Department of Pharmacology, Howard University College of Medicine, Washington, District of Columbia 20059, USA.
Prostate. 2010 May 15;70(7):755-64. doi: 10.1002/pros.21108.
Class IA PI 3-kinases produce phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 is bound by AKT which facilities its activation by PDK1. Activated AKT promotes cell survival and stimulates cell proliferation. Class IA PI 3-kinases are heterodimers consisting of a regulatory subunit p85 and a catalytic subunit p110. The p110alpha isoform has been shown to be mutated in a number of tumor types. A number of recent studies suggest that the p110beta isoform may be functionally relevant in prostate cancer. In this study we extend this work to include the examination of the expression and functional properties of p110alpha and p110beta in three different prostate cancer cell lines, DU145, LNCaP, PC3, as well as the non-tumorigenic but immortalized RWPE1 prostate epithelial cell line.
Western blot analysis was used to measure protein expression and quantitative real-time PCR was used to measure mRNA levels. After targeted knockdown using isoform-specific siRNAs to reduce PI 3-kinase p110alpha or p110beta isoform expression, we measured downstream signally events such as phosphorylation of AKT, ERK 1/2, PDK, and FOXO, as well as biological consequences such as changes in apoptosis, and alterations in cell cycle progression.
In all three prostate cancer cell lines examined, targeted knockdown of p110beta, and not p110alpha, resulted in significantly reduced AKT, PDK, and FOXO phosphorylation. While knockdown of either p110 isoform resulted in an increase in apoptosis and a cell cycle arrest in G1 in the remaining non-apoptotic cells, these effects were much more pronounced with knockdown of p110beta.
Our results support the concept that p110beta appears to be the predominant functional class I PI 3-kinase isoform in prostate cancer cells.
IA 类 PI 3-激酶产生磷脂酰肌醇（3,4,5）-三磷酸（PIP3）。PIP3 被 AKT 结合，从而促进其被 PDK1 激活。激活的 AKT 促进细胞存活并刺激细胞增殖。IA 类 PI 3-激酶是由调节亚基 p85 和催化亚基 p110 组成的异源二聚体。已证实 p110alpha 同工型在多种肿瘤类型中发生突变。最近的一些研究表明，p110beta 同工型在前列腺癌中可能具有功能相关性。在这项研究中，我们将这项工作扩展到包括检查三种不同的前列腺癌细胞系（DU145、LNCaP、PC3）以及非致瘤但永生化的 RWPE1 前列腺上皮细胞系中 p110alpha 和 p110beta 的表达和功能特性。
使用 Western blot 分析测量蛋白表达，使用定量实时 PCR 测量 mRNA 水平。使用针对同工型特异性 siRNA 进行靶向敲低以降低 PI 3-激酶 p110alpha 或 p110beta 同工型的表达后，我们测量了下游信号事件，如 AKT、ERK 1/2、PDK 和 FOXO 的磷酸化，以及生物后果，如凋亡的变化和细胞周期进程的改变。
在所有三种检查的前列腺癌细胞系中，靶向敲低 p110beta 而不是 p110alpha 导致 AKT、PDK 和 FOXO 磷酸化显著减少。虽然敲低任一同工型都会导致剩余非凋亡细胞中的细胞凋亡增加和细胞周期停滞在 G1 期，但这种影响在 p110beta 敲低时更为明显。
我们的结果支持这样一种概念，即 p110beta 似乎是前列腺癌细胞中主要的功能性 IA 类 PI 3-激酶同工型。
