Screening Of MDR1 Expression Inhibitors And Investigate Its Reversal Effect On Drug Resistance In Tumor

Malignant tumors are a serious global health issue with increasing incidence rates.The rapid development of medical technology has significantly improved the overall survival of some types of cancer patients.However,the existence of tumor stem cells has led to the gradual development of drug resistance in tumor cells exposed to long-term stress induced by chemotherapy drugs,resulting in treatment failure.Chemotherapy resistance has become a significant problem that cannot be ignored in anti-tumor therapy.Recent studies have found that tumor cells can develop a broad range of drug resistance to chemotherapy drugs with completely different structural and pharmacological mechanisms,leading to clinical treatment failure.The current study has demonstrated that the multi-drug resistance(MDR)effect of tumors mediated by P-glycoprotein(P-gp)encoded by the MDR1 gene can be effectively reversed by blocking its drug efflux pump function.Therefore,downregulation of its expression and function can reverse MDR in tumors,thereby increasing their sensitivity to chemotherapy drugs.To this end,a screening model was established by targeting the MDR1 promoter to identify an MDR1 expression inhibitor.More than 600 natural small-molecule compounds were screened for MDR1 inhibitors with better inhibitory effects and less toxicity.The target compound was further investigated for its reversing effect on drug-resistant cells.1.Screening and identification of MDR1 expression inhibitorsTo search for potential compounds that can inhibit MDR1 expression,we transfected MDR1-luc plasmids containing the MDR1 promoter into HEK-293 T cells and treated them with over 600 small-molecule natural compounds respectively.We used a luciferase assay to assess the effect of these compounds on MDR1 transcription.The results showed that compounds such as T2-70,T4-41,and T5-17 significantly inhibited the activity of the MDR1 promoter.2.Identification of drug resistance characteristics of drug-resistant cellsWe used the MCF-7/ADR drug-resistant cell line and the MCF-7 parental cell line as experimental cells.To verify the drug resistance of the parental and drug-resistant cells,we used the MTT method to measure cell viability after treating the cells with varying concentrations of doxorubicin for 48 hours.We also measured the IC50 values of doxorubicin for both cell types.The results showed that the survival rate of MCF-7/ADR cells was significantly higher than that of MCF-7 cells after treatment with different concentrations of doxorubicin,and the IC50 value of doxorubicin for MCF-7/ADR cells was higher than that for MCF-7 cells.3.The effect of T2-70 on MDR1 expressionTo determine the effect of T2-70 on intracellular MDR1 expression,we initially used the MTT assay to evaluate the non-toxic concentration of the compound T2-70 on MCF-7/ADR cells and MCF-7 cells.The results indicated that the cell viability was almost unaffected when the concentration of T2-70 was less than 7μg/m L,and therefore,we selected a concentration of 6μg/m L for subsequent experiments.Real-time PCR and Western blot methods were employed to detect the effects of T2-70 on MDR1 expression.The results demonstrated that T2-70 can inhibit the expression of MDR1 at both the m RNA and protein levels.4.Drug resistance reversal effect of T2-70 on MCF-7/ADR cellsTo determine the reversal effect of T2-70 on tumor drug resistance,we first used the MTT assay to evaluate whether T2-70 affects the sensitivity of MCF-7/ADR cells to doxorubicin.Subsequently,flow cytometry was used to detect the effect of T2-70 on cell apoptosis.The results showed that T2-70 can enhance the sensitivity of MCF-7/ADR cells to doxorubicin.5.The impact of T2-70 on P-gp function in MCF-7/ADR cellsTo detect the effect of T2-70 on the function of P-gp protein in cells,we used two dyes,ADR and Rho-123,as P-gp substrates to detect the effect of the compound on drug efflux.We observed the fluorescence intensity inside cells under a fluorescence microscope.The results showed that after the addition of T2-70,the fluorescence accumulation of ADR and Rho-123 inside the cells was significantly enhanced.These results suggest that T2-70 reduces the drug efflux pump function of P-gp protein,increases the intracellular drug concentration,and enhances the sensitivity of tumors to chemotherapy drugs.In summary,this study identified T2-70 as a compound that can inhibit MDR1 expression,suppress P-gp protein expression at the cellular level,and reverse tumor drug resistance,providing a new lead compound for the development of tumor chemotherapy sensitizers.