Changes On The Proliferation Of Colon Cancer Cells Under Hypoxia Over Long Term In Vitro

Objective：One of the characteristics of solid tumor is hypoxia，different hypoxia conditions may produce different effects on tumor cellapoptosis and proliferation. We establish hypoxia environment in vitroinvestigate the effect of long term hypoxic environment in vitro on thechange of proliferation of colon carcinoma cell lines and detect the possiblemechanism.Methods：After14~28d cultured in hypoxia environment, MTT andColony-forming assay were performed to detect the effect of hypoxiaadaption on the cell proliferation of SW480and HCT116cell lines. Laserscanning confocal microscope(LSCM) were performed to detect theexpression of hypoxia-inducible factor-1(HIF-1α) and β-catenin in the twocell lines. Western blot were performed to detect the HIF-1α, andproliferating cell nuclear antigen(PCNA). To elucidate the underlyingmechanisms of this condition, western blot were performed to detect theprotein levels of β-catenin and glycogen synthase kinase-3β(GSK-3β),phosphorylation of GSK-3β on the Ser9residue (pGSK--3β(ser9)), which are two key factors of wnt/β-catenin signal pathway. All of these detectionswere compared with control group which was the same cell line cultured innormoxic condition.Results：The results of MTT and Colony-forming assay were bothindicated that the cells cultured in hypoxic environment after2weeks werechanged growed faster than cells cultured in normoxic condition. LSCMindicated that HIF-1α expressed both in cytoplasm and nucleus, while innormoxic expressed main in nucleus. Western blot results indicated that thecells in hypoxia condition expressed HIF-1α, β-catenin and pGSK-3β(ser9)increased (HIF-1α: P<0.05; β-catenin: P<0.01; pGSK-3β(ser9), P<0.01), butthe total GSK-3β have no significant difference（P>0.05）. The PCNA alsoexpressed increased(SW480: P<0.05; HCT116: P<0.01).Conclusion： Hypoxic adaptation promote colon carcinoma cell linesproliferation and it’s maybe the results of HIF-1α interaction with increasedactivity of Wnt/β-catenin.