Radiosensitizing Effect Of Silibinin On The Model Of Lung Cancer In Advanced Stage And Mechanism

Objective:According to investigation,lung cancer occupies the highest percentage in the morbidity and mortality of malignant tumors in our country.There are two-thirds of patients with non-small cell lung cancer?NSCLC?in advanced stage,which shows one weak 5-year survival rate.Radiotherapy is considered as one of treatments for NSCLC.However,due to hypoxic cells in tumor tissues forming the refractory effect to ionizing radiation,the curative effect of lung cancer in advanced stage is siginificant reduced.¹²C ions for lung cancer show more effectiveness in local control rate and five-year survival rate for tumor,compared with low linear energy transfer?LET?.Hypoxia inducible factor-1??HIF-1??plays one critical role in the resistance of hypoxic tumor cells to radiotherapy by promoting the transcription of B-cell lymphoma-2?BCL-2?.In present study,the models of Sprague-Dawley?SD?rats burdened lung cancer in advanced stage established by walker-256 ascites tumor cells were used to observe the regulation of silibinin to HIF-1?/BCL-2 signaling in animals exposure to radiation and suival life of animals.Meanwhile,H1299 cells,one human NSCLC cell line,were utilized to investigate the radiosentization of silibinin on NSCLC cells and mechanism.Methods:Total experiments were divided into two parts,including in vitro and in vivo.H1299 cells with high expression of HIF-1?plasmid were used in this experiment.H1299/M-HIF-1?cells with the mutant HIF-1?plasmid were over-expressed in normoxic environment.H1299/W-HIF-1?cells with wild-type HIF-1?plasmid were over-expressed under hypoxic condition.H1299 cells with free pcDNA3.0 plasmids?H1299/F?and parental H1299 cells were served as control in vitro.Western Blot assay was utilized to detect the expression of HIF-1?and BCL-2 protein in 4 types of cells under normoxic and hypoxic environment or by silibinin pretreated.Furthermore,the expression of HIF-1?and BCL-2 was measured after irradiation of X-ray or¹²C ions in different doses.Meanwhile,after silibinin treatment,the survival rates of 4 types of cells by 2Gy of X-ray irradiation were measured by MTT method.5-week-old male SD rats were used the present experiment in vivo.The models for advanced lung cancer in SD rats were established by injecting walker-256 ascites tumor cells through tail vein,and healthy rats were control.After YC-1,HA14-1 and silibinin treatment,immunohistochemistry was used to detect the proteins of HIF-1?and BCL-2in SD rat burdened lung cancer.Moreover,the levels of HIF-1?and BCL-2 were observed in irradiated tissues.3-[50-hydroxymethyl-20furyl]-1-benzyl indazole?YC-1?,one HIF-1?-targeted inhibitor,was used in present experiment and each rat was administrated in 10 mg/kg of dose.2-Amino-6-bromo-?-cyano-3-?ethoxycarbonyl?-4H-1-benzopyran-4-acetic acid ethyl ester?HA14-1?,one BCL-2-targeted inhibitor,also used in this study and its dose was 25 mg/kg for each rat.The administration dose of silibinin was 35 mg/kg for each rat.Right lung of animal model was exposed to X-rays in 15Gy of fractionated irradiation owing to evading heart exposure to ionizing radiation.Prior to 12 hours,the different drugs were administrated in rats exposure to irradiation.Meanwhile,the survival time of rat burdened lung cancer with different treatment was observed in this study.Results:The results from Western Blot assay showed that the levels of HIF-1?and BCL-2 were reduced n 4 types of cells pretreated with silibinin.However,their expression was found in those cells after irradiation.The increasing levels of HIF-1?and BCL-2 by radiation were inhibited again by silibinin treatment.Sensitive enhancement ratios?SERs?of silibinin to H1299/M-HIF-1?,H1299/W-HIF-1?,H1299/F and H1299 cells were 1.32,1.42,2.13,2.15,respectively.In addition,¹²C ions could interrupt HIF-1?/BCL-2 signaling in four types of cells.The results of immunohistochemistry showed that the expression of HIF-1?and BCL-2 was decreased significantly in tumor tissue of SD rats pretreated by YC-1,HA14-1 and silibinin.Of note,there was the significant elevation of HIF-1?and BCL-2 protein in tumor tissues of the irradiated animal and such increasing levels of HIF-1?and BCL-2 protein were reduced again by three inhibitors,including YC-1,HA14-1 and silibinin.Meanwhile,silibinin could observably extend the survival life of irradiated tumor-bearing rats.Conclusion:Silibinin can significantly inhibit the HIF-1?/BCL-2 signaling pathway in NSCLC cells,and increase the radiosensitivity of NSCLC cells.In the irradiated animal model of advanced lung cancer,silibinin also significantly reduced the high expression of HIF-1?and BCL-2 in radiation-induced tumor tissues and effectively extended the survival life of irradiated rats.In addition,¹²C ions showed one strong blocking effect on the HIF-1?/BCL-2 signaling,which partially explained the lethal effect of¹²C ions on lung cancer cells.