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Background and purpose: Many drugs associated with acquired long QT syndrome (LQTS) directly block human ether-a-gogo-related gene (hERG) K þ channels. Recently, disrupted trafficking of the hERG channel protein was proposed as a new mechanism underlying LQTS, but whether this defect coexists with the hERG current block remains unclear. This study investigated how ketoconazole, a direct hERG current inhibitor, affects the trafficking of hERG channel protein.Experimental approach: Wild-type hERG and SCN5A/hNa v 1.5 Na þ channels or the Y652A and F656C mutated forms of the hERG were stably expressed in HEK293 cells. The K þ and Na þ currents were recorded in these cells by using the whole-cell patch-clamp technique (231C). Protein trafficking of the hERG was evaluated by Western blot analysis and flow cytometry. Key results: Ketoconazole directly blocked the hERG channel current and reduced the amount of hERG channel protein trafficked to the cell surface in a concentration-dependent manner. Current density of the hERG channels but not of the hNa v 1.5 channels was reduced after 48 h of incubation with ketoconazole, with preservation of the acute direct effect on hERG current. Mutations in drug-binding sites (F656C or Y652A) of the hERG channel significantly attenuated the hERG current blockade by ketoconazole, but did not affect the disruption of trafficking. Conclusions and implications: Our findings indicate that ketoconazole might cause acquired LQTS via a direct inhibition of current through the hERG channel and by disrupting hERG protein trafficking within therapeutic concentrations. These findings should be considered when evaluating new drugs.

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BackgroundExosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients.MethodsExosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4–2 and bone metastatic C4–2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody.ResultsAmong proteins upregulated in C4–2 and C4–2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4–2 and C4–2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues.ConclusionsOur results suggest that serum exosomal GGT activity could be a useful biomarker for PC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3301-x) contains supplementary material, which is available to authorized users.

Abstract. Treatment with taxanes for castration-resistant prostate cancer often leads to the development of resistance. It has been recently demonstrated that exosomes present in the body fluids contain proteins and RNAs in the cells from which they are derived and could serve as a diagnostic marker for various diseases. In the present study, we aimed to identify proteins contained in exosomes that could be markers for progression and taxane-resistance of prostate cancer. Exosomes were isolated by differential centrifugation from the culture medium of taxane-resistant human prostate cancer PC-3 cells (PC-3R) and their parental PC-3 cells. Isolated exosomes were subjected to iTRAQ-based quantitative proteomic analysis. Exosomes were also isolated from the culture medium by using anti-CD9 antibody-conjugated magnetic beads. Protein expression was knocked down by siRNA transfection followed by analysis of the silencing effects. Proteomic analysis showed that integrin β4 (ITGB4) and vinculin (VCL) were upregulated in exosomes derived from PC-3R cells compared to PC-3 cells. The elevation of ITGB4 and VCL was confirmed in exosomes captured by anti-CD9 antibody from the culture medium of PC-3R cells. Silencing of ITGB4 and VCL expression did not affect proliferation and taxane-resistance of PC-3R cells, but ITGB4 knockdown attenuated both cell migration and invasion and VCL knockdown reduced invasion. Our results suggest that ITGB4 and VCL in exosomes could be useful markers for progression of prostate cancer associated with taxane-resistance, providing the basis for development of an exosome-based diagnostic system. IntroductionProstate cancer is one of the most common male cancers and is the second-leading cause of cancer death among men in the United States (1). Most patients with prostate cancer well respond to androgen deprivation therapy, but 10-20% of those develop castration-resistant prostate cancer (CRPC) (2). Taxanes, a class of microtubule-targeting anticancer agents such as paclitaxel and docetaxel, have been administered to CRPC patients and docetaxel is currently used as the first-line chemotherapy (3). Docetaxel therapy demonstrated an overall survival benefit for CRPC patients, but there is a finite amount of time before acquiring resistance (3,4).Development of resistance to anticancer drugs is associated with more malignant and aggressive phenotype and acceleration of tumor growth in various types of cancer including prostate cancer (5-7). Although prostate-specific antigen (PSA) has been commonly used as a marker for tumor growth, there are no clinically useful markers for diagnosing progression and aggressiveness as well as taxane-resistance of prostate cancer. Given that such markers were available, one could select CRPC patients sensitive to docetaxel, avoid administration to drug-resistant patients and also monitor the drug efficacy, minimizing the incidence of adverse effects as well. Furthermore, prediction of progression and prognosis of CRPC patients might be possible.Exosomes are microvesi...

1 Miconazole, an imidazole antifungal agent, is associated with acquired long QT syndrome and ventricular arrhythmias. Miconazole increases the plasma concentration of QT-prolonging drugs by inhibiting the hepatic cytochrome P450 metabolic pathway, but whether it has direct effects on cardiac ion channels has not been elucidated. 2 To determine the mechanism underlying these clinical findings, we investigated the effect of miconazole on human ether-a-go-go-related gene (HERG) K þ channels. 3 HERG channels were heterologously expressed in human embryonic kidney 293 (HEK293) cells and whole-cell currents were recorded using a patch-clamp technique (231C). 4 Miconazole inhibited HERG peak tail current in a concentration-dependent manner (0.4-40 mM) with an IC 50 of 2.1 mM (n ¼ 3-5 cells at each concentration, Hill coefficient 1.2). HERG block was not frequency-dependent. It required channel activation, occurred rapidly, and had very slow dissociation properties. 5 The activation curve was shifted in a negative direction (V 1/2 : À9.572.3 mV in controls and À15.372.4 mV after 4 mM miconazole, Po0.05, n ¼ 6). Miconazole did not change other channel kinetics (activation, deactivation, onset of inactivation, recovery from inactivation, steady-state inactivation). 6 The S6 domain mutation, F656C, abolished the inhibitory action of miconazole on HERG current indicating that miconazole preferentially binds to an aromatic amino-acid residue within the pore-S6 region. 7 Our findings indicate that miconazole causes HERG channel block by binding to a common drug receptor, and this involves preferential binding to activated channels. Thus, miconazole prolongs the QT interval by direct inhibition of HERG channels.