Effects of allopurinol and febuxostat on uric acid transport and transporter expression in human umbilical vein endothelial cells.

Uric acid induces radical oxygen species formation, endothelial inflammation, and endothelial dysfunction which contributes to the progression of atherosclerosis. Febuxostat inhibits BCRP- and allopurinol stimulates MRP4-mediated uric acid efflux in human embryonic kidney cells. We hypothesized that endothelial cells express uric acid transporters that regulate intracellular uric acid concentration and that modulation of these transporters by febuxostat and allopurinol contributes to their different impact on cardiovascular mortality. The aim of this study was to explore a potential difference between the effect of febuxostat and allopurinol on uric acid uptake by human umbilical vein endothelial cells. Febuxostat increased intracellular uric acid concentrations compared with control. In contrast, allopurinol did not affect intracellular uric acid concentration. In line with this observation, febuxostat increased mRNA expression of GLUT9 and reduced MRP4 expression, while allopurinol did not affect mRNA expression of these uric acid transporters. These findings provide a possible pathophysiological pathway which could explain the higher cardiovascular mortality for febuxostat compared to allopurinol but should be explored further.

Interactions between corn starch and lingonberry polyphenols and their effects on starch digestion and glucose transport.

This study investigated the interaction mechanism between corn starch (CS) and lingonberry polyphenols (LBP) during starch gelatinization, focusing on their effects on starch structure and physicochemical properties. Moreover, it explored the effect of this interaction on starch digestion and glucose transport. The results indicated that LBP interacted non-covalently with CS during starch gelatinization, disrupted the short-range ordered structure of starch, decreased gelatinization enthalpy of starch, and formed a dense network structure. Furthermore, the incorporation of LBP remarkably reduced the digestibility of CS. In particular, the addition of 10 % LBP decreased the terminal digestibility (C∞) from 77.87 % to 60.43 % and increased the amount of resistant starch (RS) by 21.63 %. LBP was found to inhibit α-amylase and α-glucosidase in a mixed manner. Additionally, LBP inhibited glucose transport in Caco-2 cells following starch digestion. When 10 % LBP was added, there was a 34.17 % decrease in glucose transport compared with starch digestion without LBP. This study helps establish the foundation for the development of LBP-containing starch or starch-based healthy foods and provides new insights into the mechanism by which LBP lowers blood glucose.

MCF-7 cell - derived exosomes were involved in protecting source cells from the damage caused by tributyltin chloride via transport function.

Tributyltin chloride (TBTC) is a ubiquitous environmental pollutant with various adverse effects on human health. Exosomes are cell - derived signaling and substance transport vesicles. This investigation aimed to explore whether exosomes could impact the toxic effects caused by TBTC via their transport function. Cytotoxicity, DNA and chromosome damage caused by TBTC on MCF-7 cells were analyzed with CCK-8, flow cytometry, comet assay and micronucleus tests, respectively. Exosomal characterization and quantitative analysis were performed with ultracentrifugation, transmission electron microscope (TEM) and bicinchoninic acid (BCA) methods. TBTC content in exosomes was detected with Liquid Chromatography-Mass Spectrometry (LC-MS). The impacts of exosomal secretion on the toxic effects of TBTC were analyzed. Our data indicated that TBTC caused significant cytotoxicity, DNA and chromosome damage effects on MCF-7 cells, and a significantly increased exosomal secretion. Importantly, TBTC could be transported out of MCF-7 cells by exosomes. Further, when exosomal secretion was blocked with GW4869, the toxic effects of TBTC were significantly exacerbated. We concluded that TBTC promoted exosomal secretion, which in turn transported TBTC out of the source cells to alleviate its toxic effects. This investigation provided a novel insight into the role and mechanism of exosomal release under TBTC stress.

The inflammatory injury of porcine small intestinal epithelial cells induced by deoxynivalenol is related to the decrease in glucose transport.

The present study aimed to investigate the effects of deoxynivalenol (DON) stimulation on inflammatory injury and the expression of the glucose transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter protein 2 (GLU2) in porcine small intestinal epithelial cells (IPEC-J2). Additionally, the study aimed to provide initial insights into the connection between the expression of glucose transporters and the inflammatory injury of IPEC-J2 cells. DON concentration and DON treatment time were determined using the CCK­8 assay. Accordingly, 1.0 µg/mL DON and treatment for 24 h were chosen for subsequent experiments. Then IPEC-J2 cells were treated without DON (CON, N = 6) or with 1 µg/mL DON (DON, N = 6). Lactate dehydrogenase (LDH) content, apoptosis rate, and proinflammatory cytokines including interleukin (IL)-1ß, Il-6, and tumor necrosis factor α (TNF-α) were measured. Additionally, the expression of AMP-activated protein kinase α1 (AMPK-α1), the content of glucose, intestinal alkaline phosphatase (AKP), and sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) activity, and the expression of SGLT1 and GLU2 of IPEC-J2 cells were also analyzed. The results showed that DON exposure significantly increased LDH release and apoptosis rate of IPEC-J2 cells. Stimulation with DON resulted in significant cellular inflammatory damage, as evidenced by a significant increase in proinflammatory cytokines (IL-1ß, IL-6, and TNF-α). Additionally, DON caused damage to the glucose absorption capacity of IPEC-J2 cells, indicated by decreased levels of glucose content, AKP activity, Na+/K+-ATPase activity, AMPK-α1 protein expression, and SGLT1 expression. Correlation analysis revealed that glucose absorption capacity was negatively correlated with cell inflammatory cytokines. Based on the findings of this study, it can be preliminarily concluded that the cell inflammatory damage caused by DON may be associated with decreased glucose absorption.

Glucose is one of the most basic nutrients necessary to sustain animal life and plays a crucial role in animal body composition and energy metabolism. Previous studies suggested a link between glucose absorption and inflammatory injury. In the present study, deoxynivalenol (DON) stimulation caused severe inflammatory injury and reduced the glucose absorption capacity of IPEC-J2 cells. Pearson's correlation analysis revealed a negative correlation between glucose absorption capacity and cell inflammatory cytokines. Ultimately, it can be speculated that the cellular inflammatory response triggered by DON may be related to the altered expression of glucose transporters.

Inhibition of human drug transporter activities by succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0-3.9 µM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 µM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 µM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.

Cysteine Donor-Based Brain-Targeting Prodrug: Opportunities and Challenges.

Overcoming blood-brain barrier (BBB) to improve brain bioavailability of therapeutic drug remains an ongoing concern. Prodrug is one of the most reliable approaches for delivering agents with low-level BBB permeability into the brain. The well-known antioxidant capacities of cysteine (Cys) and its vital role in glutathione (GSH) synthesis indicate that Cys-based prodrug could potentiate therapeutic drugs against oxidative stress-related neurodegenerative disorders. Moreover, prodrug with Cys moiety could be recognized by the excitatory amino acid transporter 3 (EAAT3) that is highly expressed at the BBB and transports drug into the brain. In this review, we summarized the strategies of crossing BBB, properties of EAAT3 and its natural substrates, Cys and its donors, and Cys donor-based brain-targeting prodrugs by referring to recent investigations. Moreover, the challenges that we are faced with and future research orientations were also addressed and proposed. It is hoped that present review will provide evidence for the pursuit of novel Cys donor-based brain-targeting prodrug.

Unexpected Enhancement of Cytotoxicity of Cisplatin in a Rat Kidney Proximal Tubular Cell Line Overexpressing Mitochondrial Glutathione Transport Activity.

In previous studies, we identified the two principal transporters that mediate the uptake of glutathione (GSH) from cytoplasm into the mitochondrial matrix of rat kidney proximal tubular cells. We hypothesized that genetic modulation of transporter expression could markedly alter susceptibility of renal proximal tubular cells to a broad array of oxidants and mitochondrial toxicants. Indeed, we previously showed that overexpression of either of these transporters resulted in diminished susceptibility to several chemicals. In the present work, we investigated the influence of overexpression of the mitochondrial 2-oxoglutarate carrier (OGC) in NRK-52E cells on the cytotoxicity of the antineoplastic drug cisplatin. In contrast to previous results showing that overexpression of the mitochondrial OGC provided substantial protection of NRK-52E cells from injury due to several toxicants, we found a remarkable enhancement of cellular injury from exposure to cisplatin as compared to wild-type NRK-52E cells. Despite the oxidative stress that cisplatin is known to cause in the renal proximal tubule, the increased concentrations of mitochondrial GSH associated with OGC overexpression likely resulted in increased delivery of cisplatin to molecular targets and increased cellular injury rather than the typical protection observed in the previous work.

Methamphetamine enhances caveolar transport of therapeutic agents across the rodent blood-brain barrier.

The blood-brain barrier (BBB) restricts clinically relevant accumulation of many therapeutics in the CNS. Low-dose methamphetamine (METH) induces fluid-phase transcytosis across BBB endothelial cells in vitro and could be used to enhance CNS drug delivery. Here, we show that low-dose METH induces significant BBB leakage in rodents ex vivo and in vivo. Notably, METH leaves tight junctions intact and induces transient leakage via caveolar transport, which is suppressed at 4°C and in caveolin-1 (CAV1) knockout mice. METH enhances brain penetration of both small therapeutic molecules, such as doxorubicin (DOX), and large proteins. Lastly, METH improves the therapeutic efficacy of DOX in a mouse model of glioblastoma, as measured by a 25% increase in median survival time and a significant reduction in satellite lesions. Collectively, our data indicate that caveolar transport at the adult BBB is agonist inducible and that METH can enhance drug delivery to the CNS.

Hepatic processing of mercuric ions facilitates delivery to renal proximal tubules.

Mercury (Hg) is a toxic heavy metal to which humans are exposed on a regular basis. Hg has a high affinity for thiol-containing biomolecules with the majority of Hg in blood being bound to albumin. The current study tested the hypothesis that circulating Hg-albumin complexes are taken up into hepatocytes and processed to form Hg-glutathione (GSH) conjugates (GSH-Hg-GSH). Subsequently, GSH-Hg-GSH conjugates are exported from hepatocytes into blood via multidrug resistance transporters (MRP) 3 and 5. To test this hypothesis, the portal vein and hepatic artery in Wistar rats were ligated to prevent delivery of Hg to the liver. Ligated and control rats were injected with HgCl2 or GSH-Hg-GSH (containing radioactive Hg) and the disposition of Hg was assessed in various organs. Renal accumulation of Hg was reduced significantly in ligated rats exposed to HgCl2. In contrast, when rats were exposed to GSH-Hg-GSH, the renal accumulation of Hg was similar in control and ligated rats. Experiments using HepG2 cells indicate that Hg-albumin conjugates are taken up by hepatocytes and additional experiments using inside-out membrane vesicles showed that MRP3 and MRP5 mediate the export of GSH-Hg-GSH from hepatocytes. These data are the first to show that Hg-albumin complexes are processed within hepatocytes to form GSH-Hg-GSH, which is, in part, exported back into blood via MRP3 and MRP5 for eventual excretion in urine.

Brassinosteroids Mitigate Cadmium Effects in Arabidopsis Root System without Any Cooperation with Nitric Oxide.

The heavy metal cadmium (Cd) affects root system development and quiescent center (QC)-definition in Arabidopsis root-apices. The brassinosteroids-(BRs)-mediated tolerance to heavy metals has been reported to occur by a modulation of nitric oxide (NO) and root auxin-localization. However, how BRs counteract Cd-action in different root types is unknown. This research aimed to find correlations between BRs and NO in response to Cd in Arabidopsis's root system, monitoring their effects on QC-definition and auxin localization in root-apices. To this aim, root system developmental changes induced by low levels of 24-epibrassinolide (eBL) or by the BR-biosynthesis inhibitor brassinazole (Brz), combined or not with CdSO4, and/or with the NO-donor nitroprusside (SNP), were investigated using morpho-anatomical and NO-epifluorescence analyses, and monitoring auxin-localization by the DR5::GUS system. Results show that eBL, alone or combined with Cd, enhances lateral (LR) and adventitious (AR) root formation and counteracts QC-disruption and auxin-delocalization caused by Cd in primary root/LR/AR apices. Exogenous NO enhances LR and AR formation in Cd-presence, without synergism with eBL. The NO-signal is positively affected by eBL, but not in Cd-presence, and BR-biosynthesis inhibition does not change the low NO-signal caused by Cd. Collectively, results show that BRs ameliorate Cd-effects on all root types acting independently from NO.

GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.

Dependence of glucose transport on autophagy and GAPDH activity.

Glucose uptake in the brain is critically important to brain health. Using two widely used cell line model systems, we have found that siramesine, a lysosomotropic agent and ligand for the sigma-2 receptor, inhibits glucose uptake and decreases pools of the GLUT1 glucose transporter at the plasma membrane. Siramesine induces autophagy but also disrupts degradation of autophagy substrates, providing a potential mechanism for its action on glucose uptake. In other cell systems, many of the effects of siramesine can be suppressed by α -tocopherol, a type of vitamin E and potent antioxidant, and α-tocopherol also suppressed the effect of siramesine on glucose uptake, suggesting a role for reactive oxygen species and membrane maintenance. We have also identified a novel mechanism for siramesine in which it inhibited plasma membrane levels of GAPDH, a key protein in glycolysis which localizes to the plasma membrane in some cell types. Indeed, GAPDH inhibitors decreased glucose uptake, like siramesine, likely through an overlapping pathway with siramesine. GAPDH inhibitors induced autophagy but inhibited degradation of autophagy targets. Thus, we have identified novel mechanisms required for glucose uptake which may have important implications in disease.

Diet-induced obesity in mice impairs host defense against Klebsiella pneumonia in vivo and glucose transport and bactericidal functions in neutrophils in vitro.

Obesity impairs host defense against Klebsiella pneumoniae, but responsible mechanisms are incompletely understood. To determine the impact of diet-induced obesity on pulmonary host defense against K. pneumoniae, we fed 6-wk-old male C57BL/6j mice a normal diet (ND) or high-fat diet (HFD) (13% vs. 60% fat, respectively) for 16 wk. Mice were intratracheally infected with Klebsiella, assayed at 24 or 48 h for bacterial colony-forming units, lung cytokines, and leukocytes from alveolar spaces, lung parenchyma, and gonadal adipose tissue were assessed using flow cytometry. Neutrophils from uninfected mice were cultured with and without 2-deoxy-d-glucose (2-DG) and assessed for phagocytosis, killing, reactive oxygen intermediates (ROI), transport of 2-DG, and glucose transporter (GLUT1-4) transcripts, and protein expression of GLUT1 and GLUT3. HFD mice had higher lung and splenic bacterial burdens. In HFD mice, baseline lung homogenate concentrations of IL-1ß, IL-6, IL-17, IFN-γ, CXCL2, and TNF-α were reduced relative to ND mice, but following infection were greater for IL-6, CCL2, CXCL2, and IL-1ß (24 h only). Despite equivalent lung homogenate leukocytes, HFD mice had fewer intraalveolar neutrophils. HFD neutrophils exhibited decreased Klebsiella phagocytosis and killing and reduced ROI to heat-killed Klebsiella in vitro. 2-DG transport was lower in HFD neutrophils, with reduced GLUT1 and GLUT3 transcripts and protein (GLUT3 only). Blocking glycolysis with 2-DG impaired bacterial killing and ROI production in neutrophils from mice fed ND but not HFD. Diet-induced obesity impairs pulmonary Klebsiella clearance and augments blood dissemination by reducing neutrophil killing and ROI due to impaired glucose transport.

Spheroidization of ultrasonic degraded corn silk polysaccharide to enhance bioactivity by the anti-solvent precipitation method.

BACKGROUND: Corn silk is a very important by-product of corn production with medicinal value. Corn silk polysaccharide (CSP) is the main active ingredient. In the present study, ultrasound and spheroidization by anti-solvent were applied to improve the biological activity of CSP. RESULTS: The results showed that ultrasonic degradation improved the α-glucosidase inhibitory activity of CSP by changing its physicochemical characteristics. As the anti-solvent ratio increased, the particle size of the nanoparticles (NPs) from the spheroidization of ultrasonic-degraded corn silk polysaccharide (UCSP) gradually increased, and NP-1 exhibited the highest inhibitory effect of α-glucosidase. Isothermal titration calorimetry (ITC) results indicated that the enhanced activity might be due to more α-glucosidase binding sites with NP-1 compared with no spheroidization. Western blotting results showed that NP-1 could improve the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) uptake in the L6 cells by regulating the phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway and the translocation of glucose transporter 4 (GLUT4). NP-1 also exhibited excellent stability in different environments. CONCLUSION: The study revealed that ultrasonic treatment and spheroidization processing showed potential applications for improving the biological activity of polysaccharides. © 2021 Society of Chemical Industry.

Targeting cholesterol homeostasis in hematopoietic malignancies.

Cholesterol is a vital lipid for cellular functions. It is necessary for membrane biogenesis, cell proliferation, and differentiation. In addition to maintaining cell integrity and permeability, increasing evidence indicates a strict link between cholesterol homeostasis, inflammation, and hematological tumors. This makes cholesterol homeostasis an optimal therapeutic target for hematopoietic malignancies. Manipulating cholesterol homeostasis by either interfering with its synthesis or activating the reverse cholesterol transport via the engagement of liver X receptors affects the integrity of tumor cells both in vitro and in vivo. Cholesterol homeostasis has also been manipulated to restore antitumor immune responses in preclinical models. These observations have prompted clinical trials involving acute myeloid leukemia to test the combination of chemotherapy with drugs interfering with cholesterol synthesis (ie, statins). We review the role of cholesterol homeostasis in hematopoietic malignancies as well as in cells of the tumor microenvironment and discuss the potential use of lipid modulators for therapeutic purposes.

Components of the phosphatidylserine endoplasmic reticulum to plasma membrane transport mechanism as targets for KRAS inhibition in pancreatic cancer.

KRAS is mutated in 90% of human pancreatic ductal adenocarcinomas (PDACs). To function, KRAS must localize to the plasma membrane (PM) via a C-terminal membrane anchor that specifically engages phosphatidylserine (PtdSer). This anchor-binding specificity renders KRAS-PM localization and signaling capacity critically dependent on PM PtdSer content. We now show that the PtdSer lipid transport proteins, ORP5 and ORP8, which are essential for maintaining PM PtdSer levels and hence KRAS PM localization, are required for KRAS oncogenesis. Knockdown of either protein, separately or simultaneously, abrogated growth of KRAS-mutant but not KRAS-wild-type pancreatic cancer cell xenografts. ORP5 or ORP8 knockout also abrogated tumor growth in an immune-competent orthotopic pancreatic cancer mouse model. Analysis of human datasets revealed that all components of this PtdSer transport mechanism, including the PM-localized EFR3A-PI4KIIIα complex that generates phosphatidylinositol-4-phosphate (PI4P), and endoplasmic reticulum (ER)-localized SAC1 phosphatase that hydrolyzes counter transported PI4P, are significantly up-regulated in pancreatic tumors compared to normal tissue. Taken together, these results support targeting PI4KIIIα in KRAS-mutant cancers to deplete the PM-to-ER PI4P gradient, reducing PM PtdSer content. We therefore repurposed the US Food and Drug Administration-approved hepatitis C antiviral agent, simeprevir, as a PI4KIIIα inhibitor In a PDAC setting. Simeprevir potently mislocalized KRAS from the PM, reduced the clonogenic potential of pancreatic cancer cell lines in vitro, and abrogated the growth of KRAS-dependent tumors in vivo with enhanced efficacy when combined with MAPK and PI3K inhibitors. We conclude that the cellular ER-to-PM PtdSer transport mechanism is essential for KRAS PM localization and oncogenesis and is accessible to therapeutic intervention.

Reducing the Periplasmic Glutathione Content Makes Escherichia coli Resistant to Trimethoprim and Other Antimicrobial Drugs.

Although glutathione (GSH) has been shown to influence the antimicrobial effects of many kinds of antibiotics, little is known about its role in relation to trimethoprim (TMP), a widely used antifolate. In this study, several genes related to glutathione metabolism were deleted in different Escherichia coli strains (i.e., O157:H7 and ATCC 25922), and their effects on susceptibility to TMP were tested. The results showed that deleting gshA, gshB, grxA, and cydD caused TMP resistance, and deleting cydD also caused resistance to other drugs. Meanwhile, deleting gshA, grxA, and cydD resulted in a significant decrease of the periplasmic glutathione content. Supplementing exogenous GSH or further deleting glutathione importer genes (gsiB and ggt) restored TMP sensitivity to ΔcydD. Subsequently, the results of quantitative-reverse transcription PCR experiments showed that expression levels of acrA, acrB, and tolC were significantly upregulated in both ΔgrxA and ΔcydD. Correspondingly, deleting cydD led to a decreased accumulation of TMP within bacterial cells, and further deleting acrA, acrB, or tolC restored TMP sensitivity to ΔcydD. Inactivation of CpxR and SoxS, two transcriptional factors that modulate the transcription of acrAB-tolC, restored TMP sensitivity to ΔcydD. Furthermore, mutations of gshA, gshB, grxA, cydC, and cydD are highly prevalent in E. coli clinical strains. Collectively, these data suggest that reducing the periplasmic glutathione content of E. coli leads to increased expression of acrAB-tolC with the involvement of CpxR and SoxS, ultimately causing drug resistance. To the best of our knowledge, this is the first report showing a linkage between periplasmic GSH and drug resistance in bacteria. IMPORTANCE After being used extensively for decades, trimethoprim still remains one of the key accessible antimicrobials recommended by the World Health Organization. A better understanding of the mechanisms of resistance would be beneficial for the future utilization of this drug. It has been shown that the AcrAB-TolC efflux pump is associated with trimethoprim resistance in E. coli clinical strains. In this study, we show that E. coli can sense the periplasmic glutathione content with the involvement of the CpxAR two-component system. As a result, reducing the periplasmic glutathione content leads to increased expression of acrA, acrB, and tolC via CpxR and SoxS, causing resistance to antimicrobials, including trimethoprim. Meanwhile, mutations in the genes responsible for periplasmic glutathione content maintenance are highly prevalent in E. coli clinical isolates, indicating a potential correlation of the periplasmic glutathione content and clinical antimicrobial resistance, which merits further investigation.

Reduction in Copper Uptake and Inhibition of Prostate Cancer Cell Proliferation by Novel Steroid-based Compounds.

BACKGROUND/AIM: Knockdown of human copper transporter 1 has been associated with reduction in copper uptake and suppression of prostate cancer cell proliferation and tumor growth. This study evaluated the effects of steroid-based compounds on copper uptake and proliferation of prostate cancer cells based on their anticancer activity and previous docking analysis of steroid-based copper transporter 1 inhibitors. MATERIALS AND METHODS: We synthesized several new steroid-based compounds and used 64Cu uptake assay and copper quantification assay with inductively coupled plasma mass spectrometry to study their effects on the cellular copper uptake by prostate cancer cells. Additionally, we used CCK-8 cell proliferation assay to study their effects on the proliferation of prostate cancer cells. RESULTS: Significant reduction in cellular copper uptake was observed in the prostate cancer cells treated with these new steroid-based compounds. Moreover, proliferation of prostate cancer cells was suppressed by treatment with the steroid-based compound 6, which had the strongest copper uptake inhibition activity. CONCLUSION: Reduction in copper uptake and inhibition of cell proliferation were demonstrated in prostate cancer cells treated with the new steroid-based compounds synthesized in this study. Steroid-based copper transporter 1 inhibitors may become novel anticancer drugs for targeted anti-copper therapy of prostate cancer and other copper hypermetabolic cancers.

Structurally screening calixarenes as peptide transport activators.

Calixarenes are reportedly excellent activators that can remarkably improve the transport efficiencies of cell penetrating peptides. We employed eight calixarenes to systematically study the influence of structure on activation efficiency, which revealed that the scaffold, head group, and alkyl chain are all significant factors for activation efficiency by affecting affinities with the peptide and membrane.

Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy.

Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in the autophagy pathway, the so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B-/-) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.