Effects Of Yc-1 On Hif-1α Transcriptional Activity In Esophageal Squmaous Carcinoma Cells

BackgroudEsophageal cancer is commonly seen in China,with high mortality and poor prognosis. Among eaophageal cancers,esophageal squmaous cell carcinoma (ESCC)account for approximately 95%.Recently many researches have revealed that hypoxia-inducible factor 1 alpha(HIF-1α) is involved in ESCC.As an important transcriptional factor, HIF-1αplays a key role in angiogenesis, invasion and metastasis in ESCC. YC-1[3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole ] shows promising anticancer effects by inhibition of HIF-1α, blockage of angiogenesis and tumor growth.ObjetivesTo investigate the effects of YC-1 on HIF-1αexpression, proliferation, apoptosis and migration in esophageal squmaous carcinoma cell line Eca109 cells. Methods1.Esophageal squamous cancer Eca109 cells were incubated under normoxic and hypoxic conditions for different time (24, 48, 72h). Expression of HIF-1αprotein was detected by Western blot, thus the most suitable hypoxic incubated time was selected.2.Eca109 cells were incubated with different concentration of YC-1(0,1,5,10,20,30,40μmol/l) for different time(24, 48, 72h).The cell viability and cytotoxicty were measured by MTT assay.3.Eca109 cells were incubated with vehicle(DMSO)and YC-1 (20μmol/l) under normoxia and hypoxia conditions for 48h. HIF-1αand MAPK p42/p44 expression were measured by RT-PCR and Western Blot assay. VEGF ,MMP2,p53,Bcl2 and Bax were detected by Western Blot assay.4.Migrations were measured by transwell chamber.100,000 Eca109 cells were seeded into the upper chamber with 100μl FBS free DMEM,and YC-1(0,20μmol/l) was added to the base well.After incubation for 24h, the migrated cells were counted under a light microscope. 5.Eca109 cells were incubated with vehicle (DMSO) and YC-1 (20μmol/l) under normoxia and hypoxia conditions for 48h. The cells were then labeled with AnnexinV-FITC and propidium iodide and detected in a fluorescence-activated cell sorter.6.Eca109 cells were incubated with vehicle (DMSO) and YC-1 (20μmol/l) under normoxia and hypoxia conditions for 48h. Samples of 10,000 cells were then analyzed for DNA content and cell cycle phase distributions were analyzed by Mod Fit software.Results1.Expression of HIF-1αin Eca109 cells under hypoxic conditions The expression of HIF-1αwas increasd after incubated in hypoxia incubater for 48h, compared to normoxia condition.Thus,48h was selected for hypoxic incubation time. 2.Effect of YC-1 on proliferation in Eca109 cells Various concentration of YC-1 showed antiproliferation effect in Eca109 cells, which was in a time and dose dependent manner.The IC50 was approximately 20μmol/l for 48h . 3. Effect of YC-1 on HIF-1αand HIF-1αtranscriptional driven genes in Eca109 cells YC-1 suppressed expression of HIF-1αand MAPKp42/p44.The expression of VEGF,MMP2,Bcl2,Survivin was decreased,while the expression of p53,Bax was increased.4. Effect of YC-1 on migration in Eca109 cells After being exposed to YC-1, number of cells migrated was significantly decreased than that treated with vehicle. There was no s ignificant difference between the numbers of migrated cells under both normoxia and hypoxia conditions.5. Effect of YC-1 on apoptosis in Eca109 cells The apoptosis rate in hypoxia condition was higher than that under normoxia condition.After exposed to 20μmol/l YC-1,apoptosis rate was higher than control group.6. Effect of YC-1 on cell cycle in Eca109 cells YC-1 induced S phase cycle arrest in Eca109 cells. After incubated with 20μmol/l YC-1 for 24,48 and 72h,the proportion of S phase was increased with a stastistical significance.Conclusions1. YC-1shows antituomr effect in Eca109 cells by inducton of apoptosis,S phase arrest,and inhibition of proliferation and migration.2.YC-1 inhibited the mRNA and protein expression of HIF-1αunder normoxia and hypoxia, suggesting that the inhibitory effect occurs at a transcriptional level.3.YC-1 inhibited the expression of VEGF and MMP2, suggesting that YC-1 has potential effect of inhibition of angiogenesis and metastasis.4.The expression of MAPKp42/p44 was suppressed by YC-1,indicating that formentioned inhibitory effect maybe through MAPKp42/p44 pathway.