Radiosensitivity Of Non-Small Cell Lung Cancer Cells Regulated By The Crosstalk Of HIF-1? With BCL-2

Objective: The morbidity and mortality of patients with lung cancer are the highest in those with tumors in China.In aging society,human will face the huge disturbance of lung cancer in health.Most of the patients with non-small cell lung cancer(NSCLC)have been in advanced stage when they were diagnosed,resulting in rather low 5-year survival rate(no more than 2%).Radiotherapy is one of the main treatments for NSCLC,but outcome of patients often is poor owing to the resistant responsiveness of hypoxic cells in NSCLC tissue to traditional radiotherapy.Hypoxia inducible factor-1?(HIF-1?)is an important transcription factor which closely related to the malignant phenotype of tumor.HIF-1? has capability to promote tumor cells generating adaptive response to hypoxic environment and tolerance to radiotherapy by regulating downstream target genes.As an important anti-apoptosis gene,B-cell lymphoma-2(BCL-2),which shows high level of expression in several malignant tumors,can keep survival of tumor cells in various adverse conditions.To radiotherapeutics,tumor cells produce radioresistance by BCL-2 inducing the inhibition of cell apoptosis.In present study,H1299 cells,one human NSCLC cell line,was used as the experiment model in order to observe the changes in radiosensitivity of irradiated NSCLC cells via HIF-1?/BCL-2 signal pathway.Methods: In this study,the recombination plasmids with high expression of mutation HIF-1? and wild HIF-1? were transfected into H1299 cells to establish two new cells respectively,namely H1299/M-HIF-1? and H1299/W-HIF-1? cells.Meanwhile,H1299 cells with free vector transfection(F cells)and parental H1299 cells were used as control.Cobalt chloride(CoCl2),one mimetic hypoxic rea gent,was used to induce expression of HIF-1?.3-[5'-hydroxymethyl-2'-furyl]-1-b enzyl indazole(YC-1)and ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate(HA14-1)were used to targeting inhibit HIF-1? and BCL-2 respectively.The levels of HIF-1? and BCL-2 proteins in the cells pretre ated by different methods were detected by Western Blot.Furthermore,four cells were exposed to irradiation of X-ray in 0,1,2,3,5,7Gy through different pre treatments.The changes of HIF-1? and BCL-2 expression in irradiated cells were also detected through Western Blot.Additionally,the survival fractions of cells were calculated using a clonogenic assay,and then survival curves were fitted wi th to obtained D0 values of different irradiated cells.Finally,the cell proliferatio n assay was used to determine the population doubling times(PDTs)of these ce lls with different treatments.Results: The results showed that under normoxic condition,the increasing levels of HIF-1? and BCL-2 expression were found in H1299/M-HIF-1? cells through Western Blot assay.After the treatment of chemical hypoxia,the up-regulation of HIF-1? and BCL-2 expressions were exhibited in H1299/W-HIF-1?,H1299/F and parental H1299 cells,H1299/W-HIF-1? cells which showed the highest levels.Through YC-1 treatment,concomitant with down-regulation of HIF-1? expression,the levels of BCL-2 proteins were decreased in four cells.And BCL-2 expression could be obviously inhibited by H14-1 treatment.After four cells were exposed to X-rays,the increasing levels of intracellular HIF-1? and BCL-2 proteins were observed under normoxic condition.However,up-regulation of HIF-1? and BCL-2 expression was absent in the cells exposure to irradiation in low or middle doses by CoCl2 or YC-1 pretreatment.Moreover,there was one significant reduction of HIF-1? and BCL-2 proteins in those cells suffering to high doses of irradiation.Additionally,treatment of the combination of irradiation with HA14-1 could evidently inhibit the accumulation of HIF-1? and BCL-2 proteins in the irradiated cells.The results from clonogenic assay showed that under normoxic condition,H1299/M-HIF-1? cell has one higher value of D0 compared with other cells,which H1299/M-HIF-1?,H1299/W-HIF-1?,H1299/F and parental H1299 cells were 4.375,3.845,3.311 and 3.021 Gy respectively.Chemical hypoxia could further raise the D0 values of four cells,which H1299/M-HIF-1?,H1299/W-HIF-1?,H1299/F and H1299 cells were 4.787,4.679,3.877 and 4.07 Gy respectively.Further calculation showed that values of the oxygen enhancement ratios(OERs)of H1299/MHIF-1?,H1299/W-HIF-1?,H1299/F and H1299 cells were 1.094,1.217,1.171 and 1.347 each.After YC-1 was used to treat these cells,values of the sensitivity enhancement ratios(SERs)of H1299/M-HIF-1?,H1299/W-HIF-1?,H1299/F and H1299 cells were 1.225,1.128,1.146 and 1.457 each.Likewise,HA14-1 could enhance the sensitivity of four cells,which SERs of H1299/M-HIF-1?,H1299/W-HIF-1?,H1299/F and H1299 cells were 1.4,1.828,1.335 and 1.221 respectively.To the changes of four cells growth,under normoxic condition,PDTs of irradiated cells were extended with enhancement of irradiation doses;conversely,extension of the PDTs of irradiated cells were not observed in hypoxic microenvironment.In addition,YC-1 did not influence PDTs of hypoxic cells exposure to irradiation in low doses.HA14-1 could significantly increase PDTs of hypoxic cells exposure to irradiation,but increasing degree of the PDTs of hypoxic cells was lower than that of normoxic cells.Conclusion: The expression of HIF-1? can be up-regulated in hypoxic NSCLC cells,followed by the generation of refractory responsiveness to irradiation of X-rays.The mechanism may be involving with the activation of HIF-1?/BCL-2 signal pathway.Simultaneously,low linear energy transfer(LET)of ionizing radiation can promote the accumulation of HIF-1?,and then induce the resistance of NSCLC cells to radiation via the trigger of HIF-1?/BCL-2 signaling.