协和医学杂志

2012, (01) 13-20

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层黏连蛋白对胰腺癌细胞内在性耐药的影响及其机制
Effect of Laminin on the Intrinsic Chemoresistance of Pancreatic Cancer Cell

吴焕文;梁智勇;师晓华;任新瑜;刘彤华;
WU Huan-wen,LIANG Zhi-yong,SHI Xiao-hua,REN Xin-yu,Liu Tong-hua Department of Pathology,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100730,China

摘要(Abstract):

目的探讨层黏连蛋白(laminin,LN)对胰腺癌细胞内在性耐药的影响,并探讨其作用机制。方法选择胰腺癌细胞系AsPC-1,检测LN对盐酸吉西他滨(健择,gemcitabine,Gem)的细胞毒性及对Gem诱导细胞凋亡能力的影响。通过Westernblot等方法检测LN对下游信号通路的影响;在AsPC-1中,过表达黏着斑激酶相关非激酶(focal adhesion kinase-related non-kinase,FRNK)或应用PF-573,228抑制黏着斑激酶(focal adhesion kinase,FAK)磷酸化与活性,检测FAK及其下游信号的抑制对LN作用的影响。结果在胰腺癌细胞系AsPC-1中,LN处理使Gem的细胞毒性及Gem诱导细胞凋亡的能力显著降低。Gem作用72h后,LN处理组细胞存活率为57.71%±6.08%,形成的克隆数目为55.33±5.01;而在无LN包被组,细胞存活率与形成的克隆数目分别为36.65%±4.14%及31.43±4.62(P均<0.05);LN处理组AnnexinV标记阳性细胞百分数(41.00%±5.46%)较无LN处理组(55.70%±3.44%)显著下降(P<0.05)。LN能够时间依赖性上调AsPC-1细胞的FAKTyr397位点及Akt磷酸化水平,LN还可以显著增加AsPC-1细胞的survivin蛋白表达水平及BADSer-136位点磷酸化水平。过表达FRNK或应用PF-573,228能够显著抑制FAK磷酸化水平及其下游通路的活化,并能够对抗LN对胰腺癌内在性耐药的效应。应用PF-228可以使LN处理后Gem诱导的AsPC-1细胞凋亡率从26.77%±0.49%升高至38.53%±2.83%(P<0.05)。结论细胞外基质蛋白LN能够诱导胰腺癌细胞系对Gem产生内在性耐药。LN影响胰腺癌细胞内在性耐药的具体机制可能与FAK磷酸化及其下游PI3K-Akt通路活化、BadSer-136位点磷酸化与survivin表达水平改变有关,FAK靶向治疗与Gem联合在胰腺癌治疗中具有重要的潜在应用价值。
Objective To determine the effect of laminin(LN) on the intrinsic chemoresistance of pancreatic cancer and whether such effect is mediated by LN-induced focal adhesion kinase(FAK) phosphorylation and subsequent downstream signal pathway.Methods The effect of LN on gemcitabine(Gem)-induced cytotoxicity and apoptosis in pancreatic cancer cell line AsPC-1 cells were determined by MTT assay,clonogenic assay and apoptosis analysis.The effect of LN on the expression and phosphorylation of FAK,Akt,and ERK1/2 were detected by Western blotting.The changes in the effects of LN in AsPC-1 cells were explored by-FAK phosphorylation inhibition through focal adhesion kinase-related non-kinase(FRNK) overexpression or specific FAK phosphorylation inhibitor PF-573,228.Results LN decreased Gem-induced cytotoxicity and apoptosis in AsPC-1 cells.After Gem treatment for 72 h,the viability was 57.71%±6.08% and the colony number was 55.33±5.01 on LN,while the viability and the colony number on plastic were 36.65%±4.14% and 31.43±4.62,respectively(both P<0.05).The Annexin V positivity of AsPC-1 cells on LN(41.00%±5.46%) was significantly lower than that on plastic(55.70%±3.44%)(P<0.05).Moreover,LN induced FAK and Akt phosphorylation in a time-dependent manner and increased the levels of survivin and pBad(pS136).Specific inhibition of LN-induced FAK phosphorylation by either FRNK overexpression or PF-573,228 suppressed the effect of LN on AsPC-1 cells.PF-573,228 increased Gem-induced apoptosis in AsPC-1 cells from 26.77%±0.49% to 38.53%±2.83% on LN(P<0.05).Conclusions LN contributes to the increased intrinsic chemoresistance of pancreatic cancer cell to Gem,which may be achieved through the regulation of FAK,Akt,and Bad phosphorylation and survivin expression.Targeted FAK inhibitors may be a promising way to enhance chemosensitivity in pancreatic cancer.

关键词(KeyWords): 层黏连蛋白;胰腺癌;化疗耐药;黏着斑激酶
laminin;pancreatic cancer;chemoresistance;focal adhesion kinase

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基金项目(Foundation): “十一五”国家科技计划支撑项目(2006BAI02A14)

作者(Author): 吴焕文;梁智勇;师晓华;任新瑜;刘彤华;
WU Huan-wen,LIANG Zhi-yong,SHI Xiao-hua,REN Xin-yu,Liu Tong-hua Department of Pathology,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100730,China

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