Multidrug resistance (MDR) is a critical problem in cancer chemotherapy. Cancer cells can develop resistance not only to a single cytotoxic drug, but also to entire classes of structurally and functionally unrelated c...Multidrug resistance (MDR) is a critical problem in cancer chemotherapy. Cancer cells can develop resistance not only to a single cytotoxic drug, but also to entire classes of structurally and functionally unrelated compounds. Several mechanisms can mediate the development of MDR, including increased drug efflux from the cells by ABC-transporters (ABCT), activation of metabolic enzymes, and defective pathways towards apoptosis. Many plant secondary metabolites (SMs) can potentially increase sensitivity of drug-resistant cancer cells to chemotherapeutical agents. The present thesis investigates the modulation of MDR by certain medicinal plants and their active compounds. The inhibition of ABCTs (P-gp/MDR1, MRP1, BCRP) and metabolic enzymes (GST and CYP3A4), and the induction of apoptosis are useful indicators of the efficacy of a potential medicinal drug. The focus of this study was the possible mechanisms of drug resistance including: expression of resistance proteins, activation of metabolic enzymes, and alteration of the apoptosis and how to overcome their resistance effect on cancer cells. The overall goal of this review was to evaluate how commonly used medicinal plants and their main active secondary metabolites modulate multidrug resistance in cancer cells in order to validate their uses as anticancer drugs, introduce new therapeutic options for resistant cancer, and facilitate the development of their anticancer strategies and/or combination therapies. In conclusion, SMs from medicinal plants exhibit multitarget activity against MDR-related proteins, metabolic enzymes, and apoptotic signaling, this may help to overcome resistance towards chemotherapeutic drugs.展开更多
The enduring discussion,why plants produce secondary metabolites with pharmacologically and toxicologically active towards mammals traces back to the eminent role of medicinal plants in the millennia-old history of ma...The enduring discussion,why plants produce secondary metabolites with pharmacologically and toxicologically active towards mammals traces back to the eminent role of medicinal plants in the millennia-old history of manhood.In recent years,the concept of an animal plant warfare emerged,which focused on the co-evolution between plants and herbivores.As a reaction to herbivory,plants developed mechanical defenses such as thorns and hard shells,which paved the way for adapted animal physiques.Plants evolved further defense systems by producing chemicals that exert toxic effects on the animals that ingest them.As a result of this selective pressure,animals developed special enzymes,e.g.cytochrome P450 monooxigenases(CYP450)that metabolize xenobiotic phytochemicals.As a next step in the evolutionary competition between plants and animals,plants evolved to produce non-toxic pro-drugs,which become toxic only after ingestion by animals through metabolization by enzymes such as CYP450.Because these sequestered evolutionary developments call to mind an arms race,the term animal plant warfare has been coined.The evolutionary competition between plants and animals may help to better understand the modes of action of medicinal plants and to foster the efficient and safe use of phytotherapy nowadays.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
In the present study we have characterized ATP-dependent transport of cAMP and cGMP in physiological, but also supraphysiological concentrations. The uptake into inside-out vesicles from human erythrocytes could be di...In the present study we have characterized ATP-dependent transport of cAMP and cGMP in physiological, but also supraphysiological concentrations. The uptake into inside-out vesicles from human erythrocytes could be dissected into two components with high and low affinity. The respective Km-values were 30.8 ± 5.2 and 352 ± 26 μM for cAMP and 2.6 ± 0.4 and 260 ± 15 μM for cGMP. The two cyclic nucleotides were unable to mutually inhibit cellular efflux for concentrations up to about 100 μM. At higher concentrations the inhibition curve showed a steep fall. The IC50-value for cAMP reduction of high affinity [3H]-cGMP transport was 695 ± 9 μM. The respective value for cGMP inhibition of [3H]-cAMP efflux was 284 ± 20 μM. These observations are compatible with two selective high affinity transport systems. Other endogenous substances such as prostaglandins did not discriminate between cyclic nucleotide transport. The IC50 values for inhibition of [3H]-cAMP and [3H]-cGMP were 4.1 and 4.2 μM for PGE1, 2.7 and 4.4 μM for PGE2, respectively. However, the prostaglandin analog misoprostol discriminated distinctly between cAMP and cGMP transport with respective IC50-values of 4.5 and 24 μM. The assumption that the specific PDE5-inhibitor sildenafil could distinguish between the two cyclic nucleotides was disproved with respective IC50 values of 3.8 and 2.9 μM for inhibition of [3H]-cAMP and [3H]-cGMP, respectively. However, at least one sildenafil analog (PHAR0099048) showed a clear difference with respective IC50 values of 2.0 and 0.52 μM. The other tested sildenafil analogs showed no or minor ability to discriminate with IC50 values of 0.16 and 0.17 μM for IS-39213, and 0.35 and 0.16 μM for IS-60049, respectively. In agreement with previous reports, the present study shows that proteins responsible for cyclic nucleotide transport are multiorganic anion pumps. However, the observation that drug analogs may discriminate between these two efflux systems makes them potential drug targets.展开更多
Aim:The present in silico study aimed to evaluate the ATP-binding cassette(ABC)transporter inhibition potential of Bulbine frutescens(B.frutescens)phytochemicals.Methods:Several previous studies and databases were use...Aim:The present in silico study aimed to evaluate the ATP-binding cassette(ABC)transporter inhibition potential of Bulbine frutescens(B.frutescens)phytochemicals.Methods:Several previous studies and databases were used to retrieve the ligands and target protein structure.The molecular docking study was performed using the Auto Dock Tools,and the GROMACS package was applied to accomplish molecular dynamics simulation.Results:Utilizing the molecular docking and simulation approach,~25 phytochemicals were screened against the ABC transporter protein.Docking score analysis revealed that B.frutescens phytochemical 4’-Demethylknipholone 2’-β-D-glucopyranoside exhibited strong binding on the ABC transporter protein with a minimum binding score-9.8 kcal/mol in comparison to the standard ABC transporter inhibitor diltiazem(-6.86 kcal/mol).Furthermore,molecular dynamics simulation for 4’-Demethylknipholone 2’-β-D-glucopyranoside showed an acceptable root mean square deviation,radius of gyration,root mean square fluctuation,and hydrogen bond,in addition to other lead compounds.Conclusion:The in-silico study demonstrated that B.frutescens phytochemical 4’-Demethylknipholone 2’-β-D-glucopyranoside possesses anti-drug resistance properties and requires further testing in preclinical settings.展开更多
Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice （Oryza sativa L.） male sterile mutant. Based on map- based clon...Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice （Oryza sativa L.） male sterile mutant. Based on map- based cloning and sequence analysis, we identified a 1,459-bp deletion in an adenosine triphosphate （ATP）-binding cassette （ABC） transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcgl5. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 （meiosis II stage） to stage 10 （late microspore stage）. Two genes CYP704B2 and WDA1, involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcgl5 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther Iocules. Consistently, histological analysis showed that osabcgl5 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.展开更多
文摘Multidrug resistance (MDR) is a critical problem in cancer chemotherapy. Cancer cells can develop resistance not only to a single cytotoxic drug, but also to entire classes of structurally and functionally unrelated compounds. Several mechanisms can mediate the development of MDR, including increased drug efflux from the cells by ABC-transporters (ABCT), activation of metabolic enzymes, and defective pathways towards apoptosis. Many plant secondary metabolites (SMs) can potentially increase sensitivity of drug-resistant cancer cells to chemotherapeutical agents. The present thesis investigates the modulation of MDR by certain medicinal plants and their active compounds. The inhibition of ABCTs (P-gp/MDR1, MRP1, BCRP) and metabolic enzymes (GST and CYP3A4), and the induction of apoptosis are useful indicators of the efficacy of a potential medicinal drug. The focus of this study was the possible mechanisms of drug resistance including: expression of resistance proteins, activation of metabolic enzymes, and alteration of the apoptosis and how to overcome their resistance effect on cancer cells. The overall goal of this review was to evaluate how commonly used medicinal plants and their main active secondary metabolites modulate multidrug resistance in cancer cells in order to validate their uses as anticancer drugs, introduce new therapeutic options for resistant cancer, and facilitate the development of their anticancer strategies and/or combination therapies. In conclusion, SMs from medicinal plants exhibit multitarget activity against MDR-related proteins, metabolic enzymes, and apoptotic signaling, this may help to overcome resistance towards chemotherapeutic drugs.
文摘The enduring discussion,why plants produce secondary metabolites with pharmacologically and toxicologically active towards mammals traces back to the eminent role of medicinal plants in the millennia-old history of manhood.In recent years,the concept of an animal plant warfare emerged,which focused on the co-evolution between plants and herbivores.As a reaction to herbivory,plants developed mechanical defenses such as thorns and hard shells,which paved the way for adapted animal physiques.Plants evolved further defense systems by producing chemicals that exert toxic effects on the animals that ingest them.As a result of this selective pressure,animals developed special enzymes,e.g.cytochrome P450 monooxigenases(CYP450)that metabolize xenobiotic phytochemicals.As a next step in the evolutionary competition between plants and animals,plants evolved to produce non-toxic pro-drugs,which become toxic only after ingestion by animals through metabolization by enzymes such as CYP450.Because these sequestered evolutionary developments call to mind an arms race,the term animal plant warfare has been coined.The evolutionary competition between plants and animals may help to better understand the modes of action of medicinal plants and to foster the efficient and safe use of phytotherapy nowadays.
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.
基金The financial support from the Norwegian Cancer Society and the kind gift of sildenafil from Pfizer Inc.is gratefully acknowledged.
文摘In the present study we have characterized ATP-dependent transport of cAMP and cGMP in physiological, but also supraphysiological concentrations. The uptake into inside-out vesicles from human erythrocytes could be dissected into two components with high and low affinity. The respective Km-values were 30.8 ± 5.2 and 352 ± 26 μM for cAMP and 2.6 ± 0.4 and 260 ± 15 μM for cGMP. The two cyclic nucleotides were unable to mutually inhibit cellular efflux for concentrations up to about 100 μM. At higher concentrations the inhibition curve showed a steep fall. The IC50-value for cAMP reduction of high affinity [3H]-cGMP transport was 695 ± 9 μM. The respective value for cGMP inhibition of [3H]-cAMP efflux was 284 ± 20 μM. These observations are compatible with two selective high affinity transport systems. Other endogenous substances such as prostaglandins did not discriminate between cyclic nucleotide transport. The IC50 values for inhibition of [3H]-cAMP and [3H]-cGMP were 4.1 and 4.2 μM for PGE1, 2.7 and 4.4 μM for PGE2, respectively. However, the prostaglandin analog misoprostol discriminated distinctly between cAMP and cGMP transport with respective IC50-values of 4.5 and 24 μM. The assumption that the specific PDE5-inhibitor sildenafil could distinguish between the two cyclic nucleotides was disproved with respective IC50 values of 3.8 and 2.9 μM for inhibition of [3H]-cAMP and [3H]-cGMP, respectively. However, at least one sildenafil analog (PHAR0099048) showed a clear difference with respective IC50 values of 2.0 and 0.52 μM. The other tested sildenafil analogs showed no or minor ability to discriminate with IC50 values of 0.16 and 0.17 μM for IS-39213, and 0.35 and 0.16 μM for IS-60049, respectively. In agreement with previous reports, the present study shows that proteins responsible for cyclic nucleotide transport are multiorganic anion pumps. However, the observation that drug analogs may discriminate between these two efflux systems makes them potential drug targets.
基金support from the Indian Council of Medical Research(ICMR),India in the form of ICMR Senior Research fellowship.SK acknowledges the University Grants Commission(UGC),India and Department of Science and Technology(DST),India for providing financial support in the form of UGC-BSR Research Start-Up-Grant[No.F.30-372/2017(BSR)]and DST-SERB Grant(EEQ/2016/000350)respectively.SK also acknowledges DST,India for providing the Departmental DST-FIST grant to the Department of Biochemistry,Central University of Punjab,India.AKS and MS acknowledges CSIR and ICMR,India funding agencies respectively for providing financial assistance in the form of a Senior Research Fellowship.KSP acknowledge DBT,India funding agencies for providing financial assistance in the form of Junior Research Fellowship.
文摘Aim:The present in silico study aimed to evaluate the ATP-binding cassette(ABC)transporter inhibition potential of Bulbine frutescens(B.frutescens)phytochemicals.Methods:Several previous studies and databases were used to retrieve the ligands and target protein structure.The molecular docking study was performed using the Auto Dock Tools,and the GROMACS package was applied to accomplish molecular dynamics simulation.Results:Utilizing the molecular docking and simulation approach,~25 phytochemicals were screened against the ABC transporter protein.Docking score analysis revealed that B.frutescens phytochemical 4’-Demethylknipholone 2’-β-D-glucopyranoside exhibited strong binding on the ABC transporter protein with a minimum binding score-9.8 kcal/mol in comparison to the standard ABC transporter inhibitor diltiazem(-6.86 kcal/mol).Furthermore,molecular dynamics simulation for 4’-Demethylknipholone 2’-β-D-glucopyranoside showed an acceptable root mean square deviation,radius of gyration,root mean square fluctuation,and hydrogen bond,in addition to other lead compounds.Conclusion:The in-silico study demonstrated that B.frutescens phytochemical 4’-Demethylknipholone 2’-β-D-glucopyranoside possesses anti-drug resistance properties and requires further testing in preclinical settings.
基金supported by a grant from the National High Technology Research and Development Program of China (2012AA10A303)
文摘Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice （Oryza sativa L.） male sterile mutant. Based on map- based cloning and sequence analysis, we identified a 1,459-bp deletion in an adenosine triphosphate （ATP）-binding cassette （ABC） transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcgl5. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 （meiosis II stage） to stage 10 （late microspore stage）. Two genes CYP704B2 and WDA1, involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcgl5 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther Iocules. Consistently, histological analysis showed that osabcgl5 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.
