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1.  The plasma membrane transporter SLC5A8 suppresses tumor progression through depletion of survivin without involving its transport function 
The Biochemical journal  2013;450(1):169-178.
Summary
SLC5A8 is a sodium-coupled transporter for monocarboxylates. Among its substrates are the HDAC inhibitors butyrate, propionate, and pyruvate. Expression of SLC5A8 is silenced in cancers via DNA methylation, and ectopic expression of SLC5A8 in cancer cells induces apoptosis in the presence of its substrates that are HDAC inhibitors. Here we show that ectopic expression of SLC5A8 in cancer cells translocates the anti-apoptotic protein survivin to plasma membrane through protein-protein interaction resulting in depletion of nuclear survivin and also decreases cellular levels of survivin through inhibition of transcription. These SLC5A8-induced changes in the location and levels of survivin result in cell cycle arrest, disruption of the chromosome passenger complex involved in mitosis, induction of apoptosis, and enhancement in chemosensitivity. These effects are seen independent of the transport function of SLC5A8 and histone acetylation status of the cell; in the presence of pyruvate, a SLC5A8 substrate and also an HDAC inhibitor, these effects are amplified. Ectopic expression of SLC5A8 in the breast cancer cell line MB231 inhibits the ability of the cell to form colonies in vitro and to form tumors in mouse xenografts in vivo. The suppression of survivin transcription occurs independent of HDAC inhibition, and the underlying mechanism is associated with decreased phosphorylation of STAT3. The observed effects are specific for survivin with no apparent changes in expression of other inhibitor-of-apoptosis proteins. These studies unravel a novel, hitherto unrecognized, mechanism for the tumor-suppressive role of a plasma membrane transporter independent of its transport function.
doi:10.1042/BJ20121248
PMCID: PMC3710455  PMID: 23167260
SLC5A8; survivin; histone deacetylases; tumor suppressor; STAT3
2.  Loss of Hfe Leads to Progression of Tumor Phenotype in Primary Retinal Pigment Epithelial Cells 
Purpose.
Hemochromatosis is a disorder of iron overload arising mostly from mutations in HFE. HFE is expressed in retinal pigment epithelium (RPE), and Hfe−/− mice develop age-related iron accumulation and retinal degeneration associated with RPE hyperproliferation. Here, the mechanism underlying the hyperproliferative phenotype in RPE was investigated.
Methods.
Cellular senescence was monitored by β-galactosidase activity. Gene expression was monitored by real-time PCR. Survivin was analyzed by Western blot and immunofluorescence. Migration and invasion were monitored using appropriate kits. Glucose transporters (GLUTs) were monitored by 3-O-methyl-D-glucose uptake. Histone deacetylases (HDACs) were studied by monitoring catalytic activity and acetylation status of histones H3/H4.
Results.
Hfe−/− RPE cells exhibited slower senescence rate and higher survivin expression than wild type cells. Hfe−/− cells migrated faster and showed greater glucose uptake and increased expression of GLUTs. The expression of HDACs and DNA methyltransferase (DNMTs) also was increased. Similarly, RPE cells from hemojuvelin (Hjv)-knockout mice, another model of hemochromatosis, also had increased expression of GLUTs, HDACs, and DNMTs. The expression of Slc5a8 was decreased in Hfe−/− RPE cells, but treatment with a DNA methylation inhibitor restored the transporter expression, indicating involvement of DNA methylation in the silencing of Slc5a8 in Hfe−/− cells.
Conclusions.
RPE cells from iron-overloaded mice exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of HDACs and DNMTs. These features are seen in Hfe−/− RPE cells as well as in Hjv−/− RPE cells, providing a molecular basis for the hyperproliferative phenotype of Hfe−/− and Hjv−/− RPE cells.
Hfe−/− and Hjv−/− retinal pigment epithelial (RPE) cells exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of Histone deacetylases and DNA methyltransferase. These biochemical parameters underlie the hyperproliferative phenotype of Hfe-/- and Hjv-/- RPE cells.
doi:10.1167/iovs.12-10312
PMCID: PMC3544423  PMID: 23169885
3.  Role of SLC5A8, a plasma membrane transporter and a tumor suppressor, in the antitumor activity of dichloroacetate 
Oncogene  2011;30(38):4026-4037.
There has been growing interest among the public and scientists in dichloroacetate as a potential anticancer drug. Credible evidence exists for the antitumor activity of this compound, but high concentrations are needed for significant therapeutic effect. Unfortunately, these high concentrations produce detrimental side effects involving nervous system, thereby precluding its use for cancer treatment. The mechanistic basis of the compound’s antitumor activity is its ability to activate pyruvate dehydrogenase complex through inhibition of pyruvate dehydrogenase kinase. Since the compound inhibits the kinase at micromolar concentrations, it is not known why therapeutically prohibitive high doses are needed for suppression of tumor growth. We hypothesized that lack of effective mechanisms for the entry of dichloroacetate into tumor cells may underlie this phenomenon. Here we show that SLC5A8 transports dichloroacetate very effectively with high affinity. This transporter is expressed in normal cells, but the expression is silenced in tumor cells via epigenetic mechanisms. The lack of the transporter makes tumor cells resistant to the antitumor activity of dichloroacetate. However, if the transporter is expressed in tumor cells ectopically, the cells become sensitive to the drug at low concentrations. This is evident in breast cancer cells, colon cancer cells, and prostate cancer cells. Normal cells, which constitutively express the transporter, are however not affected by the compound, indicating the tumor cell-selective therapeutic activity. The mechanism of the antitumor activity of the compound is still its ability to inhibit pyruvate dehydrogenase kinase and force mitochondrial oxidation of pyruvate. Since the silencing of SLC5A8 in tumors involves DNA methylation and its expression can be induced by treatment with DNA methylation inhibitors, our findings suggest that combining dichloroacetate with a DNA methylation inhibitor would offer a means to reduce the doses of dichloroacetate to avoid detrimental effects associated with high doses but without compromising antitumor activity.
doi:10.1038/onc.2011.113
PMCID: PMC3140604  PMID: 21499304
SLC5A8; dichloroacetate; anticancer drug; Warburg effect; pyruvate dehydrogenase kinase; mitochondrial oxidation in cancer
4.  Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish 
PLoS ONE  2012;7(1):e29916.
Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current “high-content” whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.
doi:10.1371/journal.pone.0029916
PMCID: PMC3251595  PMID: 22238673
5.  Transport via SLC5A8 (SMCT1) Is Obligatory for 2-Oxothiazolidine-4-Carboxylate to Enhance Glutathione Production in Retinal Pigment Epithelial Cells 
The present study describes the role of the sodium-dependent monocarboxylate transporter SLC5A8 (SMCT1) in the transport of the cysteine prodrug 2-oxothiazolidine-4-carboxylate (OTC), and the resultant augmentation of glutathione production in RPE cells. Given the key causative role oxidative damage to RPE plays in the pathogenesis of AMD, the present study is critically important and highly clinically relevant.
Purpose.
To evaluate the role of SLC5A8 in the transport of 2-oxothiazolidine-4-carboxylate (OTC) and to determine whether OTC augments glutathione production in RPE cells, thereby providing protection against oxidative stress.
Methods.
SLC5A8-mediated transport of OTC was monitored in Xenopus laevis oocytes by electrophysiological means. Saturation kinetics, Na+-activation kinetics, and inhibition by ibuprofen were analyzed by monitoring OTC-induced currents as a measure of transport activity. Oxidative stress was induced in ARPE-19 cells and primary RPE cells isolated from wild type and Slc5a8-/- mouse retinas using H2O2, and the effects of OTC on cell death and intracellular glutathione concentration were examined.
Results.
Heterologous expression of human SLC5A8 in X. laevis oocytes induced Na+-dependent inward currents in the presence of OTC under voltage-clamp conditions. The transport of OTC via SLC5A8 was saturable, with a Kt of 104 ± 3 μM. The Na+-activation kinetics was sigmoidal with a Hill coefficient of 1.9 ± 0.1, suggesting involvement of two Na+ in the activation process. Ibuprofen, a blocker of SLC5A8, inhibited SLC5A8-mediated OTC transport; the concentration necessary for half-maximal inhibition was 17 ± 1 μM. OTC increased glutathione levels and protected ARPE-19 and primary RPE cells isolated from wild type mouse retinas from H2O2-induced cell death. These effects were abolished in primary RPE isolated from Slc5a8-/- mouse retinas.
Conclusions.
OTC is a transportable substrate for SLC5A8. OTC augments glutathione production in RPE cells, thereby protecting them from oxidative damage. Transport via SLC5A8 is obligatory for this process.
doi:10.1167/iovs.10-6825
PMCID: PMC3176078  PMID: 21508099
6.  Role of C/EBP homologous protein (CHOP) in Panobinostat-mediated potentiation of Bortezomib-induced lethal ER stress in Mantle Cell Lymphoma cells 
PURPOSE
Bortezomib (BZ) induces unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, as well as exhibits clinical activity in patients with relapsed and refractory Mantle Cell Lymphoma (MCL). Here, we determined the molecular basis of the improved in vitro and in vivo activity of the combination of pan-histone deacetylase (HDAC) inhibitor (HDI) panobinostat (PS) and BZ against human, cultured and primary MCL cells.
EXPERIMENTAL DESIGN
Immunoblot analyses, RT-PCR and immunofluorescent and electron microscopy were utilized to determine the effects of PS on BZ-induced aggresome formation and ER stress in MCL cells.
RESULTS
Treatment with PS treatment induced heat shock protein (hsp) 90 acetylation, depleted the levels of hsp90 client proteins, CDK4, c-RAF and AKT, as well as abrogated BZ-induced aggresome formation in MCL cells. PS also induced lethal UPR, associated with induction of CHOP. Conversely, knockdown of CHOP attenuated PS-induced cell death of MCL cells. Compared to each agent alone, co-treatment with PS increased BZ-induced CHOP and NOXA expressions, as well as increased BZ-induced UPR and apoptosis of cultured and primary MCL cells. Co-treatment with PS also increased BZ-mediated in vivo tumor growth inhibition and improved survival of mice bearing human Z138C MCL cell xenograft.
CONCLUSION
These findings suggest that increased UPR and induction of CHOP are involved in enhanced anti-MCL activity of the combination of PS and BZ.
doi:10.1158/1078-0432.CCR-10-0529
PMCID: PMC2948590  PMID: 20647473
Panobinostat; ER stress; MCL; Bortezomib; CHOP
7.  The Marine Sponge Metabolite Mycothiazole: A Novel Prototype Mitochondrial Complex I Inhibitor 
Bioorganic & medicinal chemistry  2010;18(16):5988-5994.
A natural product chemistry-based approach was applied to discover small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1). A Petrosaspongia mycofijiensis marine sponge extract yielded mycothiazole (1), a solid tumor selective compound with no known mechanism for its cell line-dependent cytotoxic activity. Compound 1 inhibited hypoxic HIF-1 signaling in tumor cells (IC50 1 nM) that correlated with the suppression of hypoxia-stimulated tumor angiogenesis in vitro. However, 1 exhibited pronounced neurotoxicity in vitro. Mechanistic studies revealed that 1 selectively suppresses mitochondrial respiration at Complex I (NADH-ubiquinone oxidoreductase). Unlike rotenone, MPP+, annonaceous acetogenins, piericidin A, and other Complex I inhibitors, mycothiazole is a mixed polyketide/peptide-derived compound with a central thiazole moiety. The exquisite potency and structural novelty of 1 suggest that it may serve as a valuable molecular probe for mitochondrial biology and HIF-mediated hypoxic signaling.
doi:10.1016/j.bmc.2010.06.072
PMCID: PMC2918693  PMID: 20637638
Hypoxia-inducible factor-1 (HIF-1); Marine natural products; Mitochondrial Complex I Inhibitor; NADH-ubiquinone oxidoreductase
8.  The Alternative Medicine Pawpaw and Its Acetogenin Constituents Suppress Tumor Angiogenesis via the HIF-1/VEGF Pathway 
Journal of natural products  2010;73(5):956-961.
Products that contain twig extracts of pawpaw (Asimina triloba, Annonaceae) are widely consumed anticancer alternative medicines. Pawpaw crude extract (CE) and purified acetogenins inhibited hypoxia-inducible factor-1 (HIF-1)-mediated hypoxic signaling pathways in tumor cells. In T47D cells, pawpaw CE and the acetogenins 10-hydroxyglaucanetin (1), annonacin (2), and annonacin A (3) inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.02 μg/mL, 12 nM, 13 nM, and 31 nM, respectively. This inhibition correlates with the suppression of the hypoxic induction of HIF-1 target genes VEGF and GLUT-1. The induction of secreted VEGF protein represents a key event in hypoxia-induced tumor angiogenesis. Both the extract and the purified acetogenins blocked the angiogenesis-stimulating activity of hypoxic T47D cells in vitro. Pawpaw extract and acetogenins inhibited HIF-1 activation by blocking the hypoxic induction of nuclear HIF-1α protein. The inhibition of HIF-1 activation was associated with the suppression of mitochondrial respiration at complex I. Thus, the inhibition of HIF-1 activation and hypoxic tumor angiogenesis constitutes a novel mechanism of action for these anticancer alternative medicines.
doi:10.1021/np100228d
PMCID: PMC2890309  PMID: 20423107
9.  The Caulerpa Pigment Caulerpin Inhibits HIF-1 Activation and Mitochondrial Respiration 
Journal of natural products  2009;72(12):2104-2109.
The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important molecular target for anticancer drug discovery. In a T47D cell-based reporter assay, the Caulerpa spp. algal pigment caulerpin (1) inhibited hypoxia-induced as well as 1,10-phenanthroline-induced HIF-1 activation. The angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF-1. Caulerpin (10 μM) suppressed hypoxic induction of secreted VEGF protein and the ability of hypoxic T47D cell-conditioned media to promote tumor angiogenesis in vitro. Under hypoxic conditions, 1 (10 μM) blocked the induction of HIF-1α protein, the oxygen-regulated subunit that controls HIF-1 activity. Reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1α protein induction and activation. Further mechanistic studies revealed that 1 inhibits mitochondrial respiration at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Under hypoxic conditions, it is proposed that 1 may disrupt mitochondrial ROS-regulated HIF-1 activation and HIF-1 downstream target gene expression by inhibiting the transport or delivery of electrons to complex III.
doi:10.1021/np9005794
PMCID: PMC2798910  PMID: 19921787

Results 1-9 (9)