Study On The Role Of Apoptosis Pathway In The Radiation Resistance Of Lung Cancer And Osteosarcoma

Radiotherapy is an effective means of treating cancer, ionizing radiation candirectly kill tumor cells, induce apoptosis or autophagy. Apoptosis is a programmedcell induced by external factors, the study on apoptosis by domestic and foreignscholars is variable and thorough. Apoptotic process involves multiple pathways,including the intrinsic apoptotic pathway (ie the mitochondrial apoptotic pathway),extrinsic apoptotic pathway (ie death ligand/receptor pathway), endoplasmicreticulum and autophagy, the different pathway can influence each other, such asexogenous and endogenous pathway may cross each other, and then magnify theapoptotic effects.Current research on cancer drugs concentrated mainly on the effect drugs ontumor cells or the increased radiosensitivity by drugs, while there are few studies ontumor cells with different radiosensitivity, particularly on the mechanism of apoptosisof radiation-resistant cancer.This study is aimed to explore the molecular mechanisms of the apoptosispathway of radiation-resistant tumor cell, and to provide a theoretical basis forimproving the efficacy of cancer radiotherapy.Objective:To observe the changes of molecular associated with the apoptosis pathway oflung cancer and osteosarcoma cell after radiation, Clarify the mechanism of apoptosisof radiation-resistant tumor cells.Methods:We select non-small cell lung cancer line (radiation-resistant A549cells andradiation-sensitive H460cells) and osteosarcoma cell line (HOS) to research. Andconstruct shNRP1-A549cell model stably expressing by means of retroviraltransfection. Flow cytometry is used to test the apoptosis and cell cycle of the tumor cells after the effects of ionizing radiation and meanwhile we use western blot andqRT-PCR to detect the expression of apoptosis-related mRNA and protein,and toinvestigate the mechanism of apoptosis pathway in radiation-resistant tumor cellsResults：1.Changes in the rate of apoptosis of radiation-resistant cells after radiation.Compared to the the sham control group, the apoptosis rate of four kinds of cellsincreased. The level of apoptosis of radiation-sensitive H460cells and shNRP1-A549cells increased significantly, compared to A549cells and HOS cells, indicating thatthe radiation resistance of A549cells and HOS cells was higher than that of H460cells and shNRP1-A549cells, and the increased level of apoptosis rate of HOS cellswas the least (significant lower than A549cells), indicating that the radiationresistance of HOS cells was the the strongest.The cell cycle progression showed that,the G2/M phase proportion of A549and shNRP1-A549cells increased comparedwith sham control group after irradiated, and the increased level of shNRP1-A549cells are more evident than the parental A549cells, indicating that the interference ofNRP1gene induced obvious G2/M phase arrest, which enhanced its sensitivity toradiation.2.The changes of related molecules of intrinsic apoptosis pathway in theradiation-resistant tumor cells on the levels of transcription and translationAfter irradiated by10Gy X-ray, The transcription level of Caspase-9gene inA549cells was significantly lower than sham control group, while that in HOS cellshad declined, but the change was not significant, the transcript level of Caspase-9gene in shNRP1-A549and H460cells was significantly higher than the sham controlgroup. The transcript level of pro-apoptotic Bax gene was increased in A549andH460cells after irradiation, but the transcript level of Bax gene in HOS cells waslower than the sham control group.The increased rate of radiation resistant A549cellswas only2-3times than the sham control group, while that in H460cells reached35times higher than the sham control group. The transcription level of anti-apoptoticBcl-xL gene in A549and HOS cells was higher than the sham control group,but thatin H460cells did not display significant change.The experimental showed that ionizing radiation could activate the intrinsic apoptosis pathway of radiation-resistanttumor cells,where the anti-apoptotic gene Bcl-xL plays a key role.After irradiated by10Gy X-ray, the changes of apoptosis-related protein levelsin the endogenous pathway were not exactly the same with that of transcription, thelevel of anti-apoptotic protein Bcl-xL expressed in H460cells was higher than shamcontrol group, while in the HOS cells, it hardly expressed,and that in shNRP1-A549cells did not change significantly; the expression level of Bax mRNA and protein inshNRP1-A549cells was higher than sham control group,but the expression level ofBax protein was lower than the parent-A549cells. The results suggested that in theapoptosis mechanism of radiation-resistant tumor cell, the endogenous apoptosispathway proteins may regulated at the post-transcriptional level.3.The changes of related molecules of exogenous apoptosis pathway in theradiation-resistant tumor cells on the levels of transcription and translationAfter irradiated by10Gy X-ray, the expression of related genes in the exogenousapoptotic pathway of radiation-resistant tumor cells was cell-specific, such as theexpression of the related genes in lung cancer cells was not consistent with theexpression of that in osteosarcoma cells, but in lung cancer cells the expression ofCaspase-8and Caspase-6gene in radiation-resistant A549cells and radio-sensitivityH460cells were on the contrary, while the levels of Caspase-3transcript areconsistent. Suggesting that the exogenous apoptosis pathway plays an important rolein the radiation-resistance of lung cancer cells, but there was no apparent effect in theradiation sensitivity of osteosarcoma (HOS) cells. After irradiated by10Gy X-ray, theexpression of apoptosis-related protein in the exogenous pathway was consistent withthe levels of transcript in radiation-resistant tumor cells. In further study,we found thatafter the interference of radiation-resistance-related genes--NRP1, the expression ofrelated protein in exogenous apoptotic pathway of irradiated shNRP1-A549cellschanged similarly with that of radiation-sensitive H460cells, which further confirmedthat the extrinsic apoptotic mechanisms play an important role in the radiationresistance of lung cancer cells.4.Changes of genes of enzymes related with apoptosis on levels of transcription and translation of radiation-resistant tumor cell.In this study, we tested the expression of apoptosis-related enzymes to explore itsmechanisms. The results showed that after irradiated by10Gy X-ray,inradiation-resistant A549and HOS cells, the level of transcription of PARP werebasically the same, while that of H460cells was lower than the sham controlgroup.The expression trend of Cleaved PARP and PARP protein in A549and HOScells was almost the same.The expression of PARP protein in H460cells wassignificantly lower than the sham control group, while the expression of CleavedPARP protein increased significantly. Cleaved PARP protein did not express inshNRP1-A549cells, but the expression level of cleaved PARP in parent-A549cellswas higher than that in the sham group, and the level of PARP protein inshNRP1-A549cells was lower than the parent-A549cells.Suggesting that after theinterference of expression of NRP1,the expression of PARP genes was inhibited, andso that the cells were more sensitive to ionizing radiation. This result indicate thatDNA repair protease PARP plays a key role in the apoptosis mechanism of portion ofthe radiation resistant tumor cells. The transcription level of MMP-2gene in radiationresistance and radiation sensitivity cells increased, but the protein level was lower,indicating that the expression of the protein was regulated in the post-transcriptionallevel, Changes in the level of transcription and translation of MMP-2gene in radiationresistance and radiation sensitivity of cells was almost the same, indicating that thechanges of MMP-2mRNAand protein expression had no obvious correlation with theradiosensitivity of tumor cell.Conclusion:1.After10Gy X-ray, the apoptosis rate of radiation and radiation-resistant lungwas higher than that in sham group cancer cells insensitive osteosarcoma cell, but theincrease rate is lower than the radiation-sensitive cancer cells2.In the apoptosis process of radiation-resistant tumor cells, endogenousapoptosis pathway related molecules Caspase-9, Bax and anti-apoptotic proteinsBcl-xL play a key role.3.The apoptosis pathway related exogenous molecule Caspase-6plays a important role in radiation-resistant lung cancer cell，but the role of Caspase-8andCaspase-6was not evident.4.The activity of PARP enzymes was inhibited in radiation-resistant tumor cells,the changes of MMP-2mRNA and protein expression had no obvious correlation withthe radiosensitivity of tumor cell.