2H-Thiopyran-2-thione sulfine, a compound for converting H2S to HSOH/H2S2 and increasing intracellular sulfane sulfur levels.

Reactive sulfane sulfur species such as persulfides (RSSH) and H2S2 are important redox regulators and closely linked to H2S signaling. However, the study of these species is still challenging due to their instability, high reactivity, and the lack of suitable donors to produce them. Herein we report a unique compound, 2H-thiopyran-2-thione sulfine (TTS), which can specifically convert H2S to HSOH, and then to H2S2 in the presence of excess H2S. Meanwhile, the reaction product 2H-thiopyran-2-thione (TT) can be oxidized to reform TTS by biological oxidants. The reaction mechanism of TTS is studied experimentally and computationally. TTS can be conjugated to proteins to achieve specific delivery, and the combination of TTS and H2S leads to highly efficient protein persulfidation. When TTS is applied in conjunction with established H2S donors, the corresponding donors of H2S2 (or its equivalents) are obtained. Cell-based studies reveal that TTS can effectively increase intracellular sulfane sulfur levels and compensate for certain aspects of sulfide:quinone oxidoreductase (SQR) deficiency. These properties make TTS a conceptually new strategy for the design of donors of reactive sulfane sulfur species.

Longevity control by supersulfide-mediated mitochondrial respiration and regulation of protein quality.

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides-to mediate mitochondrial function and to regulate longevity.

Proteomic alterations in the brain and blood-brain barrier during brain Aß accumulation in an APP knock-in mouse model of Alzheimer's disease.

BACKGROUND: Blood-brain barrier (BBB) dysfunction is supposed to be an early event in the development of Alzheimer's disease (AD). This study aimed to investigate the relationship between BBB alterations and AD progression in terms of amyloid-ß peptide (Aß) accumulation in the brains of humanized amyloid precursor protein knock-in (APP-KI) mice. METHODS: Brain Aß accumulation was examined using immunohistochemical analysis. Alterations in differentially expressed proteins were determined using sequential window acquisition of all theoretical fragment ion mass spectroscopy (SWATH-MS)-based quantitative proteomics, and Metascape, STRING, Gene Ontology, and KEGG were used for network analyses of altered biological pathways and processes. Statistical significance was determined using the unpaired two-tailed Student's t-test and Welch's t-test for two groups and one-way analysis of variance followed by Tukey's test for more than two groups. Correlations between two groups were determined using Pearson's correlation analysis. RESULTS: Brain Aß accumulation in APP-KI mice was detectable at 2 months, increased significantly at 5 months, and remained elevated at 12 months of age. The levels of differentially expressed proteins in isolated brain capillaries were higher in younger mice, whereas those in the brain were higher in older mice. Network analyses indicated changes in basement membrane-associated and ribosomal proteins in the brain capillaries. There were no significant changes in key proteins involved in drug or Aß transport at the BBB. In contrast, solute carrier transporter levels in astrocytes, microglia, and neurons were altered in the brain of older mice. Moreover, the levels of the lipid transporters Apoe and Apoj were upregulated in both the brain and isolated brain capillaries after Aß accumulation. CONCLUSIONS: Our results suggest that changes in the brain occurred after advanced Aß accumulation, whereas initial Aß accumulation was sufficient to cause alterations in the BBB. These findings may help elucidate the role of BBB alterations in AD progression and predict the distribution of drugs across the BBB in the brain of patients with AD.

Persulfide Biosynthesis Conserved Evolutionarily in All Organisms.

Significance: Persulfides/polysulfides are sulfur-catenated molecular species (i.e., R-Sn-R', n > 2; R-Sn-H, n > 1, with R = cysteine, glutathione, and proteins), such as cysteine persulfide (CysSSH). These species are abundantly formed as endogenous metabolites in mammalian and human cells and tissues. However, the persulfide synthesis mechanism has yet to be thoroughly discussed. Recent Advances: We used ß-(4-hydroxyphenyl)ethyl iodoacetamide and mass spectrometry to develop sulfur metabolomics, a highly precise, quantitative analytical method for sulfur metabolites. Critical Issues: With this method, we detected appreciable amounts of different persulfide species in biological specimens from various organisms, from the domains Bacteria, Archaea, and Eukarya. By using our rigorously quantitative approach, we identified cysteinyl-tRNA synthetase (CARS) as a novel persulfide synthase, and we found that the CysSSH synthase activity of CARS is highly conserved from the domains Bacteria to Eukarya. Because persulfide synthesis is found not only with CARS but also with other sulfotransferase enzymes in many organisms, persulfides/polysulfides are expected to contribute as fundamental elements to substantially diverse biological phenomena. In fact, persulfide generation in higher organisms-that is, plants and animals-demonstrated various physiological functions that are mediated by redox signaling, such as regulation of energy metabolism, infection, inflammation, and cell death, including ferroptosis. Future Directions: Investigating CARS-dependent persulfide production may clarify various pathways of redox signaling in physiological and pathophysiological conditions and may thereby promote the development of preventive and therapeutic measures for oxidative stress as well as different inflammatory, metabolic, and neurodegenerative diseases. Antioxid. Redox Signal. 39, 983-999.

Supersulfide catalysis for nitric oxide and aldehyde metabolism.

Abundant formation of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiols and proteins (supersulfidation), has been observed. We found here that supersulfides catalyze S-nitrosoglutathione (GSNO) metabolism via glutathione-dependent electron transfer from aldehydes by exploiting alcohol dehydrogenase 5 (ADH5). ADH5 is a highly conserved bifunctional enzyme serving as GSNO reductase (GSNOR) that down-regulates NO signaling and formaldehyde dehydrogenase (FDH) that detoxifies formaldehyde in the form of glutathione hemithioacetal. C174S mutation significantly reduced the supersulfidation of ADH5 and almost abolished GSNOR activity but spared FDH activity. Notably, Adh5C174S/C174S mice manifested improved cardiac functions possibly because of GSNOR elimination and consequent increased NO bioavailability. Therefore, we successfully separated dual functions (GSNOR and FDH) of ADH5 (mediated by the supersulfide catalysis) through the biochemical analysis for supersulfides in vitro and characterizing in vivo phenotypes of the GSNOR-deficient organisms that we established herein. Supersulfides in ADH5 thus constitute a substantial catalytic center for GSNO metabolism mediating electron transfer from aldehydes.

Supersulphides provide airway protection in viral and chronic lung diseases.

Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF). We develop a method for breath supersulphur-omics and demonstrate that levels of exhaled supersulphides increase in people with COVID-19 infection and in a hamster model of SARS-CoV-2 infection. Lung damage and subsequent lethality that result from oxidative stress and inflammation in mouse models of COPD, IPF, and ageing were mitigated by endogenous supersulphides production by CARS2/CPERS or exogenous administration of the supersulphide donor glutathione trisulphide. We revealed a protective role of supersulphides in airways with various viral or chronic insults and demonstrated the potential of targeting supersulphides in lung disease.

Development of a method for isolating brain capillaries from a single neonatal mouse brain and comparison of proteomic profiles between neonatal and adult brain capillaries.

BACKGROUND: The functions and protein expressions of the blood-brain barrier are changed throughout brain development following birth. This study aimed to develop a method to isolate brain capillaries from a single frozen neonatal mouse brain and elucidate the enrichment of brain capillaries by quantitative proteomic analysis. We further compared the expression profile of proteins between neonatal and adult brain capillary fractions. METHODS: The brain capillary fraction was prepared by the optimized method from a single frozen mouse neonatal brain on postnatal day 7. The brain capillary fractions and brain lysates were digested by trypsin and analyzed by liquid chromatography-mass spectrometry for quantitative proteomics. RESULTS: By optimizing the isolation method, we observed brain capillaries in the fraction prepared from a single neonatal mouse brain (nBC fraction). A protein amount of 31.5 µg, which is enough for proteomic analysis, was recovered from the nBC fraction. By proteomics analysis, the brain capillary selective proteins, including Abcb1a/Mdr1, Slc2a1/Glut1, Claudin-5, and Pecam-1, were found to be concentrated > 13.4-fold more in nBC fractions than in whole brain lysates. The marker proteins for neurons and astrocytes were not concentrated in nBC fractions, while those of pericytes and microglia were concentrated. Compared to adult mouse brain capillary fractions (aBC fractions), the expressions of Abcb1a/Mdr1a, Abcc4/Mrp4, and Slc2a1/Glut1 were significantly lower in nBC fractions than in aBC fractions, whereas those of Slc1a4/Asct1, Slc1a5/Asct2, Slc7a1/Cat1, and Slc16a1/Mct1 were significantly higher. Amino acid transporters, Slc38a5/Snat5, showed the greatest nBC-to-aBC ratio among transporters (9.83-fold). Network analysis of proteins expressed differentially between nBC and aBC fractions revealed that the proteins with terms related to the extracellular matrix were enriched. CONCLUSIONS: We succeeded in isolating brain capillaries from a single frozen brain of a neonatal mouse at postnatal day 7. Proteomic analysis revealed the differential expression in brain capillaries between neonatal and adult mice. Specifically, amino acid transporters, including Slc1a5/Asct2 and Slc38a5/Snat5, were found to be induced in neonatal brain capillaries. The present isolation method will promote the study of the function and expression of the neonatal blood-brain barrier.

Influence of MDR1 gene polymorphism (2677G>T) on expression and function of P-glycoprotein at the blood-brain barrier: utilizing novel P-glycoprotein humanized mice with mutation.

P-glycoprotein, the encoded product of the MDR1 / ABCB1 gene in humans, is expressed in numerous tissues including brain capillary endothelial cells and restricts the distribution of xenobiotics into the brain as an efflux pump. Although a large number of single nucleotide polymorphisms in the MDR1 gene have been identified, the influence of the nonsynonymous 2677G>T/A single nucleotide polymorphism on P-glycoprotein at the blood-brain barrier has remained unclear. In the present study, we developed a novel P-glycoprotein humanized mouse line carrying the 2677G>T mutation by utilizing a mouse artificial chromosome vector constructed by genetic engineering technology and we evaluated the influence of 2677G>T on the expression and function of P-glycoprotein at the blood-brain barrier in vivo . The results of this study showed that the introduction of the 2677G>T mutation does not alter the expression levels of P-glycoprotein protein in the brain capillary fraction. On the other hand, the brain penetration of verapamil, a representative substrate of P-glycoprotein, was increased by the introduction of the 2677G>T mutation. These results suggested that the 2677G>T single nucleotide polymorphism may attenuate the function of P-glycoprotein, resulting in increased brain penetration of P-glycoprotein substrates, without altering the expression levels of P-glycoprotein protein in the blood-brain barrier. This mutant mouse line is a useful model for elucidating the influence of an MDR1 gene single nucleotide polymorphism on the expression and function of P-glycoprotein at the blood-brain barrier.

Diurnal Changes in Protein Expression at the Blood-Brain Barrier in Mice.

Circadian rhythms influence the transport function of the blood-brain barrier (BBB) and peripheral organs. However, the influence of circadian rhythms on protein expression in the BBB remains to be completely elucidated. Therefore, we aimed to investigate diurnal changes in protein expression in the mouse BBB using quantitative proteomics. Quantitative proteomics showed that the expression of 67, 10, and 20 proteins in the isolated mouse brain capillary fraction changed significantly at zeitgeber time (ZT) 6, 12, and 18, respectively, compared to ZT0. Among them, the levels of 44 proteins were significantly increased at ZT6 and then returned to the same level as ZT0 at ZT12 and ZT18. Gene ontology analysis indicated that the proteins significantly increased at ZT6 were majorly related to translation. The brain capillary endothelial cell-selective proteins sepiapterin reductase and vascular endothelial growth factor receptor 2 showed diurnal variation. In contrast, the expression of ABC transporters, SLC transporters, and receptors associated with receptor-mediated transcytosis, and tight junction proteins did not change within a day. The present findings demonstrated that protein expression related to transport function and physical barrier at the BBB was maintained throughout the day, although the proteins involved in some biological processes exhibited diurnal variation at the BBB.

Targeted proteomics for cancer biomarker verification and validation.

Targeted proteomics is a method that measures the amount of target proteins via liquid chromatography-tandem mass spectrometry and is used to verify and validate the candidate cancer biomarker proteins. Compared with antibody-based quantification methods such as ELISA, targeted proteomics enables rapid method development, simultaneous measurement of multiple proteins, and high-specificity detection of modifications. Moreover, by spiking the internal standard peptide, targeted proteomics detects the absolute amounts of marker proteins, which is essential for determining the cut-off values for diagnosis and thus for multi-institutional validation. With these unique features, targeted proteomics can seamlessly transfer cancer biomarker candidate proteins from the discovery phase to the verification and validation phases, thereby resulting in an accelerated cancer biomarker pipeline. Furthermore, understanding the basic principles, advantages, and disadvantages is necessary to effectively utilize targeted proteomics in cancer biomarker pipelines. This review aimed to introduce the technical principles of targeted proteomics for cancer biomarker verification and validation.

Knockdown of Podocalyxin Post-Transcriptionally Induces the Expression and Activity of ABCB1/MDR1 in Human Brain Microvascular Endothelial Cells.

Podocalyxin (PODXL) is a highly sialylated transmembrane protein that is expressed on the luminal membrane of brain microvascular endothelial cells. To clarify the role of PODXL in the blood-brain barrier (BBB), the present study aimed to investigate the effect of PODXL-knockdown on protein expression, especially the expression of ABCB1/MDR1, in human microvascular endothelial cells (hCMEC/D3). By quantitative proteomics, gene ontology enrichment with differentially expressed proteins showed that PODXL-knockdown influenced the immune response and intracellular trafficking. Among transporters, the protein expression of ABCB1/MDR1 and ABCG2/BCRP was significantly elevated by approximately 2-fold in the PODXL-knockdown cells. In the knockdown cells, the efflux activity of ABCB1/MDR1 was significantly increased, while its mRNA expression was not significantly different from that of the control cells. As receptors and tight junction proteins, levels of low-density lipoprotein receptor-related protein 1 and occludin were significantly increased, while those of transferrin receptor and claudin-11 were significantly decreased in the knockdown cells. The present results suggest that PODXL functions as a modulator of BBB function, including transport, tight junctions, and immune responses. Furthermore, PODXL post-transcriptionally regulates the protein expression and efflux activity of ABCB1/MDR1 at the BBB, which may affect drug distribution in the brain.

Acetylation of the influenza A virus polymerase subunit PA in the N-terminal domain positively regulates its endonuclease activity.

The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 in vitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity in vitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.

Oral Coadministration of Zn-Insulin with d-Form Small Intestine-Permeable Cyclic Peptide Enhances Its Blood Glucose-Lowering Effect in Mice.

Oral delivery of insulin remains a challenge owing to its poor permeability across the small intestine and enzymatic digestion in the gastrointestinal tract. In a previous study, we identified a small intestine-permeable cyclic peptide, C-DNPGNET-C (C-C disulfide bond, cyclic DNP peptide), which facilitated the permeation of macromolecules. Here, we showed that intraintestinal and oral coadministration of insulin with the cyclic DNP derivative significantly reduced blood glucose levels by increasing the portal plasma insulin concentration following permeation across the small intestine of mice. We also found that protecting the cyclic DNP derivative from enzymatic digestion in the small intestine of mice using d-amino acids and by the cyclization of DNP peptide was essential to enhance cyclic DNP derivative-induced insulin absorption across the small intestine. Furthermore, intraintestinal and oral coadministration of insulin hexamer stabilized by zinc ions (Zn-insulin) with cyclic D-DNP derivative was more effective in facilitating insulin absorption and inducing hypoglycemic effects in mice than the coadministration of insulin with the cyclic D-DNP derivative. Moreover, Zn-insulin was more resistant to degradation in the small intestine of mice compared to insulin. Intraintestinal and oral coadministration of Zn-insulin with cyclic DNP derivative also reduced blood glucose levels in a streptozotocin-induced diabetes mellitus mouse model. A single intraintestinal administration of the cyclic D-DNP derivative did not induce any cytotoxicity, either locally in the small intestine or systemically. In summary, we demonstrated that coadministration of Zn-insulin with cyclic D-DNP derivative could enhance oral insulin absorption across the small intestine in mice.

Efficient isolation of brain capillary from a single frozen mouse brain for protein expression analysis.

Isolated brain capillaries are essential for analyzing the changes of protein expressions at the blood-brain barrier (BBB) under pathological conditions. The standard brain capillary isolation methods require the use of at least five mouse brains in order to obtain a sufficient amount and purity of brain capillaries. The purpose of this study was to establish a brain capillary isolation method from a single mouse brain for protein expression analysis. We successfully isolated brain capillaries from a single frozen mouse brain by using a bead homogenizer in the brain homogenization step and combination of cell strainers and glass beads in the purification step. Western blot and proteomic analysis showed that proteins expressed at the BBB in mouse brain capillaries isolated by the developed method were more enriched than those isolated from a pool of five mouse brains by the standard method. By using the developed method, we further verified the changes in expression of BBB proteins in Glut1-deficient mouse. The developed method is useful for the analysis of various mice models with low numbers and enables us to understand, in more detail, the physiology and pathology of BBB.

Proteomic Evaluation of Plasma Membrane Fraction Prepared from a Mouse Liver and Kidney Using a Bead Homogenizer: Enrichment of Drug-Related Transporter Proteins.

Quantifying the protein levels of drug transporters in plasma membrane fraction helps elucidate the function of these transporters. In this study, we conducted a proteomic evaluation of enriched drug-related transporter proteins in plasma membrane fraction prepared from mouse liver and kidney tissues using the membrane protein extraction kit and a bead homogenizer. Crude and plasma membrane fractions were prepared using either the Dounce or bead homogenizer, and protein levels were determined using quantitative proteomics. In liver tissues, the plasma membrane fractions were more enriched in transporter proteins than the crude membrane fractions; the average enrichment ratios of plasma-to-crude membrane fractions were 3.31 and 6.93 using the Dounce and bead homogenizers, respectively. The concentrations of transporter proteins in plasma membrane fractions determined using the bead homogenizer were higher than those determined using the Dounce homogenizer. Meanwhile, in kidney tissues, the plasma membrane fractions were enriched in transporters localized in the brush-border membrane to the same degree for both the homogenizers; however, the membrane fractions obtained using either homogenizer were not enriched in Na+/K+-ATPase and transporters localized in the basolateral membrane. These results indicate that fractionation, using the bead homogenizer, yielded transporter-enriched plasma membrane fractions from mouse liver and kidney tissues; however, no enrichment of basolateral transporters was observed in plasma membrane fractions prepared from kidney tissues.

Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro.

Cell-surface proteins that can endocytose into brain microvascular endothelial cells serve as promising candidates for receptor-mediated transcytosis across the blood-brain barrier (BBB). Here, we comprehensively screened endocytic cell-surface proteins in hCMEC/D3 cells, a model of human brain microvascular endothelial cells, using surface biotinylation methodology and sequential window acquisition of all theoretical fragment-ion spectra-mass spectrometry (SWATH-MS)-based quantitative proteomics. Using this method, we identified 125 endocytic cell-surface proteins from hCMEC/D3 cells. Of these, 34 cell-surface proteins were selectively internalized into human brain microvascular endothelial cells, but not into human umbilical vein endothelial cells (HUVECs), a model of human peripheral microvascular endothelial cells. Two cell-surface proteins, intercellular adhesion molecule-1 (ICAM1) and podocalyxin (PODXL), were identified as BBB-localized endocytic cell-surface proteins in humans, using open mRNA and protein databases. Immunohistochemical evaluation confirmed PODXL expression in the plasma membrane of hCMEC/D3 cells and revealed that anti-PODXL antibody-labeled cell-surface PODXL internalized into hCMEC/D3 cells. Immunohistochemistry further revealed that PODXL is localized at the luminal side of human brain microvessels, supporting its potential suitability for translational applications. In conclusion, our findings highlight novel endocytic cell-surface proteins capable of internalizing into human brain microvascular endothelial cells. ICAM1 or PODXL targeted antibody or ligand-labeled biopharmaceuticals and nanocarriers may provide effective targeted delivery to the brain across the BBB for the treatment of central nervous system (CNS) diseases.

Changes of Blood-Brain Barrier and Brain Parenchymal Protein Expression Levels of Mice under Different Insulin-Resistance Conditions Induced by High-Fat Diet.

PURPOSE: The purpose of the present study was to investigate changes of blood-brain barrier (BBB) and brain parenchymal protein expression due to type II diabetes mellitus (T2DM) induced by a high-fat diet (HFD) by using SWATH-based quantitative proteomics. METHODS: Mice were fed a HFD for 2 or 10 weeks, and then SWATH-based quantitative proteomic analysis, western blot analysis, immunohistochemistry and functional transport studies were performed. RESULTS: In brain capillaries, expression levels of BBB transporters (Glut1, P-glycoprotein) and tight-junction proteins (claudin-5, occludin) were significantly reduced in HFD mice at 2 weeks, but recovered to the levels in the normal diet (ND) group at 10 weeks. P-glycoprotein function at the BBB was reduced at 2 weeks. In the cerebral cortex and hippocampus, neurofilament, which is important for neuronal function, was decreased in HFD mice at 2 weeks, but recovered at 10 weeks. CONCLUSION: Our results suggest that changes in the status of insulin resistance influence expression of BBB transporters, which in turn may alter the expression of cognitive function-related proteins.

Large-Scale Quantitative Comparison of Plasma Transmembrane Proteins between Two Human Blood-Brain Barrier Model Cell Lines, hCMEC/D3 and HBMEC/ciß.

Transmembrane (TM) proteins localized at the plasma membrane, such as transporters and receptors, play important roles in regulating the selective permeability of the blood-brain barrier (BBB). The purpose of the present study was to clarify the differences in the expression levels of TM proteins in the plasma membrane between two established human BBB model cell lines, hCMEC/D3 and HBMEC/ciß, in order to assist researchers in selecting the most appropriate cell line for particular purposes. We first confirmed that plasma membranes could be enriched sufficiently for a quantitative proteomics study by using the Plasma Membrane Protein Extraction Kit provided by BioVision with a modified protocol. This method was applied to hCMEC/D3 and HBMEC/ciß cells, and fractions were used for untargeted quantitative proteomics based on sequential window acquisition of all theoretical fragment-ion spectra. In the plasma membrane fractions, 345 TM proteins were quantified, among which 135 showed significant expression differences between the two cell lines. In hCMEC/D3 cells, amino acid transporters SNAT1, SNAT2, SNAT5, ASCT1, CAT1, and LAT1; adenosine 5'-triphosphate-binding cassette transporters P-gp and MRP4; and GLUT1 were more highly expressed. The transferrin receptor expression was also 4.56-fold greater in hCMEC/D3 cells. In contrast, HBMEC/ciß cells expressed greater levels of IgG transporter neonatal Fc receptor, as well as tight-junction proteins PECAM1, JAM1, JAM3, and ESAM. Our results suggest that hCMEC/D3 cells have greater efflux transport, amino acid transport, and transferrin receptor-mediated uptake activities, whereas HBMEC/ciß cells have greater IgG-transport activity and tight-junction integrity.

Knockdown of Orphan Transporter SLC22A18 Impairs Lipid Metabolism and Increases Invasiveness of HepG2 Cells.

PURPOSE: The aim of this work is to investigate the roles of solute carrier family 22 member 18 (SLC22A18) in lipid metabolism and in establishing the tumor phenotype of HepG2 cells. METHODS: SLC22A18-knockdown HepG2 cells were established by stable transfection with shRNA. Protein expression levels were measured by quantitative proteomics and Western blot analysis. Cell growth was examined by cell counting kit. Accumulation of triglyceride-rich lipid droplets was measured by Oil-Red O staining. Cell migration and invasion were examined by Transwell assays. RESULTS: SLC22A18-knockdown HepG2 cells accumulated triglyceride-rich lipid droplets and showed decreased expression levels of lysosomal/autophagic proteins, suggesting that lipid degradation is suppressed. Growth of HepG2 cells was decreased by SLC22A18 knockdown, but was restored by free fatty acid supplementation. In addition, SLC22A18 knockdown decreased the expression of insulin-like growth factor-binding protein 1 (IGFBP-1) and increased the invasion ability of HepG2 cells. Exogenous IGFBP-1 blocked the increase of invasion activity induced by SLC22A18 knockdown. CONCLUSION: Our results suggest that suppression of SLC22A18 decreased the supply of intracellular free fatty acids from triglyceride-rich lipid droplets by impairing the lysosomal/autophagy degradation pathway and reduced the invasive activity of HepG2 cells by decreasing IGFBP-1 expression.

Involvement of an Orphan Transporter, SLC22A18, in Cell Growth and Drug Resistance of Human Breast Cancer MCF7 Cells.

The SLC22A18 gene, which encodes an orphan transporter, is located at the 11p15.5 imprinted region, an important tumor suppressor gene region. However, the role of SLC22A18 in tumor suppression remains unclear. Here, we investigated the involvement of SLC22A18 in cell growth, invasion, and drug resistance of MCF7 human breast cancer cell line. Western blot analysis indicated that SLC22A18 is predominantly expressed at intracellular organelle membranes. Quantitative proteomics showed that knockdown of SLC22A18 significantly altered the expression of 578 (31.0%) of 1867 proteins identified, including proteins related to malignancy and poor prognosis of breast cancer. SLC22A18 knockdown (1) increased MCF7 cell growth concomitantly with a >7-fold increase of annexin A8 (involved in cell growth and migration; a predictor of poor prognosis), (2) induced spherical morphology of MCF7 cells concomitantly with a nearly 3-fold increase of CD44 (involved in regulation of malignant phenotypes), and (3) increased chemosensitivity to vinca alkaloids concomitantly with a >80% reduction of doublecortin-like kinase 1 (involved in regulation of microtubule polymerization). Our results suggest that SLC22A18 may act as a tumor suppressor by regulating the expression levels of cell growth-related proteins, and vinca alkaloids might show therapeutic efficacy against low-SLC22A18-expressing breast cancer.