Role And Mechanism Of5-aza-2’-deoxycytidine Reverse The Resistance Of Ovarian Carcinoma

Drug resistance of ovarian cancer chemotherapy is the main reason for the failure ofthe treatment of ovarian cancer. Drug resistance influence the prognosis of patients withovarian cancer at a large extent.Moreover,MDR1and MRP gene overexpression isconsidered to be a common mechanism for tumor cells to produce the phenomenon ofmultidrug resistance. Recent studies revealed that apparent genetic factors have importantroles in regulation of primary and acquired resistance to malignant tumors including DNAmethylation, gene mutations, and deletions of tumor suppressor gene inactivation. Themechanism of regulating the expression of MDR1gene is very complicated. Manytranscription factors and histones involved in the regulation of MDR1expression, andMDR1promoter also play an important role in the regulation. Moreover, other proteins,such as multidrug resistance-associated protein (MRP), are also involved. In thisexperiment, to observe the cisplatin chemotherapy sensitivity and resistance protein P-gP,MRP expression changes as well as gene methylation status of CP70cells, various concentrations of demethylation agent5-aza-2’-deoxycytidine acted on drug-resistanthuman ovarian carcinoma cell line CP70cells. Finally, the mechanism of5-aza-dCreversing the cisplatin-resistant ovarian cancer cell line CP70resistance is discussed.[Objective]1. To observe the effects of demethylation agent5-aza-2’-deoxycytidine on multidrugresistance-ovarian cancer cell lines CP70cells.2. To observe the possible mechanism of demethylation agent5-aza-2’-deoxycytidinereversed the multidrug resistance-ovarian cancer cell lines CP70cells tolerance.[Methods]1. Cisplatin sensitive ovarian cancer cell lines A2780and cisplatin-resistant ovariancancer cell lines the CP70were cultured in cell medium. Immunocytochemistry, westernblot and fluorescent quantitation PCR method were used to observe the expression ofmulti-drug resistant (MDR) gene and multi-drug resistance associated protein(MRP). Theactivation of signaling pathway which associated with multidrug resistance gene andprotein was detected by western blot. Finally, the correlation between the signal pathwayand platinum-resistant of ovarian cancer is explored.2. Application of different concentrations (1.25μmol/l,2.5μmol/l,5μmol/l) of5-aza-dC acted to cisplatin-resistant ovarian cancer cells CP70, and MTT method wasused to detect the reversal of drug resistance role.3. Methylation-specific PCR was used to detect the expression of multidrugresistance (MDR1) and multidrug resistance associated protein (MRP) gene methylationstatus and their promoter region methylation status before and after the use of5-aza-dC inCP70cells. Fluorescent quantitative PCR and western blot assay were used to examineMDR1the MRP gene mRNA and protein expression changes before and afteradministration of5-aza-dC. Western blot was also used to detect the activation of thePI3K-AKT signaling pathway, and to explore the possible mechanism of5-aza-dCreversal of ovarian cancer resistance to chemotherapy.[Results]1. The expression of P-gP protein and MRP protein in cisplatin-sensitive ovarian cancer cell line A2780and cisplatin-resistant ovarian cancer cell line CP70were bothdetected. However, the expression in ovarian cancer resistant strain CP70was higher thanthat of cisplatin sensitive ovarian cancer cell line A2780.The results of immunocytochemistry, western blot and fluorescent quantitative PCRassays indicated that the protein and mRNA expressions of MDR1, MRP gene wereup-regulated in cisplatin-resistant ovarian cancer cell line CP70. These results suggestedthat the expressions of P-gP and MRP protein were associated with cisplatin-resistant ofovarian cancer.2. Western blot analysis showed that the protein expressions of MDR1and MRP inthe CP70cells were regulated, and simultaneously, the PI3K-AKT signaling pathway wasactivated.3. Various concentrations of5-aza-dC acted in cisplatin-resistant ovarian cancer cellline CP70, and CP70cells sensitivity to cisplatin was increased.MTT assay was used to detect the effect of5-aza-dC on CP70cells, the resultsshowed that multiple of cisplatin resistance decreased significantly, and showed adose-dependent manner, These results indicated that5-aza-dC could reverse the resistanceof CP70to cisplatin.4. MDR1and MRP gene methylation in A2780cells were lower than those of CP70cells. MDR1and MRP gene methylation degree was decreased after using5-Aza-dC. Wefurther detects the methylation status of the promoter region of the two genes, and theresults showed that there has gene methylation in promoter region of A2780cells.Whereas, there has low degree gene methylation in the resistant cell lines CP70cellspromoter region. Moreover, the methylation level of the promoter region does not changein CP70cells after using5-Aza-dC. P-GP and MRP protein levels in CP70cells havedecreased significantly after adding5-aza-dC (P <0.05), but its expression was notsignificantly different in the different concentrations of5-Aza-dC (P>0.05). The proteinand mRNA eapressions of MDR1and MRP decreased by5-Aza-dC in cisplatin-resistantovarian carcinoma cells. In conclusion, the expressions of MDR1and MRP in A2780cellsare decreased, whereas their expressions are unregulated in CP70cells. There has gene methylation in promoter region of A2780cells, whereas, there has low degree genemethylation in CP70cells promoter region. MRP and MDR expressions decreasedsignificantly in CP70cells after administrating5-Aza-dC.5. Various concentrations of5-Aza-dC acted in cisplatin-resistant ovarian cancer cellline CP70, PI3K-AKT signaling pathway is inhibited.Western blot assay was used to detect the effect of5-Aza-dC on PI3K-AKT signalingpathway in CP70cells, the results showed that cisplatin resistance of CP70decreasedsignificantly through the inhibition of5-Aza-dC on PI3K-AKT signaling pathway.[Conclusion]The present study demonstrated that high expression of MDR1and MRP proteinwere related to the cisplatin resistance of ovarian cancer, and P-GP and MRP highexpressions were associated with their gene methylated modification in promoter region.5-Aza-dC could inhibit the expressions of P-GP and MRP, and reverse CP70cells resistant.Moreover,5-Aza-dC could affect gene methylation level, but could not change of themethylated modification in promoter region. Further studies showed that5-Aza-dCinhibited the activation of the PI3K-AKT signaling pathway, and activation of thesignaling pathway was related with high expressions of MDR1and MRP. Therefore, wespeculated that5-Aza-dC may induce the change of gene methylation status, or inhibit theactivity of PI3K-AKT to regulate the expression of resistance-associated protein in someway, and then reverse CP70resistant cells, but the exact mechanism needs furtherexperimental to validation.