PKCε在肺癌細胞株抵抗TRAIL誘導細胞凋亡之角色探討

The role of PKCε in non-small cell lung cancer cells against TRAIL-induced apoptosis

楊雨寰(Yu-Huan Yang)

指導教授 ： 吳文俊

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2007年)

摘要

Tumor nercrosis factor-related apoptosis-inducing ligand (TRAIL)可以專一性的毒殺腫瘤細胞，而不會影響正常細胞，所以被認為是一個具有研究價值的抗癌藥物。但是如果腫瘤細胞對TRAIL產生抗藥性，則會大幅限制了TRAIL在臨床上的治療效果。TRAIL所引起的凋亡作用是透過與腫瘤細胞表面DR4和DR5等死亡受器的結合，但是存在於腫瘤表面的DcR1與DcR2等干擾受器(decoy receptor)則會降低TRAIL對腫瘤細胞的毒殺作用。許多文獻指出，多種癌細胞株對TRAIL具有抗藥性，且此抗藥性的表現與干擾受器的數量並沒有明顯的相關性。許多研究已經證實PKC活化對於保護細胞抵抗TRAIL引起的細胞凋亡扮演著重要的角色。因此我們分析了九株人類非小型肺癌細胞株對於human recombinant TRAIL的敏感程度。我們發現在H1355和H520對於TRAIL具有抗藥性的細胞株之中其PKCε有高度的表現量，然而在對TRAIL較具敏感性的H460和H358細胞株之中，其PKCε的表現量則相對降低。為了更了解PKCε在TRAIL抗藥性中所扮演的角色，我們利用PKCε siRNA和PKCε shRNA干擾載體來抑制PKCε的表現量。並觀察PKCε被抑制後細胞對TRAIL抗藥性的變化。在H1355和H520細胞株中抑制PKCε的表現量之後，會增加細胞對於TRAIL的敏感性。有文獻指出PKCε是一個致癌基因。在本實驗之中，我們想了解PKCε在肺癌細胞株中所扮演的角色。首先我們以shRNA干擾方式將PKCε抑制時，會降低肺癌細胞株的生長能力。而從軟培養基非附著性細胞生長試驗的結果顯示，干擾PKCε的表現之後，則會降低肺癌細胞株的轉型能力。在H1355細胞株中抑制PKCε的表現量會降低Bcl-2蛋白的表現量。另一方面在H520細胞株之中抑制PKCε的表現後，會使Bcl-xL和Mcl-1的表現量下降。由動物腫瘤實驗中，我們也得到類似的結果。綜合本實驗的結果推論，在H1355和H520非小型肺癌細胞株中PKCε可能經由調控抗細胞凋亡分子Bcl-2家族而使細胞對TRAIL產生抗藥性。

關鍵字

肺癌

並列摘要

Tumor nercrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent as it selectively kills tumor cells but spares normal cells. Resistance to TRAIL by tumor cells limits its therapeutic use in clinic. TRAIL-induced apoptosis is mediated by binding with death receptors, DR4 and DR5, but the decoy receptors ( DcR1 and DcR2 ) on the surface of tumor cells collapse TRAIL-mediated apoptosis. Recently, several articles have demonstrated that some tumor cell lines exhibit TRAIL-resistance and this resistance may be not associated with decoy receptors. Some reports indicated that PKC activation was an important factor for cell to protect apoptosis from TRAIL. Therefore, we analyzed the sensitivity of nine human non-small lung cancer ( NSCLC) cell lines to TRAIL. We found the expression of PKCε is higher in TRAIL-resistant cell lines (H1355 and H520) than those of in TRAIL-sensitive cell lines (H460 and H358). In order to investigate the role of PKCε in TRAIL-resistant cell lines, we used PKCε siRNA and PKCε shRNA to block PKCε activity, and then observed their effect on TRAIL-induced apoptosis. Knockdown of PKCε expression by siRNA resulted in enhanced sensitivity to TRAIL in H1355 and H520 cell lines. Some references pointed out that PKCε is an oncogene. In this research, we investigated the role of PKCε gene in lung cancer cells. First of all, we found out when used PKCε shRNA to suppress PKCε expression, it would decrease cell growth. According to anchorage-independent growth assay, suppressed PKCε expression would decrease cell transformation. Overexpression of PKCε shRNA was associated with a decrease in Bcl-2 protein level in H1355 cell line, and a decrease in Bcl-xL and Mcl-1 in H520 cell line. Moreover, the similar results were observed in animal tumor model. Thus, our results suggest that PKCε may act upstream of mitochondria and mediate TRAIL resistance via Bcl-2 family in H1355 and H520 cells.

並列關鍵字

TRAIL ； PKCε ； lung cancer

參考文獻

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