A facile synthesis and biological activity of novel tetrahydrobenzo[4′,5′]thieno[3′,2′:5,6]pyrido[4,3-d]pyrimidin-4(3H)-ones

The synthesis and biological activity of 2-substituted-8,9,10,11-tetrahydrobenzo[4′,5′]thieno[3′,2′:5,6] pyrido[4,3-d]pyrimidin-4(3H)-ones are described. Bioassay results indicated that these compounds have antifungal activity against Botrytis cinerea at a concentration of 50 mg/L. In addition, compounds 5m and 5n were effective to both KB cells and their parent multidrug resistant KBv200 cells with the overexpression of ABCB1. For example, compound 5m showed the best inhibition against KB and KBv200 cells with IC₅₀ values of 17.4 and 25.4 μM, respectively.     

靶向干扰Chkl增强阿霉素抗人乳腺癌MCF-7/adr细胞的作用机制研究

Chkl的高表达可能是肿瘤对化疗药物的敏感性降低的重要因素之一,本研究的目的是观察siRNA干扰Chk1对人乳腺癌耐药细胞株MCF-7/adr(耐阿霉素)生长及细胞周期的影响,探讨Chk1在乳腺癌细胞耐药中的作用机制。采用RNAi技术抑制MCF-7/adr细胞中Chk1的表达。Westernblot检测转染前后细胞内Chk1蛋白表达情况,经阿霉素作用后,流式细胞术(FCM)检测其细胞周期分布及细胞凋亡率,MTT法检测细胞增殖。Western blot结果显示,Chk1 siRNA转染24h后,MCF-7/adr细胞中Chk1蛋白表达下降了67%,明显低于对照组和空载体转染组(P<0.05)。FCM法检测结果显示,同时,抑制Chk1的表达可解除阿霉素引起的G_2/M期阻滞;使阿霉素诱导的细胞凋亡率由转染前的(5.54±0.15)%上升到(22.24±0.13)%(P<0.05);在阿霉素浓度为0.4mg/L、4mg/L时,细胞的增殖活性分别下降13%、34%。提示siRNA干扰Chk1能够通过调控MCF-7/adr细胞周期及增殖从而增强乳腺癌细胞对阿霉素的敏感性,为临床上克服乳腺癌化疗耐药提供了新的作用靶点。     

Estimation of ABCB1 concentration in plasma membrane

The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples. In this study, we demonstrate an approach, which enables us to estimate the concentration of ABCB1 molecules within plasma membranes. For this purpose, human leukemia K562 cells with varying expression levels of ABCB1 were used: drug selected K562/Dox and K562/HHT cells with very high transporter expression, and K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with gradually decreased expression of ABCB1 derived from K562/Dox cells using RNA interference technology. First, we determined the absolute amount of ABCB1 in cell lysates using immunoblotting and recombinant ABCB1 as a standard. We then determined the relative portion of transporter residing in the plasma membrane using immunohistochemistry in nonpermeabilized and permeabilized cells. These results enabled us to estimate the concentration of ABCB1 in the plasma membrane in resistant cells. The ABCB1 concentrations in the plasma membrane of drug selected K562/Dox and K562/HHT cells containing the highest amount of transporter reached millimolar levels. Concentrations of ABCB1 in the plasma membrane of resistant K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with lower transporter expression were proportionally decreased.     

Effects of plant sterols on human multidrug transporters ABCB1 and ABCC1

The effects of dietary plant sterols on human drug efflux transporters P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-gp-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytosterols found in foods, herbs, and dietary supplements such as β-sitosterol, campesterol, stigmasterol, fucosterol, and z-guggulsterone were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-gp, increased in the presence of guggulsterone in KB-C2 cells. The efflux of rhodamine 123 from KB-C2 cells was inhibited by guggulsterone. Guggulsterone also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-gp and MRP1 were stimulated by guggulsterone. These results suggest that guggulsterone, a natural dietary hypolipidemic agent have dual inhibitory effects on P-gp and MRP1 and the potencies to cause food-drug interactions.     

ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis

Auxin transport is mediated at the cellular level by three independent mechanisms that are characterised by the PIN-formed (PIN), P-glycoprotein (ABCB/PGP) and AUX/LAX transport proteins. The PIN and ABCB transport proteins, best represented by PIN1 and ABCB19 (PGP19), have been shown to coordinately regulate auxin efflux. When PIN1 and ABCB19 coincide on the plasma membrane, their interaction enhances the rate and specificity of auxin efflux and the dynamic cycling of PIN1 is reduced. However, ABCB19 function is not regulated by the dynamic cellular trafficking mechanisms that regulate PIN1 in apical tissues, as localisation of ABCB19 on the plasma membrane was not inhibited by short-term treatments with latrunculin B, oryzalin, brefeldin A (BFA) or wortmannin--all of which have been shown to alter PIN1 and/or PIN2 plasma membrane localisation. When taken up by endocytosis, the styryl dye FM4-64 labels diffuse rather than punctuate intracellular bodies in abcb19 (pgp19), and some aggregations of PIN1 induced by short-term BFA treatment did not disperse after BFA washout in abcb19. Although the subcellular localisations of ABCB19 and PIN1 in the reciprocal mutant backgrounds were like those in wild type, PIN1 plasma membrane localisation in abcb19 roots was more easily perturbed by the detergent Triton X-100, but not other non-ionic detergents. ABCB19 is stably associated with sterol/sphingolipid-enriched membrane fractions containing BIG/TIR3 and partitions into Triton X-100 detergent-resistant membrane (DRM) fractions. In the wild type, PIN1 was also present in DRMs, but was less abundant in abcb19 DRMs. These observations suggested a rationale for the observed lack of auxin transport activity when PIN1 is expressed in a non-plant heterologous system. PIN1 was therefore expressed in Schizosaccharomyces pombe, which has plant-like sterol-enriched microdomains, and catalysed auxin transport in these cells. These data suggest that ABCB19 stabilises PIN1 localisation at the plasma membrane in discrete cellular subdomains where PIN1 and ABCB19 expression overlaps.     

miR-200c-3p靶向FOSL1增加乳腺癌细胞化疗敏感性

化疗耐受是乳腺癌复发转移率居高不下、综合治疗效果难以提高的主要瓶颈。前期研究证实,miR-200c-3p在乳腺癌敏感细胞MCF-7中的表达量显著高于耐药细胞MCF-7/5Fu,提示miR-200c-3p可能参与乳腺癌化疗增敏,但是具体机制不详。生物信息学预测联合双荧光素酶报告基因实验证实,miR-200c-3p靶向调控FOSL1,且在多种肿瘤中miR-200c-3p与FOSL1表达负相关。实时荧光定量PCR技术和Western印迹技术证实,FOSL1在耐药细胞MCF-7/5Fu中的表达量显著高于亲本细胞MCF-7。在MCF-7细胞中,过表达FOSL1能够显著提高该细胞对5-Fu的化疗耐受;在MCF-7/5Fu中,使用siRNA技术沉默FOSL1,将提高该细胞对5-Fu的化疗敏感性。此外,MTT实验还发现,miR-200c-3p抑制剂能够显著上调MCF-7细胞对5-Fu的耐受,但是在此细胞中干扰FOSL1的表达,又可以增加其对5-Fu的化疗敏感性;miR-200c-3p mimics显著增加MCF-7/5Fu细胞的化疗敏感性,上调FOSL1表达后又可逆转miR-200c-3p mimics的化疗增敏作用。总之,miR-200-3p能够通过靶向FOSL1增加乳腺癌细胞对5-fluorouridine化疗敏感性。     

Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells

Development of agents to overcome multidrug resistance (MDR) is important in cancer chemotherapy. Up to date, few chemicals have been reported to down-regulate MDR1 gene expression. We evaluated the effect of tryptanthrin on P-glycoprotein (P-gp)-mediated MDR in a breast cancer cell line MCF-7. Tryptanthrin could depress overexpression of MDR1 gene. We observed reduction of P-gp protein in parallel with decreases in mRNA in MCF-7/adr cells treated with tryptanthrin. Tryptanthrin suppressed the activity of MDR1 gene promoter. Tryptanthrin also enhanced interaction of the nuclear proteins with the negatively regulatory CAAT region of MDR1 gene promoter in MCF-7/adr. It might result in suppression of MDR1 gene. In addition, tryptanthrin decreased the amount of mutant p53 protein with decreasing mutant p53 protein stability. It might contribute to negative regulation of MDR1 gene. In conclusion, tryptanthrin exhibited MDR reversing effect by down-regulation of MDR1 gene and might be a new adjuvant agent for chemotherapy.     

BIRB796, the Inhibitor of p38 Mitogen-Activated Protein Kinase,Enhances the Efficacy of Chemotherapeutic Agents in ABCB1 Overexpression Cells

ATP-binding-cassette family membrane proteins play an important role in multidrug resistance. In this study, we investigated BIRB796, an orally active inhibitor of p38 mitogen-activated protein kinase, reversed MDR induced by ABCB1, ABCG2 and ABCC1. Our results showed that BIRB796 could reverse ABCB1-mediated MDR in both the drug selected and transfected ABCB1-overexpressing cell models, but did not enhance the efficacy of substrate-chemotherapeutical agents in ABCC1 or ABCG2 overexpression cells and their parental sensitive cells. Furthermore, BIRB796 increased the intracellular accumulation of the ABCB1 substrates, such as rhodamine 123 and doxorubicin. Moreover, BIRB796 bidirectionally mediated the ATPase activity of ABCB1, stimulating at low concentration, inhibiting at high concentration. However, BIRB796 did not alter the expression of ABCB1 both at protein and mRNA level. The down-regulation of p38 by siRNA neither affected the expression of ABCB1 nor the cytotoxic effect of paclitaxel on KBV200. The binding model of BIRB796 within the large cavity of the transmembrane region of ABCB1 may form the basis for future lead optimization studies. Importantly, BIRB796 also enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBV200 cell xenografts in nude mice. Overall, we conclude that BIRB796 reverses ABCB1-mediated MDR by directly inhibiting its transport function. These findings may be useful for cancer combinational therapy with BIRB796 in the clinic.     

Discovery of a non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) that modulates ABCB1-mediated multidrug resistance (MDR)

Multidrug resistance (MDR) has been shown to reduce the effectiveness of chemotherapy. Strategies to overcoming MDR have been widely explored in the last decades, leading to a generation of numerous small molecules targeting ABC and MRP transporters. Among the ABC family, ABCB1 plays key roles in the development of drug resistance and is the most well studied. In this work, we report the discovery of a non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) from our structurally diverse in-house compound collection that selectively modulates ABCB1-mediated multidrug resistance. WS-10 enhanced the intracellular accumulation of paclitaxel in SW620/Ad300 cells, but did not affect the expression of ABCB1 Protein and ABCB1 localization. The cellular thermal shift assay (CETSA) showed that WS-10 was able to bind to ABCB1, which could be responsible for the reversal effect of WS-10 toward paclitaxel and doxorubicin in SW620/Ad300 cells. Docking simulations were performed to show the possible binding modes of WS-10 within ABCB1 transporter. To conclude, WS-10 could be used as a template for designing new ABCB1 modulators to overcome ABCB1-mediated multidrug resistance.     

Discovery of a non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) that modulates ABCB1-mediated multidrug resistance (MDR)

Multidrug resistance (MDR) has been shown to reduce the effectiveness of chemotherapy. Strategies to overcoming MDR have been widely explored in the last decades, leading to a generation of numerous small molecules targeting ABC and MRP transporters. Among the ABC family, ABCB1 plays key roles in the development of drug resistance and is the most well studied. In this work, we report the discovery of non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) from our structurally diverse in-house compound collection that selectively modulates ABCB1-mediated multidrug resistance. WS-10 enhanced the intracellular accumulation of paclitaxel in SW620/Ad300 cells, but did not affect the expression of ABCB1 Protein and ABCB1 localization. The cellular thermal shift assay (CETSA) showed that WS-10 was able to bind to ABCB1, which could be responsible for the reversal effect of WS-10 toward paclitaxel and doxorubicin in SW620/Ad300 cells. Docking simulations were performed to show the possible binding modes of WS-10 within ABCB1 transporter. To conclude, WS-10 could be used as a template for designing new ABCB1 modulators to overcome ABCB1-mediated multidrug resistance.     

The phosphodiesterase-5 inhibitor vardenafil is a potent inhibitor of ABCB1/P-glycoprotein transporter

One of the major causes of chemotherapy failure in cancer treatment is multidrug resistance (MDR) which is mediated by the ABCB1/P-glycoprotein. Previously, through the use of an extensive screening process, we found that vardenafil, a phosphodiesterase 5 (PDE-5) inhibitor significantly reverses MDR in ABCB1 overexpressing cancer cells, and its efficacy was greater than that of tadalafil, another PDE-5 inhibitor. The present study was designed to determine the reversal mechanisms of vardenafil and tadalafil on ABC transporters-mediated MDR. Vardenafil or tadalafil alone, at concentrations up to 20 μM, had no significant toxic effects on any of the cell lines used in this study, regardless of their membrane transporter status. However, vardenafil when used in combination with anticancer substrates of ABCB1, significantly potentiated their cytotoxicity in ABCB1 overexpressing cells in a concentration-dependent manner, and this effect was greater than that of tadalafil. The sensitivity of the parenteral cell lines to cytotoxic anticancer drugs was not significantly altered by vardenafil. The differential effects of vardenafil and tadalafil appear to be specific for the ABCB1 transporter as both vardenafil and tadalafil had no significant effect on the reversal of drug resistance conferred by ABCC1 (MRP1) and ABCG2 (BCRP) transporters. Vardenafil significantly increased the intracellular accumulation of [(3)H]-paclitaxel in the ABCB1 overexpressing KB-C2 cells. In addition, vardenafil significantly stimulated the ATPase activity of ABCB1 and inhibited the photolabeling of ABCB1 with [(125)I]-IAAP. Furthermore, Western blot analysis indicated the incubation of cells with either vardenafil or tadalafil for 72 h did not alter ABCB1 protein expression. Overall, our results suggest that vardenafil reverses ABCB1-mediated MDR by directly blocking the drug efflux function of ABCB1.     

Long non‐coding RNA FTH1P3 activates paclitaxel resistance in breast cancer through miR‐206/ABCB1

Emerging evidence has indicated the important function of long non‐coding RNAs (lncRNAs) in tumour chemotherapy resistance. However, the underlying mechanism is still ambiguous. In this study, we investigate the physiopathologic role of lncRNA ferritin heavy chain 1 pseudogene 3 (FTH1P3) on the paclitaxel (PTX) resistance in breast cancer. Results showed that lncRNA FTH1P3 was up‐regulated in paclitaxel‐resistant breast cancer tissue and cells (MCF‐7/PTX and MDA‐MB‐231/PTX cells) compared with paclitaxel‐sensitive tissue and parental cell lines (MCF‐7, MDA‐MB‐231). Gain‐ and loss‐of‐function experiments revealed that FTH1P3 silencing decreased the 50% inhibitory concentration (IC50) value of paclitaxel and induced cell cycle arrest at G2/M phase, while FTH1P3‐enhanced expression exerted the opposite effects. In vivo, xenograft mice assay showed that FTH1P3 silencing suppressed the tumour growth of paclitaxel‐resistant breast cancer cells and ABCB1 protein expression. Bioinformatics tools and luciferase reporter assay validated that FTH1P3 promoted ABCB1 protein expression through targeting miR‐206, acting as a miRNA “sponge.” In summary, our results reveal the potential regulatory mechanism of FTH1P3 on breast cancer paclitaxel resistance through miR‐206/ABCB1, providing a novel insight for the breast cancer chemoresistance.     

MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential

MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.     

Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation

Polar transport of the plant hormone auxin is controlled by PIN- and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co-IP) and shotgun LC-MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. In-vitro and yeast expression analyses indicated that PID specifically modulates ABCB1-mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co-transfection in tobacco revealed that PID enhances ABCB1-mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho-proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter-active impact on ABCB1 activity that is coordinated by TWD1-PID interaction.     

The liposomal daunorubicin plus tamoxifen: improving the stability, uptake, and biodistribution of carriers

The synergistic effects of tamoxifen on the sensitivity of MCF-7 cells to daunorubicin have been reported. Whether the effects of daunorubicin on MCF-7/adr cells can be improved by tamoxifen in liposomes and how tamoxifen changes daunorubicin's behavior in vivo remains unclear. The aim of this study was to investigate the effects of tamoxifen on the uptake and biodistribution of daunorubicin liposomes by breast-cancer-resistant MCF-7/adr cells in vitro and in vivo. The uptake of liposomes by MCF-7/adr cells in vitro studies was measured using flow cytometry and laser confocal microscopy. The biodistributions of carriers and free drugs were evaluated by DiR dye using in vivo imaging. Tamoxifen obviously enhanced the cellular uptake of liposomes by MCF-7/adr cells in time-dependent manners. According to the results from in vivo imaging analysis, the mean fluorescence intensity of DiR liposomes with tamoxifen in the tumor regions of MCF-7/adr tumor-bearing nude mice was much stronger than that of DiR liposomes alone (16,450 ± 1,331 versus 3,666 ± 321; n = 3). Pegylated liposomes elongated the existence of daunorubicin in the circulatory system and the enhanced permeability and retention effect enhanced its concentration in local tumor tissues, which may provide the precondition for tamoxifen further promoting the uptake by MCF-7/Adr cells in vivo. Using daunorubicin liposomes and tamoxifen together generates better biodistribution profiles in tumor tissue than using daunorubicin liposomes only, which contributes to improving the therapeutic effect of breast cancer treatment.     

ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity

Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1(-) bulk tumor cells. ABCB1(+) cells also exhibited enhanced clonogenicity, anchorage-independent growth, tumorigenicity and mitochondrial activity compared to ABCB1(-) cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1(+) cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma.     

Increased ABCB1 Expression in TP-110-Resistant RPMI-8226 Cells

TP-110, a novel proteasome inhibitor, has been found to possess potent growth inhibition in human multiple myeloma cells. To enhance its therapeutic effects, we established TP-110-resistant RPMI-8226 (RPMI-8226/TP-110) cells and elucidated their resistance mechanisms. The IC₅₀ value for TP-110 cytotoxicity in the RPMI-8226/TP-110 cells was about 10-fold higher than that of the parental sensitive cells. The RPMI-8226/TP-110 cells exhibited distinct drug resistance to other proteasome inhibitors. Furthermore, they showed high cross-resistance to the cytotoxic effects of doxorubicin, etoposide, taxol, and vincristine. P-glycoprotein (MDR1), encoded by ABCB1, was elevated in the RPMI-8226/TP-110 cells, and the MDR1 inhibitor verapamil overcame their resistance to TP-110. The results of DNA microarray and RT-PCR suggested that the expression of ABCB1 is significantly elevated in RPMI-8226/TP-110 cells. This indicates that resistance in RPMI-8226/TP-110 cells is involved in the expression of P-glycoprotein, a drug-efflux pump.