The Roles Of Autophagy In The Killing Effect Of Radiation Combined With Cisplatin In Non-small Cell Lung Cancer

Radiation therapy and chemotherapy is an effective method for clinical treatmentof non-small cell lung cancer, ionizing radiation and chemotherapy drugs can led tothe death of non-small cell lung cancer through a variety of signalingpathway.Autophagy is also named as typeⅡprogrammed cell death, in this processcells degrade its proteins and organelles, and recycle amino acids and proteins toachieve the goal of maintenance of steady-state process under stress or damage byforming autolysosome.Autophagy is associated with a variety of human diseases,such as cancer, heart muscle disease, neurasthenia, and hepatic disease,gastrointestinal disorders, etc.Autophagy can be formed through adaptive response tomaintain cell survival; it can also lead to autophagic cell death when excessiveautophagy occurs.This study focus on the roles of autophagy in non-small cell lungcancer cells and its mechanism,and provide theoretical basis for clinical treatmentplan.ObjectiveTo explore the role of Autophagy in different radiation scheme or combination ofcisplatin with radiation in the treatment of non-small cell lung cancer and its possiblemechanism.Methods(1) Cell lines:human non-small cell lung cancer cell lines,A549and H1299wereused.(2)Animal model preparation:the concentration of the logarithmic phase Lewislung cancer cells were adjusted and1×106cells(0.2ml) were injected into right hindleg in C57BL/6mice.(3) H1299cells were divided into control group (0Gy) and theirradiation group (4Gy,8Gy);A549cells were divided into control group (0Gy) and theirradiation group (2Gy,8Gy).The tumor-bearing mice were divided into5groups: thecontrol group (0Gy);routine exposure group(2Gy everytime, five days,10Gy in total);super segmentation groups(1Gy everytime,2times/day,4h intervals, five days,10Gy in total);joint group(2Gy everytime, five days,10Gy in total, giving mice celiacinjection of cis-platinum for the first time before irradiation (3mg/kg));singleexposure groups(give10Gy irradiation at a time).(4) index detection: MDC stainingand flow cytometry were used to detect the changes in autophagy and apoptosisrespectively, MTT assay was used to detect cell survival,western blot was used to testthe changes of the expression of BECLIN1and MAPLC3,immunohistochemicalmethod was used to detect the changes of related gene expression.Results1Ionizing radiation-induced autophagy in H1299and A549cellsMDC staining and flow cytometry was performed in this experiment afterdifferent treatments (0Gy,4Gy,8Gy).It was found that green autophagy puncta wasappeared in the cells, the number of autophagic vesicles significantly increased withthe increase of ionizing radiation (P<0.05), ionizing radiation can induce H1299autophagy.Flow cytometry test showed that the necrosis rate of H1299cells in eachgroup were4.2%，7.5%，14.3%;Apoptosis rate was3.4%，5.3%,7.7%,it has significantdifference between groups (P <0.05), suggesting ionizing radiation associated withH1299cell necrosis and apoptosis.2The effect of cisplatin on the survival and autophagyTo detect the influence of cisplatin on A549cells, MTT method wasperformed.when the concentrations of cisplatin was less than5μmol/L, it has noobviously effect on cell survival. With the increase of concentration of cisplatin, thesurvival of A549cells was reduced obviously.Cisplatin can affect autophagy of A549cells.The MDC staining was used afterdifferent treatments (0Gy,2Gy×5,2Gy×5+cis).The results showed that MDC positivecells in different experimental groups were obviously higher compared with thecontrol group,and cisplatin treatment group is higher than2Gy×5group(P<0.05).Suggesting that the effects of cisplatin on the survival of non-small cell lungcancer might associate with autophagy. 3Different radiation and combined radiation effects on mice with lungcancer tumor sizeConcentration of the logarithmic phase Lewis lung cancer cells was adjusted into1×106,0.2ml of cells were inject into right hind leg in C57BL/6mice.The mice weredivided into several groups: control group (0Gy);routine exposure group:2Gyeverytime,five days,10Gy in total;hyperfractionation groups:1Gy everytime,2times/day,4h intervals, five days,10Gy in total;joint group:2Gy everytime, fivedays,10Gy in total, giving mice celiac injection of cis-platinum for the first timebefore irradiation (3mg/kg);single exposure groups: give10Gy irradiation at atime.Since then, the daily measurement of tumors size changes was performed.6daysafter radiation the mice were sacrified,the size of tumor was measured, joint group <super segmentation group <conventional segmentation group <control group. Inaddition, cisplatin combined with radiotherapy is the most effective methodscompared with the other groups.4The changes of autophagy related proteins in NSCLC cells after differentradiation and radiation combined with chemotherapy treatmentTo confirm autophagy played a role in the process of radiotherapy.The totalproteins were extracted and western blot was used to detect Beclin and MAPLC3expression.Compared with control group,the expression of MAPLC3in thehyperfractionation group、 the routine exposure group and joint group were obviouslyhigher, increased by1.34,2.76, and1.59times respectively, and the expression levelof beclin1increased2.26,1.5, and1.6times respectively. The results indicate thatautophagy is involved in the process of killing tumor cells, and played an importantrole.Tumors were also fixed by10%formaldehyde.And immunohistochemicalstaining was made.A large amount of brown granules can be seen in the cells (F=34.04, P <0.001), compared with other treatment group it was most pronounced in thejoint radiotherapy group.The protein expression of MAPLC3II was significantlyincreased, and4.2times for the control group,3.2times for the control group in theroutine exposure group.PI3KIII and BECLIN1protein expression was increased, and the most obvious difference among which the expression level of combinedradiotherapy and3.6times as the control group (F=19.79, P <0.001; F=3.509, P <0.05).Autophagy plays an important role in the treatment of displacementof transplanted tumor in the process of radiotherapy, especially in the radiationcombined with chemotherapy treatment.PI3KIII and BECLIN1protein expressionwere significantly increased, prompting the role of PI3KIII/BECLIN1in autophagyinduction.Conclusion1, Ionizing radiation and chemotherapy can induce autophagy in non-small celllung cancers, A549and H1299cell lines.2, Conventional radiotherapy and radiotherapy combined cisplatin can obviouslyinhibit displacement of transplanted tumor of non-small cell lung cancer tumorgrowth.3, Conventional radiotherapy and radiotherapy combined cisplatin play a rolein tumor suppression through autophagy.4, The effect of tumor destruction for divided radiotherapy is better thanconventional radiotherapy.