Experimental Study On Expression Property And Anti-tumor Effect Of PcEgr-hp53 Induced By Ionizing Radiation

Radiotherapy is an important way in the treatment of many human cancers. It is animportant project to enhance the radiotherapeutic effects and reduce radiotherapeutic sideeffect of tumors. According to the mechanism that ionizing radiation can activate earlygrowth response-1 (Egr-1) gene promoter and induce the expression of downstream genes,the researchers in the Weichselbaum's laboratory first combined gene therapy withradiotherapy in order to enhance radiotherapeutic effects. Gene-radiotherapy has beenconsidered as an effective way of cancer treatment now. In this study, a recombinant plasmidpcEgr-hp53 containing wild type human suppressor gene p53 (wt-p53) and radio-sensitivepromoter (Egr-1) was constructed. It has been demonstrated that the expression of p53 hadenhanced property induced by irradiation in vitro. The anti-tumor effect and mechanism ofpcEgr-hp53 recombinant plasmid combined with X-ray irradiation has been studied. Theresults demonstrate that pcEgr-hp53 recombinant plasmid combined with X-ray irradiationcould inhibit the growth of A549 with wt-p53 and SKOV-3 with mt-p53 cell lines. Themechanism may correlated with the enhancement of p53 expression, G₁ arrest of A549 andSKOV-3 cell lines, the expression increases of apoptotic regulatory protein Bax andcaspase-3 and the expression decrease of apoptotic regulatory protein Bcl-2 after genetransfer combined with irradiation. These researchs would provide a theoretical andexperimental basis in order to enhance radiotherapeutic effects of malignant tumors.1 Construction of pcEgr-hp53 recombinant plasmid1.1 Construction and identification of pcEgr-hp53 and pCMV-hp53 plasmidHuman p53 cDNA was inserted into the multiple cloning sites (MCS) of pcDNA3.1 toconstruct pCMV-hp53 recombinant plasmid. The CMV promoter of pCMV-hp53 wasreplaced by radio-sensitive promoter Egr-1 to construct pcEgr-hp53 recombinant plasmid.The electrophoresis showed that the recombination was right.1.2 The measurement of recombinant plasmid pcEgr-hp53 sequenceThere was a mutant point in p53 encoding sequence and the point was corrected.2 Transfection of recombinant plasmid and selection of stable expressing cellspcEgr-hp53 plasmids were packed with liposome to transfect human lungadenocarcinoma A549 cells with wt-p53 and human ovarian carcinoma SKOV-3 cells withmt-p53, 48 – 72 h later, and the cells were selected in medium containing 400 μg/ml G418.After selection for 10 – 20 days, the stable expressing cells were observed. Through theamplification of cell culture, the cells which could stably express p53 were gained and namedA549-hp53 and SKOV -3-hp53.3 Expression properties of recombinant plasmid induced by irradiation3.1 Changes of p53 mRNA and protein expressions of A549-hp53 after X-rayirradiation with different dosesThe results showed that p53 mRNA levels in A549-vect group decreased and increasedsignificantly in A549-hp53 group with the augment of irradiation doses, and the proteinexpression of p53 in A549-vect/0.5 – 5 Gy groups was higher than that in A549-vect/0 Gygroup. The protein expression of p53 in A549-hp53/2 – 5 Gy groups was significantly higherthan that in A549-hp53/0 Gy group. The protein expression of p53 in A549-hp53/2 – 5 Gygroups increased significantly, but that in A549-hp53/0.5 Gy group was remarkably lowerthan that in A549-vect control after X-ray irradiation with the same dose. The resultsindicate that there was the enhancement property of p53 expression in A549 cellstransfected recombinant plasmid pcEgr-hp53 induced by radiationin.3.2 Changes of p53 mRNA and protein expressions of SKOV-3-hp53 after X-rayirradiation with different dosesThe results showed that p53 mRNA level in SKOV-3-vect/0.5 – 5 Gy groups decreasedand increased significantly in SKOV-3-hp53/0.5 – 5 Gy groups with the increase ofirradiation doses.The results also showed that the protein expression of p53 in SKOV-3-vect/0.5 – 5 Gygroups was lower than that in SKOV-3-vect/0 Gy group, especially remarkable lower inSKOV-3-vect/2 Gy group. The protein expression of p53 in SKOV-3-hp53/0.5 – 5 Gy groupswas significantly higher than that in SKOV-3-hp53 /0 Gy group. The protein expression ofp53 in SKOV-3-hp53/0 – 5 Gy group was higher than that in SKOV-3-vect/0 – 5 Gy groupsafter X-ray irradiation with the same dose. The results indicate that there was theenhancement property of p53 expression in SKOV-3 cells transfected recombinantplasmid pcEgr-hp53 induced by radiationin.3.3 The effect of recombinant plasmid pcEgr-hp53 transfection combined withX-ray irradiation on the proliferation of tumorr cell lines3.3.1 The effect of recombinant plasmid pcEgr-hp53 transfection combined withX-ray irradiation on the proliferation of A5499 cell linesThe stable expressing A549-vect and A549-hp53 cells were divided into 4 differentdoses groups and the cells were again divided into 2 groups in each dose group, A549-vect/0Gy, A549-vect/0.5 Gy, A549-vect/2 Gy, A549-vect/5 Gy and A549-hp53/0 Gy,A549-hp53/0.5 Gy, A549-hp53/2 Gy, A549-hp53/5 Gy groups. The results showed that thecell growth in A549-hp53/0 Gy group was not significantly lower than that in A549-vect/0Gy group. The growth speed of cells at first day after irradiation in A549-hp53/0.5 – 5 Gygroups was markedly lower than that in A549-hp53/0 Gy group and the cells at 8 days afterirradiation was only 4.5% and 23.7% of those, respectively, in A549-hp53/0 Gy group. Thegrowth speed in A549-hp53 group also was markedly lower than that in A549-vect controlafter X-ray irradiation with the same dose. These results indicate that pcEgr-hp53 transfectioncombined with X-ray irradiation could inhibit the growth of A549 cell line and the inhibitingeffect was dose-dependent.3.3.2 Effects of recombinant plasmid pcEgr-hp53 transfection combined with X-rayirradiation on proliferation of SKOV-33 cell linesStable expressing cells SKOV-3-vect and SKOV-3-hp53 were divided into 4 differentdoses groups and the cells were again divided into 2 groups in each dose group,SKOV-3-vect /0 Gy, SKOV-3-vect /0.5 Gy, SKOV-3-vect /2 Gy, SKOV-3-vect /5 Gy andSKOV-3-hp53/0 Gy, SKOV-3-hp53/0.5 Gy, SKOV-3-hp53/2 Gy, SKOV-3-hp53/5 Gygroups. The results showed that the cell growth in SKOV-3-hp53/0 Gy group was lower thanthat in A549-vect/0 Gy group and especially significant lower at the 6 day after transfection(P < 0.05). The growth speed of cells at first day after irradiation in SKOV-3-hp53/0.5 – 5Gy groups was markedly lower than that in SKOV-3-hp53/0 Gy group and the cells at 8 daysafter irradiation was only 6.4% and 6.8% of those, respectively, in SKOV-3-hp53/0 Gy group(P < 0.05 – P < 0.001). The growth speed in SKOV-3-hp53 group also was markedly lowerthan that in SKOV-3-vect control after X-ray irradiation with the same dose. These resultsindicate that pcEgr-hp53 transfection combined with X-ray irradiation could inhibit thegrowth of SKOV-3 cell line and the inhibiting effect was dose-dependent.3.4 Effect of pcEgr-hp53 transfection combined with X-ray irradiation ontumor cell apoptosis3.4.1 Effect of pcEgr-hp53 transfection combined with X-ray irradiation on apoptosisof A549 cellsTUNEL assay was used to detect the apoptosis of A549-vect and A549-hp53 cells. Theresults showed that the percentage of cell apoptosis in A549-vect/0.5 – 5 Gy groups wassignificantly higher than that in A549-vect/0 Gy group. The percentage of cell apoptosis inA549-hp53/0.5 – 5 Gy group were significantly higher than that in A549-hp53/0 Gy group.the percentage of cell apoptosis in A549-hp53/2 – 5 Gy group also was markedly higher thanthat in A549-vect/2 – 5 Gy after X-ray irradiation with the same dose. These results suggestthat the Egr-1 promoter can be activated by X-ray irradiation and enhance the gene proteinexpression of p53 downstream which in turn induce cell apoptosis, and wt-p53 genetransfection can markedly sensitize A549 cells to irradiation-induced apoptosis signal.3.4.2 Effect of pcEgr-hp53 transfection combined with X-ray irradiation on apoptosisof SKOV-3 cellsTUNEL assay was used to detect the apoptosis of SKOV-3-vect and SKOV-3-hp53 cells.The results showed that the percentage of cell apoptosis in SKOV-3-vect/2 – 5 Gy groupswas higher than that in SKOV-3-hp53/0Gy group. The percentage of cell apoptosis inSKOV-3-hp53/0.5 – 5 Gy groups was significantly higher than that in SKOV-3-hp53/0 Gygroup. The percentage of cell apoptosis in SKOV-3-hp53/0 – 5 Gy groups also was markedlyhigher than that in SKOV-3-vect/0 – 5 Gy after X-ray irradiation with the same dose. Theresults suggest that the Egr-1 promoter can be activated by X-ray irradiation and enhance thegene protein expression of p53 downstream which in turn induce cell apoptosis, and wt-p53gene transfection can markedly sensitize SKOV-3 cells to irradiation-induced apoptosissignal.3.5 Molecular mechanism of growth inhibiting effect in tumor cellstransfected by pcEgr-hp53 plasmid combined with X-ray irradiation3.5.1 Effect of pcEgr-hp53 transfection combined with X-ray irradiation on cellcycle progression of A549 cellsThe results detected with flow cytometry method showed that the percentage of G0/G1phase cells in A549-vect/0.5 – 5 Gy groups was markedly higher than that in A549-vect/0 Gygroup, the percentage in A549-hp53/2 – 5 Gy groups higher than that in A549-hp53/0 Gygroup, the percentage in A549-hp53/0 – 5 Gy groups also was markedly higher than that inA549-vect/0 – 5 Gy after X-ray irradiation with the same dose. The percentage of G2/Mphase cells in A549-vect/5 Gy group was markedly higher than that in A549-vect /0 Gygroup, and the percentage in A549-hp53/5 Gy group was markedly higher than that inA549-hp53/0 Gy group. The percentage of S phase cells in A549-vect /2 – 5 Gy groups wasmarkedly lower than that in A549-vect /0 Gy group, the percentage in A549-hp53/2 – 5 Gygroups was markedly lower than that in A549-hp53 /0 Gy group, and the percentage inA549-hp53/0 – 5 Gy groups also was markedly lower than that in A549-vect/0 – 5 Gy afterX-ray irradiation with the same dose.These results suggest that the gene transfection combined with irradiation exerts acoordinate effect, which not only can lead to p53-induced G1 arrest but also can lead toradiation-induced G2 arrest in A549 cells.3.5.2 Effect of pcEgr-hp53 transfection combined with X-ray irradiation on cell cycleprogression of SKOV-3 cellsThe results detected with flow cytometry method showed that the percentage of G0/G1phase cells in SKOV-3-vect/5 Gy group was significant lower than that in SKOV-3-vect /0Gy group, the percentage in SKOV-3-hp53/0.5 – 5 Gy groups was markedly higher than thatin SKOV-3-hp53/0 Gy group, and the percentage in SKOV-3-hp53/0 – 5 Gy groups was alsomarkedly higher than that in SKOV-3-vect/0 – 5 Gy groups after X-ray irradiation with thesame dose. The percentage of G2/M phase cells in SKOV-3-vect/2 – 5 Gy groups wasmarkedly higher than that in SKOV-3-vect/0 Gy group, the percentage in SKOV-3-hp53/0.5 2 Gy groups was markedly lower than that in SKOV-3-hp53/0 Gy group, and thepercentage in SKOV-3-vect/0 – 2 Gy groups changed not obviously as compared with that inSKOV-3-vect/0 Gy group. The percentage of S phase cells of SKOV-3-hp53/2 – 5 Gy groupswas markedly lower than that in SKOV-3-hp53/0 Gy group, the percentage inSKOV-3-hp53/0.5 Gy group also was markedly higher than that in SKOV-3-vect/0.5 Gygroup,and the percentage in SKOV-3-hp53/2 – 5 Gy groups was markedly lower than that inSKOV-3-vect/2 5 Gy groups. These results suggest that the gene transfection combinedwith irradiation exerts a coordinate effect.3.5.3 Changes in Bcl-2 protein expression of A549-hp53 and SKOV-3-hp53 after X-rayirradiation with different dosesThe results showed that the change of Bcl-2 protein expression in A549-vect group wasnot obviously with the enhancement of irradiation doses. The expression of Bcl-2 proteinA549-hp53/0 – 5 Gy groups was significantly lower than that in A549-hp53/0 Gy group, theexpression in A549-hp53/0.5 – 5 Gy groups was lower than that in A549-vect/0.5 – 5 Gyafter X-ray irradiation with the same dose, and the expression in SKOV-3-vect groupchanged not obvously with the enhancement with irradiation doses. The expression inSKOV-3-hp53 /0.5 Gy group was significantly hgher in SKOV-3-hp53/0.5 Gy and that inSKOV-3-hp53/5 Gy group was significantly lower than that in SKOV-3-hp53/5 Gy group.The expression in SKOV-3-hp53/2 – 5 Gy groups was lower than that in SKOV-3-vect/2 – 5Gy control after X-ray irradiation with the same dose. The results suggest that pcEgr-hp53recombinant plasmid transfection in vitro combinated with ionizing radiation could cause theexpression decrease of Bcl-2 protein in A549 and SKOV-3 cells to inhibite the growth oftumor cells.3.5.4 Changes in Bax protein expression of A549-hp53 and SKOV-3-hp53 cells afterX-ray irradiation with different dosesThe results showed that the expression of Bax protein in A549-vect group was lowerwith the enhancement of radiation doses, but not significant. The expression inA549-hp53/0.5 – 5 Gy groups was significantly higher than that in A549-hp53/0 Gy group.The expression of A549-hp53/0.5 – 5 Gy was higher than that in A549-vect/0.5 – 5 Gy groupafter X-ray irradiation with the same dose. The expression in SKOV-3-vect/0.5 Gy group wasmarkedly higher and that in SKOV-3-vect/2 Gy lower than that in SKOV-3-vect/0 Gy group.The expression in SKOV-3-hp53/2 Gy and /5 Gy groups of was significantly higher than thatin SKOV-3-hp53/0 Gy group. The expression in SKOV-3-hp53/0.5 – 5 Gy groups was higherthan that in SKOV-3-vect control after X-ray irradiation with the same dose.3.5.5 Changes of caspase-3 mRNA and protein expressions in A549-hp53 cells afterX-ray irradiation with different dosesThe results showed that the caspase-3 protein expression in A549-vect/0.5 – 5 Gygroups was lower than that in A549-vect/0 Gy group, but not significant. The expression inA549-hp53/2 – 5 Gy groups was significantly higher than that in A549-hp53/0 Gy group. Theexpression in A549-hp53/0.5 – 5 Gy groups was significantly higher than that inA549-vect/0.5 – 5 Gy after X-ray irradiation with the same dose. The results of caspase-3mRNA level in these two groups were coincident with the protein expression.3.5.6 Changes of caspase-3 mRNA and protein expressions in SKOV-3-hp53 cells afterX-ray irradiation with different dosesThe results showed that the caspase-3 protein expression in SKOV-3-vect/0.5 – 5 Gygroups was lower than that of SKOV-3-vect/0 Gy group, but not significant. The expressionin SKOV-3-hp53/2 – 5 Gy groups was significantly higher than that in SKOV-3-hp53/ 0 Gygroup. The expression in SKOV-3-hp53/0.5 – 5 Gy groups was markedly higher than that inSKOV-3-vect/0.5 – 5 Gy groups after X-ray irradiation with the same dose. The results ofcaspase-3 mRNA level in these two groups were coincident with the protein expression. Theabove-mentioned results suggest that pcEgr-hp53 recombinant plasmid transfection in vitrocombinated with ionizing radiation could induce the apoptosis in SKOV-3 cells, whichdown-regulate Bcl-2 protein expression and up-regulate Bax protein expression, openinreversibly PTP in mitochondria, enter smaller molecules in adventitia into intramembrane,loss the electric potential in mitochondron transmembrane, damage adventitia, releasecytochrome c and to bind to Apaf 1, and stimulate caspase-9 and caspase-3.