A New Proteasome Inhibitor,TP-110, Induces Apoptosis in Human Prostate Cancer PC-3 Cells

Proteasome inhibitors are useful in the treatment of cancer. Recently, we found a new proteasome inhibitor, TP-110, derived from tyropeptin A produced by Kitasatospora sp. Here we report that TP-110 induces apoptosis in human prostate cancer PC-3 cells. TP-110 showed strong cytotoxicity to PC-3 cells (IC₅₀=0.05 μM). It increased the number of cells in the G₂-M phase and increased the accumulated amounts of the p21 and p27 proteins, which are negative regulators of cell cycle progression. Furthermore, it induced apoptosis along with chromatin condensation and DNA fragmentation in PC-3 cells, and TP-110-induced apoptosis appeared to be associated with caspase activation. Additionally, TP-110 inhibited not only the degradation of IκB and the nuclear translocation of nuclear factor-κB (NF-κB), but also the DNA binding activity of NF-κB. These results indicate that TP-110 shows a strong growth inhibition and apoptosis in PC-3 cells.     

ABCB1基因C3435T多态性与耐药性癫痫的关系

目的:探讨中国粤西地区汉族人群ABCB1基因C3435T (rs1045642)的单核苷酸多态性与耐药性癫痫的关系.方法:研究对象128例,包括正常对照组60例和癫痫组68例.根据患者对抗癫痫药物的反应性将癫痫组分为耐药组(30例)和药物敏感组(38例).提取所有研究对象外周血基因组DNA,采用PCR扩增后基因测序鉴定ABCB1基因C3435T多态性.测定该位点基因型频率和等位基因频率,并进行统计学分析.结果:各组ABCB1基因C3435T基因型频率的分布符合Hardy-Weinburg平衡,提示其来自同一孟德尔群体.正常对照组与药物敏感组及耐药组,药物敏感组与耐药组间基因型频率比较,差异均无统计学意义(P＞0.05).正常对照组与药物敏感组及耐药组,药物敏感组与耐药组间等位基因型频率比较,差异亦无统计学意义(P＞0.05).结论:本研究结果初步证实ABCB1基因C3435T位点多态性分布与耐药性癫痫之间无相关性.     

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.     

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.     

ABCB1基因多态性与难治性癫痫

近年来,难治性癫痫的病因与多药耐药基因以及多药耐药基因与抗癫痫治疗的因果关系越来越受到关注。P糖蛋白(P-glycopretein,P-gp)是由ATP结合盒B亚家族成员1转运蛋白基因(ATP-binding cassette subfamily B member 1 transporter gene,ABCB1)编码的产物。它不仅可以限制抗癫痫药物(antiepileptic drug,AED)的消化道吸收,而且可以在细胞和亚细胞水平调控药物在中枢神经系统的运输过程。除了生理和环境因素的影响,P-gp的功能和表达的变化可能主要取决于ABCB1基因的多态性,这是目前研究得最广泛、最深入的多药耐药机制。本文就目前ABCB1基因多态性与难治性癫痫的相关性研究进展作一综述。     

Association of a polymorphism of ABCB1 with obesity in Japanese individuals

The aim of the present study was to identify gene polymorphisms that confer susceptibility to obesity. A total of 5448 unrelated Japanese individuals from two independent populations were examined: subject panel A comprised 4252 individuals who visited participating hospitals; subject panel B comprised 1196 community-dwelling elderly individuals. The genotypes for 95 polymorphisms of 67 candidate genes were determined. The χ² test revealed that six polymorphisms were related (p < 0.05) to the prevalence of obesity in subject panel A; after application of Bonferroni's correction, however, only the 2677G → A/T polymorphism (rs2032582) of the ATP-binding cassette, subfamily B, member 1 gene (ABCB1) was significantly associated (p = 0.0003) with obesity. Subsequent multivariable logistic regression analysis also revealed that the 2677G → A/T polymorphism of ABCB1 was significantly associated with obesity. For validation of this association, the 2677G → A/T polymorphism of ABCB1 was examined in subject panel B and again found to be significantly associated with obesity. Body mass index was significantly (p = 0.01) greater for individuals with the variant T allele of this polymorphism than for those with the GG genotype in the combined subject panels A and B. Our results suggest that the ABCB1 genotype may prove informative for assessment of genetic risk for obesity in Japanese individuals.     

药物基因组学相关P450和ABCB1多态性及SNP检测技术

药物基因组学（phamacogenomics）是临床检测遗传差异引起药物应答个体性差异的学科,它涉及药物代谢和有害的药物反应的预测等方面的内容。个性化药物和个性化治疗发展的关键条件是能够快速简便的检测出病人的遗传多态性。文章综述了药物基因相关问题,细胞色素酶1）450和ABCB1转运蛋白的遗传多态性以及检测遗传多态性的相关技术。     

Structural insights into P-glycoprotein (ABCB1) by small angle X-ray scattering and electron crystallography

P-glycoprotein (ABCB1) is an ATP-binding cassette protein that is associated with the acquisition of multi-drug resistance in cancer and the failure of chemotherapy in humans. Structural insights into this protein are described using a combination of small angle X-ray scattering data and cryo-electron crystallography data. We have compared the structures with bacterial homologues, and discuss the development of homology models for P-glycoprotein based on the bacterial Sav1866 structure.     

Inhibition of Proteasome Activity by Tyropeptin A in PC12 Cells

Tyropeptin A, a potent proteasome inhibitor not reported before, was produced by Kitasatospora sp. MK993-dF2. In this study, we investigated the effects of tyropeptin A on proteasome activity in PC12 cells. Tyropeptin A inhibited the intracellular proteasome activity in a dose-dependent way and seemed to cause neurite outgrowth. As expected, ubiquitinated proteins that should be substrates for the proteasome accumulated in cells treated with tyropeptin A. Hence, it appears that tyropeptin A can permeate into cells and there inhibit the intracellular proteasome activity.     

Decreased Plakophilin-1 Expression Promotes Increased Motility in Head and Neck Squamous Cell Carcinoma Cells

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues, including the oral epithelium. The transmembrane core of the desmosome is composed of the desmosomal cadherins that interact extracellularly to mediate cell-cell adhesion. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with the keratin intermediate filament cytoskeleton. Plakophilin 1 is a major desmosomal plaque component that functions to recruit intermediate filaments to sites of cell-cell contact via interactions with desmoplakin. Decreased assembly of desmosomes has been reported in several epithelial cancers. We examined plakophilin-1 expression in an esophageal squamous cell carcinoma tissue microarray and found that plakophilin-1 expression inversely correlates with tumor grade. In addition, we sought to investigate the effect of plakophilin-1 expression on desmosome assembly and cell motility in oral squamous cell carcinoma cell lines. Cell lines expressing altered levels of plakophilin-1 were generated and the ability of these cells to recruit desmoplakin to sites of cell-cell contact was examined. Our results show that decreased expression of plakophilin-1 results in decreased desmosome assembly and increased cell motility and invasion. These data lead us to propose that loss of plakophilin-1 expression during head and neck squamous cell carcinoma (HNSCC) progression may contribute to an invasive phenotype.     

Nitensidine A,a guanidine alkaloid from Pterogyne nitens,is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.     

The multidrug resistance pump ABCB1 is a substrate for the ubiquitin ligase NEDD4-1

Abstract

The ATP Binding Cassette transporter ABCB1 can export the neurotoxic peptide β-amyloid from endothelial cells that line the blood-brain barrier (BBB). This has the potential to lower cerebral levels of β-amyloid, but ABCB1 expression in the BBB appears to be progressively reduced in patients with Alzheimer’s disease. The surface density of many membrane proteins is regulated by ubiquitination catalyzed by ubiquitin E3 ligases. In brain capillaries of mice challenged with β-amyloid ex vivo, we show that the level of the ubiquitin ligase Nedd4 increases concomitant with reduction in Abcb1. In vitro we show that human ABCB1 is a substrate for human NEDD4-1 ligase. Recombinant ABCB1 was purified from Sf21 insect cells and incubated with recombinant NEDD4-1 purified from Escherichia coli. The treated ABCB1 had reduced mobility on SDS-PAGE, and mass spectrometry identified eight lysine residues, K271, K272, K575, K685, K877, K885, K887 and K1062 that were ubiquitinated by NEDD4-1. Molecular modelling showed that all of the residues are exposed on the surface of the intracellular domains of ABCB1. K877, K885 and K887 in particular, are located in the intracellular loop of transmembrane helix 10 (TMH10) in close proximity, in the tertiary fold, to a putative NEDD4-1 binding site in the intracellular helix extending from TMH12 (PxY motif, residues 996–998). Transient expression of NEDD4-1 in HEK293 Flp-In cells stably expressing ABCB1 was shown to reduce the surface density of the transporter. Together, the data identify this ubiquitin ligase as a potential target for intervention in the pathophysiology of Alzheimer’s disease.     

Absence of a general association between ABCB1 genetic variants and response to antiepileptic drugs in epilepsy patients

Over-expression of efflux transporter P-glycoprotein (PgP) encoded by ABCB1 gene has been implicated in poor responsive epilepsy. Several genetic variants have been shown to influence the expression levels of P-glycoprotein. The aim of the present study was to investigate the role of ABCB1 polymorphisms: C1236T, G2677T/A and C3435T in determining drug response to first line antiepileptic drugs (AEDs) namely phenobarbitone, phenytoin, carbamazepine and valproate in North Indian cohort of epilepsy patients. DNA samples were obtained from 392 consecutive epilepsy patients, out of which 228 had completed follow-up evaluation at 12 months. After attaining steady state of the AEDs in the first two months of study, 133 patients showed complete freedom from seizures (no-seizure group) and 95 patients continued to have seizures (recurrent-seizures group) in the remaining period of study. Comparison of “no-seizure” and “recurrent-seizures” groups revealed no significant differences in allelic, genotypic and haplotypic frequencies for all the studied variants. In conclusion, our finding disproves a general association between ABCB1 polymorphisms and drug response in epilepsy patients.     

The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo

The mitochondrial transporter ATP binding cassette mitochondrial erythroid (ABC-me/ABCB10) is highly induced during erythroid differentiation by GATA-1 and its overexpression increases hemoglobin production rates in vitro. However, the role of ABC-me in erythropoiesis in vivo is unknown. Here we report for the first time that erythrocyte development in mice requires ABC-me. ABC-me-/- mice die at day 12.5 of gestation, showing nearly complete eradication of primitive erythropoiesis and lack of hemoglobinized cells at day 10.5. ABC-me-/- erythroid cells fail to differentiate because they exhibit a marked increase in apoptosis, both in vivo and ex vivo. Erythroid precursors are particularly sensitive to oxidative stress and ABC-me in the heart and its yeast ortholog multidrug resistance-like 1 have been shown to protect against oxidative stress. Thus, we hypothesized that increased apoptosis in ABC-me-/- erythroid precursors was caused by oxidative stress. Within this context, ABC-me deletion causes an increase in mitochondrial superoxide production and protein carbonylation in erythroid precursors. Furthermore, treatment of ABC-me-/- erythroid progenitors with the mitochondrial antioxidant MnTBAP (superoxide dismutase 2 mimetic) supports survival, ex vivo differentiation and increased hemoglobin production. Altogether, our findings demonstrate that ABC-me is essential for erythropoiesis in vivo.     

AQP1真核表达载体的构建及其在FRT细胞的表达

目的：构建小鼠AQP1基因真核表达质粒并观察其在FRT细胞中的表达。方法：采用RT—PCR方法从小鼠肾脏组织的总cDNA中扩增出小鼠的AQP1基因,采用基因重组技术将AQP1的cDNA片段插入真核表达载体pCAGGS,构建小鼠AQP1的真核表达质粒,脂质体转染FRT细胞进行表达。结果：酶切和测序结果证实AQP1真核表达质粒构建成功,经脂质体转染FRT细胞后,免疫荧光检测证明AQPI蛋白在真核细胞中成功表达。结论：成功构建真核表达质粒pCAGGS—AQP1-myc,并在FRT细胞中得以表达。为进一步研究小鼠AQP1过表达时的功能及机制奠定了实验基础。     

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.     

Evidence for the involvement of FAM110C protein in cell spreading and migration

A number of factors, including protein kinases, Rho GTPases and actin and microtubule cytoskeletons play a crucial role in cell migration and spreading. We have recently shown that ectopic expression of FAM110C can alter cellular morphology by mechanisms yet to be determined. In this study, a FAM110C antiserum has been developed and used to study endogenously expressed FAM110C. Our data show that FAM110C is expressed by different cell lines and it can be detected throughout the cell. Interestingly, depletion of FAM110C by short interfering RNA reduced integrin-mediated filopodia formation, hepatocyte growth factor-induced migration, and phosphorylation of the Akt1 kinase in the epithelial cell line HepG2. Furthermore, co-immunoprecipitation and co-localization studies show that both ectopically and endogenously expressed FAM110C interact, or is part of a protein complex, with the Akt1 kinase. This interaction is transient and follows the activation of Akt1. In addition, we show that alpha-tubulin co-precipitates with FAM110C which further supports an interaction with the microtubule cytoskeleton. Collectively, these findings suggest a new function for FAM110C in the regulation of cell spreading, migration and filopodia induction.