Publications by authors named "Vadivel Ganapathy"

248 Publications

SLC6A14 and SLC38A5 Drive the Glutaminolysis and Serine-Glycine-One-Carbon Pathways in Cancer.

Pharmaceuticals (Basel) 2021 Mar 4;14(3). Epub 2021 Mar 4.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.

The glutaminolysis and serine-glycine-one-carbon pathways represent metabolic reactions that are reprogramed and upregulated in cancer; these pathways are involved in supporting the growth and proliferation of cancer cells. Glutaminolysis participates in the production of lactate, an oncometabolite, and also in anabolic reactions leading to the synthesis of fatty acids and cholesterol. The serine-glycine-one-carbon pathway is involved in the synthesis of purines and pyrimidines and the control of the epigenetic signature (DNA methylation, histone methylation) in cancer cells. Methionine is obligatory for most of the methyl-transfer reactions in the form of S-adenosylmethionine; here, too, the serine-glycine-one-carbon pathway is necessary for the resynthesis of methionine following the methyl-transfer reaction. Glutamine, serine, glycine, and methionine are obligatory to fuel these metabolic pathways. The first three amino acids can be synthesized endogenously to some extent, but the need for these amino acids in cancer cells is so high that they also have to be acquired from extracellular sources. Methionine is an essential amino acid, thus making it necessary for cancer cells to acquire this amino acid solely from the extracellular milieu. Cancer cells upregulate specific amino acid transporters to meet this increased demand for these four amino acids. SLC6A14 and SLC38A5 are the two transporters that are upregulated in a variety of cancers to mediate the influx of glutamine, serine, glycine, and methionine into cancer cells. SLC6A14 is a Na/Cl -coupled transporter for multiple amino acids, including these four amino acids. In contrast, SLC38A5 is a Na-coupled transporter with rather restricted specificity towards glutamine, serine, glycine, and methionine. Both transporters exhibit unique functional features that are ideal for the rapid proliferation of cancer cells. As such, these two amino acid transporters play a critical role in promoting the survival and growth of cancer cells and hence represent novel, hitherto largely unexplored, targets for cancer therapy.
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http://dx.doi.org/10.3390/ph14030216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000594PMC
March 2021

α-Methyl-l-tryptophan as a weight-loss agent in multiple models of obesity in mice.

Biochem J 2021 Apr;478(7):1347-1358

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, U.S.A.

α-Methyl-L-tryptophan (α-MLT) is currently in use as a tracer in its 11C-labeled form to monitor the health of serotonergic neurons in humans. In the present study, we found this compound to function as an effective weight-loss agent at pharmacological doses in multiple models of obesity in mice. The drug was able to reduce the body weight when given orally in drinking water (1 mg/ml) in three different models of obesity: normal mice on high-fat diet, Slc6a14-null mice on high-fat diet, and ob/ob mice on normal diet. Only the l-enantiomer (α-MLT) was active while the d-enantiomer (α-MDT) had negligible activity. The weight-loss effect was freely reversible, with the weight gain resuming soon after the withdrawal of the drug. All three models of obesity were associated with hyperglycemia, insulin resistance, and hepatic steatosis; α-MLT reversed these features. There was a decrease in food intake in the treatment group. Mice on a high-fat diet showed decreased cholesterol and protein in the serum when treated with α-MLT; there was however no evidence of liver and kidney dysfunction. Plasma amino acid profile indicated a significant decrease in the levels of specific amino acids, including tryptophan; but the levels of arginine were increased. We conclude that α-MLT is an effective, reversible, and orally active drug for the treatment of obesity and metabolic syndrome.
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http://dx.doi.org/10.1042/BCJ20210100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038855PMC
April 2021

Consequences of NaCT/SLC13A5/mINDY deficiency: good versus evil, separated only by the blood-brain barrier.

Biochem J 2021 Feb;478(3):463-486

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, U.S.A.

NaCT/SLC13A5 is a Na+-coupled transporter for citrate in hepatocytes, neurons, and testes. It is also called mINDY (mammalian ortholog of 'I'm Not Dead Yet' in Drosophila). Deletion of Slc13a5 in mice leads to an advantageous phenotype, protecting against diet-induced obesity, and diabetes. In contrast, loss-of-function mutations in SLC13A5 in humans cause a severe disease, EIEE25/DEE25 (early infantile epileptic encephalopathy-25/developmental epileptic encephalopathy-25). The difference between mice and humans in the consequences of the transporter deficiency is intriguing but probably explainable by the species-specific differences in the functional features of the transporter. Mouse Slc13a5 is a low-capacity transporter, whereas human SLC13A5 is a high-capacity transporter, thus leading to quantitative differences in citrate entry into cells via the transporter. These findings raise doubts as to the utility of mouse models to evaluate NaCT biology in humans. NaCT-mediated citrate entry in the liver impacts fatty acid and cholesterol synthesis, fatty acid oxidation, glycolysis, and gluconeogenesis; in neurons, this process is essential for the synthesis of the neurotransmitters glutamate, GABA, and acetylcholine. Thus, SLC13A5 deficiency protects against obesity and diabetes based on what the transporter does in hepatocytes, but leads to severe brain deficits based on what the transporter does in neurons. These beneficial versus detrimental effects of SLC13A5 deficiency are separable only by the blood-brain barrier. Can we harness the beneficial effects of SLC13A5 deficiency without the detrimental effects? In theory, this should be feasible with selective inhibitors of NaCT, which work only in the liver and do not get across the blood-brain barrier.
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http://dx.doi.org/10.1042/BCJ20200877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868109PMC
February 2021

SLC6A14 deficiency is linked to obesity, fatty liver, and metabolic syndrome but only under conditions of a high-fat diet.

Biochim Biophys Acta Mol Basis Dis 2021 May 26;1867(5):166087. Epub 2021 Jan 26.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA. Electronic address:

SLC6A14 is a Na/Cl-coupled transporter for neutral/cationic amino acids, expressed in ileum and colon. A single-nucleotide polymorphism (SNP), rs2011162 (-22,510C > G), in SLC6A14 coding for the 3'-untranslated region (3'-UTR) is associated with obesity in humans. But the impact of this polymorphism on the transporter expression and its connection to obesity are not known. Our objective was to address these issues. The impact of rs2011162 (-22,510C > G) on SLC6A14 expression was monitored using a luciferase reporter. The link between Slc6a14 and obesity was investigated in wild type and Slc6a14 mice when fed a normal diet or a high-fat diet. The obesity-associated 3'-UTR polymorphism reduced SLC6A14 expression. With a high-fat diet, Slc6a14 mice gained more weight than wild type mice. With normal diet, there was no difference between the two genotypes. The gain in body weight with the high-fat diet in Slc6a14 mice was accompanied with metabolic syndrome. With the high-fat diet, Slc6a14 mice showed increased food intake, developed fatty liver, and altered plasma amino acid profile. The high-fat diet-associated hepatic steatosis in Slc6a14 mice showed male preponderance. We conclude that the 3'-UTR SNP in SLC6A14 associated with obesity decreases the expression of SLC6A14 and that the deficiency of SLC6A14 is linked to obesity. This is supported by the findings that Slc6a14 mice develop obesity, fatty liver, and metabolic syndrome. This connection is evident only with a high-fat diet. Therefore, dietary/pharmacologic interventions that induce SLC6A14 expression in the intestinal tract might have potential for obesity prevention..
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http://dx.doi.org/10.1016/j.bbadis.2021.166087DOI Listing
May 2021

Extracellular Citrate Fuels Cancer Cell Metabolism and Growth.

Front Cell Dev Biol 2020 4;8:602476. Epub 2020 Dec 4.

Department of Surgery, University Medical Center Regensburg, Regensburg, Germany.

Cancer cells need excess energy and essential nutrients/metabolites not only to divide and proliferate but also to migrate and invade distant organs for metastasis. Fatty acid and cholesterol synthesis, considered a hallmark of cancer for anabolism and membrane biogenesis, requires citrate. We review here potential pathways in which citrate is synthesized and/or supplied to cancer cells and the impact of extracellular citrate on cancer cell metabolism and growth. Cancer cells employ different mechanisms to support mitochondrial activity and citrate synthesis when some of the necessary substrates are missing in the extracellular space. We also discuss the different transport mechanisms available for the entry of extracellular citrate into cancer cells and how citrate as a master metabolite enhances ATP production and fuels anabolic pathways. The available literature suggests that cancer cells show an increased metabolic flexibility with which they tackle changing environmental conditions, a phenomenon crucial for cancer cell proliferation and metastasis.
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http://dx.doi.org/10.3389/fcell.2020.602476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793864PMC
December 2020

Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina.

Antioxidants (Basel) 2020 Dec 24;10(1). Epub 2020 Dec 24.

Department of Biochemistry and Molecular Biology, Augusta University, Augusta, GA 30912, USA.

Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina; retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, northern and western blots, and immunofluorescence were used to analyze the expression of xc-, Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in the control, an oxidatively stressed RPE. The Se-Met effect was selective; under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- with a consequent increase in glutathione.
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http://dx.doi.org/10.3390/antiox10010009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823377PMC
December 2020

Metformin, valproic acid, and starvation induce seizures in a patient with partial SLC13A5 deficiency: a case of pharmaco-synergistic heterozygosity.

Psychiatr Genet 2021 02;31(1):32-35

Department of Psychiatry, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

SLC13A5/NaCT is a sodium-coupled citrate transporter expressed in the plasma membrane of the liver, testis, and brain. In these tissues, SLC13A5 has important functions in the synthesis of fatty acids, cholesterol, and neurotransmitters. In recent years, patients homozygous for recessive mutations in SLC13A5, known as SLC13A5 deficiency [early infantile epileptic encephalopathy-25 (EIEE-25)], exhibit severe global developmental delay, early-onset intractable seizures, spasticity, and amelogenesis imperfecta affecting tooth development. Although the pathogenesis of SLC13A5 deficiency remains not clearly understood, cytoplasmic citrate deficits, decreased energy status in neurons, and citrate-zinc chelation are hypothesized to explain the neurological deficits. However, no study has examined the possibility of specific pharmacological drugs and/or lifestyle changes synergizing with heterozygosity of SLC13A5 deficiency to increase the risk of EIEE-25 clinical phenotype. Here, we report on a heterozygous SLC13A5-deficient patient who demonstrated evidence of pharmaco-synergistic heterozygosity upon administration of metformin, valproic acid, and starvation. The report illustrates the importance of careful consideration of the potential adverse effects of specific pharmacological treatments in patients with heterozygosity for disease-causing recessive mutations in SLC13A5.
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http://dx.doi.org/10.1097/YPG.0000000000000269DOI Listing
February 2021

Functional analysis of a species-specific inhibitor selective for human Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY).

Biochem J 2020 11;477(21):4149-4165

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock 79430, Texas, U.S.A.

The Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY) in the liver delivers citrate from the blood into hepatocytes. As citrate is a key metabolite and regulator of multiple biochemical pathways, deletion of Slc13a5 in mice protects against diet-induced obesity, diabetes, and metabolic syndrome. Silencing the transporter suppresses hepatocellular carcinoma. Therefore, selective blockers of NaCT hold the potential to treat various diseases. Here we report on the characteristics of one such inhibitor, BI01383298. It is known that BI01383298 is a high-affinity inhibitor selective for human NaCT with no effect on mouse NaCT. Here we show that this compound is an irreversible and non-competitive inhibitor of human NaCT, thus describing the first irreversible inhibitor for this transporter. The mouse NaCT is not affected by this compound. The inhibition of human NaCT by BI01383298 is evident for the constitutively expressed transporter in HepG2 cells and for the ectopically expressed human NaCT in HEK293 cells. The IC50 is ∼100 nM, representing the highest potency among the NaCT inhibitors known to date. Exposure of HepG2 cells to this inhibitor results in decreased cell proliferation. We performed molecular modeling of the 3D-structures of human and mouse NaCTs using the crystal structure of a humanized variant of VcINDY as the template, and docking studies to identify the amino acid residues involved in the binding of citrate and BI01383298. These studies provide insight into the probable bases for the differential effects of the inhibitor on human NaCT versus mouse NaCT as well as for the marked species-specific difference in citrate affinity.
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http://dx.doi.org/10.1042/BCJ20200592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657661PMC
November 2020

Functional Distinction between Human and Mouse Sodium-Coupled Citrate Transporters and Its Biologic Significance: An Attempt for Structural Basis Using a Homology Modeling Approach.

Chem Rev 2021 May 11;121(9):5359-5377. Epub 2020 Oct 11.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, United States.

NaCT (SLC13A5; mINDY), a sodium-coupled citrate transporter, is the mammalian ortholog of INDY. Loss-of-function mutations in human NaCT cause severe complications with neonatal epilepsy and encephalopathy (EIEE25). Surprisingly, mice lacking this transporter do not have this detrimental brain phenotype. The marked differences in transport kinetics between mouse and human NaCTs provide at least a partial explanation for this conundrum, but a structural basis for the differences is lacking. Neither human nor mouse NaCT has been crystallized, and any information known on their structures is based entirely on what was inferred from the structure of VcINDY, a related transporter in bacteria. Here, we highlight the functional features of human and mouse NaCTs and provide a plausible molecular basis for the differences based on a full-length homology modeling approach. The transport characteristics of human NaCT markedly differ from those of VcINDY. Therefore, the modeling with VcINDY as the template is flawed, but this is the best available option at this time. With the newly deduced model, we determined the likely locations of the disease-causing mutations and propose a new classification for the mutations based on their location and potential impact on transport function. This new information should pave the way for future design and development of novel therapeutics to restore the lost function of the mutant transporters as a treatment strategy for patients with EIEE25.
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http://dx.doi.org/10.1021/acs.chemrev.0c00529DOI Listing
May 2021

Transporter-Targeted Nano-Sized Vehicles for Enhanced and Site-Specific Drug Delivery.

Cancers (Basel) 2020 Oct 1;12(10). Epub 2020 Oct 1.

Department of Pharmacy, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Zhejiang 325027, China.

Nano-devices are recognized as increasingly attractive to deliver therapeutics to target cells. The specificity of this approach can be improved by modifying the surface of the delivery vehicles such that they are recognized by the target cells. In the past, cell-surface receptors were exploited for this purpose, but plasma membrane transporters also hold similar potential. Selective transporters are often highly expressed in biological barriers (e.g., intestinal barrier, blood-brain barrier, and blood-retinal barrier) in a site-specific manner, and play a key role in the vectorial transfer of nutrients. Similarly, selective transporters are also overexpressed in the plasma membrane of specific cell types under pathological states to meet the biological needs demanded by such conditions. Nano-drug delivery systems could be strategically modified to make them recognizable by these transporters to enhance the transfer of drugs across the biological barriers or to selectively expose specific cell types to therapeutic drugs. Here, we provide a comprehensive review and detailed evaluation of the recent advances in the field of transporter-targeted nano-drug delivery systems. We specifically focus on areas related to intestinal absorption, transfer across blood-brain barrier, tumor-cell selective targeting, ocular drug delivery, identification of the transporters appropriate for this purpose, and details of the rationale for the approach.
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http://dx.doi.org/10.3390/cancers12102837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599460PMC
October 2020

Hereditary hemochromatosis promotes colitis and colon cancer and causes bacterial dysbiosis in mice.

Biochem J 2020 10;477(19):3867-3883

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, U.S.A.

Hereditary hemochromatosis (HH), an iron-overload disease, is a prevalent genetic disorder. As excess iron causes a multitude of metabolic disturbances, we postulated that iron overload in HH disrupts colonic homeostasis and colon-microbiome interaction and exacerbates the development and progression of colonic inflammation and colon cancer. To test this hypothesis, we examined the progression and severity of colitis and colon cancer in a mouse model of HH (Hfe-/-), and evaluated the potential contributing factors. We found that experimentally induced colitis and colon cancer progressed more robustly in Hfe-/- mice than in wild-type mice. The underlying causes were multifactorial. Hfe-/- colons were leakier with lower proliferation capacity of crypt cells, which impaired wound healing and amplified inflammation-driven tissue injury. The host/microflora axis was also disrupted. Sequencing of fecal 16S RNA revealed profound changes in the colonic microbiome in Hfe-/- mice in favor of the pathogenic bacteria belonging to phyla Proteobacteria and TM7. There was an increased number of bacteria adhered onto the mucosal surface of the colonic epithelium in Hfe-/- mice than in wild-type mice. Furthermore, the expression of innate antimicrobial peptides, the first-line of defense against bacteria, was lower in Hfe-/- mouse colon than in wild-type mouse colon; the release of pro-inflammatory cytokines upon inflammatory stimuli was also greater in Hfe-/- mouse colon than in wild-type mouse colon. These data provide evidence that excess iron accumulation in colonic tissue as happens in HH promotes colitis and colon cancer, accompanied with bacterial dysbiosis and loss of function of the intestinal/colonic barrier.
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http://dx.doi.org/10.1042/BCJ20200392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7557149PMC
October 2020

Therapeutic application and construction of bilirubin incorporated nanoparticles.

J Control Release 2020 12 1;328:407-424. Epub 2020 Sep 1.

Department of Pharmacy, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325027, China. Electronic address:

Bilirubin is a yellow-colored metabolite of heme degradation (a bile pigment), once believed to be toxic, but recently recognized as a powerful endogenous antioxidant of physiologic importance. During the past two decades, several studies have demonstrated the potential of bilirubin in theranostic applications. Here this paper summarizes the current state of the field, providing a detailed review of the published literature on the theranostic applications of bilirubin-conjugated nanoparticles and the basis and mechanisms underlying their efficacy. This review covers the analytical description of the construction of the nanoparticulate bilirubin system, primary mechanisms of therapeutic action, drug delivery, and imaging potential. It also lays out the possible translational future of bilirubin-conjugated nanoparticles in therapy and diagnosis.
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http://dx.doi.org/10.1016/j.jconrel.2020.08.054DOI Listing
December 2020

Deficiency Reduces Tumor Growth by Targeting Stem Cell Self-Renewal.

Cancer Res 2020 09 14;80(18):3855-3866. Epub 2020 Jul 14.

Department of Biochemistry and Molecular Biology, Augusta University, Augusta, Georgia.

RAD51-associated protein 1 (RAD51AP1) plays an integral role in homologous recombination by activating RAD51 recombinase. Homologous recombination is essential for preserving genome integrity and RAD51AP1 is critical for D-loop formation, a key step in homologous recombination. Although RAD51AP1 is involved in maintaining genomic stability, recent studies have shown that RAD51AP1 expression is significantly upregulated in human cancers. However, the functional role of RAD51AP1 in tumor growth and the underlying molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been fully understood. Here, we use Rad51ap1-knockout mice in genetically engineered mouse models of breast cancer to unravel the role of RAD51AP1 in tumor growth and metastasis. RAD51AP1 gene transcript was increased in both luminal estrogen receptor-positive breast cancer and basal triple-negative breast cancer, which is associated with poor prognosis. Conversely, knockdown of RAD51AP1 (RADP51AP1 KD) in breast cancer cell lines reduced tumor growth. Rad51ap1-deficient mice were protected from oncogene-driven spontaneous mouse mammary tumor growth and associated lung metastasis. , limiting dilution studies provided evidence that Rad51ap1 plays a critical role in breast cancer stem cell (BCSC) self-renewal. RAD51AP1 KD improved chemotherapy and radiotherapy response by inhibiting BCSC self-renewal and associated pluripotency. Overall, our study provides genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis by increasing BCSC self-renewal and may serve as a novel target for chemotherapy- and radiotherapy-resistant breast cancer. SIGNIFICANCE: This study provides evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating breast cancer stem cell self-renewal.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-3713DOI Listing
September 2020

Rescue of mutant gonadotropin-releasing hormone receptor function independent of cognate receptor activity.

Sci Rep 2020 06 29;10(1):10579. Epub 2020 Jun 29.

Department of Molecular Medicine, The Scripps Research Molecular Screening Center, Scripps Research Florida, 130 Scripps Way #1A1, Jupiter, FL, 33458, USA.

Molecules that correct the folding of protein mutants, restoring their functional trafficking, are called pharmacoperones. Most are clinically irrelevant and possess intrinsic antagonist or agonist activity. Here, we identify compounds capable of rescuing the activity of mutant gonadotropin-releasing hormone receptor or GnRHR which, is sequestered within the cell and if dysfunctional leads to Hypogonadotropic Hypogonadism. To do this we screened the E90K GnRHR mutant vs. a library of 645,000 compounds using a cell-based calcium detection system. Ultimately, we identified 399 compounds with EC ≤ 5 µM with no effect in counterscreen assays. Medicinal chemistry efforts confirmed activity of 70 pure samples and mode of action studies, including radioligand binding, inositol phosphate, and toxicity assays, proved that we have a series of tractable compounds that can be categorized into structural clusters. These early lead molecules rescue mutant GnRHR function and are neither agonist nor antagonists of the GnRHR cognate receptor, a feature required for potential clinical utility.
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http://dx.doi.org/10.1038/s41598-020-67473-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324376PMC
June 2020

The Hepatic Plasma Membrane Citrate Transporter NaCT (SLC13A5) as a Molecular Target for Metformin.

Sci Rep 2020 05 22;10(1):8536. Epub 2020 May 22.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, 79430, Texas, United States.

Metformin is the first-line treatment for type 2 diabetes. Inhibition of hepatic gluconeogenesis is the primary contributor to its anti-diabetic effect. Metformin inhibits complex I and α-glycerophosphate shuttle, and the resultant increase in cytoplasmic NADH/NAD ratio diverts glucose precursors away from gluconeogenesis. These actions depend on metformin-mediated activation of AMP kinase (AMPK). Here we report on a hitherto unknown mechanism. Metformin inhibits the expression of the plasma membrane citrate transporter NaCT in HepG2 cells and decreases cellular levels of citrate. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, elicits a similar effect. The process involves a decrease in maximal velocity with no change in substrate affinity. The decrease in NaCT expression is associated with decreased mRNA levels. AMPK inhibits mTOR, and the mTOR inhibitor rapamycin also decreases NaCT expression. The transcription factor downstream of AMPK that is relevant to cAMP signaling is CREB; decreased levels of phospho-CREB seem to mediate the observed effects of metformin on NaCT. Citrate is known to suppress glycolysis by inhibiting phosphofructokinase-1 and activate gluconeogenesis by stimulating fructose-1,6-bisphophatase; therefore, the decrease in cellular levels of citrate would stimulate glycolysis and inhibit gluconeogenesis. These studies uncover a novel mechanism for the anti-diabetic actions of metformin.
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http://dx.doi.org/10.1038/s41598-020-65621-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244496PMC
May 2020

Involvement of a Na-coupled Oligopeptide Transport System for β-amyloid Peptide (Aβ) in Brain Cells.

Pharm Res 2020 May 17;37(6):98. Epub 2020 May 17.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, Texas, 79430-6540, USA.

Purpose: A Na-coupled transport system in mammalian cells is responsible for the uptake of oligopeptides consisting of 5 or more amino acids. Here we investigated if this transport system is expressed in brain cells and transports the 42-amino-acid β-amyloid peptide (Aβ).

Methods: The human and mouse neuronal cell lines SK-N-SH and HT22, human microglial cell line HMC-3, and human blood-brain barrier endothelial cell line hCMEC/D3 were used to monitor the uptake of [H]-deltorphin II (a heptapeptide) and fluorescence-labeled Aβ.

Results: All four cell lines exhibited Na-coupled uptake of deltorphin II. Aβ competed with deltorphin II for the uptake. Uptake of fluorescence-labeled Aβ was detectable in these cell lines, and the uptake was Na-dependent and inhibitable by deltorphin II. The Na-coupled uptake disappeared at high concentrations of Aβ due to oligomerization of the peptide. Exposure of the cells to excess iron abolished the uptake. In hCMEC/D3 cells cultured on Transwell filters, the uptake was localized preferentially to the abluminal membrane.

Conclusion: A Na-coupled transport system mediates the uptake of Aβ monomers in neuronal and microglial cells. The same system is also responsible for the uptake of Aβ from brain into blood-brain barrier endothelial cells. These findings have relevance to Alzheimer's disease.
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http://dx.doi.org/10.1007/s11095-020-02835-7DOI Listing
May 2020

Synergism between SLC6A14 blockade and gemcitabine in pancreactic cancer: a 1H-NMR-based metabolomic study in pancreatic cancer cells.

Biochem J 2020 05;477(10):1923-1937

Department of Pharmacy, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325027, China.

Gemcitabine is the first-line chemotherapy for pancreatic cancer. To overcome the often-acquired gemcitabine resistance, other drugs are used in combination with gemcitabine. It is well-known that cancer cells reprogram cellular metabolism, coupled with the up-regulation of selective nutrient transporters to feed into the altered metabolic pathways. Our previous studies have demonstrated that the amino acid transporter SLC6A14 is markedly up-regulated in pancreatic cancer and that it is a viable therapeutic target. α-Methyltryptophan (α-MT) is a blocker of SLC6A14 and is effective against pancreatic cancer in vitro and in vivo. In the present study, we tested the hypothesis that α-MT could synergize with gemcitabine in the treatment of pancreatic cancer. We investigated the effects of combination of α-MT and gemcitabine on proliferation, migration, and apoptosis in a human pancreatic cancer cell line, and examined the underlying mechanisms using 1H-NMR-based metabolomic analysis. These studies examined the intracellular metabolite profile and the extracellular metabolite profile separately. Combination of α-MT with gemcitabine elicited marked changes in a wide variety of metabolic pathways, particularly amino acid metabolism with notable alterations in pathways involving tryptophan, branched-chain amino acids, ketone bodies, and membrane phospholipids. The metabolomic profiles of untreated control cells and cells treated with gemcitabine or α-MT were distinctly separable, and the combination regimen showed a certain extent of overlap with the individual α-MT and gemcitabine groups. This represents the first study detailing the metabolomic basis of the anticancer efficacy of gemcitabine, α-MT and their combination.
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http://dx.doi.org/10.1042/BCJ20200275DOI Listing
May 2020

Chronic exposure to excess iron promotes EMT and cancer via p53 loss in pancreatic cancer.

Asian J Pharm Sci 2020 Mar 5;15(2):237-251. Epub 2020 Mar 5.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.

Based on the evidence that hemochromatosis, an iron-overload disease, drives hepatocellular carcinoma, we hypothesized that chronic exposure to excess iron, either due to genetic or environmental causes, predisposes an individual to cancer. Using pancreatic cancer as our primary focus, we employed cell culture studies to interrogate the connection between excess iron and cancer, and combined and studies to explore the connection further. Ferric ammonium citrate was used as an exogenous iron source. Chronic exposure to excess iron induced epithelial-mesenchymal transition (EMT) in normal and cancer cell lines, loss of p53, and suppression of p53 transcriptional activity evidenced from decreased expression of p53 target genes (p21, cyclin D1, Bax, SLC7A11). To further extrapolate our cell culture data, we generated - () mouse (pancreatic neoplastic mouse model) expressing and genetic background. p53 target gene expression decreased in / mouse pancreas compared to / mouse pancreas. Interestingly, the incidence of acinar-to-ductal metaplasia and cystic pancreatic neoplasms (CPN) decreased in / mice, but the CPNs that did develop were larger in these mice than in / mice. In conclusion, these and studies support a potential role for chronic exposure to excess iron as a promoter of more aggressive disease via p53 loss and SLC7A11 upregulation within pancreatic epithelial cells.
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http://dx.doi.org/10.1016/j.ajps.2020.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193456PMC
March 2020

Pharmacologic inducers of the uric acid exporter ABCG2 as potential drugs for treatment of gouty arthritis.

Asian J Pharm Sci 2020 Mar 7;15(2):173-180. Epub 2019 Nov 7.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, United States.

Uric acid is the end product of purine catabolism and its plasma levels are maintained below its maximum solubility in water (6-7 mg/dl). The plasma levels are tightly regulated as the balance between the rate of production and the rate of excretion, the latter occurring in urine (kidney), bile (liver) and feces (intestinal tract). Reabsorption in kidney is also an important component of this process. Both excretion and reabsorption are mediated by specific transporters. Disruption of the balance between production and excretion leads to hyperuricemia, which increases the risk of uric acid crystallization as monosodium urate with subsequent deposition of the crystals in joints causing gouty arthritis. Loss-of-function mutations in the transporters that mediate uric acid excretion are associated with gout. The ATP-Binding Cassette exporter ABCG2 is important in uric acid excretion at all three sites: kidney (urine), liver (bile), and intestine (feces). Mutations in this transporter cause gout and these mutations occur at significant prevalence in general population. However, mutations that are most prevalent result only in partial loss of transport function. Therefore, if the expression of these partially defective transporters could be induced, the increased number of the transporter molecules would compensate for the mutation-associated decrease in transport function and hence increase uric acid excretion. As such, pharmacologic agents with ability to induce the expression of ABCG2 represent potentially a novel class of drugs for treatment of gouty arthritis.
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http://dx.doi.org/10.1016/j.ajps.2019.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193448PMC
March 2020

Hereditary hemochromatosis disrupts uric acid homeostasis and causes hyperuricemia via altered expression/activity of xanthine oxidase and ABCG2.

Biochem J 2020 04;477(8):1499-1513

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, U.S.A.

Hereditary hemochromatosis (HH) is mostly caused by mutations in the iron-regulatory gene HFE. The disease is associated with iron overload, resulting in liver cirrhosis/cancer, cardiomegaly, kidney dysfunction, diabetes, and arthritis. Fe2+-induced oxidative damage is suspected in the etiology of these symptoms. Here we examined, using Hfe-/- mice, whether disruption of uric acid (UA) homeostasis plays any role in HH-associated arthritis. We detected elevated levels of UA in serum and intestine in Hfe-/- mice compared with controls. Though the expression of xanthine oxidase, which generates UA, was not different in liver and intestine between wild type and Hfe-/- mice, the enzymatic activity was higher in Hfe-/- mice. We then examined various transporters involved in UA absorption/excretion. Glut9 expression did not change; however, there was an increase in Mrp4 and a decrease in Abcg2 in Hfe-/- mice. As ABCG2 mediates intestinal excretion of UA and mutations in ABCG2 cause hyperuricemia, we examined the potential connection between iron and ABCG2. We found p53-responsive elements in hABCG2 promoter and confirmed with chromatin immunoprecipitation that p53 binds to this promoter. p53 protein was reduced in Hfe-/- mouse intestine. p53 is a heme-binding protein and p53-heme complex is subjected to proteasomal degradation. We conclude that iron/heme overload in HH increases xanthine oxidase activity and also promotes p53 degradation resulting in decreased ABCG2 expression. As a result, systemic UA production is increased and intestinal excretion of UA via ABCG2 is decreased, causing serum and tissue accumulation of UA, a potential factor in the etiology of HH-associated arthritis.
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http://dx.doi.org/10.1042/BCJ20190873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200644PMC
April 2020

SLC6A14, a Na+/Cl--coupled amino acid transporter, functions as a tumor promoter in colon and is a target for Wnt signaling.

Biochem J 2020 04;477(8):1409-1425

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, U.S.A.

SLC6A14 is a Na+/Cl--coupled transporter for neutral and cationic amino acids. It is expressed at basal levels in the normal colon but is up-regulated in colon cancer. However, the relevance of this up-regulation to cancer progression and the mechanisms involved in the up-regulation remain unknown. Here, we show that SLC6A14 is essential for colon cancer and that its up-regulation involves, at least partly, Wnt signaling. The up-regulation of the transporter is evident in most human colon cancer cell lines and also in a majority of patient-derived xenografts. These findings are supported by publicly available TCGA (The Cancer Genome Atlas) database. Treatment of colon cancer cells with α-methyltryptophan (α-MT), a blocker of SLC6A14, induces amino acid deprivation, decreases mTOR activity, increases autophagy, promotes apoptosis, and suppresses cell proliferation and invasion. In xenograft and syngeneic mouse tumor models, silencing of SLC6A14 by shRNA or blocking its function by α-MT reduces tumor growth. Similarly, the deletion of Slc6a14 in mice protects against colon cancer in two different experimental models (inflammation-associated colon cancer and genetically driven colon cancer). In colon cancer cells, expression of the transporter is reduced by Wnt antagonist or by silencing of β-catenin whereas Wnt agonist or overexpression of β-catenin shows the opposite effect. Finally, SLC6A14 as a target for β-catenin is confirmed by chromatin immunoprecipitation. These studies demonstrate that SLC6A14 plays a critical role in the promotion of colon cancer and that its up-regulation in cancer involves Wnt signaling. These findings identify SLC6A14 as a promising drug target for the treatment of colon cancer.
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http://dx.doi.org/10.1042/BCJ20200099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7182441PMC
April 2020

The lactate receptor GPR81 promotes breast cancer growth via a paracrine mechanism involving antigen-presenting cells in the tumor microenvironment.

Oncogene 2020 04 19;39(16):3292-3304. Epub 2020 Feb 19.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA.

GPR81 is a G-protein-coupled receptor for lactate, which is upregulated in breast cancer and plays an autocrine role to promote tumor growth by tumor cell-derived lactate. Here we asked whether lactate has any paracrine role via activation of GPR81 in cells present in tumor microenvironment to help tumor growth. First, we showed that deletion of Gpr81 suppresses breast cancer growth in a constitutive breast cancer mouse model (MMTV-PyMT-Tg). We then used a syngeneic transplant model by monitoring tumor growth from a mouse breast cancer cell line (AT-3, Gpr81-negative) implanted in mammary fat pad of wild-type mice and Gpr81-null mice. Tumor growth was suppressed in Gpr81-null mice compared with wild-type mice. There were more tumor-infiltrating T cells and MHCII-immune cells in tumors from Gpr81-null mice compared with tumors from wild-type mice. RNA-seq analysis of tumors indicated involvement of immune cells and antigen presentation in Gpr81-dependent tumor growth. Antigen-presenting dendritic cells expressed Gpr81 and activation of this receptor by lactate suppressed cell-surface presentation of MHCII. Activation of Gpr81 in dendritic cells was associated with decreased cAMP, IL-6 and IL-12. These findings suggest that tumor cell-derived lactate activates GPR81 in dendritic cells and prevents presentation of tumor-specific antigens to other immune cells. This paracrine mechanism is complementary to the recently discovered autocrine mechanism in which lactate induces PD-L1 in tumor cells via activation of GPR81 in tumor cells, thus providing an effective means for tumor cells to evade immune system. As such, blockade of GPR81 signaling could boost cancer immunotherapy.
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http://dx.doi.org/10.1038/s41388-020-1216-5DOI Listing
April 2020

Endocytosis of ATB(SLC6A14)-targeted liposomes for drug delivery and its therapeutic application for pancreatic cancer.

Expert Opin Drug Deliv 2020 03 5;17(3):395-405. Epub 2020 Feb 5.

Department of Pharmacy, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China.

: SLC6A14 (ATB), a Na/Clcoupled transporter for neutral/cationic amino acids, is overexpressed in many cancers; It has been investigated as a target for improved liposomal drug delivery to treat liver cancer.: Here we explored the mechanism of ATB-mediated entry of such liposomes. As ATB is highly expressed in pancreatic cancer, we also examined the therapeutic utility of ATB-targeted liposomal drug delivery to treat this cancer.: The uptake of lysine-conjugated liposomes (LYS-LPs) was greater in ATB-positive MCF7 cells. The uptake process consisted of two steps: binding and internalization. The binding of LYS-LPs to MCF7 cells was higher than that of bare liposomes, and the process was dependent on Na and Cl, and inhibitable by ATB substrates or blocker. In contrast, the internalization step was independent of lysine. The cellular entry of LYS-LPs facilitated by ATB occurred via endocytosis with transient endosomal degradation of ATB protein with subsequent recovery. Moreover, LYS-LPs also enhanced the uptake and cytotoxicity of gemcitabine in these cells in an ATB-dependent manner.: We conclude that ATB could be exploited for targeted drug delivery in the form of lysine-conjugated liposomes and that the approach represents a novel strategy for enhanced pancreatic cancer therapy.
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http://dx.doi.org/10.1080/17425247.2020.1723544DOI Listing
March 2020

Deficiency of Dietary Fiber in -Null Mice Promotes Bacterial Dysbiosis and Alters Colonic Epithelial Transcriptome towards Proinflammatory Milieu.

Can J Gastroenterol Hepatol 2019 27;2019:2543082. Epub 2019 Dec 27.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the intestinal tract due to disruption of the symbiotic relationship between the host immune system and microbiota. Various factors alter the gut microbiota which lead to dysbiosis; in particular, diet and dietary fibers constitute important determinants. Dietary fiber protects against IBD; bacteria ferment these dietary fibers in colon and generate short-chain fatty acids (SCFAs), which mediate the anti-inflammatory actions of dietary fibers. SLC5A8 is a high-affinity transporter in the apical membrane of colonic epithelium which mediates the entry of SCFAs from the lumen into cells in Na-coupled manner. Due to the unique transport kinetics, the function of the transporter becomes important only under conditions of low dietary fiber intake. Here, we have examined the impact of dietary fiber deficiency on luminal microbial composition and transcriptomic profile in colonic epithelium in wild-type (WT) and -null (KO) mice. We fed WT and KO mice with fiber-containing diet (FC-diet) or fiber-free diet (FF-diet) and analyzed the luminal bacterial composition by sequencing 16S rRNA gene in feces. Interestingly, results showed significant differences in the microbial community depending on dietary fiber content and on the presence or absence of Slc5a8. There were also marked differences in the transcriptomic profile of the colonic epithelium depending on the dietary fiber content and on the presence or absence of Slc5a8. We conclude that absence of fiber in diet in KO mice causes bacterial dysbiosis and alters gene expression in the colon that is conducive for inflammation.
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http://dx.doi.org/10.1155/2019/2543082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949682PMC
July 2020

OCTN2-targeted nanoparticles for oral delivery of paclitaxel: differential impact of the polyethylene glycol linker size on drug delivery , , and .

Drug Deliv 2020 Dec;27(1):170-179

Department of Pharmacy, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China.

Targeted nanocarriers have shown great promise in drug delivery because of optimized drug behavior and improved therapeutic efficacy. How to improve the targeting efficiency of nanocarriers for the maximum possible drug delivery is a critical issue. Here we developed L-carnitine-conjugated nanoparticles targeting the carnitine transporter OCTN2 on enterocytes for improved oral absorption. As a variable, we introduced various lengths of the polyethylene glycol linker (0, 500, 1000, and 2000) between the nanoparticle surface and the ligand (CNP, C5NP, C10NP and C20NP) to improve the ligand flexibility, and consequently for more efficient interaction with the transporter, to enhance the oral delivery of the cargo load into cells. An increased absorption was observed in cellular uptake and in intestinal perfusion assay when the polyethylene glycol was introduced to link L-carnitine to the nanoparticles; the highest absorption was achieved with C10NP. In contrast, the linker decreased the absorption efficiency . As the presence or absence of the mucus layer was the primary difference between / versus , the presence of this layer was the likely reason for this differential effect. In summary, the size of the polyethylene glycol linker improved the absorption and , but interfered with the absorption . Even though this strategy of increasing the ligand flexibility with the variable size of the polyethylene glycol failed to increase oral absorption , this approach is likely to be useful for enhanced cellular uptake following intravenous administration of the nanocarriers.
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http://dx.doi.org/10.1080/10717544.2019.1710623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6968687PMC
December 2020

Lactate/GPR81 signaling and proton motive force in cancer: Role in angiogenesis, immune escape, nutrition, and Warburg phenomenon.

Pharmacol Ther 2020 02 10;206:107451. Epub 2019 Dec 10.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA. Electronic address:

Reprogramming of biochemical pathways is a hallmark of cancer cells, and generation of lactic acid from glucose/glutamine represents one of the consequences of such metabolic alterations. Cancer cells export lactic acid out to prevent intracellular acidification, not only increasing lactate levels but also creating an acidic pH in extracellular milieu. Lactate and protons in tumor microenvironment are not innocuous bystander metabolites but have special roles in promoting tumor-cell proliferation and growth. Lactate functions as a signaling molecule by serving as an agonist for the G-protein-coupled receptor GPR81, involving both autocrine and paracrine mechanisms. In the autocrine pathway, cancer cell-generated lactate activates GPR81 on cancer cells; in the paracrine pathway, cancer cell-generated lactate activates GPR81 on immune cells, endothelial cells, and adipocytes present in tumor stroma. The end result of GPR81 activation is promotion of angiogenesis, immune evasion, and chemoresistance. The acidic pH creates an inwardly directed proton gradient across the cancer-cell plasma membrane, which provides driving force for proton-coupled transporters in cancer cells to enhance supply of selective nutrients. There are several molecular targets in the pathways involved in the generation of lactic acid by cancer cells and its role in tumor promotion for potential development of novel anticancer therapeutics.
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http://dx.doi.org/10.1016/j.pharmthera.2019.107451DOI Listing
February 2020

Ambidextrous Approach To Disrupt Redox Balance in Tumor Cells with Increased ROS Production and Decreased GSH Synthesis for Cancer Therapy.

ACS Appl Mater Interfaces 2019 Jul 18;11(30):26722-26730. Epub 2019 Jul 18.

Department of Pharmacy , The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University , Wenzhou 325027 , China.

An effective steady-state redox balance is maintained in cancer cells, allowing for protection against oxidative stress and thereby enhancing cell proliferation and tumor growth. Disruption of this redox balance would increase the cellular content of reactive oxygen species (ROS) and potentiate oxidative stress-induced cell death in tumor cells, thus representing an effective strategy for cancer treatment. Glutathione (GSH) is a major reducing agent, and its cellular levels are determined at least partly by the availability of cysteine via xCT (SLC7A11)-mediated entry of cystine into cells. We developed a nanoplatform using ZnO nanoparticles (NPs) as a carrier, loaded with salicylazosulfapyridine (SASP), and stabilized with DSPE-PEG, to form ultra-small NPs (SASP/ZnO NPs). The goal of this NP strategy is to disrupt the redox balance in cells by two mechanisms: increased generation of ROS and decreased synthesis of GSH. Such an approach would be effective in killing tumor cells. As expected, the SASP/ZnO NPs enhanced ROS production because of ZnO and impaired GSH synthesis because of SASP-induced inhibition of xCT (SLC7A11) transport function. As a consequence, treatment of tumor cells with SASP/ZnO NPs in vitro and in vivo resulted in a synergistic disruptive effect on redox balance in tumor cells and induced cell death and decreased tumor growth. This ambidextrous approach has potential in cancer therapy by combining two complementary pathways to disrupt the redox balance in tumor cells.
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http://dx.doi.org/10.1021/acsami.9b09784DOI Listing
July 2019

Renal iron accelerates the progression of diabetic nephropathy in the HFE gene knockout mouse model of iron overload.

Am J Physiol Renal Physiol 2019 08 12;317(2):F512-F517. Epub 2019 Jun 12.

Department of Ophthalmology and Department of Biochemistry and Molecular Biology, Saint Louis University, St. Louis, Missouri.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease associated with high mortality worldwide. Increases in iron levels have been reported in diabetic rat kidneys as well as in human urine of patients with diabetes. In addition, a low-iron diet or iron chelators delay the progression of DN in patients with diabetes and in animal models of diabetes. Possible maladaptive mechanisms of organ damage by tissue iron accumulation have not been well studied. We recently reported that iron induced the retinal renin-angiotensin system (RAS) and accelerated the progression of diabetic retinopathy. However, whether iron regulates the systemic RAS is unknown. To explore if iron alters the expression of intrarenal RAS and its role in the progression of DN, we used the high Fe iron (HFE) knockout mouse, a genetic model of systemic iron overload. We found that diabetes upregulated the expression of iron regulatory proteins and augmented tissue iron accumulation in the kidneys of both type 1 and type 2 diabetic mouse models. Iron accumulation in the kidneys of HFE knockout mice was associated with increase in serum and intrarenal renin expression. Induction of diabetes in HFE knockout mice using streptozotocin caused a much higher accumulation of renal iron and accelerated the progression of nephropathy compared with diabetic wild-type mice. Treatment of diabetic mice with the iron chelator deferiprone reversed the renin upregulation and reduced kidney injury. Thus, our results establish a new link between renal iron and RAS activity. Exploring the mechanisms of iron-induced RAS activation further may have a significant therapeutic impact on hypertension and DN.
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http://dx.doi.org/10.1152/ajprenal.00184.2019DOI Listing
August 2019

Development and radiosynthesis of the first F-labeled inhibitor of monocarboxylate transporters (MCTs).

J Labelled Comp Radiopharm 2019 06;62(8):411-424

Department of Neuroradiopharmaceuticals, Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Research Site Leipzig, Leipzig, Germany.

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT-mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [ F]FACH ((E)-2-cyano-3-{4-[(3-[ F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylic acid), as a novel radiolabeled MCT1/4 inhibitor for imaging with PET. A fluorinated analog of α-cyano-4-hydroxycinnamic acid (FACH) was synthesized, and the inhibition of MCT1 and MCT4 was measured via an L-[ C]lactate uptake assay. Radiolabeling was performed by a two-step protocol comprising the radiosynthesis of the intermediate (E)/(Z)-[ F]tert-Bu-FACH (tert-butyl (E)/(Z)-2-cyano-3-{4-[(3-[ F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylate) followed by deprotection of the tert-butyl group. The radiofluorination was successfully implemented using either K[ F]F-K -carbonate or [ F]TBAF. The final deprotected product [ F]FACH was only obtained when [ F]tert-Bu-FACH was formed by the latter procedure. After optimization of the deprotection reaction, [ F]FACH was obtained in high radiochemical yields (39.6 ± 8.3%, end of bombardment (EOB) and radiochemical purity (greater than 98%).
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http://dx.doi.org/10.1002/jlcr.3739DOI Listing
June 2019

Transport Mechanisms for the Nutritional Supplement β-Hydroxy-β-Methylbutyrate (HMB) in Mammalian Cells.

Pharm Res 2019 Apr 17;36(6):84. Epub 2019 Apr 17.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX, 79430-6540, USA.

Purpose: β-Hydroxy-β-methylbutyrate (HMB), a nutritional supplement, elicits anabolic activity in muscle. Here we investigated the mechanism of HMB uptake in muscle cells.

Methods: Murine muscle cells (C2C12) and human mammary epithelial cells (MCF7) were used for uptake. As HMB is a monocarboxylate, focus was on monocarboxylate transporters, monitoring interaction of HMB with H-coupled lactate uptake, and influence of H directly on HMB uptake. Involvement of MCT1-4 was studied using selective inhibitors and gene silencing. Involvement of human Na/monocarboxylate transporter SMCT1 was also assessed using Xenopus oocytes.

Results: H-coupled lactate uptake was inhibited by HMB in both mammalian cells. HMB uptake was H-coupled and inhibited by lactate. C2C12 cells expressed MCT1 and MCT4; MCF7 cells expressed MCT1-4; undifferentiated C2C12 cells expressed SMCT1. SMCT1 mediated Na-coupled HMB transport. Inhibitors of MCT1/4, siRNA-mediated gene silencing, and expression pattern showed that MCT1-4 were responsible only for a small portion of HMB uptake in these cells.

Conclusion: HMB uptake in C2C12 and MCF7 cells is primarily H-coupled and inhibited by lactate, but MCT1-4 are only partly responsible for HMB uptake. SMCT1 also transports HMB, but in a Na-coupled manner. Other, yet unidentified, transporters mediate the major portion of HMB uptake in C2C12 and MCF7 cells.
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http://dx.doi.org/10.1007/s11095-019-2626-3DOI Listing
April 2019