Restoration Of Chemosensitivity In Cancer Cells With MDR Phenotype By Deoxyribozyme, Compared With Ribozyme

Objective:One of the main mechanisms for multidrug resistance (MDR) involves multidrug resistance gene1(MDR1) which encodes P-glycoprotein (Pgp). Pgp acts as a drug efflux pump and exports chemotherapeutic agents from cancer cells. Specific inhibition of Pgp expression by gene therapy is considered a well-respective strategy having less innate toxicities. At present, the investigation of DRz in reversal MDR is scarce. In the study, phosphorothioate DRz that targets to the translation initiation codon AUG was synthesized and transfected into breast cancer cells and leukemia cells with MDR phenotype. ASODN (antisense oligonucleotide) and ribozyme targets to the same region were also synthesized for comparison analysis.Methods:Alterations in MDR1mRNA and Pgp were determined by RT-PCR, flow cytometry and Rh123retention tests. Chemosensitivity of the treated cells was determined by MTT assay. The phosphorothioate DRz that targets to the translation initiation codon AUG was synthesized and transfected into breast cancer cells MCF-7and leukemia cells K562which are sensitive to Adriamycin and the cells with MDR phenotype MCF-7/ADM and K562/ADR. ASODN (antisense oligonucleotide) and ribozyme targets to the same region were also synthesized for comparison analysis. Alterations in MDR1mRNA and Pgp and Rh123retention were compared to untreated group, the mock control, the unspecific ASODN, unspecific ribozyme and unspecific DRz group.Results: 1. In RT-PCR analysis, the amplification products of MDR1and β-actin were396bp and206bp. Thirty-six hours after transfection with DRz, ASODN, ribozyme and the controls, respectively, at concentration of5μ g/ml, significant decrease of MDR1mRNA expression was observed in the cells treated with DRz, ASODN or ribozyme compared with the untreated group. No changes were observed in the unspecific control groups and no change was observed on the expression level of GSTπ:in all groups.2. The amount of Pgp was analyzed quantitatively by flow cytometry. And it indicates that DRz could inhibit Pgp expression even at concentration of0.5μg/ml.The inhibition effect of DRz on Pgp expression appeared12hours after transfection and reached its climax36hours after transfection, with better inhibition efficiency than that of either the ribozyme plasmid or ASODN. A longer inhibitory effect on Pgp synthesis by the ribozyme plasmid was also observed.3. The function of Pgp was measured using the Rh123retention test. Thirty-six hours after transfection with DRz, ASODN or ribozyme at a concentration of5μg/ml, intracellular Rh123retention in DRz group was significantly higher than that observed in the ASODN group and ribozyme group. These data indicate that the efflux function of Pgp in the treated cells was inhibited, especially in the DRz group.4. In the MTT assay, compared with the ASODN or ribozyme group, a significant reduction in drug resistance to Adriamycian and Vinblastin was found in cells treated with DRz. The best level of restoration of chemosensitivity was observed36hours after transfection in the DRz group.Conclusions:DRz targeted to the translation initiation codon AUG can reverse MDR phenotype in cancer cells and restore their chemosensitivity. Moreover, the reversal efficiency of DRz is better than that of ribozyme and ASODN targets to the same region of MDR1mRNA.