Study On Anti-tumor Effects Of CRAd.pEgr1-Smac In Combination With Irradiation On MDA-MB-231 Cells

Tumor is one of disease which seriously threaten human health. Now, the primary ways for tumor therapy mainly include operation, radiotherapy, chemotherapy and biotherapy. Furthermore, there are some auxiliary ways, including endocrine therapy, the traditional Chinese medicine treatment, thermotherapy and radiofrequency ablation therapy. But these ways can't thoroughly cure tumor. Turner is a kind of complicated disease involved in mulpile steps, multiple stages and mulpile factors. It is difficult to achieve satisfactory results by solo way. So, the combined treatment for tumor is imperative.Nowadays, with the rapid development of molecular biology and genetic engineering and technology, the gene-radiotherapy is intended to attract in the research of tumor therapy. The method, which gene therapy and radiotherapy together combine and effectively cooperate, would have good application prospect for the treatment of regional tumor. The discovery of radiation-sensitive early growth response-1 (Egr-1) promoter provides an opportunity for the effective combination of radiotherapy and gene therapy.In order that radiotherapy combines with gene therapy together to cure cancer, the conditionally replicative adenovirus of CRAd.pEgrl-Smac, which expresses Smac gene, was constructed successfully by taking advantage of multiple-gene introduction system in this study. There were three features in it:(1) the CRAd replication could target in tumer cells; (2) the radiation could kill tumor cells and activate transcription of the Egr-1 promoter to induce the expression of the Smac genes; (3) the Smac gene could promote apoptosis of tumor cells. In this way, the radiation dose could be lowed, and the damage to normal tissues could be alleviated or avoided. The combination of radiotherapy, promoting-apoptosis and targeted replication achieved the purpose of inhibiting tumor growth and killing tumor cells. The study would provide a promising way to treat tumors. 1 Construction of recombinant plasmid1.1 Acquisition of E1B55K geneThe specific PCR primers were designed and synthesized with the following primers according to the sequence of E1B55K gene:upstream 5'-gtcgacaaggataaatggagcgaaga-3', including Sal I enzyme digestion sites, downstream 5'-ggaagatctttcattcccgagggt-3', including Bgl II enzyme digestion sites. The E1B55K gene was cloned and amplified from cDNA of 293T cells as the template, and identified by the cleavage of endonucleases and sequencing process. The results of sequencing analysis confirmed that the E1B55K gene was correctly coloned.1.2 Acquisition of ElA-ElBp (deleted CR2 region) geneThe specific PCR primers were designed and synthesized with the following primers according to the sequence of E1A-E1Bp gene:upstream 5'-cggcaagcttatgagacatattatctgcca-3', including HindⅢenzyme digestion sites; downstream 5'-gcctcgaggaggtcagatgtaaccaaga-3', including Xho I enzyme digestion sites. The E1A-ElBp gene was amplified from cDNA of 293T cells as the template, and ligated to T vector for sequencing. The result of sequencing analysis was in coincidence with the anticipated result. Then E1A gene deleted CR2 region was amplified by overlap-PCR, and ligated to T vector for sequencing. The results of sequencing analysis confirmed that the E1A-E1Bp gene deleted CR2 region was correctly coloned.1.3 Acquisition of hTert promter geneThe specific PCR primers were designed and synthesized with the following primers according to the sequence of hTert promter gene:upstream 5'-gatatcccagtggattcgcgggcaca-3', including EcoRV enzyme digestion sites, downstream 5'-aagaagcttcgcgggggtggccgggg-3', including HindⅢenzyme digestion sites. The hTert promter gene was cloned and amplified from cDNA of 293T cells as the template, and identified by the cleavage of endonucleases and sequencing process. The results of sequencing analysis confirmed that the hTert promter gene was correctly coloned.1.4 Acquisition of hSmac geneThe specific PCR primers were designed and synthesized with the following primers according to the sequence of hSmac gene:upstream 5'-gctctagaatggcggctctgaagagttg-3', including XhoⅠenzyme digestion sites, downstream 5'-gatatctcaggccctcaatcctcacgc-3', including EcoRⅤenzyme digestion sites. The hSmac gene was cloned and amplified from cDNA of placent as the template, and identified by the cleavage of endonucleases and sequencing process. The results of sequencing analysis confirmed that the hSmac gene was correctly coloned.1.5 Acquisition of Egr-1 promter geneThe specific PCR primers were designed and synthesized with the following primers according to the sequence of Egr-1 promter gene:upstream 5'-gcggccgcgacccggaaacgccatataa-3', including XhoⅠenzyme digestion sites, downstream 5'-ctcgagccaagttctgcgcgctggga-3', including NotⅠenzyme digestion sites. The Egr-1 promter gene was cloned and amplified from T-Egr1 as the template, and identified by the cleavage of endonucleases and sequencing process. The results of sequencing analysis confirmed that the Egr-1 promter gene was correctly coloned.1.6 Construction of recombinant plasmidThe E1B55K, E1A-ElBp (deleted CR2 region), hTert, hSmac, and Egr-1 genes were respectively ligated to pShuttle vector to construct the recombinant plasmid, pShuttle-Egr1-Smac-hTert-E1A-E1Bp-E1B55K. selectively delated CR2 region of E1A gene by using the technique of genetic engineering.2 Package and identification of conditionally replicative adenovirusRcombinant plasmid pShuttle-Egr1-Smac-hTert-E1A-E1Bp-E1B55K was linearized with the restriction endonulease PmeⅠ, then cotransformed into the Escherichia coli strain BJ5183 (Adeasy-1+) to produce recombinant adenovirus plasmid of pAd.Egr1-Smac-hTert-E1A-ElBp-E1B55K. HEK293 cells were transformed with recombinant adenovirus plasmid. The adenovirus was harvested when CPE was found in culture plate. The recombinant adenovirus of CRAd.pEgr1-Smac was amplimfied and identified. In the following experiments, the effects of CRAd.pEgr1-Smac combined with irradiation on the cellular growth and apoptosis of MDA-MB-231 cells, and the expression rule of gene mRNA and protein in the cells was detected. 3 Experimental grouping and index detectionThe six groups in the experiment were the control, CRAd.p, CRAd.pEgrl-Smac. IR (irradiation), CRAd.p+IR and CRAd.pEgrl-Smac+IR groups. In the time-course experiment, selected time points of detecting mRNA (protein) exprossion were 4,8,12 and 24 h (6,12,24 and 48 h) after irradiation. In the dose-effect experiment, the selected irradiation doses were 0, 0.5,1.0,2.0 and 5.0 Gy. In the time-effect the irradiation dose was 2.0 Gy. CCK-8 assay was used to detect the cell proliferation, and flow cytometry to the apoptosis. Real-time PCR was used to detect the expressions of Smac, caspase-9 and-3 genes in mRNA level, and Weatern blot was used to detecte the expressions of Smac, Cyt c. caspase-9 and-3 on protein level.4 Selection of Sensitive cell linesAfter infected 24 h with recombinant adenovirus, the cells of MCF-7, MDA-MB-231, HCT-8 and HepG2 were irradiated with 2.0 Gy. The cellular proliferation was detected by CCK-8 assay 48 h after irradiation. The results showed that the inhibition effects of the recombinant adenovirus of CRAd.pEgrl-Smac on the MCF-7. MDA-MB-231 and HepG2 cells were significantly. When it combinated with X-ray irradiation, the inhibition can be signioficantly enchanced to the MCF-7 and MDA-MB-231 cells, especially to later. The HepG2 cells were ineffective to irradiation. The inhibition of the recombinant adenovirus on HCT-8 cells was irregularity, and was worse under irradiation. Considerring these results, the MDA-MB-231 cells were selected to discuss the anti-turmor effects of of the recombinant adenovirus in combination with irradiation on them.5 Growth inhibitory effects of CRAd.pEgrl-Smac and X-irradiation on MDA-MB-231 cells5.1 Growth inhibitory effects of recombinant adenovirus on MDA-MB-231 cellsThe cells were infected with different titers of recombinant adenovirus, and the cellular proliferation was detected by CCK-8 assay at 24,48 and 72 h after infectation. Their results showed that along with the enlargement of the adenovirus doses, the inhibition of cellular growth increased gradually. There was significant difference in the inhibition of cellular growth between the 0 and 5 MOI groups only 72 h after infection (P<0.05). also that between the 10,50 and 250 MOI groups and the 0 MOI group (P<0.05 or P<0.01) at different time. Among these, the inhibition of cell growth in the 250 MOI group was the strongest.5.2 Dose-effect relationship of the growth inhibitory effects on MDA-MB-231 cells infected with CRAd.pEgrl-Smac by X-irradiationThe cells were irradiated with different doses 24 h after infected with recombinant adenovirus, and the cellular proliferation was detected by CCK-8 assay 24 h after irradiation. The results showed that along with the enlargement of the irradiation doses, the inhibition of cellular growth increased gradually. Under the identical irradiation dose, there was significant difference in the inhibition of cellular growth between the control and CRAd.pEgrl-Smac groups after irradiated (P<0.05 or P<0.01). When the irradiation doses were 2.0 or 5.0 Gy, the cellular growth in the CRAd.pEgrl-Smac group was inhibited significantly as compared with that in the control and CRAd.p groups respectively (P<0.05 or P<0.01). Among these, the inhibition of cell growth in the CRAd.pEgrl-Smac+5.0 Gy group was the strongest.5.3 Time-effect of CRAd.pEgrl-Smac in combination with X-irradiation on growth inhibitory effects of MDA-MB-231 cellsThe cells 24 h after infected with recombinant adenovirus were irradiated with 2.0 Gy. The cellular proliferation was detected by CCK-8 assay at the different time-points after irradiation. The results showed that the growth rates of the MDA-MB-231 cells infected with the adenovirus after irradiation declined, especially in the CRAd.pEgrl-Smac+2.0 Gy group. Under the same time-point, as compared with that in the control group, the growth rate in the CRAd.pEgrl-Smac +2.0 Gy group declined significantly from the beginning of 6 h after irradiation (P<0.05 or P< 0.01). There were significant differences in the growth rate between the CRAd.pEgrl-Smac+2.0 Gy and others groups 24 and 48 h after irradiation (P<0.05 or P<0.01).5.4 Effects of CRAd.pEgrl-Smac in combination with X-irradiation on apoptosis of MDA-MB-231 cellsThe cells 24 h after infected with recombinant adenovirus were irradiated with 2.0 Gy, and harvested 24 h later. Then the cell apoptotic percentages were detected by FCM. The results showed that as compared with that in the control group, the apoptotic percentage in others irradation groups increased significantly (P＜0.05 or P<0.01), especially that in the CRAd.pEgrl-Smac+2.0 Gy group was the highest 48 h after irradiation, there was significant difference in the cell apoptotic percentages as compared with that in other groups under the same time-point. The results show that the effects of CRAd.pEgr1-Smac in combination with X-irradiation on apoptosis of MDA-MB-231 cells surpass those in the irradiation, gene therapy (CRAd.pEgrl-Smac) and CRAd.p+irradiation groups. The results suggest that the synergism in the promoting apoptosis of Smac gene controlled by Egr-1 with radiotherapy achieves the goal of killing tumor cells.6 Radiation-induced expression rule of CRAd.pEgrl-Smac in MDA-MB-231 cells6.1 Time-course effects of CRAd.pEgrl-Smac in combination with 2.0 Gy X-irradiation on Smac, Cyt c, caspase-3 and -9 mRNA expressions in MDA-MB-231 cellsThere were six groups in the experiment:the control, CRAd.p, CRAd.pEgrl-Smac,2.0 Gy, CRAd.p+2.0 Gy and CRAd.pEgrl-Smac+2.0 Gy groups. The cells were irradiated with 2.0 Gy X-ray 24 h after infected with 5 MOI adenovirus titers, and harvested 4,8,12, and 24 h later. The expressions of four kinds of genes mRNA in MDA-MB-231 cells were detected by real-time PCR.6.1.1 Expression of Smac mRNAThe results showed that the expression of Smac mRNA in the CRAd.pEgrl-Smac+2.0 Gy group increased significantly from 4 h after irradiation, and arrived to the top 8 h later, then to decline., There were significant differences in the Smac expressions between the control and CRAd.pEgrl-Smac+2.0 Gy groups and other groups at the same time-point (P<0.01).6.1.2 Expression of Cyt c mRNAThe results showed that the expressions of Cyt c mRNA in the CRAd.pEgrl-Smac+2.0 Gy group increased significantly from 4 h after irradiation and arrived to the top 8 h later, then to decline. There were significant differences in the Cyt c expressions between the control and CRAd.pEgr1-Smac+2.0 Gy groups and other groups at the same time-point (P<0.01).6.1.3 Expression of caspase-9 mRNAThe results showed that the expressions of caspase-9 mRNA in the CRAd.pEgrl-Smac 2.0 Gy group increased significantly from 4 h after irradiation and arrived to the top 8 h later, then to decline. There were significant differences in the caspase-9 expressions between the control and CRAd.pEgrl-Smac+2.0 Gy groups and other groups at the same time-point (P< 0.01).6.1.4 The expression of caspase-3 mRNAThe results showed that the expressions of caspase-3 mRNA in the CRAd.pEgrl-Smac+ 2.0 Gy group increased significantly from 4 h after irradiation and arrived to the top 8 h later, then to decline. There were significant differences in the caspase-3 expressions between the control and CRAd.pEgrl-Smac+2.0 Gy groups and other groups at the same time-point (P< 0.01).6.2 Time-course effects of CRAd.pEgrl-Smac in combination with 2.0-Gy X-irradiation on Smac, Cyt c, caspase-3 and -9 protein expressions in MDA-MB-231 cellsThere were six groups in the experiment:the control, CRAd.p, CRAd.pEgrl-Smac,2.0 Gy, CRAd.p+2.0 Gy and CRAd.pEgrl-Smac+2.0 Gy groups. The cells were irradiated with 2.0 Gy X-ray 24 h after infected with 5 MOI adenovirus titers, and harvested 6,12,24 and 48 h later. The expressions of four kinds of proteins in MDA-MB-231 cells were detected by Western blot.6.2.1 Expression of Smac proteinThe results showed that the Smac protein expression increased significently after irradiation, especially that in CRAd.pEgrl-Smac+2.0 Gy group. The Smac protein expression in CRAd.pEgrl-Smac+2.0 Gy group increased significantly from 6 h after irradiation and to reached the top 12 h later, then to decline. But, at the same time-point, the Smac protein expression in CRAd.pEgrl-Smac+2.0 Gy group was the strongest.6.2.2 Expression of Cyt c proteinThe results showed that the Cyt c protein expression increased significently after irradiation, especially that in CRAd.pEgrl-Smac+2.0 Gy group. The Cyt c protein expression in CRAd.pEgrl-Smac+2.0 Gy group began to increase significantly from 6 h after irradiation and to reached the top at 12 h, then to decline. But at the same time-point, the Cyt c protein expression in CRAd.pEgrl-Smac+2.0 Gy group was the strongest. 6.2.3 Expression of caspase-9 proteinThe results showed that the caspase-9 protein expression increased significently after irradiation, especially that in CRAd.pEgrl-Smac+2.0 Gy group. The caspase-9 protein expression in CRAd.pEgrl-Smac+2.0 Gy group began to increase significantly from 6 h after irradiation and reached to the top 12 h later, then to decline. The expression increased slightly 24 h after irradiation and there were not significant differences between every groups 48 h later.6.2.4 Expression of caspase-3 proteinThe results showed that the caspase-3 protein expression increased significently after irradiation, especially that in CRAd.pEgrl-Smac+2.0 Gy group. The caspase-3 protein in CRAd.pEgr1-Smac+2.0 Gy group began to increase significantly from 6 h after irradiation and to reached to the top at 12 h, then to decline. But, under the same time-point, the caspase-3 protein expression in CRAd.pEgrl-Smac+2.0 Gy group was the strongest.In summary, the conditionally replicative adenovirus possessed double targeting to turner cells of CRAd.pEgrl-Smac was constructed successfully for the first time in this study. The recombinant adenovirus possessed the characteristics of irradiation inducibility and targeting replication in tumor cells. The effects of CRAd.pEgrl-Smac and radiotherapy on the inhibiting growth and promoting apoptosis on MDA-MB-231 cells were superior to others groups. It is thoughted to be the combining action of the targeting replication of CRAd, the promoting apoptosis effect of Smac. and the directly killing tumor cells by irradiation. The study opens up a new way to improve the effects of gene-radiotherapy, and provides the new way and experimental bases for the clinical application of tumer gene-radiotherapy.