Studies On The Reversal Of Multidrug Resistance Of Cancer By P53 Gene Therapy Combined With Radiotherapy

Ovarian cancer incidence remains high in gynecological tumors. Currently, the primary ways for tumor therapy include surgery, radiotherapy and chemotherapy. During the course of treatments, the main reason which limited the cure rate and decreased the survival in ovarian cancer is the phenomenon of multidrug resistance(MDR). The development of multidrug resistance progression is a complex process with the involvement of multi-gene, multi-channel and multi-factor, thus the limitations or side effects of monotherapy lead to poor efficacy. Therefore, comprehensive treatments of cancer multidrug resistance are used in basic research and clinical treatment of cancer. With the development of molecular biology and the functional genomics, comprehensive treatment of gene-radiotherapy would provide a promising way for tumor treatment. In this study, cellular as well as biological methods were adopted to detect the difference of drug sensitivity in human ovarian cancer cell lines SKOV3 and SKVCR2.0, in which wtp53 or mtp53 were transfected. Multi-drug gene MDR1 and its product P-gp glycoprotein expression were detected after the treatments of fractionated or single irradiation. Studies on the reversal of multidrug resistance of ovarian cancer by p53 gene therapy combined with radiotherapy and exploring the relevant mechanisms would provide experimental evidence for the research of MDR.1 The detection of biological parameters and drug sensitivity in SKOV3 and SKVCR2.0 cells1.1 Cell doubling time of SKOV3 and SKVCR2.0 cellsCells were counted at 24h, 48 h, 72 h, 96 h, 120 h, 144h, 168h, 192h after planting and the growth curve was obtained. The cell doubling time of SKVCR2.0 was 53h and that of SKOV3 was 29h. The doubling time in resistant cell line SKVCR2.0 was significantly longer than parental cell line and it was about 1.8 fold higher. Results showed that SKVCR2.0 grew slowly in the growth period of logarithmic growth phase.1.2 The expression of P-gp,p53 in SKOV3 and SKVCR2.0 cells Western Blotting was applied to detect the change of P-gp and p53 expression in two p53- deficient cell lines, SKOV3 and SKVCR2.0. Results showed that in SKVCR2.0 cells the expression of P-gp was detectable, while there was no P-gp expression in SKOV3.1.3 The differences of drug sensitivity between SKOV3 and SKVCR2.0 cells MTT was applied to detect the change of survival rate in SKOV3 and SKVCR2.0, which were treated with different doses of vincristine(VCR), cisplatin(DDP), pirarubicin(THP) and etoposide(VP-16) respectively for 48h. As treated with VCR, VP-16,THP respectively, the survival rate of SKVCR2.0 was significantly higher than SKOV3 (P <0.05); When treated with DDP, there was no significant difference between two cell lines in the lower concentration, the survival rate of SKVCR2.0 increased significantly in the higher concentration. The results showed that the main reason cross-resistance of ovarian cancer cells was the overexpression of P-gp.2 The establishment of cell models with wtp53 and mtp53 in SKOV3 cell lines2.1 The establishment of SKOV3-wtp53, SKOV3-175H cell models Liposome transfection method was applied to establish SKOV3-wtp53, SKOV3-175H in vitro models. Western Blotting detection showed that wtp53 and mtp53-175H were introuduced and the models with diffeent p53 expression background were successfully constructed, i.e., SKOV3-wtp53 and SKOV3-175H.2.2 The differences of drug sensitivity in SKOV3, SKOV3-wtp53, SKOV3-175H MTT was applied to detect the change of drug sensitivity in SKOV3, SKOV3-wtp53, SKOV3-175H. After the treated with different doses of VCR, DDP, THP and VP-16 respectively for 48h, there was no significant difference among the three cells (P>0.05). The results showed that mtp53-175H could not induce multidrug resistance of parental cell line SKOV3.3 The establishment of cell models with wtp53 and mtp53 in SKVCR2.03.1 The establishment of SKVCR-wtp53, SKVCR-175H cell models Liposome transfection method was applied to establish SKVCR-wtp53, SKVCR-175H cell models. Western Blotting detection showed that wtp53 and mtp53-175H were introuduced and the models with diffeent p53 expression background were successfully constructed, i.e., SKVCR-wtp53, SKVCR-175H. Western Blotting was used to detect P-gp expression. The overexpression of P-gp was detectable in SKVCR2.0 cells, while the decreased expression P-gp was shown in SKVCR-wtp53 and SKVCR-175H cells.3.2 The differences of drug sensitivity in SKVCR2.0, SKVCR-wtp53, SKVCR-175HMTT was applied to detect the change of drug sensitivity in SKVCR2.0, SKVCR-wtp53, SKVCR-175H. Cells were cultured in 96 well plate with the concentration of 4×103 per well. We have selected four chemotherapeutics which were commonly used in clinical treatments: VCR, VP-16, THP, and DDP. Each one was used in 6 concentration grades, each concentration has three wells, the cell viability was detected 48h after the administration of chemicals. In VCR group: cell viability of SKVCR-wtp53 was significantly lower than SKVCR2.0 in low concentration of VCR (P<0.05), but there was no difference in high concentration. Within 0.2-2μg/ml of VCR, cell viability of SKVCR-175H decreased significantly, lower than SKVCR2.0 (P <0.05).In THP group: with the increase of drug concentration, the cell viability of three cells decreased, especially of SKVCR-175H. At 0.5μg/ml of THP, SKVCR-wtp53 and SKVCR-175H were at the lowest level of cell viability (P<0.05). Within 0.5-1.5μg/ml of THP, cell viability of SKVCR-175H was lower than control significantly (P <0.05).In DDP group: There was no significantly change in SKVCR-175H cell viability compared with the control. In low concentration, survival rate of SKVCR-wtp53 decreased as compared with SKVCR2.0 (P<0.05), while in high concentration, survival rate of SKVCR-wtp53 increased significantly (P<0.05).In VP-16 group: at 4μg/ml of VP-16, survival rate of SKVCR-wtp53 and SKVCR-175H were lower than SKVCR2.0 (P<0.05), while there was no change observed in other concentrations. These results suggested that wtp53 and mtp53-175H could reverse multidrug resistance.4 The expression of MDR associated proteins in different cell models Western Blotting was applied to detected the expression of MDR associated proteins in different models. TopoⅡαdecreased in SKVCR2.0 as compared with parental cell SKOV3; TopoⅡαprotein increased when transfected with wtp53 and 175H. AKT1 protein increased in SKVCR2.0 as compared with SKOV3, when transfected with wtp53 and 175H, AKT1 expression increased. PI3K-Ⅲexpression decreased in SKVCR2.0 as compared with SKOV3, when transfected with wtp53, PI3K-Ⅲexpression increased.The results suggested that p53 could up-regulate TopoⅡαexpression and triggered cell death; wtp53 could up-regulate PI3K-Ⅲand LC3 expression which involved in autophagosome formation, suggesting that autophagy might be involved in this process and the sensitivity to chemotherapeutic drugs increased.5 Effects of fractionated and single irradiation on drug resistance Fractionated irradiation program has been performed with 2Gy per fraction/day for five days. However, the single irradiation group was set with 10Gy. Western Blotting and RT-PCR were applied to detected MDR/P-gp expression in SKVCR2.0. P-gp expression decreased at 8h, 16h and 32h after fractionated irradiation as compared with control. P-gp expression was lower at 8h and up to a maximum at 16h and decreased at 32h after 10Gy irradiation. In each time points P-gp expression was lower than fractionated irradiation and control groups. RT-PCR results showed that MDR1 gene expression decreased after 10Gy or 2Gy×5 irradiation. From above-mentioned it showed that both two irradiation programs could inhibit MDR1 gene and P-gp glycoprotein expression and increase the sensitivity of ovarian cancer cells to chemotherapeutic drugs.This study will provide the evidence for supporting the combined application of gene therapy, radiotherapy for the reversal of MDR, consequently promote the treatment efficency of malignant tumors.