Curcumin enhances the membrane trafficking of the sodium iodide symporter and augments radioiodine uptake in dedifferentiated thyroid cancer cells via suppression of the PI3K-AKT signaling pathway.

Radioactive iodine (RAI) is commonly used to treat differentiated thyroid cancer (DTC). A major challenge is the dedifferentiation of DTC with the loss of radioiodine uptake. Patients with distant metastases have persistent or recurrent disease and develop resistance to RAI therapy due to tumor dedifferentiation. Hence, tumor redifferentiation to restore sensitivity to RAI therapy is considered a promising strategy to overcome RAI resistance. In the present study, curcumin, a natural polyphenolic compound, was found to re-induce cell differentiation and increase the expression of thyroid-specific transcription factors, TTF-1, TTF-2 and transcriptional factor paired box 8 (PAX8), and iodide-metabolizing proteins, including thyroid stimulating hormone receptor (TSHR), thyroid peroxidase (TPO) and sodium iodide symporter (NIS) in dedifferentiated thyroid cancer cell lines, BCPAP and KTC-1. Importantly, curcumin enhanced NIS glycosylation and its membrane trafficking, resulting in a significant improvement of radioiodine uptake in vitro. Additionally, AKT knockdown phenocopied the restoration of thyroid-specific gene expression; however, ectopic expressed AKT inhibited curcumin-induced up-regulation of NIS protein, demonstrating that curcumin might improve radioiodine sensitivity via the inhibition of the PI3K-AKT-mTOR signaling pathway. Our study demonstrates that curcumin could represent a promising adjunctive therapy for restoring iodide avidity and improve radioiodine therapeutic efficacy in patients with RAI-refractory thyroid carcinoma.

The flavonoid quercetin reduces cell migration and increases NIS and E-cadherin mRNA in the human thyroid cancer cell line BCPAP.

Thyroid cancer is the most frequent cancer of the endocrine system. Most patients are treated with thyroidectomy followed by radioiodine therapy. However, in part of the patients, a reduction of the sodium-iodide symporter (NIS) occurs, rendering radioiodine therapy ineffective. Moreover, epithelial-mesenchymal transition (EMT) may occur, leading to more aggressive and invasive features. Herein, we evaluated the effect of the flavonoid quercetin on EMT and NIS expression in BCPAP, a papillary thyroid carcinoma cell line. BCPAP was treated with 100 µM quercetin for 24 h and cell viability, apoptosis, EMT markers and NIS were evaluated. Quercetin decreased cell viability by enhancing apoptosis. The flavonoid also reduced matrix metalloproteinase 9 and increased E-cadherin mRNA levels, inhibiting BCPAP adhesion and migration. Additionally, quercetin increased NIS expression and function. Thus, our results suggest that quercetin could be useful as adjuvant in thyroid cancer therapy, inducing apoptosis, reducing invasion and increasing the efficacy of radioiodine therapy.

Apical Sodium-Dependent Bile Acid Cotransporter, A Novel Transporter of Indocyanine Green, and Its Application in Drug Screening.

Bile acid plays critical roles in the elimination of inorganic compounds such as bilirubin, heavy metals, and drug metabolites. Apical sodium-dependent bile acid cotransporter (ASBT), a solute carrier membrane transport protein, transports bile acids. Several inhibitors of ASBT have been evaluated in clinical trials. Sodium taurocholate cotransporting polypeptide (NTCP), belonging to the same family as ASBT, has fluorescein 5(6)-isothiocyanate (FITC) and indocyanine green (ICG) transportability. ICG, a Food and Drug Administration-approved fluorophore at near-infrared range, has perfect optical characteristics, so it can be applied in cell tracking and drug screening. In this study, ASBT and NTCP were transduced into the HT-1080 cell line. Nude mice were subcutaneously xenografted with control and ASBT-expressing cells. ICG transportability was observed through flow cytometry, fluorescent microscopy, multi-mode plate readers, and an in vivo imaging system. Several molecules, including taurocholate, sodium deoxycholate, cyclosporine A, nifedipine, and Primovist, were used to evaluate an in vitro drug-screening platform by using the combination of ICG and ASBT through flow cytometry. ICG and FITC were validated and shown to be transported by ASBT. NTCP had a higher ICG intensity compared with ASBT. For cell tracking, the ASBT xenograft had similar ICG signals as the control. For a drug-screening platform, the ICG intensity decreased with 186 µM taurocholate (56.8%), deoxycholate (83.8%), and increased with nifedipine (133.2%). These findings are suggestive of opportunities for the high-throughput drug screening of cholestasis and other diseases that are related to the dynamics of bile acid reabsorption.

The Expanding Therapeutic Potential of Neuronal KCC2.

Dysfunctions in GABAergic inhibitory neural transmission occur in neuronal injuries and neurological disorders. The potassium-chloride cotransporter 2 (KCC2, SLC12A5) is a key modulator of inhibitory GABAergic inputs in healthy adult neurons, as its chloride (Cl-) extruding activity underlies the hyperpolarizing reversal potential for GABAA receptor Cl- currents (EGABA). Manipulation of KCC2 levels or activity improve symptoms associated with epilepsy and neuropathy. Recent works have now indicated that pharmacological enhancement of KCC2 function could reactivate dormant relay circuits in an injured mouse's spinal cord, leading to functional recovery and the attenuation of neuronal abnormality and disease phenotype associated with a mouse model of Rett syndrome (RTT). KCC2 interacts with Huntingtin and is downregulated in Huntington's disease (HD), which contributed to GABAergic excitation and memory deficits in the R6/2 mouse HD model. Here, these recent advances are highlighted, which attest to KCC2's growing potential as a therapeutic target for neuropathological conditions resulting from dysfunctional inhibitory input.

Kinetic studies of K-Cl cotransport in cultured rat vascular smooth muscle cells.

During aging, and development of atherosclerosis and cardiovascular disease (CVD), aortic vascular smooth muscle cells (VSMCs) transition from healthy contractile to diseased synthetic phenotypes. K-Cl cotransport (KCC) maintains cell volume and ion homeostasis in growth and differentiation, and hence is important for VSMC proliferation and migration. Therefore, KCC activity may play a role in the contractile-to-synthetic VSMC phenotypic transition. Early, medium, and late synthetic passage VSMCs were tested for specific cytoskeletal protein expression. KCC-mediated ouabain- and bumetanide-insensitive Rb+ (a K+ congener) influx was determined as Cl--dependent Rb+ influx at different external Rb+ and Cl- ion concentrations, [Rb+]o and [Cl-]o. Expressions of the cytoskeletal proteins α-actin, vimentin, and desmin fell from early through late synthetic VSMCs. KCC kinetic parameters, such as maximum velocity ( Vm), and apparent Cl- and Rb+ affinities ( Km), were calculated with Lineweaver-Burk, Hanes-Woolf, and Hill approximations. Vm values of both Rb+- and Cl--dependent influxes were of equal magnitude, commensurate with a KCC stoichiometry of unity, and rose threefold from early to late synthetic VSMCs. Hill coefficients for Rb+ and Cl- correlated with cell passage number, suggesting increased KCC ligand cooperativity. However, Km values for [Cl-]o were strikingly bimodal with 60-80 mM in early, ~20-30 mM in medium, and 60 mM in late passage cells. In contrast, Km values for [Rb+]o remained steady at ~17 mM. Since total KCC isoform expression was similar with cell passage, structure/function changes of the KCC signalosome may accompany the transition of aortic VSMCs from a healthy to a diseased phenotype.

Reactivation of Dormant Relay Pathways in Injured Spinal Cord by KCC2 Manipulations.

Many human spinal cord injuries are anatomically incomplete but exhibit complete paralysis. It is unknown why spared axons fail to mediate functional recovery in these cases. To investigate this, we undertook a small-molecule screen in mice with staggered bilateral hemisections in which the lumbar spinal cord is deprived of all direct brain-derived innervation, but dormant relay circuits remain. We discovered that a KCC2 agonist restored stepping ability, which could be mimicked by selective expression of KCC2, or hyperpolarizing DREADDs, in the inhibitory interneurons between and around the staggered spinal lesions. Mechanistically, these treatments transformed this injury-induced dysfunctional spinal circuit to a functional state, facilitating the relay of brain-derived commands toward the lumbar spinal cord. Thus, our results identify spinal inhibitory interneurons as a roadblock limiting the integration of descending inputs into relay circuits after injury and suggest KCC2 agonists as promising treatments for promoting functional recovery after spinal cord injury.

Chemical array system, a platform to identify novel hepatitis B virus entry inhibitors targeting sodium taurocholate cotransporting polypeptide.

Current anti-hepatitis B virus (HBV) agents including interferons and nucleos(t)ide analogs efficiently suppress HBV infection. However, as it is difficult to eliminate HBV from chronically infected liver, alternative anti-HBV agents targeting a new molecule are urgently needed. In this study, we applied a chemical array to high throughput screening of small molecules that interacted with sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for HBV. From approximately 30,000 compounds, we identified 74 candidates for NTCP interactants, and five out of these were shown to inhibit HBV infection in cell culture. One of such compound, NPD8716, a coumarin derivative, interacted with NTCP and inhibited HBV infection without causing cytotoxicity. Consistent with its NTCP interaction capacity, this compound was shown to block viral attachment to host hepatocytes. NPD8716 also prevented the infection with hepatitis D virus, but not hepatitis C virus, in agreement with NPD8716 specifically inhibiting NTCP-mediated infection. Analysis of derivative compounds showed that the anti-HBV activity of compounds was apparently correlated with the affinity to NTCP and the capacity to impair NTCP-mediated bile acid uptake. These results are the first to show that the chemical array technology represents a powerful platform to identify novel viral entry inhibitors.

Excitatory GABAergic Action and Increased Vasopressin Synthesis in Hypothalamic Magnocellular Neurosecretory Cells Underlie the High Plasma Level of Vasopressin in Diabetic Rats.

Diabetes mellitus (DM) is associated with increased plasma levels of arginine-vasopressin (AVP), which may aggravate hyperglycemia and nephropathy. However, the mechanisms by which DM may cause the increased AVP levels are not known. Electrophysiological recordings in supraoptic nucleus (SON) slices from streptozotocin (STZ)-induced DM rats and vehicle-treated control rats revealed that γ-aminobutyric acid (GABA) functions generally as an excitatory neurotransmitter in the AVP neurons of STZ rats, whereas it usually evokes inhibitory responses in the cells of control animals. Furthermore, Western blotting analyses of Cl- transporters in the SON tissues indicated that Na+-K+-2Cl- cotransporter isotype 1 (a Cl- importer) was upregulated and K+-Cl- cotransporter isotype 2 (KCC2; a Cl- extruder) was downregulated in STZ rats. Treatment with CLP290 (a KCC2 activator) significantly lowered blood AVP and glucose levels in STZ rats. Last, investigation that used rats expressing an AVP-enhanced green fluorescent protein fusion gene showed that AVP synthesis in AVP neurons was much more intense in STZ rats than in control rats. We conclude that altered Cl- homeostasis that makes GABA excitatory and enhanced AVP synthesis are important changes in AVP neurons that would increase AVP secretion in DM. Our data suggest that Cl- transporters in AVP neurons are potential targets of antidiabetes treatments.

Iodinated TG in Thyroid Follicles Regulate TSH/TSHR Signaling for NIS Expression.

Our previous research has suggested that high degree of iodinated thyroglobulin (TG) may inhibit the expression and function of sodium iodide symporter (NIS), but the underlying mechanism remains unclear. In present study, we discuss a newly constructed follicle model in vitro, which was used to simulate the follicular structure of the thyroid and explore the regulatory roles of iodinated TG in the follicular lumen on NIS expression. The results showed that both NIS expression and PKA activity were increased in lowly iodinated TG group, while decreased NIS expression with increased PKC activity was found in highly iodinated TG group. Also, NIS expression was increased in PKA agonist-treated group, while decreased NIS was found in PKC agonist-treated group. Moreover, when the PLC-PKC pathway was blocked by PKC-specific inhibitor, highly iodinated TG significantly promoted the expression of NIS. However, when the cAMP-PKA pathway was blocked by a PKA-specific blocker, highly iodinated TG slightly suppressed NIS expression. TG with a low degree of iodination had the reverse effect on NIS. When the PLC-PKC pathway was blocked, TG with a low degree of iodination slightly promoted NIS expression. However, when the cAMP-PKA pathway was blocked, TG with a low degree of iodination greatly inhibited NIS expression. All these suggested that iodinated TG inhibited the expression of NIS by PLC-PKC pathway and promoted NIS expression via the cAMP-PKA pathway. When highly iodinated TG was present, the PLC-PKC pathway became dominant. In the presence of lowly iodinated TG, the cAMP-PKA became the major pathway.

Thyroid endocrine disruption of acetochlor on zebrafish (Danio rerio) larvae.

The herbicide acetochlor is widely used and detected in the environment and biota, and has been suspected to disrupt the thyroid endocrine system, but underlying mechanisms have not yet been clarified. In the present study, zebrafish larvae (7 days post-fertilization) were exposed to a series concentration of acetochlor (0, 1, 3, 10, 30, 100 and 300 µg l(-1) ) within a 14-day window until 21 days post-fertilization. Thyroid hormones and mRNA expression profiles of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis were analyzed. Exposure to the positive control, 3,5,3'-triiodothyronine (T3 ), altered the mRNA expression, suggesting that the HPT axis in the critical window of zebrafish responded to chemical exposure and could be used to evaluate the effects of chemicals on the thyroid endocrine system. The mRNA expressions of genes involved in thyroid hormone synthesis (tshß, slc5a5 and tpo) were upregulated significantly with acetochlor treatment, which might be responsible for the increased thyroxine concentrations. The downregulation of genes related to thyroid hormone metabolism (dio1 and ugt1ab) and transport (ttr) in zebrafish larvae exposed to acetochlor might further explain the increased thyroxine levels and decreased T3 levels. The mRNA expression of the thyroid hormone receptor (trα) was also upregulated upon acetochlor exposure. Results suggested that acetochlor altered mRNA expression of the HPT axis-related genes and changed the whole body thyroid hormone levels in zebrafish larvae. It demonstrated that acetochlor could cause endocrine disruption of the thyroid system by simulating the biological activity of T3 . Copyright © 2015 John Wiley & Sons, Ltd.

Atorvastatin induces bile acid-synthetic enzyme Cyp7a1 by suppressing FXR signaling in both liver and intestine in mice.

Statins are effective cholesterol-lowering drugs to treat CVDs. Bile acids (BAs), the end products of cholesterol metabolism in the liver, are important nutrient and energy regulators. The present study aims to investigate how statins affect BA homeostasis in the enterohepatic circulation. Male C57BL/6 mice were treated with atorvastatin (100 mg/kg/day po) for 1 week, followed by BA profiling by ultra-performance LC-MS/MS. Atorvastatin decreased BA pool size, mainly due to less BA in the intestine. Surprisingly, atorvastatin did not alter total BAs in the serum or liver. Atorvastatin increased the ratio of 12α-OH/non12α-OH BAs. Atorvastatin increased the mRNAs of the BA-synthetic enzymes cholesterol 7α-hydroxylase (Cyp7a1) (over 10-fold) and cytochrome P450 27a1, the BA uptake transporters Na⁺/taurocholate cotransporting polypeptide and organic anion transporting polypeptide 1b2, and the efflux transporter multidrug resistance-associated protein 2 in the liver. Noticeably, atorvastatin suppressed the expression of BA nuclear receptor farnesoid X receptor (FXR) target genes, namely small heterodimer partner (liver) and fibroblast growth factor 15 (ileum). Furthermore, atorvastatin increased the mRNAs of the organic cation uptake transporter 1 and cholesterol efflux transporters Abcg5 and Abcg8 in the liver. The increased expression of BA-synthetic enzymes and BA transporters appear to be a compensatory response to maintain BA homeostasis after atorvastatin treatment. The Cyp7a1 induction by atorvastatin appears to be due to suppressed FXR signaling in both the liver and intestine.

Therapeutic restoration of spinal inhibition via druggable enhancement of potassium-chloride cotransporter KCC2-mediated chloride extrusion in peripheral neuropathic pain.

Peripheral neuropathic pain, typified by the development of spontaneous pain or pain hypersensitivity following injury to the peripheral nervous system, is common, greatly impairs quality of life, and is inadequately treated with available drugs. Maladaptive changes in chloride homeostasis due to a decrease in the functional expression of the potassium-chloride cotransporter KCC2 in spinal cord dorsal horn neurons are a major contributor to the central disinhibition of γ-aminobutyric acid type A receptor- and glycine receptor-mediated signaling that characterizes neuropathic pain. A compelling novel analgesic strategy is to restore spinal ionotropic inhibition by enhancing KCC2-mediated chloride extrusion. We review the data on which this theory of alternative analgesia is based, discuss recent high-throughput screens that have searched for small-molecule activators of KCC2, and propose other strategies of KCC2 activation based on recent developments in the basic understanding of KCC2's functional regulation. Exploiting the chloride-dependent functional plasticity of the γ-aminobutyric acid and glycinergic system by targeting KCC2 may be a tenable method of restoring ionotropic inhibition not only in neuropathic pain but also in other "hyperexcitable" diseases of the nervous system such as seizures and spasticity.

Chloride extrusion enhancers as novel therapeutics for neurological diseases.

The K(+)-Cl(-) cotransporter KCC2 is responsible for maintaining low Cl(-) concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABA(A) and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl(-) transport. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl(-)]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl(-)]i. CLP257 restored impaired Cl(-) transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.

Manipulation of PBF/PTTG1IP phosphorylation status; a potential new therapeutic strategy for improving radioiodine uptake in thyroid and other tumors.

CONTEXT: The clinical effectiveness of ablative radioiodine treatment of thyroid tumors is limited by the availability of the sodium iodide symporter (NIS) at the plasma membrane (PM) for uptake of ¹³¹I. A significant proportion of well-differentiated thyroid tumors are unable to concentrate sufficient radioiodine for effective therapy, and in other tumor models such as breast tumors, where radioiodine uptake would be an attractive therapeutic option, uptake is insufficient. OBJECTIVE: Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is overexpressed in multiple cancers and significantly decreases NIS expression at the PM. The goal of this study was to identify a method by which PBF repression of NIS may be overcome in human tumors. RESULTS: Here, we identify PBF as a tyrosine phosphoprotein that specifically binds the proto-oncogene tyrosine protein kinase Src in mass spectrometry, glutathione S-transferase pulldown and coimmunoprecipitation assays. Src induction leads to phosphorylation at PBF residue Y174. Abrogation of this residue results in PM retention and a markedly reduced ability to bind NIS. The Src inhibitor PP1 inhibits PBF phosphorylation in multiple cell lines in vitro, including human primary thyroid cells. Of direct clinical importance to the treatment of thyroid cancer, PP1 stimulates iodide uptake by transfected NIS in TPC1 thyroid carcinoma cells and entirely overcomes PBF repression of iodide uptake in human primary thyroid cells. CONCLUSIONS: We propose that targeting PBF phosphorylation at residue Y174 via tyrosine kinase inhibitors may be a novel therapeutic strategy to enhance the efficacy of ablative radioiodine treatment in thyroid and other endocrine and endocrine-related tumors.

N-ethylmaleimide activates a Cl(-)-independent component of K(+) flux in mouse erythrocytes.

The K-Cl cotransporters (KCCs) of mouse erythrocytes exhibit higher basal activity than those of human erythrocytes, but are similarly activated by cell swelling, by hypertonic urea, and by staurosporine. However, the dramatic stimulation of human erythroid KCCs by N-ethylmaleimide (NEM) is obscured in mouse erythrocytes by a prominent NEM-stimulated K(+) efflux that lacks Cl(-)-dependence. The NEM-sensitivity of Cl(-)-independent K(+) efflux of mouse erythrocytes is lower than that of KCC. The genetically engineered absence of the K-Cl cotransporters KCC3 and KCC1 from mouse erythrocytes does not modify Cl(-)-independent K(+) efflux. Mouse erythrocytes genetically devoid of the Gardos channel KCNN4 show increased NEM-sensitivity of both Cl(-)-independent K(+) efflux and K-Cl cotransport. The increased NEM-sensitivity and stimulation magnitude of Cl(-)-independent K(+) efflux in mouse erythrocytes expressing transgenic hypersickling human hemoglobin SAD (HbSAD) are independent of the presence of KCC3 and KCC1, but absence of KCNN4 reduces the stimulatory effect of HbSAD. NEM-stimulated Cl(-)-independent K(+) efflux of mouse red cells is insensitive to ouabain and bumetanide, but partially inhibited by chloroquine, barium, and amiloride. The NEM-stimulated activity is modestly reduced at pH6.0 but not significantly altered at pH8.0, and is abolished at 0°C. Although the molecular identity of this little-studied K(+) efflux pathway of mouse erythrocytes remains unknown, its potential role in the pathophysiology of sickle red cell dehydration will be important for the extrapolation of studies in mouse models of sickle cell disease to our understanding of humans with sickle cell anemia.

The PARP inhibitor PJ34 modifies proliferation, NIS expression and epigenetic marks in thyroid cancer cell lines.

Since PARP-1 is supposed to be part of a multimeric repressor of sodium iodide symporter (NIS) expression, in this study the effect of the PARP inhibitor PJ34 on several properties of thyroid cancer cell lines was investigated. In TPC1, BCPAP, FRO, WRO cell lines PJ34 induced a strong increase in NIS mRNA levels. In BCPAP and TPC1 cells also significant increase of radio-iodine uptake was induced. Accordingly, in transfection experiments performed in TPC1 cells, treatment with PJ34 increased NIS promoter activity without affecting PARP-1 binding to the promoter sequence. We also investigated the epigenetic status of NIS promoter after PJ34 treatment in TPC1 cell line: in addition to an increase of histone modification activation marks (H3K9K14ac, H3K4me3), surprisingly we observed also an increase of H3K27me3, a classical repressive mark. Our data demonstrate that in various thyroid cancer cell lines PARP inhibition increases NIS gene expression through a particular modulation of transcriptional regulatory mechanisms. Therefore, we suggest that PARP inhibitors may deserve future investigations as tools for medical treatment of thyroid cancer.

Ascorbic acid participates in a general mechanism for concerted glucose transport inhibition and lactate transport stimulation.

In this paper, we present a novel function for ascorbic acid. Ascorbic acid is an important water-soluble antioxidant and cofactor in various enzyme systems. We have previously demonstrated that an increase in neuronal intracellular ascorbic acid is able to inhibit glucose transport in cortical and hippocampal neurons. Because of the presence of sodium-dependent vitamin C transporters, ascorbic acid is highly concentrated in brain, testis, lung, and adrenal glands. In this work, we explored how ascorbic acid affects glucose and lactate uptake in neuronal and non-neuronal cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression of glucose and ascorbic acid transporters in non-neuronal cells was studied. Like neurons, HEK293 cells expressed GLUT1, GLUT3, and SVCT2. With radioisotope-based methods, only intracellular ascorbic acid, but not extracellular, inhibits 2-deoxyglucose transport in HEK293 cells. As monocarboxylates such as pyruvate and lactate, are important metabolic sources, we analyzed the ascorbic acid effect on lactate transport in cultured neurons and HEK293 cells. Intracellular ascorbic acid was able to stimulate lactate transport in both cell types. Extracellular ascorbic acid did not affect this transport. Our data show that ascorbic acid inhibits glucose transport and stimulates lactate transport in neuronal and non-neuronal cells. Mammalian cells frequently present functional glucose and monocarboxylate transporters, and we describe here a general effect in which ascorbic acid functions like a glucose/monocarboxylate uptake switch in tissues expressing ascorbic acid transporters.

Hydrocortisone and purinergic signaling stimulate sodium/iodide symporter (NIS)-mediated iodide transport in breast cancer cells.

The sodium/iodide symporter (NIS) mediates a remarkably effective targeted radioiodide therapy in thyroid cancer; this approach is an emerging candidate for treating other cancers that express NIS, whether endogenously or by exogenous gene transfer. Thus far, the only extrathyroidal malignancy known to express functional NIS endogenously is breast cancer. Therapeutic efficacy in thyroid cancer requires that radioiodide uptake be maximized in tumor cells by manipulating well-known regulatory factors of NIS expression in thyroid cells, such as TSH, which stimulates NIS expression via cAMP. Similarly, therapeutic efficacy in breast cancer will likely depend on manipulating NIS regulation in mammary cells, which differs from that in the thyroid. Human breast adenocarcinoma MCF-7 cells modestly express endogenous NIS when treated with all-trans-retinoic acid (tRa). We report here that hydrocortisone and ATP each markedly stimulates tRa-induced NIS protein expression and plasma membrane targeting in MCF-7 cells, leading to at least a 100% increase in iodide uptake. Surprisingly, the adenyl cyclase activator forskolin, which promotes NIS expression in thyroid cells, markedly decreases tRa-induced NIS protein expression in MCF-7 cells. Isobutylmethylxanthine increases tRa-induced NIS expression in MCF-7 cells, probably through a purinergic signaling system independent of isobutylmethylxanthine's action as a phosphodiesterase inhibitor. We also observed that neither iodide, which at high concentrations down-regulates NIS in the thyroid, nor cAMP has a significant effect on NIS expression in MCF-7 cells. Our findings may open new strategies for breast-selective pharmacological modulation of functional NIS expression, thus improving the feasibility of using radioiodide to effectively treat breast cancer.

Roles for KCC transporters in the maintenance of lens transparency.

PURPOSE: To determine whether the potassium chloride cotransporter (KCC) family is expressed in the rat lens and to ascertain whether the transporters are involved in the regulation of lens volume and transparency. METHODS: RT-PCR was performed on RNA extracted from fiber cells to identify members of the KCC family expressed in the lens. Western blot analysis and immunocytochemistry, using KCC isoform-specific antibodies, were used to verify expression at the protein level and to localize KCC isoform expression. Organ-cultured rat lenses were incubated in isotonic artificial aqueous humor (AAH) that contained either the KCC-specific inhibitor [(dihydronindenyl)oxy] alkanoic acid (DIOA), the KCC activator N-ethylmaleimide (NEM), or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) for up to 18 hours. Lens wet weight was monitored, and lens transparency and tissue morphology were recorded with dark-field and confocal microscopy, respectively. RESULTS: Molecular experiments to characterize KCC isoform expression showed that KCC1, -3, and -4 were all expressed in the lens at both the transcript and protein levels and that KCC2 was not. Immunocytochemistry indicated that the three KCC isoforms exhibited distinct differentiation-dependent expression patterns, with KCC1 and -3 being restricted to the lens cortex, whereas KCC4 was found throughout the entire lens, including the lens core. In the lens cortex, most of the labeling for all KCC isoforms was cytoplasmic, whereas in the lens core, KCC4 labeling was associated with the membrane. Incubation of lenses in 100 microM DIOA for 18 hours caused lenses to increase their wet weight and induced a cortical opacity that was caused by extensive damage to peripheral fiber cells located up to 150 microm in from the lens capsule, whereas deeper fiber cells appeared unaffected by DIOA exposure. Lower concentrations of DIOA (10 microM) revealed that this damage was initiated primarily by the swelling of peripheral fiber cells. In contrast, NPPB-treated lenses exhibited a deeper zone (>100 microm) of cell damage that was initiated by the dilation of the extracellular space between fiber cells. Exposure of lenses to the KCC activator NEM caused cell shrinkage in peripheral fiber cells but extensive cell swelling in deeper fiber cells. Peripheral cell swelling caused a differential recruitment of KCC isoforms from a cytoplasmic pool to the plasma membrane. DIOA-induced cell swelling increased the association of KCC4 with membrane, whereas hypotonic cell swelling dramatically increased the association of KCC1 with the membrane. CONCLUSIONS: The rat lens expresses three KCC transporter isoforms (KCC1, -3, and -4) in a differentiation-dependent manner. Modulation of transporter activity and subcellular localization suggests that multiple KCC transporters mediate KCl efflux in peripheral fiber cells in a dynamic fashion. These results indicate that, in addition to Cl- channels, KCC transporters play a role in mediating a circulating flux of Cl- ions, which contributes to the maintenance of lens transparency through controlling the steady state volume of lens fiber cells.