Killing Effects Of Triple Targeting CRAd.pE-Smac Combined With X-ray Irradiation On MDA-MB-231Cells In Vitro

The common treatments for cancer included surgery, radiotherapy and chemotherapy. Somestudies showed that40%cured cancer cases made use of radiotherapy, or comprehensivetreatment based on radiotherapy. Single radiotherapy drawbacks were mainly inevitable radiationdamage to normal tissues, while obtaining certain therapeutic effects. And solid tumor has hypoxiccharacteristics, which leads that tumor cells tolerate to radiotherapy and affects the therapeuticresults. Gene-radiotherapy for tumors is a new concept, and has the enhancing role of targetingtherapeutic effects; at same time, it could make the decrease of treatment dose, reduces radiationside injury, and achieves the dual effects of radiotherapy and gene therapy. Two problems aboutresearch of tumor gene-radiotherapy must lead to attention: targeting and efficient aiming gene. Inthe study, we made use of the characteristics of hypoxia response element (HRE) and humantelomerase reverse transcriptase (hTERT) to transform adenovirus vetor, enhance the replicatecapability of these vetors in hypoxic tumor cells, and improve targeting. In addition, we made useof radiation-inducing characteristics of Egr-1promoter to achieve radiation targeting. So our studycould achieve triple targeting from three different views. Smac is a kind of pro-apoptotic proteinreleased from mitochondria, its regulation mechanism is to get rid of the inhibiting role of IAPs oncaspases family by Smac specifically binding IAPs. Thereby, Smac could promote cell apoptosis.When the cells receive apoptotic stimuli signal, the Smac stored in mitochondria is released intocytoplasm to activate corresponding signaling pathways and promote cell apoptosis. Radiotherapyitself play therapeutic role by promoting cell apoptosis. Therefore, in the study we made use ofSmac as targeting gene to enhance pro-apoptotic role of radiotherapy and suppress effectivelytumor growth. In the study we designed triple targeting conditionally replicative adenovirus,explored its expression regulatory induced by radiation and anti-tumor effects, and investigatedthe relative mechanism. The study has opened up a new way for improving gene-radiotherapy, andprovided a promising way for treating tumors and experimental basis.1. Construction of conditionally replicative adenovirus vector and adenovirus packageIn this study, the PCR and nested PCR techniques were used to fish E1A-E1Bp, E1B55K geneand hTERT promoter sequences from human embryonic kidney293cells and Smac gene sequencefrom human cervical cancer HeLa cells, clone Egr-1promoter sequence from T-Egr-1palsmid,and synthesize HRE promoter sequence. In present study, we used HRE/hTERT chimericpromoter to replace essential gene promoter of adenovirus replication by gene recombinationtechnology, placed Egr-1promoter into upstream of Smac gene fragment, constructed tumor,hypoxia and radiation triple targeting conditionally replicative adenovirus vetor pshuttle-Egr1-Smac-HRE-hTERT-E1A-E1Bp-E1B55k, and successfully conducted adeveriuspackaging. The virus was amplificated using293cells to achieved stable virus titer adenovirus,and identificated correctlly.2. Experimental groupingThere were six groups in the experiment to include the control, CRAd.pE-Smac, Hypoxia (H),CRAd.p, CRAd.pE-Smac+H and CRAd.p+H groups, and the cells in every group were treatedaccording to unirradiation (Null) and4Gy irradiation.3. Smac expression regulatorySmac mRNA and protein were measured by real time PCR and Western blotting, respectively.After MDA-MB-231cells were infected by CRAd.pE-Smac adenovirus, they were treated usingCOCl2to imitate hypxia. Without irradiation, Smac mRNA expression in each group increasedwith time prolongation between4–24h, reached to peak value at12h, expressed mostly in theCRAd.pE-Smac+H group, and was120times as those in the control group. After4Gyirradiation, Smac mRNA expression regulatory was similar with that without irradiation, but itwas more than that without irradiation (P <0.05or P <0.01). In addition, Smac proteinexpression showed that it was not signifecantly among each group without irradiation at6h, but itincreased in the CRAd.pE-Smac, hypoxia and CRAd.pE-Smac+H groups12,24and48h afterirradiation, especially, it was most signifecantly24h after irradiation. Smac protein expression didnot increase6h after4Gy irradiation, did in the CRAd.pE-Smac group12,24and48h afterirradiation, and did more than that without irradiation, especially, did most signifecantly in theCRAd.pE-Smac+H group at24and48h. These rusults showed that virus, hypoxia and radiationall could induce the increase of Smac mRNA and protein expressions, and the combination of thethree could achieve maximum efficiency.4. Proliferation inhibition of CRAd.pE-Smac combined with radiation in MDA-MBA-231cellsMTT assay was used to measure MDA-MB-231cell proliferation. The results showed thatafter CRAd.pE-Smac infection, cells were treated using COCl2to imitate hypxia. Withoutirradiation, hypoxic MDA-MBA-231cell proliferation decreased at6–48h, and CRAd.pE-Smacinfection and CRAd.p infection combined hypoxia cells were inhibited more seriously. After4Gyirradiation, hypoxic MDA-MBA-231cell proliferation without virus infection did not changesignifecantly6h after irradiation, and decreased12–48h after irradiation, even was inhibited,and the inhibition was more than that withour irradiation. The results showed that Smacoverexpression, hypoxia and radiation all could inhibit cell proliferation, and there were somedifferences in time-course effects of inhibiting cell proliferation.5. Effects of CRAd.pE-Smac combined with irradiation on MDA-MB-231cells cycle phaseand apoptosisAfter CRAd.pE-Smac adenovirus infected MDA-MB-231cells, they were treated usingCOCl2to imitate hypxia to explore the changes of cell cycle phase and apoptosis without and with 4Gy irradiation. The results showed that the cell percentage in G2/M phase increased at12h ascompared with that in the control group, it all increased significantly in each group (P <0.05or P<0.01), especially, that in the CRAd.pE-Smac, CRAd.pE-Smac+H and CRAd.p+H groupsincreased signifecantly. After4Gy irradiation, as compared with that in the control group, the cellpercentage in G2/M phage all increased significantly in each group (P <0.05or P <0.01),especially, those in the CRAd.pE-Smac, CRAd.pE-Smac+H and CRAd.p+H groups increasedsignificantly; but there was no signifecant difference between unirradiation and4Gy irradiation.In addition, the cell percentage in S phage all increased without and wih4Gy irradiation at24h.And the cell percentage in G2/M phage all increased significantly in each group at24and48h, itsregulatory was same to that at12h. These results showed that Smac overexpression and radiationcould cause G2/M arrest, it combined with hypoxia also could induce S delay, which were theindication of cell radiosensitivity, and enhanced cell killing effects of radiation.6. The molecular mechanism of MDA-MB-231cell apoptosis induced by CRAd.pE-Smaccombined with radiationCyt c, caspase-9and-3mRNAs and proteins were measured by real time PCR and Westernblotting, respectively. The results showed that in the MDA-MB-231cells infected only byCRAd.pE-Smac adenovirus, Cyt c, caspase-9and-3mRNAs increased without irradiation, andhypoxia could enchance these protein expressions (except for Cyt c and caspase-9mRNAs), andreached to peak value at12h. After4Gy irradiation, in the MDA-MB-231cells infected only byCRAd.pE-Smac adenovirus, Cyt c, caspase-9and-3mRNAs all increased, and adenovirus,hypoxia and radiation had duplicating effects except for Cyt c mRNA, and other factors also hadthis duplicating effect characteristics, which reflect the triple targeting features. Furthermore, Cytc, caspase-9and-3protein expressions had been detected in the present experiments. The resultsshowed that these proteins had similar regulatory with their mRNA, and there were activecaspase-9and-3fragements in MDA-MB-231cells.In summary, in the study we achieved tumor-specific, hypoxia and radiation triple targetingSmac overexpression, which had proliferation inhibition and pro-apoptosis effects on breastcancer MDA-MB-231cells, and did the ability that arrested the cells in G2/M phase―aradiosensitive period, then was advantageous for subsequent radiotherapy. The study provided anew breast cancer gene-radiotherapy program, which not only could take advantage of hypoxicmicroenviroment in solid tumor to enhance Smac overexpression, and overcome the inefficiencycaused by hypoxia, but also achieve a multi-regulation gene-radiotherapy, and fully integrate thefavorable and unfavorable condition of radiotherapy to maximize the benefits ofgene-radiotherapy. The study provided the new way for the clinical application of breast cancer.