生育酚结合蛋白通过磷酸肌醇3激酶途径抑制前列腺癌的生长  

α-Tocopherol-associated Protein Suppresses Prostate Cancer Proliferation by Phosphoinositide 3-Kinase Pathway

作　　者：温星桥[1] 李小娟[2] 罗云[3] 王林[1] 周祥福[1] 蔡育彬[1] 温机灵[1] 高新[1] 

机构地区：[1]中山大学附属第三医院泌尿外科,广东广州510630 [2]中山大学附属第三医院保健科,广东广州510630 [3]中山大学附属第三医院肾移植科,广东广州510630

出　　处：《中山大学学报（医学科学版）》2007年第4期367-372,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(30600620);广东省自然科学基金(05001762)

摘　　要：【目的】探讨生育酚结合蛋白(TAP)对前列腺癌细胞的生长调节及其分子机制。【方法】四甲基偶氮唑盐生长试验(MTT)测定细胞增殖情况,体外集落形成试验测定各细胞的致癌能力,高效液相层析法检测细胞内维生素E的浓度,利用基因转染、基因沉默、免疫印迹、Northern blot,RT-PCR、荧光定量PCR、免疫沉淀等方法研究TAP对前列腺细胞生长的作用及机制。【结果】TAP mRNA水平在前列腺癌细胞LNCaP、PC-3、DU-145、CWR22R中均较正常前列腺细胞RWPE-1低。TAP可促进癌细胞保留维生素E并增强其抗前列腺癌增殖的效应。无维生素E作用下,转染TAP可抑制前列腺癌细胞LNCaP、DU-145的生长,第6天细胞数比对照组分别减少35.8%与42.4%;LNCaP细胞克隆形成率下降54.3%(P<0.05)。利用siRNA在良性前列腺细胞HPr-1中沉默TAP基因,培养9d后,细胞数比对照组增加124.3%(P<0.01)。TAP通过抑制磷酸肌醇PI3激酶信号,而非通过影响细胞周期或雄激素受体信号发挥作用。免疫沉淀实验表明TAP通过抑制PI3K的亚单位p110与p85的相互作用,进而干扰PI3K-Akt信号通路;持续激活Akt的活性可削弱TAP对前列腺癌细胞生长的抑制能力。【结论】TAP不仅能促进前列腺癌细胞摄取和增强维生素E的抗前列腺癌效应,还可通过非维生素E途径发挥作用,可能是防治前列腺癌的有价值的分子靶点。[Objective ] To investigate the effect of α-tocopherol-associated protein （TAP） gene on the proliferation of prostate cancer cells and its mechanisms. [Methods] 3- （4, 5-Dimethyhhiazol-2-yl）-2, 5- diphenyhetrazolium bromide （MTY） assay was performed to assess the cell growth rate. Colony forming assays were carried out to evaluate the carcinogenesis of different cells. Measurements of vitamin E in the prostate cancer cells were performed in high-performance liquid chromatography （HPLC）. Gene transfection, small interfering RNA, Western blot, Northern blot, RT-PCR, real-time PCR, coimmunoprecipitation assays were performed to detect the effect and mechanisms of TAP in prostate cells. [Results] The mRNA levels of TAP in LNCaP, PC-3, DU-145, and CWR22R cells were reduced compared with the normal prostate cell RWPE-1. Without treatment of vitamin E, over-expression of TAP in prostate cancer cells significantly suppressed cell growth by 35.8% and 42.4% at 6th day after transfection, and could down-regulate the colony forming ability by 54.3%. Knock-down of endogenous TAP by TAP small interfering RNA （siRNA） in nonmalignant prostate HPr-1 cells increased the cell growth by 124.3%. The tumor suppressor function of TAP was via down-regulation of phosphoinositide 3-kinase （PI3K）/Akt signaling, but not by modulating cell cycle arrest or androgen receptor signaling. Immunoprecipitation results indicated that TAP inhibited the interaction of PI3K subunits, p110 with p85, and subsequently disturbed the signal pathway of PI3K-Akt. Constitutively active Akt negated the TAP-suppressive activity on prostate cancer cell growth. [Conclusions ] TAP not only mediates vitamin E absorption and facilitate antiproliferation effect in prostate cancer cells, but also suppresses cancer cell viability through a non-vitamin E manner. TAP may be a valuable molecular target for prevention and therapy of prostate cancer.

关 键 词：生育酚结合蛋白:前列腺癌 磷酸肌醇3激酶 

分 类 号：R697.31[医药卫生—泌尿科学]