Efflux Assay Research Articles

Octyl gallate reduces ABC multidrug transporter CaCdr1p expression and leads to its mislocalisation in azole-resistant clinical isolates of Candida albicans

ObjectivesFungal pathogens pose a serious threat to public health. Widespread and prolonged use of antifungal drugs has led to the development of multidrug resistance in the human fungal pathogen Candida albicans. Among several mechanisms leading to drug resistance in C. albicans, overexpression of drug efflux transporters remains by far the leading cause of multidrug resistance, facilitated by overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters. Hence, targeting efflux pumps still represents a promising approach to combat multidrug resistance. In this study, the effect of octyl gallate (OG), a natural food additive, on drug efflux pump activity of C. albicans was analysed. MethodsDrug efflux pump activity was determined by rhodamine 6G (R6G) efflux and Nile red accumulation assay in a Candida drug resistance protein 1 (CaCdr1p)-overexpressing strain. Gene expression and protein expression and localisation were studied by RT-PCR, Western blot and confocal microscopy. Ergosterol content was measured by the alcoholic KOH method. ResultsOG specifically inhibits the activity of CaCdr1p, belonging to the ABC superfamily. The underlying mechanism was confirmed as competitive mode of inhibition by OG as revealed by Lineweaver–Burk plot. Furthermore, OG leads to reduced expression of CDR1 and CaCdr1p and mislocalisation of CaCdr1p. Additionally, OG sensitises azole-susceptible and -resistant clinical matched-pair isolates Gu4 & Gu5 and leads to impeded R6G efflux and depleted ergosterol content. ConclusionThe ability of OG as a potent inhibitor of CaCdr1p that chemosensitises drug-resistant C. albicans warrants further studies to be exploited as an effective antifungal agent.

Efflux pump inhibitory activity of biologically synthesized silver nanoparticles against multidrug-resistant Acinetobacter baumannii clinical isolates.

This study was carried out to investigate the possible efflux pump inhibitory activity of biologically synthesized silver nanoparticles (AgNPs) against multidrug-resistant (MDR) Acinetobacter baumannii isolates. In this study, the physicochemical characteristics of synthesized AgNPs were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectrophotometer (FTIR) methods. Subsequently, MDR A. baumannii isolates were recovered from clinical samples and the phenotypic cartwheel efflux assay and polymerase chain reaction (PCR) were used to elucidate the possible presence of efflux pump in MDR strains. After treatment of MDR strains with sub-minimum inhibitory concentration (MIC) concentration of AgNPs, the expression level of efflux pump genes was evaluated using a quantitative real-time PCR technique. The synthesized AgNPs had a spherical nanostructure, with mean size 38.89 nm, according to SEM and TEM data. XRD and FTIR results confirmed the synthesis of AgNPs. The results of PCR revealed that among 50 strains, 12 A. baumannii strains had efflux pump genes and the expression level of AdeA, AdeC, AdeS, AdeR, AdeI, AdeJ, and AdeK efflux pump genes was downregulated significantly after the treatment with AgNPs. In addition, the inhibitory effect of AgNPs on efflux pumps can be detected when the MIC of ethidium bromide (EtBr) with AgNPs is lower than that of EtBr alone. According to the results, the biologically synthesized AgNPs exhibit efflux pumps inhibitory activity, which may be one of the possible mechanisms of their antibacterial activity against MDR A. baumannii strains.

Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations

BackgroundThe human Caco-2 cell line is a common in vitro model of the intestinal epithelial barrier. As the intestine is a major interface in cholesterol turnover and represents a non-biliary pathway for cholesterol excretion, Caco-2 cells are also a valuable model for studying cholesterol homeostasis, including cholesterol uptake and efflux. Currently available protocols are, however, either sketchy or not consistent among different laboratories. Our aim was therefore to generate a collection of optimized protocols, considering the different approaches of the different laboratories and to highlight possibilities and limitations of measuring cholesterol transport with this cell line.ResultsWe developed comprehensive and quality-controlled protocols for the cultivation of Caco-2 cells on filter inserts in a single tight monolayer. A cholesterol uptake as well as a cholesterol efflux assay is described in detail, including suitable positive controls. We further show that Caco-2 cells can be efficiently transfected for luciferase reporter gene assays in order to determine nuclear receptor activation, main transcriptional regulators of cholesterol transporters (ABCA1, ABCB1, ABCG5/8, NPC1L1). Detection of protein and mRNA levels of cholesterol transporters in cells grown on filter inserts can pose challenges for which we highlight essential steps and alternative approaches for consideration. A protocol for viability assays with cells differentiated on filter inserts is provided for the first time.ConclusionsThe Caco-2 cell line is widely used in the scientific community as model for the intestinal epithelium, although with highly divergent protocols. The herein provided information and protocols can be a common basis for researchers intending to use Caco-2 cells in the context of cellular cholesterol homeostasis.

Ospemifene displays broad-spectrum synergistic interactions with itraconazole through potent interference with fungal efflux activities

Azole antifungals are vital therapeutic options for treating invasive mycotic infections. However, the emergence of azole-resistant isolates combined with limited therapeutic options presents a growing challenge in medical mycology. To address this issue, we utilized microdilution checkerboard assays to evaluate nine stilbene compounds for their ability to interact synergistically with azole drugs, particularly against azole-resistant fungal isolates. Ospemifene displayed the most potent azole chemosensitizing activity, and its combination with itraconazole displayed broad-spectrum synergistic interactions against Candida albicans, Candida auris, Cryptococcus neoformans, and Aspergillus fumigatus (ΣFICI = 0.05–0.50). Additionally, in a Caenorhabditis elegans infection model, the ospemifene-itraconazole combination significantly reduced fungal CFU burdens in infected nematodes by ~75–96%. Nile Red efflux assays and RT-qPCR analysis suggest ospemifene interferes directly with fungal efflux systems, thus permitting entry of azole drugs into fungal cells. This study identifies ospemifene as a novel antifungal adjuvant that augments the antifungal activity of itraconazole against a broad range of fungal pathogens.

Clarithromycin effectively enhances doxorubicin-induced cytotoxicity and apoptosis in MCF7 cells through dysregulation of autophagy

PurposeUse of autophagy inhibitors in combination with chemotherapy has become a novel chemotherapeutic strategy. In this study, we aimed to determine whether the effectiveness of doxorubicin (DOX) is augmented by clarithromycin (CAM) in MCF7 cells and the molecular mechanisms involved. Materials and methodsCombined cytotoxicity of CAM and DOX was assessed by MTT assay and was analyzed using the Chou-Talalay's method. To clarify the underlying mechanisms, several factors, including apoptosis (Annexin V/propidium iodide staining), intracellular level of DOX (spectrofluorimetry) and P-glycoprotein activity (Rhodamin 123 efflux assay) were measured. In addition, autophagy was evaluated by intracellular labeling with anti-LC3II and LysoTrackerGreen (LTG) staining and analyzed by flowcytometry. ResultsThe anti-proliferation effect of DOX was synergistically enhanced by CAM in MCF7 cells and was associated with an increase in the apoptotic cell death. However, the intracellular level of DOX remained unchanged in the presence of CAM. Based on the findings, 100 μM of CAM did not exhibit any inhibitory effects on P-glycoprotein activity. Flow cytometric analysis indicated that DOX at IC20 concentration induced the autophagy flux, as confirmed by the increased level of LC3II and LTG signals. Moreover, combined treatment with DOX and CAM resulted in more pronounced LTG signals, but no change in LC3II. These results indicate that CAM blocks the autophagy flux induced by DOX. ConclusionsThese findings suggest that suppression of autophagy by CAM may promote chemotherapeutic outcome in breast cancer. However, further investigations are needed to evaluate the application of CAM in adjuvant breast cancer therapy.

Analysis of compensatory mechanisms involved in response to genetic perturbation of drug transporters

A diverse repertoire of xenobiotic transporters from the ABC and SLC families are expressed in clinically relevant cell types. Structural and functional similarity of these transporters’ specificities is known to complicate efforts to perturb them. Given the therapeutic importance of these transporters, it is important to uncover the pathways that could lead to compensation for transporter loss‐of‐function. CRISPR/Cas9 based gene editing is extensively utilized for creating genetic lesions to analyze loss‐of‐function mutations. When applied in embryos a commonly observed outcome is genetic compensation. In the present study we take advantage of CRISPR/Cas9 based knockout of xenobiotic transporters in sea urchin embryos. Sea urchins express orthologs of several ABC transporters including P‐glycoprotein (ABCB1) and MRP1 (ABCC1). The fluorescent substrates and inhibitors of these transporters are also well‐defined providing simple system for analyzing changes in transporter activity in response to mutations. We have designed CRISPR single guide RNAs (sgRNAs) targeting orthologs of P‐glycoprotein (ABCB1, ABCB4), multidrug resistance proteins (ABCC1, ABCC4, ABCC5) and breast cancer resistance protein (ABCG2). Using a known CRISPR target in sea urchins, Nodal, we have successfully created small indels resulting in robust phenotypes. Using a bioinformatics tool called Inference of CRISPR Edits (ICE), we measured a cutting efficiency of 77.16% ±26.5 across four biological replicates. Knockdown of ABCB1a expression by antisense morpholino and inhibition of ABCB1a transporter activity with Valspodar (PSC833) was used to standardize the ABCB1a transporter activity in controls. We observed 2.5‐fold and 10‐fold increase in ABCB‐specific substrate, bodipy‐verapamil (b‐ver), in ABCB1a morphants and PSC inhibited embryos, respectively, while ABCC‐specific substrate, fluorescein diacetate (FDA) showed no change. Using microinjection of single as well as multiplex sgRNA, we now have successfully created cuts in ABCB1a loci resulting in small indels and large deletions, respectively. Currently, ABCB1a crispants are being analyzed for phenotypes using efflux assays. These crispants sometimes show increased accumulation of b‐ver, and strong efflux of FDA indicating compensation by ABCC1. We are also currently working to understand how variation in Cas9 cutting efficiency could relate to phenotypic variability. Recent studies have identified the role of Nonsense Mediated Decay (NMD) of mutant transcripts as one of the triggers for compensation which can be reversed by inhibiting the NMD pathway. As such, future work will focus on the role of mRNA stability and NMD on sensing of lesions in transporter loci. Results from this study could have impact on understanding the emergence of drug resistance and help design more effective ways to overcome it.Support or Funding InformationSupported by R01ES027921 and R01ES030318 to Amro Hamdoun

Resensitization of Fluconazole-Resistant Urinary Candida spp. Isolates by Amikacin through Downregulation of Efflux Pump Genes.

The contribution of fluconazole-resistant Candida spp. isolates to urinary tract infections in Egypt has become a nationwide problem. A recent approach to overcome such disaster is combining conventional antifungals with non-antifungals. This study investigated the interaction of amikacin with fluconazole against resistant Candida strains isolated from the urine culture of patients admitted to Alexandria Main University Hospital. Among the collected Candida spp. isolates, 42.9% were resistant to fluconazole with MICs ranging between 128 and 1,024 μg/ml. The resistance-modifying activity of amikacin (4,000 μg/ml) was studied against fluconazole-resistant isolates where amikacin sensitized 91.7 % of resistant Candida spp. isolates to fluconazole with a modulation factor ranging between 32 and 256. The rhodamine efflux assay was performed to examine the impact of amikacin on efflux pump activity. After 120 minutes of treatment, amikacin affected the efflux pump activity of the isolates tested with a percentage of reduction in the fluorescence intensity of 8.9%. Quantitative real-time PCR was applied to assess the amikacin effect on the expression of the efflux pump genes MDR1, CDR1, and CDR2. The downregulatory effect of amikacin on the expression of the studied genes caused a percentage of reduction in the expression level ranging between 42.1 and 94%. In conclusion, amikacin resensitized resistant Candida spp. isolates to fluconazole and could be used in combination in the management of candiduria with a higher efficiency or at lower administration doses. To the best of our knowledge, this is the first study evaluating the enhancement of fluconazole activity in combination with amikacin against Candida spp.

Novel ABCB1 and ABCC Transporters Are Involved in the Detoxification of Benzo(α)pyrene in Thick Shell Mussel, Mytilus coruscus

ATP-binding cassette (ABC) transporters play a role in xenobiotic resistance through transporting xenobiotics into or out of cells. However, their functional role in molluscs defense against polycyclic aromatic hydrocarbons (PAHs) remains unclear. In the present study, novel McoABCB1 and McoABCC transporters were identified from the thick shell mussel Mytilus coruscus, and their benzo(α)pyrene (Bap) detoxification function in gills was determined by qRT-PCR and efflux assays. McoABCB1 and McoABCC transcripts were constitutively expressed in all examined tissues, suggesting a primary physiological function. Bap, used as a chemical stress agent, induced expression of McoABCB1 and McoABCC mRNA in gills, however at high concentration, it inhibited their expression at the late stage. Efflux activities in M. coruscus gills showed a dose-dependent elevation in the Bap pre-exposure group, compared with that in the reversin 205 or MK571 single exposure groups. These results demonstrated the presence of two types of ABC transporters and their involvement in cellular Bap detoxification.

The Pyrazolo[3,4-d]pyrimidine Derivative, SCO-201, Reverses Multidrug Resistance Mediated by ABCG2/BCRP.

ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells.

Reversal Effect of ALK Inhibitor NVP-TAE684 on ABCG2-Overexpressing Cancer Cells.

Failure of cancer chemotherapy is mostly due to multidrug resistance (MDR). Overcoming MDR mediated by overexpression of ATP binding cassette (ABC) transporters in cancer cells remains a big challenge. In this study, we explore whether NVP-TAE684, a novel ALK inhibitor which has the potential to inhibit the function of ABC transport, could reverse ABC transporter-mediated MDR. MTT assay was carried out to determine cell viability and reversal effect of NVP-TAE684 in parental and drug resistant cells. Drug accumulation and efflux assay was performed to examine the effect of NVP-TAE684 on the cellular accumulation and efflux of chemotherapeutic drugs. The ATPase activity of ABCG2 transporter in the presence or absence of NVP-TAE684 was conducted to determine the impact of NVP-TAE684 on ATP hydrolysis. Western blot analysis and immunofluorescence assay were used to investigate protein molecules related to MDR. In addition, the interaction between NVP-TAE684 and ABCG2 transporter was investigated via in silico analysis. MTT assay showed that NVP-TAE684 significantly decreased MDR caused byABCG2-, but not ABCC1-transporter. Drug accumulation and efflux tests indicated that the effect of NVP-TAE684 in decreasing MDR was due to the inhibition of efflux function of ABCG2 transporter. However, NVP-TAE684 did not alter the expression or change the subcellular localization of ABCG2 protein. Furthermore, ATPase activity analysis indicated that NVP-TAE684 could stimulate ABCG2 ATPase activity. Molecular in silico analysis showed that NVP-TAE684 interacts with the substrate binding sites of the ABCG2 transporter. Taken together, our study indicates that NVP-TAE684 could reduce the resistance of MDR cells to chemotherapeutic agents, which provides a promising strategy to overcome MDR.

Hesperetin reverses P‑glycoprotein‑mediated cisplatin resistance in DDP‑resistant human lung cancer cells via modulation of the nuclear factor‑κB signaling pathway.

Lung cancer is the leading cause of cancer-associated mortality worldwide. Cisplatin (DDP) is a first-line chemotherapeutic drug for the treatment of lung cancer; however, the majority of patients develop resistance to DDP. P-glycoprotein (P-gp), also referred to as multidrug resistance (MDR) protein 1, is associated with an MDR phenotype, which results in failure of cancer chemotherapy; thus, identifying effective MDR pump inhibitors may improve the outcomes of patients who develop resistance to treatment. Hesperetin is a derivative of hesperidin, which is extracted from tangerine peel and exhibits multiple antitumor properties. In the present study, human lung adenocarcinoma A549 and A549/DDP cells were treated with different concentrations of hesperetin and DDP, respectively. Furthermore, rhodamine 123 efflux assays, Cell Counting Kit-8 assays, immunofluorescence, reverse transcription-quantitative PCR and western blot analysis were used to elucidate the mechanisms underlying the effects of hesperetin On A549/DDP cells. Additionally, a xenograft model of lung cancer in nude mice was established to explore the effects of hesperetin on A549/DDP cell growth in vivo. The results demonstrated that hesperetin sensitized A549/DDP cells to DDP. In vivo, hesperetin pretreatment significantly inhibited tumor growth. Mechanistically, hesperetin markedly decreased the expression of P-gp and increased the intracellular accumulation of the P-gp substrate, rhodamine 123, in A549/DDP cells. In addition, pretreatment of A549/DDP cells with hesperetin significantly inhibited nuclear factor (NF)-κB (p65) activity and its nuclear translocation. Taken together, the results of the present study suggest that hesperetin reversed P-gp-mediated MDR by decreasing P-gp expression in A549/DDP cells, which was associated with inhibition of the NF-κB signaling pathway. These findings may provide the basis for the use of hesperetin clinically to reverse MDR.

Regulation of breast cancer resistance protein and P-glycoprotein by ezrin, radixin and moesin in lung, intestinal and renal cancer cell lines.

Ezrin (Ezr), radixin (Rdx) and moesin (Msn) (ERM) proteins anchor other proteins to the cell membrane, serving to regulate their localization and function. Here, we examined whether ERM proteins functionally regulate breast cancer resistance protein (BCRP) and P-glycoprotein in cell lines derived from lung, intestinal and renal cancers. ERM proteins were each silenced with appropriate siRNA. BCRP and P-gp functions were evaluated by means of efflux and uptake assays using 7-ethyl-10-hydroxycamptothecin (SN-38) and rhodamine123 (Rho123) as specific substrates, respectively, in non-small cell lung cancer HCC827 cells, intestinal cancer Caco-2 cells and renal cancer Caki-1 cells. In HCC827 cells, the efflux rates of SN-38 and Rho123 were significantly decreased by knockdown of Ezr or Msn, but not Rdx. However, BCRP function was unaffected by Ezr or Rdx knockdown in Caco-2 cells, which do not express Msn. In Caki-1 cells, Rdx knockdown increased the intracellular SN-38 concentration, while knockdown of Ezr or Msn had no effect. Our findings indicate that regulation of BCRP and P-gp functions by ERM proteins is organ-specific. Thus, if the appropriate ERM protein(s) are functionally suppressed, accumulation of BCRP or P-gp substrates in lung, intestine or kidney cancer tissue might be specifically increased.

Fengycins From Bacillus amyloliquefaciens MEP218 Exhibit Antibacterial Activity by Producing Alterations on the Cell Surface of the Pathogens Xanthomonas axonopodis pv. vesicatoria and Pseudomonas aeruginosa PA01.

Bacillus amyloliquefaciens MEP218 is an autochthonous bacterial isolate with antibacterial and antifungal activities against a wide range of phytopathogenic microorganisms. Cyclic lipopeptides (CLP), particularly fengycins, produced by this bacterium; are the main antimicrobial compounds responsible for the growth inhibition of phytopathogens. In this work, the CLP fraction containing fengycins with antibacterial activity was characterized by LC-ESI-MS/MS. In addition, the antibacterial activity of these fengycins was evaluated on the pathogens Xanthomonas axonopodis pv. vesicatoria (Xav), a plant pathogen causing the bacterial spot disease, and Pseudomonas aeruginosa PA01, an opportunistic human pathogen. In vitro inhibition assays showed bactericidal effects on Xav and PA01. Atomic force microscopy images revealed dramatic alterations in the bacterial surface topography in response to fengycins exposure. Cell damage was evidenced by a decrease in bacterial cell heights and the loss of intracellular content measured by potassium efflux assays. Furthermore, the viability of MRC-5 human normal lung fibroblasts was not affected by the treatment with fengycins. This study shows in vivo evidence on the less-known properties of fengycins as antibacterial molecules and leaves open the possibility of using this CLP as a novel antibiotic.

First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation.

The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the thermodynamic barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which facilitate helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize this helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.

A Practical Perspective on the Evaluation of Small Molecule CNS Penetration in Drug Discovery.

The separation of the brain from blood by the blood-brain barrier and the bloodcerebrospinal fluid (CSF) barrier poses unique challenges for the discovery and development of drugs targeting the central nervous system (CNS). This review will describe the role of transporters in CNS penetration and examine the relationship between unbound brain (Cu-brain) and unbound plasma (Cu-plasma) or CSF (CCSF) concentration. Published data demonstrate that the relationship between Cu-brain and Cu-plasma or CCSF can be affected by transporter status and passive permeability of a drug and CCSF may not be a reliable surrogate for CNS penetration. Indeed, CCSF usually over-estimates Cu-brain for efflux substrates and it provides no additional value over Cu-plasma as the surrogate of Cu-brain for highly permeable non-efflux substrates. A strategy described here for the evaluation of CNS penetration is to use in vitro permeability, P-glycoprotein (Pgp) and breast cancer resistance protein efflux assays and Cu-brain/Cu-plasma in preclinical species. Cu-plasma should be used as the surrogate of Cu-brain for highly permeable non-efflux substrates with no evidence of impaired distribution into the brain. When drug penetration into the brain is impaired, we recommend using (total brain concentration * unbound fraction in the brain) as Cu-brain in preclinical species or Cu-plasma/in vitro Pgp efflux ratio if Pgp is the major limiting mechanism for brain penetration.

Vanillin confers antifungal drug synergism in Candida albicans by impeding CaCdr2p driven efflux

AimAmong the most common mechanisms of multidrug resistance (MDR) in prevalent human fungal pathogen, Candida albicans, overexpression of drug efflux pumps remains the predominant mechanism. Hence to inhibit efflux pumps and chemosensitize C. albicans against traditional antifungal drugs still represents an attractive approach. The present study aimed to analyze the effect of Vanillin (Van), a natural food flavoring agent, on drug efflux pump activity of Candida albicans. Methods and resultsWe observed that Van specifically inhibits Candida drug resistance protein 2 (CaCdr2p) activity belonging to ATP Binding Cassette (ABC) superfamily as revealed by abrogated rhodamine 6G efflux and nile red accumulation assay with CaCdr2p over expressing strain. Insight studies into the mechanisms suggested that abrogated efflux by CaCdr2p is due to competitive mode of inhibition by Van as depicted by Lineweaver-Burk plot. RT-PCR, western blot and confocal microscopy further unraveled that Van leads to reduced expression of CDR2 and CaCdr2p mislocalization respectively. Furthermore, Van sensitizes the azole sensitive and resistant clinical matched pair of isolates Gu4/Gu5 and led to abrogated rhodamine 6G efflux and depleted ergosterol. Furthermore, Van synergizes with membrane targeting drugs fluconazole and amphotericin B as their fractional inhibitory coefficient index was less than 0.5. ConclusionVan being a potent inhibitor of CaCdr2p and chemosensitizing of drug resistant C. albicans warrants further studies to be exploited as effective antifungal agent.

Metformin Disrupts Bile Acid Efflux by Repressing Bile Salt Export Pump Expression.

The bile salt export pump (BSEP), a key player in hepatic bile acid clearance, has been the center of research on drug-induced cholestasis. However, such studies focus primarily on the direct inhibition of BSEP, often overlooking the potential impact of transcriptional repression. This work aims to explore the disruption of bile acid efflux caused by drug-induced BSEP repression. BSEP activity was analyzed in human primary hepatocytes (HPH) using a traditional biliary-clearance experiment and a modified efflux assay, which includes a 72-h pretreatment prior to efflux measurement. Relative mRNA and protein expressions were examined by RT-PCR and Western blotting, respectively. Metformin concentration-dependently repressed BSEP expression in HPH. Although metformin did not directly inhibit BSEP activity, longer metformin exposure reduced BSEP transport function in HPH by down-regulating BSEP expression. BSEP repression by metformin was found to be AMP-activated protein kinase-independent. Additional screening of 10 reported cholestatic non-BSEP inhibitors revealed that the anti-cancer drug tamoxifen also markedly repressed BSEP expression and reduced BSEP activity in HPH. Repression of BSEP alone is sufficient to disrupt hepatic bile acid efflux. Metformin and tamoxifen appear to be prototypes of a class of BSEP repressors that may cause drug-induced cholestasis through gene repression instead of direct BSEP inhibition.

The ABCA1 blocking agent probucol decreases capacitation in ejaculated dog spermatozoa

BackgroundThe ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid transport within and through cell membranes of many cells including spermatozoa. Cholesterol efflux is important for capacitation of spermatozoa. ABCA1 expression has been assessed in canine spermatozoa previously but its role in capacitation still has to be determined. The aim of the study was to test whether inhibition of ABCA1 (1) decreases capacitation in ejaculated and epididymal canine sperm samples and (2) decreases cholesterol efflux in the same samples. Twenty-one ejaculates and sperm from 22 epididymal tails were collected from healthy dogs. Motility was measured by CASA and viability assessed after staining with SYBR-14/PI. Samples from ejaculated sperm and sperm from epididymal tails were aliquoted. One part was incubated with the ABCA1 inhibitor probucol, the other served as a negative control. In all samples, capacitation was evaluated by chlortetracyclin (CTC) assay and cholesterol was measured by cholesterol efflux assay and colorimetric enzymatic assay.ResultsIn ejaculated sperm, blockade of ABCA1 with 100 µM of probucol/mL of sample resulted in a significantly higher percentage of uncapacitated and acrosome reacted spermatozoa (P < 0.001 and P = 0.031), capacitation was significantly decreased (35% in probucol samples vs 54.2% in controls, P < 0.001). In probucol inhibited sperm samples from epididymal tails, the percentage of capacitated spermatozoa did not differ between groups but the percentage of acrosome reacted spermatozoa increased significantly (P = 0.014). The cholesterol measurement revealed significantly lower cholesterol concentration in the probucol group when compared to the controls (P = 0.035), however only in ejaculated sperm samples.ConclusionsCTC assay and cholesterol measurement revealed significant differences between groups; we conclude that inhibition of ABCA1 significantly decreased capacitation and cholesterol efflux in ejaculated canine spermatozoa. The inhibition was not complete but ABCA1 is supposed to contribute to capacitation in canine ejaculated spermatozoa. ABCA1 is probably not important for capacitation of epididymal spermatozoa but might exert other functions during spermatozoa ripening.

K+ Release Assay and K+ Measurement in Oocyte Assay.

The Xenopus oocyte is a powerful system for the exogenous expression and functional characterization of plant membrane transport proteins. Until now, a number of potassium transporters and channels have been identified in oocytes expression system by the two-electrode voltage clamp technology. It is difficult to characterize K+/H+ anti-transporters, especially, electroneutral transporter. The K+ efflux assay system enables easy, fast, large-scale measurement of the transporters activity without two-electrode voltage clamp technology. This protocol describes a technique to measure the efflux activity of potassium transporter in oocytes expressing system.

Protective Effect of Naringin on In Vitro Gut-Vascular Barrier Disruption of Intestinal Microvascular Endothelial Cells Induced by TNF-α.

Naringin is a polymethoxylated flavonoid commonly found in citrus species and has therapeutic potential in intestinal disorders. However, the effect and mechanism of naringin on gut-vascular barrier disruption has not yet been reported. This study aimed to investigate the distinguishing and selectively protective effects of naringin on tumor necrosis factor (TNF)-α-induced gut-vascular barrier disruption and elucidate the potential mechanism. In the present study, an in vitro gut-vascular barrier model composed of rat intestinal microvascular endothelial cells (RIMVECs) was studied. Evans blue-albumin efflux assay showed that naringin (50 μM) evidently protected the integrity of RIMVEC monolayer barriers against TNF-α-induced disruption. Naringin maintained the expression and distribution of tight junction proteins including zona occludin-1, occludin, claudin-1, and claudin-2. Additionally, naringin protected RIMVECs from TNF-α-induced apoptosis and cell migration suppression (41.1 ± 2.2 vs 51.1 ± 3.5%; 61.0 ± 5.1 vs 72.2 ± 6.2%). Our results indicate that naringin effectively ameliorates gut-vascular barrier disruption.