Effect Of Hypoxia-inducible Factor-1α On Angiopoietin-2 Expression In Non-small Cell Lung Cancer

Objective1.To investigate the clinical significance and the relationship among the expressions of HIF-1αand angiopoietin-2 in non-small cell lung cancer (NSCLC).2. To determine the role of HIF-1αin the induction of angiopoietin-2 expression in A549 cells during hypoxia.3. To demonstrate the relationship between the expression of HIF-1αand angiopoietin-2 in the NSCLC and tumor angiogenesis.MethodsThe expressions of HIF-1αand angiopoietin-2 were detected in 46 human NSCLC samples by SP immunohistochemistry;The A549 cells were cultured in DMEM with 10% new bovine serum, hypoxia-like condition was chemically created by exposure of cells to cobalt chloride, the productions of HIF-1αprotein and angiopoietin-2 mRNA were observed at different hypoxia culture phases (6h, 12h, and 24h) and in the presence of different doses genestein which can inhibit tyrosine kinase activity. Of the total, the HIF-1αprotein was tested by Western Blot analysis; the expression of angiopoietin-2 mRNA was shown using the reverse transcription PCR (RT-PCR).Results1.The expression rates of HIF-1αand angiopoietin-2 were 73.9%, 63.0% respectively in human NSCLC tissues, significantly higher than those in paraneoplastic tissues which were 39.1%, 23.9% respectively(P<0.05); Moreover, the expression rates of such molecules showed no correlation to age, gender, smoking, lymph node metastasis, differentiations and histological classification(P>0.05); The HIF-1αexpression was positively related to the expression of angiopoietin-2(contingency coefficient r=0.343, P<0.05).2. Both HIF-1αprotein and angiopoietin-2 mRNA were hypoxia-induced in a time-dependent manner, for that HIF-1αprotein expression was peaked at 6h (0.2964±0.0227), meanwhile, angiopoietin-2 expression also increased after 6h treatment, and reached the maximum at 12h post-treatment(0.0998±0.0068). The expressions of HIF-1αprotein and angiopoietin-2 mRNA of hypoxia group were significantly higher than those of control group which were 0.0758±0.0084 , 0.0495±0.0035 respectively(P<0.05), although the expressions levels of HIF-1αprotein(0.1315±0.0179) and angiopoietin-2 mRNA(0.0635±0.0023) were down-regulated during prolonged hypoxia for 24 h, both were still higher than those of control group(P<0.05).3. The expression of HIF-1αprotein decreased in the presence of 50 uM, 100 uM, and 200 uM genestein respectively, compared to that of control group during hypoxia for 12 h. Moreover, dose-dependent inhibitory effect of genestein on hypoxia-induced angiopoietin-2 expression showed at 200 uM>100 uM>50 uM. A significant correlation between HIF-1αprotein and angiopoietin-2 mRNA was found by Spearman'rank correlation test(r=0.852, P<0.05).Conclusion1. The positive rates of HIF-1αand angiopoietin-2 expression in 46 human NSCLC tissues are significantly higher than those in paraneoplastic tissues (P<0.05), the HIF-1αexpression was positively related to the expression of angiopoietin-2, these finding implicate abnormal expressions of HIF-1αand angiopoietin-2 are correlated with development of NSCLC.2. The expressions of HIF-1αprotein and angiopoietin-2 mRNA which displayed similar induction by acute hypoxia can be increased in A549 cells, the HIF-1αprotein may regulate the expression of angiopoietin-2 under hypoxic condition, and the increase expressions of HIF-1αprotein and angiopoietin-2 mRNA are correlated with tumor angiogenesis and contribute to development of NSCLC together.