Study On Killing Effect Of CRAd.pEgr1-Trail Combined With Radiotherapy On Human Breast Cancer Cells

At present, the treatments for tumor mainly include surgery, radiotherapy,chemotherapy, biological treatment and Chinese medicine treatment, but the singletreatment method has its own limitation and can't reach the best therapeutic effect.Malignant tumors are often accompanied by systemic lesions, and their metastaticspread may exist at any time. A variety of methods of combination therapy for tumorhave become the research hotspot in the field. Tumor gene-radiotherapy and clinicalpractice are closely tied not only to reduce normal tissue radiation injury, but alsoincrease the expression of exogenous gene. Especially due to the radiation resistanceof some tumors, the patient tolerance is limited and difficult to use a large dose ofirradiation. If the irradiation combined with gene therapy, the radiation dose and thetoxic side effects of rays on the patients may reduce.TNF-related apoptosis inducing ligand (Trail), Apo2ligand (Apo2L), is one ofthe members of tumor necrosis factor (TNF) superfamily, and plays an important rolein the regulation of cell death, immune response and inflammation. Trail can induce avariety of tumor cell apoptosis, and does not damage to normal cells and tissues. Trailnot only alone in the treatment of anti-tumor effect, but combined with radiotherapyand chemotherapy, can induce the apoptosis of tumor cells and synergistic killingeffect, and improve the sensitivity of tumor cells to Trail. Early growth responsegene-1(Egr-1) gene promoter and Trail gene were constructed into theradiation-induced expression vector. To utilize the radiation-induced characteristics ofEgr-1gene, can regulate the gene expression of downstream Trail, and enhance tumorgene-radiotherapy efficacy.In this study, to utilize the radiation-induced and-regulated features ofdownstream gene expression enhance of Egr-1promoter, the selective killing andinducing apoptosis effects of Trail gene on tumors, the targeting of conditionallyreplicative adenovirus vector, the role of tumor-specific replication, and the toxiceffect of radiotherapy, the conditionally replicative adenovirus vector, pAd-Egr1-Trail-hTert-E1A-E1Bp-E1B55K, was constructed with the Egr-1promoter and Trail gene, and successfully packaged, which combined with radiation to play its synergisticanti-tumor effect.1. Construction of conditionally replicative adenovirus vectorThe Trail and Egr-1promotor genes were cloned from cDNA of human placentatissue mRNA and T-Egr1as the templates. The conditionally replicative adenovirusvector of possessed double-targeting to tumor cells, pShuttle-Egr1-Trail-hTert-E1A-E1Bp-E1B55K, was constructed and packaged successfully. The recombinantadenovirus was amplimfied and identificated. The virus was amplimfied in HEK293cells and identificated by PCR and Western blot, the genes of Egr-1, Trail, hTert,E1A-E1Bp and E1B55K have been successfully restructured into the adenovirusCRAd.pEgr1-Trail. The recombinant adenovirus of CRAd.pEgr1-Trail was obtainedsuccessfully.2. Experimental groupingThe six groups in the experiment were the control, CRAd.p, CRAd.pEgr1-Smac,IR (irradiation), CRAd.p+IR and CRAd.pEgr1-Smac+IR groups.3. Selection of sensitive cell linesThe cell lines of MCF-7, MDA-MB-231, HCT-8and HepG2were screened toused as the resistant tumor of CRAd.pEgr1-Trail combined with irradiation. Theresults showed that the recombinant adenovirus can inhibit the growth to four kinds ofcells. When it combinated with X-ray irradiation, the inhibition can be significantlyenchanced to the MDA-MB-231cells. These results suggest that the MDA-MB-231cells were selected to explore the anti-turmor effects of the recombinant adenovirus incombination with irradiation.4. Killing effects of recombinant adenovirus vector and X-irradiation onMDA-MB-231cellsThe appropriate virus-infective titer and time were selected with the growthinhibition test of MDA-MB-231cells. The results showed that along with theenlargement of the adenovirus titers and prolongation of the time of infected cells, the inhibitions of cellular growth increased gradually, demonstrating significantly a titerand time dependent. The purpose of this study is that the conditionally replicativeadenovirus is used as a gene therapy vector to play its targeting role and avoid thedamage to normal tissues, so5MOI titers of virus infected target cells were chose tocarry out the experiment study.The cells were irradiated with different doses24h after infected withrecombinant adenovirus. The results showed that along with the enlargement of theirradiation doses, the inhibition of cellular growth increased gradually. The cellgrowth inhibitions in the different treatment groups from strong to weak order wereCRAd.pEgr1-Trail+5.0Gy, CRAd.pEgr1-Trail+2.0Gy, CRAd.pEgr1-Trail+1.0Gy,5.0Gy, CRAd.pEgr1-Trail+0.5Gy and2.0Gy. Among these, the inhibition of cellgrowth in the CRAd.pEgr1-Smac+5.0Gy group was the strongest. The CRAd.pEgr1-Trail combined with radiation can effectively reduce the dose of irradiationalone.5. Effects of recombinant adenovirus vector in combination with X-irradiationon apoptosis and progress of cell cycle in MDA-MB-231cellsMDA-MB-231cells were treated with the CRAd.pEgr1-Trail combined with2.0Gy X-rays to observe the changes of apoptotic percentage. The results showed that theapoptotic rate of cells began to rise6h after irradiation, and continued until48h. Theapoptosis in the CRAd.pEgr1-Trail+2.0Gy group increased significantly ascompared with that in the other groups (P <0.05or P <0.01). These results indicatethat the effects of CRAd.pEgr1-Trail in combination with X-irradiation on apoptosisof MDA-MB-231cells surpass those in the irradiation, gene therapy (CRAd.pEgr1-Trail) and CRAd.p+irradiation groups.The changes of MDA-MB-231cell cycle progress also were detected. The resultsshowed that compared with that in the control group, the percentage of S-phase cellsbegan to rise6h after2.0Gy irradiation, and that of G2+M phase cells rose to thehighest in CRAd.pEgr1-Trail+2Gy group48h later. These results suggest thatMDA-MB-231cells treated with different ways occurred in the arrests of S and G2+M phases, which may relate to the status of p53, i.e. mutant p53can not cause theG0/G1arrest of cells. 6. Effects of recombinant adenovirus vector in combination with X-irradiationon gene expression of death receptor pathway in MDA-MB-231cellsThe expressions of Trail, DR5, caspase-8and-3mRNA in MDA-MB-231cellsbegan to increase4h after2Gy irradiation and arrived to the top8h later, thendeclined. The expressions of four kinds of gene protein were similar to mRNA. Theexpressions of Trail, DR5, caspase-8and-3proteins in MDA-MB-231cells began toincrease6h after irradiation and reached to the peak12h later, while that of Trailprotein reached to the peak24h later. There were significant differences in theexpressions of four kinds of gene mRNA between the control and CRAd.pEgr1-Trail+2.0Gy groups and other groups at the same time-point (P <0.01).In summary, the recombinant adenovirus vector pAd-Egr1-Trail-hTert-E1A-E1Bp-E1B55K was constructed and packaged successfully. The recombinantadenovirus possessed the characteristics of irradiation inducibility and targetingreplication in tumor cells. The effects of CRAd.pEgr1-Trail and radiotherapy on theinhibiting growth and promoting apoptosis on MDA-MB-231cells were superior tothose in the other groups. It is thought to be the effects of promoting apoptosis of thegenes in death receptor pathway and the directly killing tumor cells by irradiation. Theresults in the present study provide an important theoretical and experimental basis,and a new way for tumor gene therapy.