Study On The Effect Of Glutathion On HIF-1α Expression In Irradiated Hypoxia Hepatoma Cells

Nowdays,radiotherapy(RT) is recognized as a potentially curative option with the development of three dimensional conformal radiotherapy(3DCRT) technique,especially on the advanced stage of HCC.However,the high proliferation of tumor cells and hepatic cirrhosis induce local hypoxia inside HCC,following by the prodution of hypoxic cells. Owing to hypoxia inducting cell resistance to radiation,the survival of these cells is considered as one of main reasons for the failure in radio-oncology and recidivation of the tumor.It is well-documented that hypoxic tumor cells could adapt to hypoxia microenvironment by modulating the expression of specific genes.Hypoxia inducible fator-1 (HIF-1) is the major transcription factor that induces the adaptive response of tumor cells during hypoxia.HIF-1 is a heterodimeric protein consisted by two subunits of the constitutively expressed HIF-1β/ARNT and the highly regulated HIF-1α.The overall activity of HIF-1 is represented by the intracellular level of HIF-1α.Moreover,HIF-1αplays an important role in tumor invasion,recidivation,metastasis,and insensitivity to chemo- and radio-therapy.Furthermore,some drugs and techniques may enhance HIF-1αexpression during tumor treatment stage,leading to poor response to oncologic therapy.It has been shown that reactive oxygen species(ROS) may regulate the expression of HIF-1α. Since irradiation affects the intracellular ROS level by adjusting cell microenviromental redox state,it is possible that irradiation may modulate the level of expression of HIF-1αunder hypoxia condition.It is therefore important that,under hypoxia condition,the change of HIF-1αexpression in irradiated tumor cells should be studied.Meanwhile,it should be clarified whether the levels of HIF-1αexpression were changed in radioresistant cells isolated by fractionated irradiation.Research on the fields mentioned above could be very helpful to the development of new radiotherapy techniques and for the searching of new radiosensitive targets.Objective1 The effect of glutathione on HIF-1αexpression in hypoxic hepatoma cellsTo study the effect of reduced glutathione(GSH) on HIF-1αexpression and the mechanism,the changes of HIF-1αexpression and the regulation role of GSH were explored in hepatoma cells with physical and chemical hypxia treatment.All experiments were carried on human hepatocellular carcinoma HepG2 cells.2 The effect of glutathione on HIF-1αin hypoxic hepatoma cells after irradiationTo clarify the effect of glutathione on HIF-1αexpression in irradiated hypoxia cells and the regulating mechanism,the changes of GSH and HIF-1αexpression were observed in hypoxic cells exposed toγ-rays.Meanwhile,the radiosensitivities of hypoxic HepG2 cells were observed with the changes of HIF-1αexpression.3 Establishment and identification of radioresistant cell sublineTo further investigate the changes of HIF-1αexpression in radioresistant cells,the radioresistant monoclonal cell subline was induced and isolated by mimic clinic fractionated irradiation,temporary named as HepG2/R60.It was further identificated by cellular morphology,biology characteristics and radioresistance correlative genes.4 Change of HIF-1αexpression in radioresistant cellsThe radiosensitivity of HepG2/R60 cells was observed under hypoxia condition.The differences in the expression levels of HIF-1αand intracellular GSH contents between HepG2/R60 and HepG2 cells were compared to preliminary study the mechanism of change of HIF-1αexpression in radioresistant cells.Methods1 The effect of glutathione on HIF-1αexpression in hypoxic hepatoma cellsInhibitor of GSH synthesis-buthionine sulfoximine(BSO) with three tolerant concentrations (50μmol/L,100μmol/L,200μmol/L) and precursor of GSH-N-acetylcysteine(NAC) with 5mM concentration were pretreated to HepG2 cells.After hypoxia for 4 hours,the contents of intracellular GSH and oxidized glutathione(GSSG) and the ratios of GSH/GSSG were measured in HepG2 cells.Additionally,the changes of HIF-1αmRNA expression were observed by RT-PCR.Meanwhile,Western blot and immunocytochemistry (ICC) techniques were used to observe the expressions of HIF-1αprotein in hypoxic HepG2 cells.The changes of vascular Endothelial Growth Factor(VEGF) mRNA,as HIF-1αtargeting,were also detected by RT-PCR.In order to study the mechanism of the change of HIF-1αby GSH,2',7'-dichlorofluorescein diacetate(DCFH-DA) was used as labeling probe to measure mean fluorescene intensity in hypoxic cells through flow cytometry (FCM),followed by reflecting the levels of intracellular ROS.2 The effect of glutathione on HIF-1αin hypoxic hepatoma cells after irradiationNormoxic and hypoxic cells were exposed toγ-rays at different doses from ¹³⁷Cs source.Survival fractions(SF) of normoxic cells,hypoxic cells and hypoxic cells with BSO were obtained by clonogenic assay.Survival curves were then fitted with the single-hit multi-target model.Furthermore,oxygen enhancement ratio(OER),D₀ and D_q of these cells under different conditions were calculated to observe the changes of hypoxic HepG2 cells radiosensitivity and the effect of hypoxic cells radiosensitivity by BSO pretreatment.In addition,GSH and GSSG test kit was used to measure intracellular GSH and GSSG contents in irradiated hypoxia cells.The ratios of GSH/GSSG were then calculated in irradiated hypoxia cells.The changes of the levels of HIF-1αmRNA and protein in irradiated hypoxia cells were detected by RT-PCR,Western blot and ICC techniques,the expressions of VEGF mRNA were observed as well.Simultaneously,intracellular ROS levels were tested by FCM.3 Establishment and identification of radioresistant cell sublineHepG2 cells were irradiated byγ-rays at the dose of 2Gy 30 times repeatedly.Total absorbed doses were 60Gy.Monoclonal cell was selected by Difco soft-agar gel method, then extensively cultured and continuously maintained＞50 generations.Cellular morphology and ultrastructure of HepG2/R60 cells were observed by inverted microscopy and transmission electron microscopy(TEM),and their characteristics were compared with parental HepG2 cells.The growth curve of HepG2/R60 and HepG2 cells was drawn by counting cell number respectively.Population doubling time(PDT) was then calculated according to Patterson formula.Meanwhile,the detection of spontaneous apoptosis and seeding efficiency of two cells was accomplished by FCM and clonogenic assay respectively,followed by comparing the biologic difference between HepG2/R60 and HepG2 cells.Survival fractions of HepG2/R60 cells exposed toγ-rays at different doses were observed by clonogenic assay.Survival curves were then fitted with the single-hit multi-target model.Furthermore,the values of D₀ and D_q of cells were calculated to observe the radiosensitivity of HepG2/R60 cells.The levels of radioresistance correlative gene expressions in HepG2/R60 cells,after exposed to 2Gy irradiation,were also observed by RT-PCR,and then compared with parental HepG2 cells.4 Change of HIF-1αexpression in radioresistant cellsSurvival fractions of HepG2/R60 cells,under hypoxia condition,exposed toγ-rays at different doses were observed and calculated by clonogenic assay.After fitting the survival curve,the values of OER,D₀ and D_q cells were calculated to observe the radiosensitivity of hypoxic HepG2/R60 cells.The changes of HIF-1αmRNA and protein expression in hypoxic HepG2/R60 cells were detected by RT-PCR and Western blot technique,and then compared with parental HepG2 cells.Meanwhile,the changes of the levels of VEGF mRNA expression were observed in HepG2/R60 cells and HepG2 cells under hypoxia and normoxia condition.Such as above technique,intracellular GSH and GSSG contents in hypoxic and normoxic HepG2/R60 cells were tested and the ratios of GSH/GSSG were calculated to observe the change of total glutathione in HepG2/R60 cells.The levels of ROS in normoxic and hypoxic HepG2/R60 cells were further reflected by mean fluorescene intensities resulted from DCFH-DA labeling probe.Results1 The effect of glutathione on HIF-1αexpression in hypoxic hepatoma cellsThe contents of GSH in hypoxic cells were significantly reduced by different concentrations of BSO,followed by the reduction of the ratios of GSH/GSSG.The contents of GSH in hypoxic HepG2 cells with 100μmol/L BSO were decreased about 70%,whereas 5mM NAC might reverse the inhibition of GSH synthesis by BSO.According to the results from RT-PCR,the level of HIF-1αmRNA might be reduced in hypoxic HepG2 cells with 100μmol/L BSO pretreatment.Down-regulation of HIF-1αmRNA expression could be partly reversed by 5mM NAC treatment.The results from Western blot and ICC showed that the expressions of HIF-1αprotein in physical and chemical hypoxia HepG2 cells were significantly reduced by 50μmol/L BSO pretreatment.Similarly,NAC was able to increase the levels of HIF-1αprotein in hypoxic HepG2 cells by BSO pretreatment.And the levels of VEGF mRNA expression could be decreased with 100μmol/L BSO.Meanwhile, Intracellular ROS levels were up-regulated by different concentration of BSO treatment; however,the high-level of ROS induced by BSO could be inhibited by NAC treatment.2 The effect of glutathione on HIF-1αin hypoxic hepatoma cells after irradiationIt is observed that cells resistance to irradiation could be greatly induced by decreasing intracellular oxygen pression with physical or chemical methods.The data from clonogenic assay showed that the OER value of HepG2 cells was 2.71 and 2.17,respectively in physical and chemical hypoxia.The radioresistance of hypoxic cells was obviously reduced with the different concentrations of BSO pretreatment.At the range of 1-5Gy,the contents of GSH in hypoxic and normoxic cells were increased at 2 hours after irradiation.Moreover, GSH contents in irradiated hypoxia cells were higher than in irradiated normoxia cells. Additionally,the levels of GSSG contents were increased in both irradiated cells,resulting to the reduction of the ratios of GSH/GSSG.However,the degree of the reduction of GSH/GSSS ratios in irradiated hypoxia cells with steady state was lower than in irradiated normoxia cells in a dose-dependent manner.The different concentrations of BSO were used to pretreat irradiated hypoxia cells,leading to the reduction of intracellular GSH contents, the enhancement of intracellular GSSG contents and the decrease of GSH/GSSG ratios.The expressions of HIF-1αmRNA were up-regulated in irradiated hypoxia cells at 1-5Gy range. Meanwhile,it was shown that the the expressions of HIF-1αprotein in irradiated hypoxia cells were higher than in non-irradiated hypoxia cells.The expressions of HIF-1αin hypoxic cells after irradiation were further obviously down-regulated by the different concentrations of BSO pretreatment.And irradiation might enhance the expression of VEGF mRNA,sequently the up-regulation inhibited by BSO.The levels of intracellular ROS were enormously enhanced in irradiated normoxia cells.However,slightly reductions of intracellular ROS levels were found in irradiated hypoxia cells.Furthermore,the levels of intracellular ROS were significantly up-regulated in irradiated hypoxia cells with BSO pretreatment.3 Establishment and identification of radioresistant cell sublineCompared to parental HepG2 cells,the morphology of HepG2/R60 cells showed higher irregularity and monstrosity,the clearer appearance of cells and the slow growth of cell colony.Ultrastructural investigations showed that there were the increases of microvillus on the surfaces of HepG2/R60 cells with plenty of rough endo-plasmic reticulum,abundance of mitochondria and viable Golgi complex.Further observation found that there were PDT prolonged,the rates of spontaneous apoptosis decreased and seeding efficiencies enhanced in HepG2/R60 cells.Moreover,the radiosensitivity of HepG2/R60 cells was lower than that of parental HepG2 cells.After irradiation at different doses, survival cuver showed that HepG2/R60 cell was 1.35 and 1.28 times the values of D₀ and D_q of HepG2 cell.Additionally,the levels of three radioresistance correlative genes(Rad51, XRCC4 and BCL-2) were increased in HepG2/R60 cells by 2Gy irradiaiton.4 Change of HIF-1αexpression in radioresistant cellsAlong with the high-values of OER,D₀ and D_q,the radiosensitivity of HepG2/R60 cell was further decreased under hypoxia condition.The up-regulations of HIF-1αexpression and VEGF expression were found in hypoxic HepG2/R60 cells.There were not,under normoxia condition,higher GSH/GSSG ratios in HepG2/R60 cells compared with HepG2 cells;however,the significant differences of GSH/GSSG ratios were shown between the hypoxic HepG2/R60 and HepG2 cells.Furthermore,the down-regulaitons of intracellular ROS levels were observed in hypoxic HepG2/R60 cells. Conclusion1.The change of glutathione can influence the expressions of HIF-1αin hypoxic hepatoma cells and the mechanism may correlate with the clearance of intracellular ROS.2.The radiosensitivity of hypoxic hepatoma cells can be regulated by the levels of HIF-1αexpression.3.Ionize radiation can increase the levels of HIF-1αexpression in hypoxic hepatoma cells,which may be correlative with the changes of glutathione by irradiation.4.Radioresistant cell subline-HepG2/R60 was successfully isolated and established by fractionated irradiation.5.Radiosensitivity of HepG2/R60 cells was further reduced after hypoxia treatment.6.Up-regulations of HIF-1αexpression in hypoxic redioresistant cells may be correlative with the change of intracellular glutathione.