St-Germain Marie-Eve, Gagnon Veronique, Mathieu Isabelle, Parent Sophie, Asselin Eric
Department of Chemistry and Biology, Medical Biology Section, University of Quebec at Trois-Rivieres, C.P. 500, Trois-Rivieres, Quebec G9A 5H7, Canada.
Int J Oncol. 2004 May;24(5):1311-24.
In human endometrial cancer, the fourth most common cancer in women, tumor suppressor phosphatase tensin homologue (PTEN) is frequently mutated. In the presence of a mutated PTEN protein, Akt phosphorylation levels are increased leading to the activation of this survival pathway. Numerous studies indicated that COX-2 is inappropriately induced and up-regulated in a number of malignant cancer cells. COX-2 plays an important role in tumor cell biology, taking part actively in angiogenesis particularly via the production of prostaglandin E2 (PGE2). The present study was undertaken to determine the involvement of PI 3-K/Akt pathway in the regulation of COXs expression and PGE2 synthesis. Three different human endometrial cancer cell lines known to have wild-type PTEN (HEC 1-A) or a mutated inactive PTEN protein (RL 95-2 and Ishikawa) were used for these studies. Results showed that Akt phosphorylation was high in mutated PTEN cells. RT-PCR studies revealed that Akt1 and Akt2 were the regulated forms whereas Akt3 mRNA was nearly undetectable. COX-2 mRNA expression and protein levels were high in these cells compared to wild-type PTEN cells as demonstrated by RT-PCR and Western analysis respectively. PGE2 production was higher in mutated-PTEN expressing phospho-Akt and COX-2 compared to wild-type PTEN cells. Inhibition of PI 3-K with Wortmannin and LY294002 blocked Akt phosphorylation and inhibited expression of COX-2 in mutated-PTEN cells. Inhibition of Akt phosphorylation with specific PI 3-K inhibitors and down-regulation of COX-2 increased apoptosis in human endometrial cancer cells. Likewise, transfection of mutated-PTEN cells with a dominant negative Akt vector, resulted in COX-2 down-regulation and activation of apoptosis, as demonstrated by Hoechst nuclear staining. On the opposite, activation of Akt using a constitutively active expression vector, resulted in the up-regulation of COX-2 protein expression. Specific inhibition of COX-2 with NS-398 induced apoptosis in COX-2 expressing human endometrial cancer cells. It is concluded that the PI 3-K/Akt survival pathway is involved in the regulation of COX-2 and PGE2 synthesis in human endometrial cancer cells.
在女性中第四常见的癌症——人类子宫内膜癌中，肿瘤抑制因子磷酸酶张力蛋白同源物（PTEN）经常发生突变。在存在突变型PTEN蛋白的情况下，Akt磷酸化水平升高，导致这条生存通路被激活。大量研究表明，COX-2在许多恶性癌细胞中被不适当诱导并上调。COX-2在肿瘤细胞生物学中发挥重要作用，尤其通过前列腺素E2（PGE2）的产生积极参与血管生成。本研究旨在确定PI 3-K/Akt通路在COXs表达调控和PGE2合成中的作用。三种已知具有野生型PTEN（HEC 1-A）或突变型无活性PTEN蛋白（RL 95-2和Ishikawa）的不同人类子宫内膜癌细胞系用于这些研究。结果显示，在突变型PTEN细胞中Akt磷酸化水平较高。逆转录聚合酶链反应（RT-PCR）研究表明，Akt1和Akt2是受调控的形式，而Akt3 mRNA几乎检测不到。分别通过RT-PCR和蛋白质免疫印迹分析表明，与野生型PTEN细胞相比，这些细胞中COX-2 mRNA表达和蛋白水平较高。与野生型PTEN细胞相比，表达磷酸化Akt和COX-2的突变型PTEN细胞中PGE2产生更高。渥曼青霉素和LY294002对PI 3-K的抑制作用阻断了Akt磷酸化，并抑制了突变型PTEN细胞中COX-2的表达。用特异性PI 3-K抑制剂抑制Akt磷酸化以及COX-2的下调增加了人类子宫内膜癌细胞的凋亡。同样，用显性负性Akt载体转染突变型PTEN细胞，如通过Hoechst核染色所示，导致COX-2下调和凋亡激活。相反，使用组成型活性表达载体激活Akt，导致COX-2蛋白表达上调。用NS-398特异性抑制COX-2可诱导表达COX-2的人类子宫内膜癌细胞凋亡。得出的结论是，PI 3-K/Akt生存通路参与了人类子宫内膜癌细胞中COX-2和PGE2合成的调控。
