Studies On The Non-small Cell Lung Cancer Cell Death Induced By Radiotherapy And Related Mechanisms

Radiotherapy is one of most effective strategy for non-small cell lung cancer,ionizing radiation induce cancer cell death through a variety of signaling pathways.Studies on apoptosis have been more thorough and complete, but the type IIprogrammed cell death, autophagy in the process which ionizing radiation inducenon-small cell lung cancer cell death, the role that played has much controversy.Someresearchers reported, in human lung cancer cell line H460,when treated with3GY,ionizing radiation induced autophagy; when treated with5GY, ionizing radiation cannot induced autophagy.Early studing on non-small cell lung cancer mainly focus on the radiationsensitizing effect when drugs is added in radiotherapy, but when non-small cell lungcancer with different p53state treated with ionizing radiation, the death mode andrelated mechanism has not been reported.At same time, when non-small cell lungcancer treated with different radiotherapy plan, the death mode and relatedmechanism has also not been reported.In this study, MDC staining, colony formation, flow cytometry were used todetect autophagy and apoptosis when human non-small cell lung cancer cell linesH1299and A549were treated with ionizing radiation, while western boltting wasused to detect the change of, autophagy and apoptosis-related genes,the aim is toexplore the relationship between autophagy and apoptosis and possible mechanismsfor the new treatment programs for cancer providing theoretical basis.1. Radiation treatment results in cell viability of human non-small cell lung cancerH1299,H1299-P53,H1299-175H.To examine ionizing radiation can induce human non-small-cell lung cancer celldeath,we used H1299cell, H1299-p53cell, H1299-175H cell. After treatment ofdifferent dose of irradiation,CCK8assay results showed that all the cells undergogrowth inhibition,but the resultss have no significant difference.This result indicatessthat short-term killing effects is not obviously. We additionally performedcolony-formation assay for long-term killing effects. After irradiation in2GY,4GY,6GY compared with sham-irradiation group, cellviability in H1299cells as93%，74%，31%, cell viability in H1299-p53cells as73%,48%,32%, cell viability in H1299-175H cells as81%,56%,36%.These resultssuggest that ionizing radiation can induce H1299, H1299-P53,H1299-175H cell death.2. Ionizing radiation induced apoptosis and autophagy in non-small cell lung cancercells H1299, H1299-P53, h1299-175HApoptosis, autophagy and necrosis are three cell death ways.To determinewhether the growth inhibition by irradiation inhibitors was associated with apoptoticcell death, we treated cells for0GY,4GY,8GY with irradiation, and then examinednuclear morphology in DAPI-stained cells. with DAPI nuclear staining, cells withcondensed and fragmented nuclei are judged to be apoptotic，the result indicates thatthe cells underwent apoptosis. To determine whether the growth inhibition byirradiation inhibitors was associated with necrosis cell death, we treated cells for0GY,4GY,8GY with irradiation, and then examined nuclear morphology in PI-stained cells. with PI nuclear staining,cells with dual-core and nuclear fragmentationare judged to be neciosis，the result indicates that the cells underwent necrosis. Theformation of autophagic vacuoles was assessed by staining cells with MDC,which ledto apuncture staining pattern when autophagy is stimulated.H1299control cellspresented diffuse staining.Irradiation treatment of cells resulted in the appearance ofpunctuate staining structures.But in H1299-p53and H1299-175H cell lines,thestaining structures are decreasing in a dose-dependent manner.Autophagy inductioncan be determined by the conversion of LC3Ⅰ into LC3Ⅱ that is incorporated ontothe autophagosome.Irradiation was shown to induce the conversion of LC3Ⅰ intoLC3Ⅱ in a dose-dependent manner in H1299cell line,but in H1299-p53andH1299-175H cell line,LC3Ⅱ/LC3Ⅰis decrease in a dose-dependent manner. Toverify the above findings, we use the autophagy inhibitor3-MA treatment for H1299cells, using the apoptosis inhibitor Z-VAD on the H1299-P53and H1299-175Hcells,16hours after irradiation, MDC detection in H1299cells, compared with theuntreated group, autophagy in3MA treatment group was reduced. In the H1299-P53and H1299-175H cells, compared with the untreated group,Z-VAD-treated groupincreased autophagy.3. Autophagy is the main death method in H1299cell line when treated with irradiation, apoptosis is the main death method in H1299-p53,H1299-175H cell linewhen treated with irradiation.While irradiation was shown to induce both apoptosis、autophagy and necrosis,no direct evidence was presented verify to which method is main death method whentheated with irradiation in H1299cells and H1299-P53cells,H1299-175H cells. Toobtain objective quantification of apoptosis and necrosis,flow cytometry (FCM) wasemployed to detect the fluorescent intensity after tumor cells were double-stained withAnnexin V and PI at16h after irradiation.The necrosis rate of H1299cells in0GY,4GY,8GY group was14.3%,4.1%,7.6%; the necrosis rate of H1299-P53cells thenecrosis rate of was3.4%,2.4%,13.2%; the necrosis rate of H1299-175H cells thenecrosis rate of is was4.1%,5.4%,4.0%, indicating that necrotic cell death is not themain way to die.The apoptotic rat of H1299cells in0GY,4GY,8GY group was7.6%,5.2%,3.8%; the apoptotic rat of H1299-P53cells in0GY,4GY,8GY group was3.5%,5.9%,28.5%; the apoptotic rat of H1299-175H cells in0GY,4GY,8GY group was3.3%,4.9%,6.7%, indicating that apoptosis is the main cell death method inH1299-P53cells and H1299-175H cells, not the main cell death method in H1299cells.To further validate these results, western bloting was used to detectapoptosis-related protein BCL-2, AKT and autophagy-related protein Beclin1, Tigar.The results show that the expression of Beclin1, BCL-2increase in a dose dependentmanner, the expression of Tigar, AKT decrease in a dose dependent manner in H1299cells, suggesting that autophagy is the main death method when H1299treated withionizing radiation. In the H1299-P53and H1299-175H in,the expression of Beclin1,BCL-2decrease in a dose dependent manner, the expression of Tigar, AKT increase ina dose dependent manner, suggesting that apoptosis is the main death method whenH1299-P53cells and H1299-175H cells treated with ionizing radiation.4.MDM2/P53/P21pathway may be involved in ionizing radiation-induced non-smallcell lung cancer cell deathTo confirm whether apoptosis induced by ionizing radiation throughMDM2/P53/P21pathway, cells treated with0GY,4GY,8GY16hours latter celllysates were analyzed by western-bloting. In H1299cells, the expression of MDM2decrease in a dose dependent manner, due to lack of P53gene in H1299cells, P53andP21are not expressed before and after irradiation, indicating that MDM2/P53/P21pathway does not work in H1299cells when treated wuth ionizing radiation; in H1299-P53cells, the expression of MDM2decrease in a dose dependent manner, theexpression of P53and P21increase in a dose-dependent manner, indicating that theMDM2/P53/P21pathway involved in ionizing radiation-induced apoptosis inH1299-p53cells; in H1299-175H cells, the expression of MDM2decrease in a dosedependent manner, the expression of P53increase in a dose dependent manner, theexpress of P21can not be detected, indicating that the MDM2/P53/P21pathway donot involved in ionizing radiation-induced apoptosis in H1299-175H cells.5.The survival rate of A549when treated with different radiotherapy planIn order to detect ionizing radiation can induce non-small cell lung cancer A549cell death, we performed clonogenic assay. A549cells weretreated with1*2*5GY,2*5GY,2*5GY+CIS and10GY, the colony number was43,37,2and1,significantly less than the control group (73). Morphological changes was observedbyfluorescence microscope (visible light), after irradiation,the cell morphologicalchanges different, cell shrinkage, the nucleolus has also been varying degrees ofdamage, chromatin condensation, we observed the micronucleus in10GY group.These results can clearly state that ionizing radiation can inhibit the growth of A549,10GY is the most effective strategy for treating non-small cell lung cancer.6. Different radiotherapy plan can induce apoptosis in A549To determine whether radiation-induced apoptosis in A549cells in differentradiation groups, cleavage of caspase-3was examined by Western blotting cleavedcaspase-3was not detected at0Gy group, but the expression of cleaved caspase-3increase from1*2*5GY to2*5+cis GY group, and the expression of cleavedcaspase-3reached the lowest point in10GY group. These data suggest that2*5+cisGY is the effective strategy for inducing apoptosis. Annexin-V flow cytometricanalysis was used to confirm the above findings,6.6%,23.9%,25.4%,59.1%,45.1%ofAnnexin V positive cells were detected in A549cells that received0GY,1*2*5GY,2*5GY,2*5+cis GY,10GY, respectively.Finally, the characteristic apoptosis path check-point protein P21indicating cellcycle arrest that resulted in apoptosis were detected,the expression of p21protein wasnot detected in sham-radiation group and10GY group, but in2*5+cis GY group thep21protein reached the hightest point, and1*2*5GY group the expression of p21protein is highter than2*5GY group,indicates in1*2*5GY group the radiation cellsmain underwent cell cycle arrest than2*5GY group but finally result in apoptosis. These results suggest2*5+cis GY strategy is the most effective strategy in inducingcell cycle arrest in A549cells.7. Different radiotherapy plan can induce autophagy in A549To determine whether radiation-induced autophagy in A549cells in differentradiation groups,, we used MDC staining to detect the volume of autophagic vacuoles,the results showed that when A549treated with2*5GY+cis, the percentage ofautophagic vacuoles was8times higher than the sham-irradiation group, in2*5GYgroup the percentage of autophagic vacuoles was higher than in1*2*5GY groupbut still lower than in2*5GY+cis group, in10GY group the percentage ofautophagic vacuoles was the lowe, but still higher than sham–irradiation group.These results indicateddifferent radiotherapy plan can induce autophagy in A549,and2*5GY+cis is the most effective radiotherapy in inducing autophagy.To confirm the above findings,western boltting was used to detect autophagyrelated gene LC3、Beclin1、Tigar. In2*5+cis GY groupLC3Ⅱ/LC3Ⅰratioreached the highest, LC3Ⅱ/LC3Ⅰ ratio and Beclin1expression occurs a positivecorrelation with autophagy, in1*2*5GY group LC3Ⅱ/LC3Ⅰis higher than that in0GY group but lower than that in2*5GY group,in10GY group LC3Ⅱ/LC3Ⅰwas the lowest. The expression of Beclin1in2*5+cis GY group reached the highest,in1*2*5GY group the expression of Beclin1was higher than in0GY group butlower than in2*5GY group, in10GY group the expression of Beclin1was thelowest. LC3Ⅱ/LC3Ⅰ ratio and Beclin1expression and autophagy occurs apositive correlation, LC3Ⅱ/LC3Ⅰ and Beclin1show the same trend. Tigar isautophagy inhibitor, the expression of Tigar in2*5+cis GY group was the lowest,the expression of Tigar in2*5GY group higher than in0GY group but lower than in1*2*5GY group, in10GY group the expression of Tigar reached highest. Theseresults indicate that2*5GY+cis is the most effective radiotherapy in inducingautophagy in A549.In this study,wy non-small cell lung cancer with different p53state treated withionizing radiation, the death mode and related mechanism were studied.At same time,non-small cell lung cancer treated with different radiotherapy plan, the death modeand related mechanism were studied too.In order to explare the new treatmentprograms for cancer and provide related theoretical basis.