| Abstract: | 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。
ZAK is a novel MLK family of mixed lineage kinase-like protein. The molecular weight of this protein is 92 kDa. The protein structure of ZAK contains three main functional motifs, including kinase domain (KD), leucine zipper (LZ) and sterile-alpha motif (SAM) domain, but its function is similar to MAP kinase kinase kinase (MAP3K). Our previous studies show that overexpression of ZAK gene enhances AP-1 activity via the activation of ERK and JNK, thereby making the cell growth slowed. In this study, we found that ZAK gene in migration and invasion of lung cancer cells plays an important role. In vitro, experiments proved that ZAK overexpression in human lung cancer cells H460 cells reduced the ability of migration and invasion. It increased the tissue inhibitor of metalloproteinase-1 (TIMP-1) expression , and thus inhibited matrix metalloproteinase -9 (MMP9) activity. At the same time, ZAK decreased protein expression downstream of Wnt / β-catenin pathway. From the experimental results also can be found when the presence of ZAK gene increased the cell adhesion ability. Lung cancer cells treated with different doses of TNF-α, the transcription capability of NF-κB into the nucleus was enhanced. Next, in order to prove its specificity, we used IKKα and IKKβ to affect the NF-κB entering the nuclear capability. We demonstrated that ZAK gene can suppess lung cancer cell growth and metastasis by reducing NF-κB transcription factor. In vivo, experiment also proved that lung cancer cells overexpression ZAK gene decreased the number of tumor migration in mouse lungs. Moreover, from the experimental results, we also found that the antiapoptotic proteins such as Bcl-2, Bcl-xL and Mcl-1, survivin, cIAP-2 and XIAP were decreased significantly in ZAK overexpressed cells. But the proapoptotic protein BAX,BAK and BAD, the amount of protein expressions were increased. From the experimental results, regardless of flow cytometry analysis or DAPI fluorescence staining, TRAIL can enhance apoptosis in ZAK overexpression cells. Taken together, ZAK gene for the H460 human lung cancer cells not only inhibited the growth and metastasis of cancer cells, while using TRAIL also increased the apoptosis of tumor cells and these results have been an important discovery in treatment of lung cancer. |
