Study On The Effect And Mechanism Of HIF-1α On The Expressions Of COX-2 And E-cadherin In Lung Adenocarcinoma Cell During Hypoxia

Lung cancer is the most prevalent malignancy in the world,especially lung adenocarcinoma,it may be metastasis in the early stage and isn't sensitive to chemotherapy and radiotherapy,so has the poor prognosis.Several studies have shown that hypoxia is associated with the survival,metastasis and poor prognosis of lung adenocarcinoma.For adaptation to hypoxia,tumors have a series of physiological change.Hypoxia-inducible factor 1α(HIF-1α)is the key role for promoting the adaptation to hypoxia.HIF-1αup-regulation of tumor cells regulates the target genes for adaptation to hypoxia,promoting survival and metastasis. Recently studies have shown the abnormal expressions of COX-2(Cyclooxygenase-2) and E-cadherin(Epithelial cadherin)may be associated with adaptation to hypoxia.COX-2 and its metabolite PGE2(prostaglandin E2)are thought to be associated wih tumor progression.PGE2 may induce the expression of HIF-1αin lung adenocarcinoma under normoxic conditions.E-cadherin plays imporant role in cell-cell adhesion and preserving normal structure.There are multiple mechanisms involved in inactivating the E-cadherin-mediated cell adhesion system in cancers.Recently studies on the correlations beween hypoxia and E-cadherin become focus.In approaching the mechanism of adaptation to hypoxia,in our study from three parts to investigate the internal relations and mechanism among HIF-1α,COX-2 and E-cadherin,so that to establish the experiment foundation for target therapy in the fure.一,Expression of HIF-1α,COX-2 and E-cadherin in lung adenocarcinoma and clinical significance in lung adenocarcinomaObjective To investigate the clinical value of the expression of HIF-1α,COX-2 and E-cadherin in lung adenocarcinoma and internal relation.Methods The expression of HIF-1α,COX-2 and E-cadherin in 45 lung adenocarcinoma and 10 normal lung tissue were examined by immunohistochemistry.Results There were no expression of HIF-1αand COX-2 in normal lung tissue.10 normal lung tissue have positive expression of E-cadherin.There were high expression of HIF-1αand COX-2 in lung adennocarcinoma(60%,40%).Down-regulated expression of E-cadherin were observed in lung adenocarcinoma(48.9%).Yhe expression of HIF-1αwasn't associated with histologic grade,stage and lymph node metastasis(P＞0.05).The expression of HIF-1αwas higher in tumor's diameter＞2cm group than that in tumor's diameter＜=2cm group(P＜0.05).COX-2 expression in positive lymph node metastasis group was higher than that in negative lymph node metastasis(P＜0.05).There was correlation between COX-2 expression and clinical stage(P＜0.05).COX-2 expression inâ…¡andâ…¢stage was higher than that inâ… sage.The expression of COX-2 was higher in tumor's diameter＞2cm group than that in tumor's diameter＜=2cm group(P＜0.05).There was no correlation between COX-2 expression and differentiation(P＞0.05).Reduced E-cadherin expression significantly correlated with poor differentiation(P＜0.05).Reduced E-cadherin expression significantly correlated with lymph node metastasis(P＜0.05).There was no correlation between E-cadherin expression and tumor size(P＞0.05).COX-2 expression was correlated with increased HIF-1αexpression(P＜0.05).There was no correlationg between E-cadherin expression and HIF-1αexpression(P＞0.05).Conclusions The expression of HIF-1α,COX-2 and E-cadherin were relationship with survival,invasion and metastasis of lung adenocarcinoma.HIF-1αwas the key role in lung adenocarcinoma.There were internal correlation between HIF-1αand COX-2.COX-2 may be up-regulaed by HIF-1αfor adapting to hypoxia.二,The effect of hypoxic inducation on the expression of HIF-1α,COX-2 and E-cadherin in lung adenocarcinoma cell A549Objective To investigate the effect of hypoxic inducation and HIF-1αinhibitor YC-1 on the expression of HIF-1α,COX-2 and E-cadherin in lung adenocarcinoma cell A549,reveal the internal relationship.Methods Human lung carcinoma A549 were incubated under hypoxic culure conditions((5%CO2,1%O2,94%N2,37℃).Western blot was used to detect HIF-1αprotein expression.Real time RT-PCR was used to detect COX-2 and E-cadherin mRNA.After observed the effect of hypoxia on HIF-1α,COX-2 and E-cadherin,YC-1 was used before hypoxic culure,then examined the effect on HIF-1α,COX-2 and E-cadherin.Results There was no expression of HIF-1αunder normoxic conditions.The expression of HIF-1αwas induced after hypoxia.The expression of HIF-1αprotein after 8 hours,16hours and 24 hours of continuous exposure to 1%O₂ respectively were 0.67±0.04,1.45±0.09,0.77±0.09. There was significantly different among groups(P＜0.01).The extent of HIF-1αinducation during hypoxia had further increased by 16 hours.The inhibiton of HIF-1αby YC-1 was observed.The expression of HIF-1αin YC-1 10μM group and 100μM group respectively were 0.51±0.06 and 0.06±0.03,compared with the group only under hypoxic conditions,there were significantly different(P＜0,01).HIF-1αproein was reduced in a dose-dependent manner.The expression of COX-2 mRNA after 0 hour,8 hours,16hours and 24 hours of continuous exposure to 1%O₂ respectively were 7.7±1.5%,25.3±7.7%,65.3±12%,55±14.3%,there was significantly different among groups(P＜0.01).The extent of COX-2 mRNA during hypoxia had further increased by 16 hours,increased 8-fold after hypoxic treatment(P＜0.05).The expression of E-cadherin mRNA after 0 hour,8 hours,16hours and 24 hours of continuous exposure to 1%O₂ respectively were 47.0±14.8%,36.6±12.5%,18.7±4.0%,15.7±10.3%,there was significantly different among groups(P＜0.01).The extent of E-cadherin mRNA during hypoxia had further reduced by 16 hours,reduced 1.5-fold after hypoxic treatment(P＜0.05).There were no change after using YC-1 under hypoxic conditions(P＞0.05).Conclusions Increased expression of HIF-1αwas observed after hypoxic inducation in lung adenocarcinoma cell A549.The up-regulation of COX-2 mRNA under hypoxic conditions was correlated with increased HIF-1αprotein during hypoxia.COX-2 up-regulation represented a pivotal cellular adaptive reponse to hypoxia.Maybe there was no relationship between E-cadherin up-regulaion under hypoxic conditions and HIF-1α.YC-1,the HIF-1αinhibitor,had the same reaction on lung adennocarcinoma cell,our study offered the experiment foundation for target therapy in lung adenocarcinoma.三,The study on the change of PGE2 level and PGE2-induced HIF-1αprotein in lung adenocarcinoma A549 cell during hypoxiaObjective To investigate the effect of COX-2 metabolite PGE2 and COX-2 selective inhibitor NS398 on the expression of HIF-1αprotein under hypoxic condition in lung adenocarcinoma A549 cell.Methods The PGE2 level afer 16 hours of continuous exposure to 1%O₂ was determined by ELISA,then compared with the PGE2 level under normoxia and the PGE2 level after exposure to NS398 during hypoxia.Western blot was used to detect HIF-1αprotein expression after treated with PGE2 100uM under normoxic and hypoxic conditions.The expression of HIF-1αwas examined after exposure to various concentrations of NS398 under hypoxic condition.Results The level of PGE2(0.35±0.05ng/ml)during hypoxia was higher than normoxia(0.22±0.03ng/ml)(P＜0.01).The inhibiton of PGE2 level by NS398 was observed (P＜0.01).The level of PGE2 was reduced in a dose-dependent manner.There was no expression of HIF-1αunder norrnoxic conditions.Exposure to 100μM PGE2 for 16 hours under normoxic conditions,the expression of HIF-1αprotein was 1.14±0.1. Exposure to 100μM PGE2 for 16 hours under hypoxic conditions the expression of HIF-1αprotein was 2.31±0.27,it was significantly higher than that only under hypoxic condition(1.45±0.09)(P＜0.01).The expression of HIF-1αin NS398 10μM group and NS398 100μM group respectively were 0.72±0.12 and 0.29±0.09,compared with the group only under hypoxic condiion,there were significantly different(P＜0,01).HIF-1αproein was reduced in a dose-dependent manner. Conclusions Maybe there were a potential positive feedback loop between HIF-1αand COX-2/PGE2,Increased level of PGE2 during hypoxia,resulting from COX-2 up-regulation,inducing the expression of HIF-1αprotein.So targeting HIF-1αand COX-2 maybe had coorperative value in lung adenocarcinoma.