Gao Ning, Nester Rebecca A, Sarkar Mohamadi A
Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298-0533, USA.
Toxicol Appl Pharmacol. 2004 Apr 1;196(1):124-35. doi: 10.1016/j.taap.2003.12.002.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1alpha and HIF-1beta subunits. HIF-1 expression is induced by hypoxia, growth factors, and activation of oncogenes. HIF-1 activates downstream target genes such as vascular endothelial growth factor A (VEGF-A), which plays an important role in tumor progression and angiogenesis. Estrogen exposure is considered to be the major risk factor for ovarian cancer. Estradiol (E2) is usually metabolized by CYP1A1/1A2 and CYP3A4 to the 2-hydroxy estradiol (2-OHE2) and 4-hydroxy estradiol (4-OHE2) in human liver. Many reports have suggested that the formation of 4-OHE2 is important for mammary carcinogenesis. However, the formation of 2-OHE2 may play an important role in exhibiting anticarcinogenic effects. In the present study, we have demonstrated that one of the catechol estrogen metabolites of E2, 4-OHE2, induces HIF-1alpha and VEGF-A expression at protein level in two human ovarian cancer cell lines, OVCAR-3 and A2780-CP70 cells, in dose- and time-dependent manners, whereas the other catechol estrogen metabolite of E2, 2-OHE2, does not alter HIF-1alpha and VEGF-A expression. To explore the mechanism of 4-OHE2-induced HIF-1alpha and VEGF-A expression, we studied whether phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase (MAPK) signaling pathways are involved in 4-OHE2-induced HIF-1alpha and VEGF-A expression. Our findings indicate that PI3K inhibitors, LY294002 and wortmannin, inhibited HIF-1alpha and VEGF-A expression, whereas MAPK inhibitor, PD98059, did not alter HIF-1alpha and VEGF-A expression induced by 4-OHE2. 4-OHE2, but not 2-OHE2, also induced Akt phosphorylation at Ser473 in dose- and time-dependent manners, and LY294002 and wortmannin inhibited Akt phosphorylation at Ser473 induced by 4-OHE2. Our results also indicated that the mTOR/FRAP inhibitor, rapamycin, inhibited 4-OHE2-induced HIF-1alpha and VEGF-A expression. These results suggest that the PI3K/Akt/FRAP signaling pathway is required for HIF-1alpha and VEGF-A expression induced by 4-OHE2, whereas the MAPK pathway is not required. The finding that induction of HIF-1alpha and VEGF-A expression occurs via the activation of the PI3K/Akt/FRAP signaling pathway could be an important mechanism of 4-OHE2-induced carcinogenesis.
缺氧诱导因子1（HIF-1）是一种由HIF-1α和HIF-1β亚基组成的异二聚体碱性螺旋-环-螺旋转录因子。HIF-1的表达可由缺氧、生长因子及癌基因激活诱导产生。HIF-1可激活下游靶基因，如血管内皮生长因子A（VEGF-A），其在肿瘤进展和血管生成中起重要作用。雌激素暴露被认为是卵巢癌的主要危险因素。雌二醇（E2）在人肝脏中通常由CYP1A1/1A2和CYP3A4代谢为2-羟雌二醇（2-OHE2）和4-羟雌二醇（4-OHE2）。许多报道表明，4-OHE2的形成对乳腺癌发生很重要。然而，2-OHE2的形成可能在发挥抗癌作用中起重要作用。在本研究中，我们已证明E2的儿茶酚雌激素代谢产物之一4-OHE2，在两种人卵巢癌细胞系OVCAR-3和A2780-CP70细胞中，以剂量和时间依赖性方式在蛋白水平诱导HIF-1α和VEGF-A表达，而E2的另一种儿茶酚雌激素代谢产物2-OHE2则不改变HIF-1α和VEGF-A表达。为探究4-OHE2诱导HIF-1α和VEGF-A表达的机制，我们研究了磷脂酰肌醇3-激酶（PI3K）或丝裂原活化蛋白激酶（MAPK）信号通路是否参与4-OHE2诱导的HIF-1α和VEGF-A表达。我们的研究结果表明，PI3K抑制剂LY294002和渥曼青霉素可抑制HIF-1α和VEGF-A表达，而MAPK抑制剂PD98059不改变4-OHE2诱导的HIF-1α和VEGF-A表达。4-OHE2而非2-OHE2，也以剂量和时间依赖性方式诱导Akt在Ser473位点磷酸化，且LY294002和渥曼青霉素可抑制4-OHE2诱导的Akt在Ser473位点的磷酸化。我们的结果还表明，mTOR/FRAP抑制剂雷帕霉素可抑制由4-OHE2诱导的HIF-1α和VEGF-A表达。这些结果表明，PI3K/Akt/FRAP信号通路是4-OHE2诱导HIF-1α和VEGF-A表达所必需的，而MAPK通路并非必需。通过PI3K/Akt/FRAP信号通路激活诱导HIF-1α和VEGF-A表达这一发现，可能是4-OHE2诱导致癌作用的重要机制。
