Experimental Study On The Anti-tumor Effects In Vitro And Its Mechanism Of HTRAIL Gene-radiotherapy

Radiotherapy is an effective way of cancer treatment nowadays. But the effectand application of radiotherapy are restricted according to the radioimpairment of normal tissue adjacent to the tumor and radiation resistant forsome kinds of tumor. To decrease local radiation dose and enhance theanti-tumor effect become a very important project for the radiation tumorresearch. Gene-radiotherapy, a new cancer treatment, has a good applicationvalue.In this study, the followed are discussed: ①According to the mechanism thationizing radiation can activate early growth response-1 (Egr-1) gene promoterand induce the expression of downstream genes and the apoptosis inducingeffect of converted cell, tumor cell and virus infected cell of gene-TNF relatedapoptosis inducing ligand, TRAIL, a recombinant plasmid which containinghuman soluble TRAIL95-281 gene and radio-sensitive promoter (Egr-1) wasconstructed. The expression induced by radiation, anti-tumor effect andmechanism of pEgr-shTRAIL recombinant plasmid combined with X-rayirradiation has been studied. ② To increase the gene transfection efficiency,adenovirus vector Ad-CMV-hTRAIL was used to study the expressionproperties and the enhanced anti-tumor effect by gene-radiotherapy in vitro.These research would provide a theoretically and experimental basis in orderto enhance radiotherapeutic effects of malignant tumor.Ⅰ Experimental study on the expression properties induced by radiationand the anti-tumor effect of pEgr-shTRAIL in vitro1. Construction of pEgr-shTRAIL recombinant plasmid1.1 Amplification and sequencing of human soluble TRAIL geneDesigned and synthesized two pairs of primer according to the publishedsequence of signal peptide of TNFαcDNA in GeneBank. Signal peptide ofTNFαwas amplified using pEgr-TNFα as template. Designed a bridgedprimer concluding terminal sequence and the amino acid of 95-98 ofTRAIL95-281. Using pACCMV-TRAIL as a template, TRAIL95-281 wasamplified with the bridged primer and the downstream primer. Then using thedescribed two PCR products as a template to amplify the shTRAIL cDNAsequence concluding the signal peptide and TRAIL95-281 sequence, fusion PCRwas done with the upstream primer of TNF and the downstream primer ofTRAIL95-281. Then it was linked to the T vector and sequenced, the result isconsistent with the designed.1.2 Construction and identification of recombinant plasmid pEgr-shTRAILHuman TRAIL95-281 cDNA was inserted into the MCS of pcDNA3.1 toconstruct pcDNA3.1-TRAIL95-281 recombinant plasmid. To constructpcDNA3.1-Egr recombinant plasmid, the CMV promoter of pcDNA3.1 wasreplaced by radio-sensitive promoter Egr-1, then shTRAILcDNA was insertedinto the MCS of pcDNA3.1-Egr, recombinant plasmid pEgr-shTRAILwassuccessfully constructed and be confirmed by enzyme restriction andelectrophoresis.2. Transfection of recombinant plasmid and selection of stable expresscellpEgr-shTRAIL plasmid were packed with liposome to transfect lung cancercell line A549, the cells were selected in medium containing G418. After 20days selection, stable express cells were observed. Through enlarged cellculture, the cells which could stable express shTRAIL protein were gained3. The enhanced expression properties of recombinant plasmid inducedby radiation3.1 The change of expression level of soluble TRAIL in lung cancer cellA549 transfected with recombinant plasmid after different doses of X-rayirradiationAfter lung cancer cell A549 were transfected with recombinant plasmidpEgr-shTRAIL 36 hours, it was irradiated with different doses of X-rays.8-hours after radiation, the supernatant of cultured cells were harvested. Theexpression level of TRAIL in different dose were detected by ELISA method.The results showed that the expression of soluble TRAIL in radiation groupare higher than that of sham-irrad group (p<0.001~ p<0.05),the highestexpression level appeared after 5 Gy radiation. It is 4.17 times higher thansham-irrad group. The expression of soluble TRAIL in 10-Gy radiation groupdecreased compared to 5-Gy radiation group owing to increased impairmentinduced by higher dose radiation. It demonstrated that recombinant plasmidpEgr-shTRAIL has the enhanced expression properties induced by radiation.3.2 The Change of time-course of the expression of soluble TRAIL in lungcancer cell A549 transfected with recombinant plasmid after 2Gyradiation.After lung cancer cell A549 were transfected with recombinant plasmidpEgr-shTRAIL 36 hours, it was irradiated by 2Gy X-rays. Different time afterirradiation, the supernatant of cultured cells were harvested and the expressionlevel of TRAIL in different time were detected by ELISA method. The resultsshowed that the expression of soluble TRAIL increased significantly after 4hour-radiation (p<0.001).And the express level increased gradually with longtime radiation. The peak is after 36-hour radiation. The expression level is 4.6times than that before radiation. It demonstrated that recombinant plasmidpEgr-shTRAIL has the enhanced expression properties induced by radiation.4.The influence on the proliferation of tumor cell A549 of recombinantplasmid transfer combined with X-ray irradiationStable express cell(sA549-wshTRAIL)and untransfected control cell(sA549)were divided into 0,2,5,10Gy different doses groups respectively. They areirradiated with different doses of X rays on the second day after incubation.The growth rate was drawn on the second day after incubation. The growthspeed of A549-wshTRAIL is significantly lower than untransfected group andsingle transfection group(p<0.001～p<0.05) on the forth day;Then theydecreased significantly on the eighth day(p<0.001)and are lower than that ofuntransfected group. Within10Gy, with the X-ray doses increased, the growthspeed significantly slowed down. At the 8 day, the cell numbers ofA549-wshTRAIL /10Gy group was only 2.7% of that of untransfectedsham-irrad group, 3.6% of that of single irradiation group, 24.7% of that ofuntransfected/10Gy group respectively. This result indicated thatpEgr-shTRAIL transfer combined with X-ray irradiation could inhibit thegrowth of A549 cell.5. Effect of pEgr-shTRAIL transfer combined with X-ray irradiation oncell apoptosis of tumor cell A549A549 cell was transfected transiently with p3.1Egr 和 pEgr-shTRAILrecombinant plasmid using GeneCompanionTM III transfection agent after thelung cancer cell A549 was incubated 24 hours. The transfected cells wereirradiated with 0Gy,1 Gy,2 Gy and 5 Gy x-ray ,8hours later, cell werecollected .Early apoptosis after plasmid transfer combined with irradiationwere detected using annexin V-FITC apoptosis kit. The result showed that thepercentage of early apoptosis cells in p3.1Egr groups after 2and 5 Gy X-raysirradiation were significantly increased(p<0.01,p<0.05);The percentage ofearly apoptosis cells in pEgr-shTRAIL groups were markedly higher than thatof p3.1Egr sham-irrad groups and pEgr-shTRAIL sham-irrad group( p<0.001 ～ p<0.01 ) . The percentage of early apoptosis cells inpEgr-shTRAIL groups were markedly higher than that of p3.1Egr groups( p<0.01 ) and increased significantly with the irradiation doseincrease(p<0.05).It suggested that on the one hand the Egr-1 promoter can be activated by X-rayirradiation and enhanced the expression of TRAIL downstream which in turninduced apoptosis, on the other hand, it also indicated that TRAIL gene tranfercan markedly sensitized A549 cells to irradiation-induced apoptosis signal.6. The mechanism of anti-tumor effect on A549 cells transfected bypEgr-shTRAIL plasmid combined with X-ray irradiation6.1 Enhanced expression of TRAIL in pEgr-shTRAIL transfer cellsinduced by irradiationDesigned the primers of human TRAIL receptor. Detected the expression ofDR4 and DR5 using RT-PCR method when the lung cancer cell wereirradiated with different doses of x ray 24 hours. The expression of DR5 in0,2,5 and 10 Gy groups were all increased, and has dose-dependent manner.The expression of DR5 in 5 Gy group was 1.5 times higher than that of 0 Gygroup, The expression of DR5 in 10Gy group was 1.9 times higher than that of0Gy group. However, the expression of DR4 hadn't been detected. Itsuggested that irradiation may enhance the expression of DR5 and sensitizedA549 cells to TRAIL.6.2 Expression of caspase-3 of pEgr-shTRAIL transfer combined withX-ray irradiationThere were 4 groups in this experiment, plasmid sham-irrad group,pEgr-shTRAIL sham-irrad group, plasmid /5 Gy group and pEgr-shTRAIL/5Gy group. A549 cell was transfected by p3.1Egr and pEgr-shTRAILrecombinant plasmid combined irradiation , cell total protein was extracted 8hours later, the expression of caspase-3 of tumor cell was detected usingWestern blot. All groups can express caspase-3(32KD), plasmid sham-irradgroup and pEgr-shTRAIL sham-irrad group had no caspase-3 active fragmentp20 expression. Plasmid /5 Gy group and pEgr-shTRAIL/5 Gy group bothexpressed caspase-3 active fragment p20 , and the expression of p20 inpEgr-shTRAIL/5 Gy group is markedly higher than that of plasmid /5 Gygroup. It suggested there is great association between cell apoptosis inducedby combination of irradiation and TRAIL and the activation of caspase-3.6.3 The expression of TRAIL in Stable express cells(A549-wshTRAIL)The expression of TRAIL protein of A549 cell and stable express cellA549-wshTRAIL was detected using immunohistochemistry. The resultsshowed that the expression of TRAIL in stable express cell A549-wshTRAILincreased significantly, it may be due to the combination of soluble TRAILand TRAIL receptor on the cell membrane.In conclusion, pEgr-shTRAIL express vector induced by irradiation wasconstructed firstly using molecular biology, and it was confirmed that it hasthe property of enhancing the treatment gene induced by irradiation. Theexpress level of gene can be controlled by the irradiation of X ray in order tofulfill the function of anti-tumor. In addition, the expression of DR5 wasincreased by irradiation, so sensitized the A549 cells to TRAIL, lowered theequivalent dose of x ray, coordinated the gene and irradiation, enhanced theradiotherapy effect.Ⅱ Experimental study on the anti-tumor effect with hTRAIL mediatedby recombinant adenovirus and irradiation in vitro.1. Preparation and identification of recombinant adenovirusAd-CMV-hTRAIL and Ad-CMV-EGFP were amplified in 293 cell in greatamount, virus was collected and be purified to acquire high virus titer. Thetiter of Ad-CMV-hTRAIL is 6.0×1012 VP/ml, the titer of Ad-CMV-EGFPis 2.6×1012VP/ml.Purification of two virus are higher than 1.3 qualified forthe experiment. Virus DNA was extracted from the supernatant,Ad-CMV-hTRAIL was confirmed through PCR amplifying with virus DNAtemplate.2. Infection efficiency of recombinant adenovirus to lung cancer cell.2.1 Study on efficiency of different titer Ad-CMV-EGFP infected tumorcell 48 hours.0,10,100,1000,2000,3000,5000 and 10000 MOI of Ad-CMV-EGFP virusinfected A549 cell.48 hours later, green fluorescent positive percentage wasdetected by flow cytometry as an infection percentage determination method.Green fluorescent expression was observed by fluorescent microscope. Theresults showed that infection efficiency of Ad-CMV-EGFP to A549 wasincreased due to the increase of MOI. It reached its peak when MOI is 5000VP/cell.98% cell are green. It decreased when MOI was 10000VP/cell. Sothe optimal MOI is 5000VP/cell. It reached its peak in 72 hours later and itdecreased in 96 hours(p<0.05).It can be deduced that the high expressionoccurred during 48-72 hours which A549 cell were infected with adenovirusvector.3.The expression of hTRAIL mediated by recombinant adenovirus3.1The expression of hTRAIL in 293T cell mediated by recombinantadenovirusA549 cell were infected with Ad-CMV-EGFP whose MOI was 5000 VP/cell.Culture medium and cell were collected in different time , frozen and meltedseveral times later, the protein of TRAIL in supernatant were detected byELISA. The results showed that the protein of TRAIL can be expressed ininfected 239T cell supernatant 12 hours later. It reached its peak in 24hours(p<0.01),the value was 131.45pg/ml, however, the expression of TRAILdecreased significantly ( p<0.01 ) . It had the tendency of decreasingcontinuously in 72 hours, it may be due to TRAIL binding to its receptor oncell membrane of 239T cell, then the soluble TRAIL decreased.3.2 The expression of hTRAIL in lung cancer cell A549 mediated byrecombinant adenovirusThe immunohistochemistry results showed that A549 cell infected with 5000VP/cell MOI. Ad-CMV-hTRAIL became circle, big and nuclei swollen. Thepositive TRAIL cell number increased significantly, and so did TRAIL proteinexpression. The expression of TRAIL increased significantly in A549 due tothe Ad-CMV-hTRAIL infection.4 The anti-tumor effect on lung cancer cell A549 of recombinant hTRAILvector mediated by adenovirus combined with X-ray irradiation in vitro4.1 The effect on the growth of lung cancer cell A549 of recombinanthTRAIL vector mediated by adenovirus combined with X-ray irradiationin vitroA549 cell infected with 5000 VP/cell Ad-CMV-hTRAIL were irradiated 5 GyX ray, 72 hours later, the survival rates were detected using MTT method. Theresults showed that the survival of Ad-CMV-hTRAIL infection combined withX-ray group was lower markedly (P<0.01), was 43% of single virus controlgroup, 51% of Ad-CMV-hTRAIL infection group and 58% of singleirradiation group. It suggested that Ad-CMV-hTRAIL infection can enhancethe effect of tumor cell inhibition by X ray.4.2 The apoptosis effect on lung cancer cell A549 of recombinant hTRAILvector mediated by adenovirus combined with X-ray irradiation4.2.1 Early cell apoptosis detection by flow cytometryA549 cell infected with 5000 VP/cell Ad-CMV-hTRAIL were irradiated 5 GyX ray, 24 hours later, cell were collected and early apoptosis rates weredetected by flow Cytometry. The results showed that the percentage of earlyapoptosis cell of x-radiation group and Ad-CMV-hTRAIL infection groupincreased (p<0.01). The percentage of early apoptosis cell ofAd-CMV-hTRAIL infection combined X ray group increased (p<0.001)compared to single radiation group and Ad-CMV-hTRAIL infection group,was 3.14% of X-ray group, 4.29% of Ad-CMV-hTRAIL infection group. Itsuggested that Ad-CMV-hTRAIL infection combined with X ray radiation canenhance the apoptosis effect of A549 cell.4.2.2 Cell apoptosis detection by AO/EBA549 cell infected with 5000 VP/cell Ad-CMV-hTRAIL 48 hours wereirradiated by 5 Gy X ray, 24 hours later, cell were collected and stained byAO/EB, cell apoptosis were observed by fluorescent microscope. The resultsshowed that the percentage of apoptosis cell of Ad-CMV-hTRAIL infectiongroup and 5 Gy irradiation group increased (p<0.01) compared to controlgroup. The percentage of apoptosis cell of Ad-CMV-hTRAIL infectioncombined X ray group increased (p<0.001) compared to control group,compared to Ad-CMV-hTRAIL infection group and 5Gy irradiation group(p<0.001 and p<0.001). It suggested that Ad-CMV-hTRAIL infectioncombined with X ray irradiation can enhance the apoptosis effect of A549 cellsignificantly.5. The mechanism of anti-tumor effect of hTRAIL vector mediated byadenovirus combined with X-ray irradiation5.1 The influence of TRAIL receptor expression of lung cancer cell A549irradiated by X rayThe expression of DR4 and DR5 were detected by RT-PCR after A549 cellwere radiated with different doses, had dose dependent manner. Theexpression of DR5 of 5Gy group was 1.5 times than control group;Theexpression of DR5 of 10Gy group was 1.9 times than control group. But forlung cancer cell A549, both irradiated and control group were not detected anyexpression of DR4.Irradiation may enhance the expression of DR5 andsensitized A549 cells to TRAIL. It suggested that Ad-CMV-hTRAILtransfection combined with X ray radiation inhibit the lung cancer cell A549and be relative to the increase of DR5 expression.5.2 The influence of cancer cell caspase-3 expression of hTRAIL vectormediated by adenovirus combined with X-ray irradiationA549 cell infected with 5000 VP/cell Ad-CMV-hTRAIL 48 hours wereirradiated by 5 Gy X ray, 24 hours later, cell were collected, cytoplasm proteinwere extracted and detected with Westernblot. All groups can expresscaspase-3(32KD), Ad-CMV-hTRAIL infection group, 5 Gy irradiation groupand Ad-CMV-hTRAIL infection combined X-ray radiation group all expresscaspase-3 active fragment p20,and the express level of Ad-CMV-hTRAILinfection combined X-ray radiation group was the highest. It suggested there isgreat association between cell apoptosis induced by combination of irradiationand TRAIL and the activation of caspase-3.This study confirmed that Ad-CMV-hTRAIL had high infection efficiency andhigh expression of hTRAIL95-281, and confirmed that Ad-CMV-hTRAILinfection combined X ray radiation had the anti-tumor effect on A549. Theexpression of DR5 of A549 was increased by irradiation, so sensitized theA549 cells to TRAIL, lowered the equivalent dose of x ray, coordinated theTRAIL gene and irradiation, enhanced the radiotherapy effect.In conclusion, the above two-part study confirmed that TRAIL gene combinedX ray had a good treatment effect on lung cancer cell A549. The possiblemechanisms are that the expression of TRAIL95-281 is enhanced induced byirradiation;the expression of TRAIL death receptor in A549 is enhanced induced byirradiation, so irradiation sensitized the A549 tumor cells to TRAIL;Tumor cellapopotosis increase is owing to the increased activity of caspase-3 in tumorcell;Irradiation can inhibit the growth of tumor cell. This study will providescientific and experimental basis for enhancing the treatment effect of tumorradiation therapy and provide a new treatment method.