ATP-binding cassette（ABC） transporter multidrug resistance protein 4（MRP4, ABCC4） is involved in multidrug resistance（MDR）, which is an increasing challenge to the treatment of cancers and infections. MRP4 is ov...ATP-binding cassette（ABC） transporter multidrug resistance protein 4（MRP4, ABCC4） is involved in multidrug resistance（MDR）, which is an increasing challenge to the treatment of cancers and infections. MRP4 is overexpressed in several types of cancers, and MRP4 inhibition shows striking effects against cancer progression and drug resistance. However, the structural knowledge of this protein remains unclear due to lack of an MRP4 X-ray structure, and homology modeling approach is an effective way to obtain three-dimensional structure of MRP4. We constructed three molecular models of human MRP4 mainly based on the inward facing Caenorhabditis elegans P-glycoprotein（P-gp）, the Thermotoga maritima heterodimeric ABC transporter TM287-TM288（TM287/288） and the outward facing Staphylococcus aureus Sav1866 crystal structures, which represented substrate uptake, transport and release state, respectively. The structures were further energy minimized and optimized by molecular dynamic simulations（MDS）. All the models were validated by various tools and servers, and the results showed that the quality of the models was reasonable and acceptable. These MRP4 models could be used as working tools for experimental studies on the structure and functions of MRP4 and designing more specific membrane transport modulating agents（MTMA）.展开更多
Chemotherapy remains the standard treatment for acute myeloid leukemia;however,the emergence of drug resistance is a major hurdle in the successful treatment of leukemia.The expression of multidrug resistance-associat...Chemotherapy remains the standard treatment for acute myeloid leukemia;however,the emergence of drug resistance is a major hurdle in the successful treatment of leukemia.The expression of multidrug resistance-associated protein 4(MRP4)induces re- sistance in the adriamycin-resistant acute myeloid leukemia cell line,K562/ADR.The aim of this study was to investigate whether knockdown of MRP4 by lentivirus-mediated siRNA could improve the sensitivity of K562/ADR cells to adriamycin.Five lenti- virus-mediated short hairpin RNAs(lv-shRNAs-MRP4)were designed to trigger the gene silencing RNA interference(RNAi) pathway.The efficiency of lentivirus-mediated siRNA infection into K562/ADR cells was determined using fluorescence mi- croscopy to observe lentivirus-mediated GFP expression.MRP4 expression in infected K562/ADR cells was evaluated by real- time PCR and Western blot analysis.The MTS assay was used to measure cell viability and flow cytometry was used to measure apoptosis.The transfection efficiency of K562/ADR cells was over 80 percent.The gene silencing efficacy of lv-shRNA1-MRP4 was superior to the other constructs.Infection of K562/ADR cells with lv-shRNA1-MRP4 led to strong inhibition of MRP4 mRNA and protein expression.Combined treatment with lv-shRNA1-MRP4 and adriamycin decreased cell growth and increased apoptosis compared to treatment with lv-shRNA1-MRP4 or adriamycin alone.These data indicate that in K562/ADR cells MRP4 is involved in drug resistance mechanisms and that lentivirus-mediated knockdown of MRP4 may enhance sensitivity to adriamycin.展开更多
基金National High Technology Research and Development Program 863(Grant No.2012AA020308)National Natural Science Foundation of China(Grant No.21272017)
文摘ATP-binding cassette（ABC） transporter multidrug resistance protein 4（MRP4, ABCC4） is involved in multidrug resistance（MDR）, which is an increasing challenge to the treatment of cancers and infections. MRP4 is overexpressed in several types of cancers, and MRP4 inhibition shows striking effects against cancer progression and drug resistance. However, the structural knowledge of this protein remains unclear due to lack of an MRP4 X-ray structure, and homology modeling approach is an effective way to obtain three-dimensional structure of MRP4. We constructed three molecular models of human MRP4 mainly based on the inward facing Caenorhabditis elegans P-glycoprotein（P-gp）, the Thermotoga maritima heterodimeric ABC transporter TM287-TM288（TM287/288） and the outward facing Staphylococcus aureus Sav1866 crystal structures, which represented substrate uptake, transport and release state, respectively. The structures were further energy minimized and optimized by molecular dynamic simulations（MDS）. All the models were validated by various tools and servers, and the results showed that the quality of the models was reasonable and acceptable. These MRP4 models could be used as working tools for experimental studies on the structure and functions of MRP4 and designing more specific membrane transport modulating agents（MTMA）.
基金supported by the Natural Science Foundation of Gansu Province(20090501)
文摘Chemotherapy remains the standard treatment for acute myeloid leukemia;however,the emergence of drug resistance is a major hurdle in the successful treatment of leukemia.The expression of multidrug resistance-associated protein 4(MRP4)induces re- sistance in the adriamycin-resistant acute myeloid leukemia cell line,K562/ADR.The aim of this study was to investigate whether knockdown of MRP4 by lentivirus-mediated siRNA could improve the sensitivity of K562/ADR cells to adriamycin.Five lenti- virus-mediated short hairpin RNAs(lv-shRNAs-MRP4)were designed to trigger the gene silencing RNA interference(RNAi) pathway.The efficiency of lentivirus-mediated siRNA infection into K562/ADR cells was determined using fluorescence mi- croscopy to observe lentivirus-mediated GFP expression.MRP4 expression in infected K562/ADR cells was evaluated by real- time PCR and Western blot analysis.The MTS assay was used to measure cell viability and flow cytometry was used to measure apoptosis.The transfection efficiency of K562/ADR cells was over 80 percent.The gene silencing efficacy of lv-shRNA1-MRP4 was superior to the other constructs.Infection of K562/ADR cells with lv-shRNA1-MRP4 led to strong inhibition of MRP4 mRNA and protein expression.Combined treatment with lv-shRNA1-MRP4 and adriamycin decreased cell growth and increased apoptosis compared to treatment with lv-shRNA1-MRP4 or adriamycin alone.These data indicate that in K562/ADR cells MRP4 is involved in drug resistance mechanisms and that lentivirus-mediated knockdown of MRP4 may enhance sensitivity to adriamycin.
