang, H., Jiang, G., Chen, M.G., Lu, L., and Xiao, L. (2007). Protein kinase C epsilon is overexpressed in primary human non-small cell lung cancers and functionally required for proliferation of non-small cell lung cancer cells in a p21/Cip1-dependent manner. Cancer Res 67, 6053-6063.

Baldwin, R.M., Parolin, D.A., and Lorimer, I.A. (2008). Regulation of glioblastoma cell invasion by PKC iota and RhoB. Oncogene 27, 3587-3595.

國際替代計量

PKCε經由Ras/Raf/ERK途徑促進人類肺癌細胞生長和轉移

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