Multidrug resistance(MDR),which significantly decreases the efficacy of anticancer drugs and causes tumor recurrence,has been a major challenge in clinical cancer treatment with chemotherapeutic drugs for decades.Seve...Multidrug resistance(MDR),which significantly decreases the efficacy of anticancer drugs and causes tumor recurrence,has been a major challenge in clinical cancer treatment with chemotherapeutic drugs for decades.Several mechanisms of overcoming drug resistance have been postulated.Well known P-glycoprotein(P-gp) and other drug efflux transporters are considered to be critical in pumping anticancer drugs out of cells and causing chemotherapy failure.Innovative theranostic(therapeutic and diagnostic) strategies with nanoparticles are rapidly evolving and are anticipated to offer opportunities to overcome these limits.In this review,we discuss the mechanisms of drug efflux-mediated resistance and the application of multiple nanoparticle-based platforms to overcome chemoresistance and improve therapeutic outcome.展开更多
Background:Despite improvements in objective response rates to cisplatin-based combination chemotherapy,the majority of advanced ovarian cancer remains suboptimal,resulting in poor survival.it has been found that non-...Background:Despite improvements in objective response rates to cisplatin-based combination chemotherapy,the majority of advanced ovarian cancer remains suboptimal,resulting in poor survival.it has been found that non-coding RNAs(ncRNAs)not only participate in the transmission of signals between various cells but also participate in tumor immunity and anti-tumor immune responses,thereby regulating tumor occurrence and development.However,the function and detailed mechanism of ultraconserved RNA(ucRNA)in ovarian cancer chemoresistance is still unclear.Methods:Western blotting assay,Quantitative real-time PCR analysis(qPCR),and Kaplan-Meier Plotter analysis were performed to analyze the expression and prognosis of uc.243 in ovarian carcinoma.Cytotoxicity assay and Annexin V assay were performed to analyze the function of uc.243 in cisplatin resistance in ovarian cancer cells.RNA pull-down and qPCR experiments were performed to explore the molecular mechanism of uc.243 enhancing cisplatin resistance in ovarian cancer cells.Results:Herein,we found that uc.243 was remarkably upregulated and correlated with patient survival in chemoresistance ovarian cancer patients compared with chemo-sensitive ovarian cancer.Functional experiment displayed that uc.243 induced cisplatin resistance on ovarian cancer cells by facilitating the efflux of cisplatin(CDDP);but inhibiting the expression of uc.243 significantly reverses this function.Mechanistically,uc.243 can inhibit the binding of RNA binding protein DGCR8 microprocessor complex subunit to pri-miR-155,thereby inhibiting the cleavage of pri-miR-155 and decrease in mature miR-155,subsequently upregulates the expression of ATP binding cassette subfamily B member(ABCB1,ABCC2).Conclusion:Our research findings indicate that uc.243 can induce chemotherapy resistance in ovarian cancer,suggesting that it may become a new prognostic biomarker for malignant ovarian cancer.展开更多
The human body consists of several physiological barriers that express a number of membrane transporters. For an orally absorbed drug the intestinal, hepatic, renal and blood-brain barriers are of the greatest importa...The human body consists of several physiological barriers that express a number of membrane transporters. For an orally absorbed drug the intestinal, hepatic, renal and blood-brain barriers are of the greatest importance. The ATP-binding cassette(ABC) transporters that mediate cellular efflux and the solute carrier transporters that mostly mediate cellular uptake are the two superfamilies responsible for membrane transport of vast majority of drugs and drug metabolites. The total number of human transporters in the two superfamilies exceeds 400, and about 40-50 transporters have been characterized for drug transport. The latest Food and Drug Administration guidance focuses on P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide 1B1(OATP1B1), OATP1B3, organic cation transporter 2(OCT2), and organic anion transporters 1(OAT1) and OAT3. The European Medicines Agency's shortlist additionally contains the bile salt export pump, OCT1, and the multidrug and toxin extrusion transporters, multidrug and toxin ex-trusion protein 1(MATE1) and MATE2/MATE2 K. A variety of transporter assays are available to test drugtransporter interactions, transporter-mediated drugdrug interactions, and transporter-mediated toxicity. The drug binding site of ABC transporters is accessible from the cytoplasm or the inner leaflet of the plasma membrane. Therefore, vesicular transport assays utilizing inside-out vesicles are commonly used assays, where the directionality of transport results in drugs being transported into the vesicle. Monolayer assays utilizing polarized cells expressing efflux transporters are the test systems suggested by regulatory agencies. However, in some monolayers, uptake transporters must be coexpressed with efflux transporters to assure detectable transport of low passive permeability drugs. For uptake transporters mediating cellular drug uptake, utilization of stable transfectants have been suggested. In vivo animal models complete the testing battery. Some issues, such as in vivo relevance, gender difference, age and ontogeny issues can only be addressed using in vivo models. Transporter specificity is provided by using knock-out or mutant models. Alternatively, chemical knock-outs can be employed. Compensatory changes are less likely when using chemical knockouts. On the other hand, specific inhibitors for some uptake transporters are not available, limiting the options to genetic knock-outs.展开更多
<strong>Background:</strong> Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds f...<strong>Background:</strong> Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. <strong>Aim:</strong> The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). <strong>Methods:</strong> DH5α competent <em>E. coli</em> cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. <strong>Results:</strong> The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. <strong>Conclusion:</strong> The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.展开更多
基金supported by the National Key Basic Research Program of China(973projects2009CB930200)the program of National Natural Science Foundation of China(30970784and81171455)the support of the Chinese Academy of Sciences(CAS)"Hundred Talents Program"and CAS Knowledge Innovation Program
文摘Multidrug resistance(MDR),which significantly decreases the efficacy of anticancer drugs and causes tumor recurrence,has been a major challenge in clinical cancer treatment with chemotherapeutic drugs for decades.Several mechanisms of overcoming drug resistance have been postulated.Well known P-glycoprotein(P-gp) and other drug efflux transporters are considered to be critical in pumping anticancer drugs out of cells and causing chemotherapy failure.Innovative theranostic(therapeutic and diagnostic) strategies with nanoparticles are rapidly evolving and are anticipated to offer opportunities to overcome these limits.In this review,we discuss the mechanisms of drug efflux-mediated resistance and the application of multiple nanoparticle-based platforms to overcome chemoresistance and improve therapeutic outcome.
文摘Background:Despite improvements in objective response rates to cisplatin-based combination chemotherapy,the majority of advanced ovarian cancer remains suboptimal,resulting in poor survival.it has been found that non-coding RNAs(ncRNAs)not only participate in the transmission of signals between various cells but also participate in tumor immunity and anti-tumor immune responses,thereby regulating tumor occurrence and development.However,the function and detailed mechanism of ultraconserved RNA(ucRNA)in ovarian cancer chemoresistance is still unclear.Methods:Western blotting assay,Quantitative real-time PCR analysis(qPCR),and Kaplan-Meier Plotter analysis were performed to analyze the expression and prognosis of uc.243 in ovarian carcinoma.Cytotoxicity assay and Annexin V assay were performed to analyze the function of uc.243 in cisplatin resistance in ovarian cancer cells.RNA pull-down and qPCR experiments were performed to explore the molecular mechanism of uc.243 enhancing cisplatin resistance in ovarian cancer cells.Results:Herein,we found that uc.243 was remarkably upregulated and correlated with patient survival in chemoresistance ovarian cancer patients compared with chemo-sensitive ovarian cancer.Functional experiment displayed that uc.243 induced cisplatin resistance on ovarian cancer cells by facilitating the efflux of cisplatin(CDDP);but inhibiting the expression of uc.243 significantly reverses this function.Mechanistically,uc.243 can inhibit the binding of RNA binding protein DGCR8 microprocessor complex subunit to pri-miR-155,thereby inhibiting the cleavage of pri-miR-155 and decrease in mature miR-155,subsequently upregulates the expression of ATP binding cassette subfamily B member(ABCB1,ABCC2).Conclusion:Our research findings indicate that uc.243 can induce chemotherapy resistance in ovarian cancer,suggesting that it may become a new prognostic biomarker for malignant ovarian cancer.
基金Supported by FP7 IMI MIP-DILI:Mechanism-based integrated systems for the prediction of drug-induced liver injuryFP7 Eustroke,Health-F2-2008-202213:European Stroke Research Network+1 种基金TUDAS-1-2006-0029,OMFB-00505/2007:Development of HTS kit for analyzing transporter-drug interactions using cholesterol treated insect-cells expressing human MXR transporterGOP-1.1.1-11-2011-0017:Integrated preclinical tools for the determination and the enhancement of drug absorption
文摘The human body consists of several physiological barriers that express a number of membrane transporters. For an orally absorbed drug the intestinal, hepatic, renal and blood-brain barriers are of the greatest importance. The ATP-binding cassette(ABC) transporters that mediate cellular efflux and the solute carrier transporters that mostly mediate cellular uptake are the two superfamilies responsible for membrane transport of vast majority of drugs and drug metabolites. The total number of human transporters in the two superfamilies exceeds 400, and about 40-50 transporters have been characterized for drug transport. The latest Food and Drug Administration guidance focuses on P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide 1B1(OATP1B1), OATP1B3, organic cation transporter 2(OCT2), and organic anion transporters 1(OAT1) and OAT3. The European Medicines Agency's shortlist additionally contains the bile salt export pump, OCT1, and the multidrug and toxin extrusion transporters, multidrug and toxin ex-trusion protein 1(MATE1) and MATE2/MATE2 K. A variety of transporter assays are available to test drugtransporter interactions, transporter-mediated drugdrug interactions, and transporter-mediated toxicity. The drug binding site of ABC transporters is accessible from the cytoplasm or the inner leaflet of the plasma membrane. Therefore, vesicular transport assays utilizing inside-out vesicles are commonly used assays, where the directionality of transport results in drugs being transported into the vesicle. Monolayer assays utilizing polarized cells expressing efflux transporters are the test systems suggested by regulatory agencies. However, in some monolayers, uptake transporters must be coexpressed with efflux transporters to assure detectable transport of low passive permeability drugs. For uptake transporters mediating cellular drug uptake, utilization of stable transfectants have been suggested. In vivo animal models complete the testing battery. Some issues, such as in vivo relevance, gender difference, age and ontogeny issues can only be addressed using in vivo models. Transporter specificity is provided by using knock-out or mutant models. Alternatively, chemical knock-outs can be employed. Compensatory changes are less likely when using chemical knockouts. On the other hand, specific inhibitors for some uptake transporters are not available, limiting the options to genetic knock-outs.
文摘<strong>Background:</strong> Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. <strong>Aim:</strong> The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). <strong>Methods:</strong> DH5α competent <em>E. coli</em> cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. <strong>Results:</strong> The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. <strong>Conclusion:</strong> The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.
