An Experimental Study On The Mechanism Of A549 Cell-line Radiotherapy Sensitivity By Autophagy Expression Modulation In NSCLC

The tumorigenesis of lung cancer is a very complex procedure.Many researches were carried out to find effective anticancer methods, which could recover the impairment of organelles at early development of cancer,or eliminate mutated and damaged organelles while lung cancer cells formed ,or even reversed cancer cells to normal cells.Datas from recent years indicated that the degradation and recycling of organelles were mainly depends on autophagy.Autophagy is a very primitive biological phenomena in the course of life evolution, which existed widely in plants and other lower creatures.Autophagy is also an important way in which protein degradated and organelles eliminated to keep homeostatic,it is a process to get rid of cancer cells in mammals.Many micromolecule protein participated in autophagy,what we understood well is Beclin1 gene and Beclin1 protein,microtubule-associated protein light chain3(MAPLC3) is another mark for autophagysome immunodetection. The basal autophagy level of cells keep lowly to maintain homeostatic situationin usually, autophagy level is up-regulated rapidly when crisis come. For example: growth factor deficiency,starvation,cell reconstruction,or too much damaged cell organelle or metabolic waste.Twe regulater of nutrition sensor regulates autophagy:target of rapamycin(TOR) kinase and eukaryotic initiation factor 2 (eIF2 ) kinase Gcn2. TOR shut off autophagy signal when nutrition adequacy;eIF2 start autophagy signal when starvation. There are many genes lies down-stream of TOR kinase, which participate autophagysome development,confluence,maturement,recycling. Rapamycin is antagon for TOR receptor and up-regulate autophagy,while 3-methyladenine 3MA inhibit autophagy formation.Autophagy level is closely related cancer cells development, transformation, and sensitivity of therepy.At early stage of cancer cell formation, provoked autophagy help to eliminate damaged cell organelle,and degrade the noxious substance in endochylema(eg: peroxidate)as well, pretect normal cells from cancerization.Autophagy level modulation might be cooperation or rivalry with chemotherapy to cancer cells compares to single use of chemotherapy drugs, apoptosis rate of cancer cells fluctuates with autophagy level.Down-regulation of autophagy level would make organelles elimination breakdown and accelerate cell death to improve therapeutic efficacy.In order to investigate the relationship between autophagy and NSCLC and the possibility sensitization effect of radiotherapy after autophagy expression alteration,we detected the autophagy-related gene expression level of clinical NSCLC samples to elucidate the relationship of autophagy level and NSCLC generation and development; tested autophagy and its up-stream signal conduction alteration of lung cancer cell line A549;investigated changes of radiotherapy sensitivity and autophagy correlated protein and some drug resistance gene when autophagy level of A549 altered. We also detect the A549 cell cycle and apoptosis rate to explain its mechanism to explain the association between autophagy and apoptosis.In our research, Fresh 72 NSCLC tissue samples were obtained immediately from the resected specimens of NSCLC patients who underwent pulmonary lobectomy at the department of Thoracic Surgery, Union Hospital, between Apr 2006 and Oct 2006. The protein expression of Beclin1 and MAPLC3 in tumor tissues,adjucent noncancerous tissues,and normal tissues from 72 specimens of NSCLC cases were observed by immunofluorescence staining and Western blot, and mRNA expression were tested by reverse transcription polymerase chain reaction (RT-PCR). Then, we cultivated lung cancer cell line A549 and detect autophagy related protein and mRNA expression after radiation exposure,at the same time, the upper stream gene akt and mTOR were tested. We altered autophagy expression with 3MA or rapamycin, detected the inhibition ratio and apoptosis rate in each group together with the cell cycle changing; cell colony essay was used to detect different degree ofγ-ray suppression at distinct autophagy levels; MAPLC3,pakt, mTOR,COX2 protein expression were detected by Western blot; MAPLC3,survivin,mTOR,Bcl-2,EIF-4E mRNA were detected by RTPCR. Immunofluorescence staining of Beclin1 and MAPLC3 was done for positive rate detection. All results were combined to analyze the relationship between autophagy and apoptosis.Immunofluorescence staining and West blot and RT-CCR indicated that the expression of Beclin1 and MAPLC3 in NSCLC was significantly lower than that in adjucent non-cancerous tissues and normal tissues .γ-ray could up-regulate the autophagy related gene of A549 which is time-dependent. The regulation effect of 3MA and rapamycin were observed, 3MA was antagonism while rapamycin was in coordination with radiotherapy. The enhanced inhibition effect ofγray to A549 could be connected with the alteration of apoptosis related protein and cell cycle blocken by 3MA.Our research group have done series of research about the relationship between autophagy and lung cancer. Also some original discovery were found which encourage us keep hardworkingon advanced work in the future. PARTⅠExpression of Autophagy-Related Beclin1 and MAPLC3 Gene in Non-Small Cell Lung CancerOBJECTIVE:Some studies have indicated the down-regulation of autophagy-related gene might result in tumorigenisis. This study was to investigate the expression and significance of autophagy-related gene Beclin1 and Microtubule-associated protein 1 light chain 3 (MAPLC3) gene in human non-small cell lung cancer (NSCLC).METHODS: The protein expression of Beclin1 and MAPLC3 in tumor tissues,adjucent noncancerous tissues,and normal tissues from 72 specimens of NSCLC cases were detected by immunofluorescence staining and Western blot, and mRNA expression were tested by reverse transcription polymerase chain reaction (RT-PCR).RESULTS: Immunofluorescence staining showed that the expression rate of Beclin1 in NSCLC (8.3%) was significantly lower than that in adjucent non-cancerous tissues (100%) and normal tissues (100%) (Χ2=199.40, p=0.00); the expression rate of MAPLC3 in NSCLC(13.9%) was also significantly lower than that in adjucent non-cancerous tissues (100%) and normal tissues (100%) (Χ2=182.75,P=0.00).The mRNA of Beclin1 levels significantly lower in NSCLC (1.30±0.07)than that in adjucent noncancerous tissues(1.69±0.10) and normal tissues (1.67±0.08)(F=6.6, P= 0. 00); the mRNA level of MAPLC3 in NSCLC (4.55±0.31)also significantly lower than in adjucent noncancerous tissues(6.73±0.37 ) and normal tissues ( 6.90±0.37 )(F=14.1,P=0.00). Western blot indicated the similar results that there was significantly down-regulation of the protein level of Beclin1 and MAPLC3 in NSCLC than that of in adjucent noncancerous tissues and normal tissues (Beclin1:3.49+0.29, 5.31+0.46, 6.33+0.58, F=9.73, p<0.01; MAPLC3:2.43+0.26, 3.12+0.28, 3.41+0.30, F=3.22, p=0.04).。CONCLUSIONS:The protein and mRNA expression of Beclin1 and MAPLC3 are significantly lower in NSCLC than in adjucent noncancerous tissues and normal tissues, there is no significantly difference in adjucent noncancerous and normal tissues. PARTⅡ:The effect of radiotherapy to autophgy-related gene and signal transduction path up-stream autophagy of A549OBJECTIVE:To investigate the effect ofγ-ionising radiation on lung cancer cell line A549, we detected the cell cycle and the expression of autophagy-related gene Beclin1 and MAPLC3 of A549 at different time spot after irradiation, and the PI3K/akt signal transduction path upstream of autophagy .METHODS:Control group and experimental groups at different time spot after irradiation were setted up.In each group, Beclin1,MAPLC3,pakt,mTOR positive cells were counted after immunofluorescence staining by flow cytometry or under laser confocal microscope, cell cycle was detected after PI staining and by flow cytometry. Beclin1,MAPLC3,pakt,mTOR mRNA and protein were detected by RT-PCR and Western blotRESULTS: Immunofluorescence staining showed that the expression rate of autophagy related gene in A549 was low(Beclin1:0.13±0.04,MAPLC3:0.25±0.05),the basal expression rate of signal transduction protein up stream of autophagy was low too(mTOR:0.09±0.03,pakt:0.03±0.01),positive rate ascensus after irradiation and was stable after 20h(Beclin1:0.39±0.09; MAPLC3:0.59±0.12; pakt:0.09±0.02,P<0.05).With time increasing after irradation, apoptosis rate raised(1.5% and 6.3% before and 20h after irradation),more G2-M stage cells(24.9% and 53.0% before and 20h after irradation)less S stage cells(22.3% and 3.3% before and 20h after irradation) were observed. Similar results were got when Beclin1,MAPLC3,pakt,mTOR were detected by flow cytometry after Immunofluorescence staining as under laser confocal microscope. Beclin1,MAPLC3,pakt protein and mRNA level of A549 raised after irradiation and more obviously with time-lapse,while akt and mTOR protein and mRNA level was stable.CONCLUSIONS: Afterγray irradiation,autophagy related gene Beclin1 and MAPLC3 levels were upregulated and more obviously with time-lapse, there also be more G2-M stage cells and higher apoptosis rate but less S stage cells after irradiation, pakt upstreams autophagy level raised too. PARTⅢ: The influence of autophagy expression modulation on A549 cell radiotherapy sensitivity and its mechanismObjective:To investigate different effect on autophagy-related gene of 3MA and rapamycin, and the radiotherapy sensitivity to A549 at different autophagy levels with 3MA or rapamycin, to discuss its mechanism.Methods:The subjects were divided into 6 groups: control group, radiotherapy group, 3MA therapy group, rapamycin therapy group, 3MA and radiotherapy group, rapamycin and radiotherapy group; cytotoxicity of 3MA and rapamycin were detected by MTT, cell colony number and survival score were count with cell colony essay; Beclin1, MAPLC3, pakt,mTOR protein expression rate were detected by flow cytometry after immunofluorescence staining; cell life cycle was too detected by flow cytometry; MAPLC3,survivin, mTOR,Bcl-2,EIF-4EmRNA expression was detected by RTPCR; MAPLC3,pakt,mTOR,COX2 protein expression was detected by Western blotResults: 3MA IC10:10Mm,rapamycin IC10:20nM by MTT. Autophagy-related gene Beclin1,MAPLC3 and pakt were inhibited and apoptosis rate was low and so was Survivin,Bcl-2,EIF-4E mRNA,COX2 protein in 3MA therapy group; Beclin1,MAPLC3 and pakt protein level was raised,cell apaptosis rate and mTOR protein were inhibited in rapamycin therapy gruop; in 3MA and radiotherapy group,the expression of Beclin1,MAPLC3 and pakt gene was inhibited and threre were high apoptosis rate and more G0-G1 cells ,less COX2 protein and less Bcl-2 and EIF-4E mRNA ,survival score descend obviously; in rapamycin and radiotherapy group, Beclin1 , MAPLC3 and pakt were up-regulated,cell apoptosis rate descend; more G2-M cells, survival score increased,less mTOR and EIF-4E, more Bcl-2 expression. Conclusions: 3MA down-regulated while rapamycin up-regulate autophagy expression; radiotherapy sensitivity was increased by 3MA while decreased by rapamycin; the mechanism may be related to autophagysome inhibition by 3MA, cell apoptosis enhancement, G0-G1 blockage.