Effects Of Chemical Inhibition Research Articles

Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells.

Mitochondrial membrane potential (ΔΨm) is a global indicator of mitochondrial function. Previous reports on heterogeneity of ΔΨm were qualitative or semiquantitative. Here, we quantified intercellular differences in ΔΨm in unsynchronized human cancer cells, cells synchronized in G1, S, and G2, and human fibroblasts. We assessed ΔΨm using a two-pronged microscopy approach to measure relative fluorescence of tetramethylrhodamine methyl ester (TMRM) and absolute values of ΔΨm. We showed that ΔΨm is more heterogeneous in cancer cells compared to fibroblasts, and it is maintained throughout the cell cycle. The effect of chemical inhibition of the respiratory chain and ATP synthesis differed between basal, low and high ΔΨm cells. Overall, our results showed that intercellular heterogeneity of ΔΨm is mainly modulated by intramitochondrial factors, it is independent of the ΔΨm indicator and it is not correlated with intercellular heterogeneity of plasma membrane potential or the phases of the cell cycle.

Experimental Study on the Optimum Concentration of Ferrocene in Composite Ultrafine Dry Powder

The elimination of halon and frequent fire accidents have caused an urgent need for the development of high-efficient fire extinguishing agents. ABC dry powder has been paid much attention, and has become one of the most extensively used fire extinguishing agents nowadays owing to its wide applications in various fire protection fields. Therefore, to enhance the fire-extinguishing properties of ABC dry powder, ferrocene was used as the additive to develop a new composite ultrafine dry powder based on an ultrafine ABC dry powder product. Through flame inhibition tests, two performance indexes of the inhibition process, namely temperature drops and flame height variations, were adopted to measure the degree of flame suppression. The experimental results revealed that the temperature drops and flame height variations both increased and then decreased. Moreover, the composite ultrafine dry powder containing 0.7% ferrocene displayed the best inhibitory effect with the maximum temperature drop (152.9°C) and variation rate of flame height (14.01%). Thermogravimetric analysis and differential scanning calorimetry were used to analyze the thermal decomposition of the sample powders. Furthermore, based on the analysis results of the thermal decomposition temperature, weight loss, and other thermodynamic parameters of the sample powders, the reasons for performance advantages of composite ultrafine dry powder containing 0.7% ferrocene were clarified as the faster pyrolysis rate, more absorption of heat, stronger suffocation and chemical inhibition effect. Finally, possible speculations on the suppression mechanisms of composite ultrafine dry powder containing 0.7% ferrocene were proposed.

Identifying small molecule probes of ENTPD5 through high throughput screening.

Ectonucleoside Triphosphate Diphosphohydrolase 5 (ENTPD5) has been shown to be important in maintaining cellular function in cancer, and its expression is upregulated through multiple, unique pathways in certain cancers, including laryngeal, glioblastoma multiforme, breast, testicular, and prostate. ENTPD5 supports cancer growth by promoting the import of UDP-glucose, a metabolite used for protein glycosylation and hence proper glycoprotein folding, into the ER by providing the counter molecule, UMP, to the ER antiporter. Despite its cancer-supporting function, no small molecule inhibitors of ENTPD5 are commercially available, and few studies have been performed in tissue culture to understand the effects of chemical inhibition of ENTPD5. We performed a high-throughput screen (HTS) of 21,120 compounds to identify small molecule inhibitors of ENPTD5 activity. Two hits were identified, and we performed a structure activity relationship (SAR) screen around these hits. Further validation of these probes were done in an orthogonal assay and then assayed in cell culture to assess their effect on prostate cancer cell lines. Notably, treatment with the novel ENTPD5 inhibitor reduced the amount of glycoprotein produced in treated cells, consistent with the hypothesis that ENTPD5 is important for glycoprotein folding. This work serves as an important step in designing new molecular probes for ENTPD5 as well as further probing the utility of targeting ENTPD5 to combat cancer cell proliferation.

Abstract 539: Pro-proliferative function of the histone deacetylase SIRT3 in prostate cancer

Abstract Worldwide, prostate cancer (PCa) is the second most frequently diagnosed cancer and fifth leading cause of cancer death in males. The existing treatments, as well as surgical approaches, have not been fully effective either for prevention or treatment of PCa. This necessitates a need to intensify our efforts towards the understanding of genetics and mechanism(s) of PCa. This may lead to the identification of the novel molecular target(s) and mechanism-based approaches for the management of this neoplasm. Sirtuin-3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase primarily located in the mitochondria and known to play important roles in the regulation of a variety of cellular processes, including transcription and programmed cell death. The fact that SIRT3 can regulate several cellular processes those are critical in cancer cell proliferation, makes it a potential therapeutic target for cancer management. Moreover, SIRT3 is a central regulator of mitochondrial adaptive responses that relate to metabolic reprogramming of cancer cells. However, the role of SIRT3 in cancer, including PCa, is not well understood and it has been shown to act both as a tumor suppressor as well as a tumor promoter. In this study, we determined the role of SIRT3 in PCa, employing in vitro and ex vivo approaches. The first step in our efforts to understand the role of SIRT3 in PCa was to check the expression profile of SIRT3 in a panel of human PCa cell line (DU145, 22Rν1, PC3, LNCaP, C4-2, MDA PCa 2b, E006AA-Par and E006AA-hT) by RT-qPCR and immunoblot analyses. Compared to normal human prostate epithelial cells (NrPEC), PCa cells showed higher expression of SIRT3, both at mRNA and protein levels. Further, we determined the expression profile of SIRT3 by immunostaining of a tissue microarray (TMA) containing paraffin-embedded sections of 40 cases of prostate adenocarcinoma and 8 normal prostate tissues. Our data demonstrated a significant upregulation of SIRT3 in cancerous prostatic tissues compared to the normal tissues. We next determined the effect of chemical inhibition of SIRT3 using a recently described SIRT3 inhibitor viz. 4'-Bromo-Resveratrol (4BR), in human PCa cells (DU145 and 22Rν1). 4BR treatment at 10, 20 and 40 μM concentrations for 48 and 72 h resulted in a dose- and time-dependent decrease in cell growth and proliferation. Further, 4BR treatment resulted in a marked decrease in clonogenic survival of DU145 and 22Rν1 PCa cells. Furthermore, 4BR treatment resulted in a marked cleavage of PARP, an indicator of apoptosis induction; and a decrease in the level of PCNA, a marker of cellular proliferation, in human PCa cells. Overall, our data suggest a possible pro-proliferative function of SIRT3 in PCa. Further studies are underway to unravel the role and functional significance of SIRT3 during PCa development and progression. Citation Format: Chandra K. Singh, Gagan Chhabra, Minakshi Nihal, Kenneth A. Iczkowski, Nihal Ahmad. Pro-proliferative function of the histone deacetylase SIRT3 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 539.

Small molecule inhibition of the CBFβ/RUNX interaction decreases ovarian cancer growth and migration through alterations in genes related to epithelial-to-mesenchymal transition

ObjectiveOvarian cancer survival and treatment have improved minimally in the past 20years. Novel treatment strategies are needed to combat this disease. This study investigates the effects of chemical inhibition of the CBFβ/RUNX protein-protein interaction on ovarian cancer cell lines. MethodsOvarian cancer cell lines were treated with CBFβ/RUNX inhibitors, and the effects on proliferation, DNA replication, wound healing, and anchorage-independent growth were measured. RNA-Seq was performed on compound-treated cells to identify differentially expressed genes. Genes altered by compound treatment were targeted with siRNAs, and effects on DNA replication and wound healing were measured. ResultsChemical inhibition of the CBFβ/RUNX interaction decreases ovarian cancer cell proliferation. Inhibitor treatment leads to an S-phase cell cycle delay, as indicated by an increased percentage of cells in S-phase, and a decreased DNA replication rate. Inhibitor treatment also reduces wound healing and anchorage-independent growth. RNA-Seq on compound-treated cells revealed changes in a small number of genes related to proliferation and epithelial-to-mesenchymal transition. siRNA-mediated knockdown of INHBA and MMP1 – two genes whose expression decreases with compound treatment – slowed DNA replication and impaired wound healing. ConclusionsChemical inhibition of the CBFβ/RUNX interaction is a viable strategy for the treatment of ovarian cancer.

Inhibition of Vascular Endothelial Growth Factor Receptor Decreases Regenerative Angiogenesis in Axolotls.

Angiogenesis is crucial for tissue growth and repair in mammals, and is chiefly regulated by vascular endothelial growth factor (VEGF) signaling. We evaluated the effect of chemical inhibition of VEGF receptor signaling in animals with superior regenerative ability, axolotl salamanders, to determine the impact on vascularization and regenerative outgrowth. Following tail amputation, treated animals (100nM PTK787) and controls were examined microscopically and measured over the month-long period of regeneration. Treatment with VEGFR inhibitor decreased regenerative angiogenesis; drug-treated animals had lower vascular densities in the regenerating tail than untreated animals. This decrease in neovascularization, however, was not associated with a decrease in regenerative outgrowth or with morphological abnormalities in the regrown tail. Avascular but otherwise anatomically normal regenerative outgrowth over 1mm beyond the amputation plane was observed. The results suggest that in this highly regenerative species, significant early tissue regeneration is possible in the absence of a well-developed vasculature. This research sets the groundwork for establishing a system for the chemical manipulation of angiogenesis within the highly regenerative axolotl model, contributing to a better understanding of the role of the microvasculature within strongly proliferative yet well-regulated environments. Anat Rec, 300:2273-2280, 2017. © 2017 Wiley Periodicals, Inc.

Effects of thyroid hormone manipulation on pre-nuptial molt, luteinizing hormone and testicular growth in male white-crowned sparrows (Zonotrichia leuchophrys gambelii)

Most seasonal species rely on the annual change in day length as the primary cue to appropriately time major spring events such as pre-nuptial molt and breeding. Thyroid hormones are thought to be involved in the regulation of both of these spring life history stages. Here we investigated the effects of chemical inhibition of thyroid hormone production using methimazole, subsequently coupled with either triiodothyronine (T3) or thyroxine (T4) replacement, on the photostimulation of pre-nuptial molt and breeding in Gambel’s white-crowned sparrows (Zonotrichia leuchophrys gambelii). Suppression of thyroid hormones completely prevented pre-nuptial molt, while both T3 and T4 treatment restored normal patterns of molt in thyroid hormone-suppressed birds. Testicular recrudescence was blocked by methimazole, and restored by T4 but not T3, in contrast to previous findings demonstrating central action of T3 in the photostimulation of breeding. Methimazole and replacement treatments elevated plasma luteinizing hormone levels compared to controls. These data are partially consistent with existing theories on the role of thyroid hormones in the photostimulation of breeding, while highlighting the possibility of additional feedback pathways. Thus we suggest that regulation of the hypothalamic pituitary gonad axis that controls breeding may be more complex than previously considered.

IFN-γ orchestrates mesenchymal stem cell plasticity through the signal transducer and activator of transcription 1 and 3 and mammalian target of rapamycin pathways

Mesenchymal stem cells (MSCs) display a therapeutic plasticity because of their ability to modulate immunity, foster tissue repair, and differentiate into mesodermal cells. IFN-γ has been described to differently affect human mesenchymal stem cell (hMSC) and mouse mesenchymal stem cell (mMSC) immunomodulation and differentiation, depending on the inflammatory milieu. We aimed at dissecting the relevant intracellular pathways through which IFN-γ affects MSC plasticity and the consequence of their manipulation on MSC functions. Modification of relevant IFN-γ-dependent pathways in mMSCs was carried out invitro through gene silencing or chemical inhibition of key components. Functional outcomes were assessed by means of Western blotting, real-time PCR, differentiation, and proliferation assays on MSCs. The effect on Tcells was addressed by T-cell proliferation assays; the effect of mammalian target of rapamycin (mTOR) manipulation in MSCs was studied invivo in a mouse model of delayed-type hypersensitivity assay. To address whether similar mechanisms are involved also in hMSCs on IFN-γ stimulation, the effect of chemical inhibition on the same intracellular pathways was assessed by means of Western blotting, and the final outcome on immunomodulatory properties was evaluated based on real-time PCR and T-cell proliferation. We revealed that in mMSCs IFN-γ-induced immunoregulation is mediated by early phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3, which is significantly enhanced by an extracellular signal-regulated kinase 1/2-dependent mTOR inhibition, thereby promoting pSTAT1 nuclear translocation. Accordingly, after intracellular mTOR inhibition, MSCs augmented their ability to inhibit T-cell proliferation and control delayed-type hypersensitivity invivo. Similarly, on mTOR blockade, hMSCs also enhanced their immunoregulatory features. Asustained exposure to IFN-γ led to inhibition of STAT3 activity, which in mMSCs resulted in an impaired proliferation and differentiation. These results provide new insights about MSC intracellular pathways affected by IFN-γ, demonstrating that pharmacologic or genetic manipulation of MSCs can enhance their immunomodulatory functions, which could be translated into novel therapeutic approaches.

An Evolutionarily Conserved PLC-PKD-TFEB Pathway for Host Defense.

The mechanisms that tightly control the transcriptionof host defense genes have not been fully elucidated. We previously identified TFEB as a transcription factor important for host defense, but the mechanisms that regulate TFEB during infection remained unknown. Here, we used C.elegans to discover a pathway that activates TFEB during infection. Gene dkf-1, which encodes a homolog of protein kinase D (PKD), was required for TFEB activation in nematodes infected with Staphylococcus aureus. Conversely, pharmacological activation of PKD wassufficient to activate TFEB. Furthermore, phospholipase C (PLC) gene plc-1 was also required for TFEB activation, downstream of Gαq homolog egl-30 and upstream of dkf-1. Using reverse and chemical genetics, we discovered a similar PLC-PKD-TFEB axis in Salmonella-infected mouse macrophages. In addition, PKCα was required in macrophages. These observations reveal a previously unknown host defense signaling pathway, which has been conserved across one billion years of evolution.

Chemical Synthesis of Phosphorylated Histone H2A at Tyr57 Reveals Insight into the Inhibition Mode of the SAGA Deubiquitinating Module.

Monoubiquitination of histone H2B plays a central role in transcription activation and is required for downstream histone-methylation events. Deubiquitination of H2B by the Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex is regulated by a recently discovered histone mark, phosphorylated H2AY57 (H2AY57p), which inhibits deubiquitination of H2B by the SAGA complex as well as restricting demethylation of H3 and increasing its acetylation. Evidence for the effect of H2AY57p, however, was indirect and was investigated in vivo by monitoring the effects of chemical inhibition of Tyr kinase CK2 or by mutating the phosphorylation site. We applied the total chemical synthesis of proteins to prepare H2AY57p efficiently and study the molecular details of this regulation. This analogue, together with semisynthetically prepared ubiquitinated H2B, enabled us to provide direct evidence for the cross-talk between those two marks and the inhibition of SAGA activity by H2AY57p.

Chemical Synthesis of Phosphorylated Histone H2A at Tyr57 Reveals Insight into the Inhibition Mode of the SAGA Deubiquitinating Module

AbstractMonoubiquitination of histone H2B plays a central role in transcription activation and is required for downstream histone‐methylation events. Deubiquitination of H2B by the Spt‐Ada‐Gcn5 acetyltransferase (SAGA) coactivator complex is regulated by a recently discovered histone mark, phosphorylated H2AY57 (H2AY57p), which inhibits deubiquitination of H2B by the SAGA complex as well as restricting demethylation of H3 and increasing its acetylation. Evidence for the effect of H2AY57p, however, was indirect and was investigated in vivo by monitoring the effects of chemical inhibition of Tyr kinase CK2 or by mutating the phosphorylation site. We applied the total chemical synthesis of proteins to prepare H2AY57p efficiently and study the molecular details of this regulation. This analogue, together with semisynthetically prepared ubiquitinated H2B, enabled us to provide direct evidence for the cross‐talk between those two marks and the inhibition of SAGA activity by H2AY57p.

Extinguishment of hydrogen laminar diffusion flames by water vapor in a cup burner apparatus

Transient computations with full hydrogen chemistry were performed to reveal the flame structure and extinguishment process of co-flow, hydrogen diffusion flame suppressed by water vapor. As the concentration of water vapor was increased, the flame detached away from the burner brim and formed an edge flame at the flame base. Water vapor showed larger chemical inhibition effect than nitrogen when extinguishing hydrogen flame, which was attributed to its enhanced third body effect in the reaction H + O2 + M = HO2 + M. The minimum extinguishing concentration (MEC) of water vapor and nitrogen was predicted by Senecal formula and perfectly stirred reactor (PSR) model respectively. The MECs predicted by PSR model agree with the MECs calculated by Fluent, which shows that 1) the flame extinction is controlled by the flame base, and 2) radiation absorption is negligible. The measured MECs are in a reasonable agreement with the values calculated by Fluent, which demonstrates the accuracy of the CFD model. A simple model was used to investigate the relative importance of extinguishing mechanisms of water vapor. The results show that in a co-flow configuration the thermal cooling and chemical inhibition effect are the main extinguishing mechanisms in suppressing hydrogen diffusion cup burner flame.

Absorption of ipratropium and l-carnitine into the pulmonary circulation of the ex-vivo rat lung is driven by passive processes rather than active uptake by OCT/OCTN transporters

The organic cation transporters OCT and OCTN have been reported to play a significant role in the cellular uptake of substrates within in vitro lung cells. However, no studies to date have investigated the effect of these transporters upon transepithelial absorption of substrates into the pulmonary circulation. We investigated the contribution of OCT and OCTN transporters to total pulmonary absorption of l-carnitine and the anti-muscarinic drug, ipratropium, across an intact isolated perfused rat lung (IPRL). The results obtained from the IPRL were contrasted with active transport in vitro using three human pulmonary cell lines and primary rat alveolar epithelial cells. Ex-vivo studies showed that OCT/OCTN transporters do not play a role in the overall pulmonary absorption of l-carnitine or ipratropium, as evidenced by the effect of chemical inhibition of these transporters upon pulmonary absorption. In contrast, in vitro studies showed that OCT/OCTN transporters play a significant role in cellular accumulation of substrates with preferential uptake of ipratropium by OCTs, and of l-carnitine uptake by OCTNs. The results show that in vitro uptake studies cannot be predictive of airway to blood absorption in vivo. Nevertheless, localised submucosal pulmonary concentrations of inhaled drugs and their pulmonary pharmacodynamic profiles may be influenced by OCT/OCTN transport activity.

Targeting ALDH1 to decrease tumorigenicity, growth and metastasis of human melanoma

Cells with aldehyde dehydrogenase activity (ALDH+) are the most tumorigenic cells in many cancers, including melanoma, making ALDH a candidate therapeutic target. We examined the effects of chemical inhibition of ALDH1 on the response of human melanoma xenografts to chemotherapy and the effects of ALDH1A1 RNA silencing on melanoma growth and metastasis. Addition of ALDH1 inhibitors (e.g. diethylaminobenzaldehyde) to dacarbazine chemotherapy, not only reduced tumor growth in vivo, but also resulted in a significant decrease in the number of residual cells capable of tumorigenesis. shRNA depletion of ALDH1A1 in melanoma cells resulted not only in a significant delay in appearance of xenograft melanomas and reduction in growth, but also significantly decreased the number of metastases and metastatic burden after lateral tail vein injections in mice. In summary, ALDH1 inhibition in combinatorial therapy with dacarbazine reduced the number of residual tumorigenic cells post-therapy and ALDH1A1 depletion had marked inhibitory effects on both melanoma growth and metastasis. These findings suggest that ALDH1 inhibition may not only be able to provide a therapeutic advantage in melanoma treatment, but may also prevent rapid relapse after therapy, as residual tumorigenic cells are fewer and metastatic ability is diminished.

Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster.

Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. We have previously shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. In this report, we show that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. Our results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis.

The study of thermal decomposition of 2-bromo-3,3,3-trifluoropropene and its fire-extinguishing mechanism

As a new kind of Halon replacement, 2-bromo-3,3,3-trifluoropropene (BTP) is highly effective at fire suppression with an extinguishment concentration lower than that of Halon 1301. Although the physical properties and extinguishing characteristics of BTP have been widely reported, there are relatively few studies on its thermal pyrolysis and extinguishing mechanisms. In this study, the thermal decomposition of BTP was studied over a temperature range of 25–800°C and the decomposition products were analyzed by GC and GC–MS. Experimental results showed that the decomposition products were mainly trifluoropropyne (CF3CCH) and/or bromotrifluoromethane (CF3Br). The calculated apparent activation energies for the thermal pyrolysis of BTP by first order reaction approximation were in excellent agreement with the theoretical calculation results by Gaussian 03. Furthermore, by analyzing decomposition products and their chemical inhibition effect, thermal decomposition mechanism of BTP and its chemical extinguishing mechanism at high temperature were then proposed.

NODAL and SHH dose-dependent double inhibition promotes an HPE-like phenotype in chick embryos

Holoprosencephaly (HPE) is a common congenital defect that results from failed or incomplete forebrain cleavage. HPE is characterized by a wide clinical spectrum, with inter- and intrafamilial variability. This heterogeneity is not well understood and it has been suggested that HPE involves a combination of multiple gene mutations. In this model, several mutated alleles or modifying factors are presumed to act in synergy to cause and determine the severity of HPE. This could explain the various clinical phenotypes. Screening for HPE-associated genes in humans suggests the involvement of NODAL or SHH signaling, or both. To test this multigenic hypothesis, we investigated the effects of chemical inhibition of these two main HPE signaling pathways in a chick embryo model. SB-505124, a selective inhibitor of transforming growth factor-B type I receptors was used to inhibit the NODAL pathway. Cyclopamine was used to inhibit the SHH pathway. We report that both inhibitors caused HPE-like defects that were dependent on the drug concentration and on the developmental stage at the time of treatment. We also investigated double inhibition of NODAL and SHH pathways from the onset of gastrulation by using subthreshold inhibitor concentrations. The inhibitors of the NODAL and SHH pathways, even at low concentration, acted synergistically to promote an HPE-like phenotype. These findings support the view that genetic heterogeneity is important in the etiology of HPE and may contribute to the phenotypic variability.

NODAL and SHH dose-dependent double inhibition promotes an HPE-like phenotype in chick embryos

SUMMARYHoloprosencephaly (HPE) is a common congenital defect that results from failed or incomplete forebrain cleavage. HPE is characterized by a wide clinical spectrum, with inter- and intrafamilial variability. This heterogeneity is not well understood and it has been suggested that HPE involves a combination of multiple gene mutations. In this model, several mutated alleles or modifying factors are presumed to act in synergy to cause and determine the severity of HPE. This could explain the various clinical phenotypes. Screening for HPE-associated genes in humans suggests the involvement of NODAL or SHH signaling, or both. To test this multigenic hypothesis, we investigated the effects of chemical inhibition of these two main HPE signaling pathways in a chick embryo model. SB-505124, a selective inhibitor of transforming growth factor-B type I receptors was used to inhibit the NODAL pathway. Cyclopamine was used to inhibit the SHH pathway. We report that both inhibitors caused HPE-like defects that were dependent on the drug concentration and on the developmental stage at the time of treatment. We also investigated double inhibition of NODAL and SHH pathways from the onset of gastrulation by using subthreshold inhibitor concentrations. The inhibitors of the NODAL and SHH pathways, even at low concentration, acted synergistically to promote an HPE-like phenotype. These findings support the view that genetic heterogeneity is important in the etiology of HPE and may contribute to the phenotypic variability.

In Vitro Study of Transporters Involved in Intestinal Absorption of Inorganic Arsenic

Inorganic arsenic (iAs) [As(III)+As(V)] is a drinking water contaminant, and human exposure to these arsenic species has been linked with a wide range of health effects. The main path of exposure is the oral route, and the intestinal epithelium is the first physiological barrier that iAs must cross in order to be absorbed. However, there is a lack of information about intestinal iAs absorption. The aim of this study was to evaluate the participation of certain transporters [glucose transporters (GLUT and SGLT), organic anion transporting polypeptides (OATPs), aquaporins (AQPs), and phosphate transporters (NaPi and PiT)] in intestinal absorption of As(V) and As(III), using the Caco-2 cell line as a model of the intestinal epithelium. For this purpose, the effects of chemical inhibition and gene silencing of the transporters of interest on iAs uptake were evaluated, and also the differential expression of these transporters after treatment with iAs. The results show that chemical inhibition using rifamycin SV (OATP inhibitor), phloridzin (SGLT inhibitor), phloretin (GLUT and AQP inhibitor), and copper sulfate (AQP inhibitor) leads to a significant reduction in the apparent permeability and cellular retention of As(III). RT-qPCR indicates up-regulation of GLUT2, GLUT5, OATPB, AQP3, and AQP10 after exposure to As(III), while exposure to As(V) increases the expression of sodium-dependent phosphate transporters, especially NaPiIIb. Gene silencing of OATPB, AQP10, and GLUT5 for As(III) and NaPiIIb for As(V) significantly reduces uptake of the inorganic forms. These results indicate that these transporters may be involved in intestinal absorption of iAs.

Abstract A30: High levels of STAT3 and Epstein-Barr virus latent membrane protein 1 are required for virus-driven proliferation and transformation of B cells

Abstract Epstein-Barr virus (EBV) is associated with lymphocyte and epithelial cell cancers in humans. EBV tumorigenesis requires expression of both viral genes, such as the oncoprotein latent membrane protein 1 (LMP1), and cellular genes. In previous tumorigenesis studies, we identified a B cell surface marker pattern (CD23hiCD58+) that predicts proliferation and subsequent transformation of B cells following EBV infection. To investigate the contribution of signal transducer and activator of transcription 3 (STAT3), a progrowth and survival signaling molecule, in EBV-driven proliferation and transformation of B cells, two approaches were taken: 1) Primary B cells from healthy donors were infected with EBV in the presence of AG490, which inhibits the function of STAT3 by interfering with Janus kinases, and 2) B cells from patients with autosomal-dominant hyper IgE syndrome (AD-HIES or Job syndrome) that have naturally occurring dominant negative mutations in the Stat3 gene were infected with EBV. The effects of chemical inhibition of STAT3 function and those of Stat3 mutations on EBV-driven B cell survival, proliferation, and transformation were examined. Following exposure to EBV, LMP1 + cells and CD23hiCD58+ cells expressed activated STAT3 and high levels of total STAT3. Chemical inhibition of STAT3 activation by AG490 inhibited the emergence of the CD23hiCD58+ population by 10- to 30-fold as compared to control infection. This was accompanied by a fall in the fraction of B cells expressing LMP1 and increased apoptosis of CD23hiCD58+ and LMP1 + cells. A similar pattern was observed following infection of B cells from AD-HIES patients, further supporting a role for STAT3 in survival of B cells following EBV infection. Both AG490 treatment during infection of healthy B cells and infection of B cells from AD-HIES patients resulted in accumulation of extant LMP1 + cells in S phase of the cell cycle. Evaluation of nuclear morphology following AG490 treatment demonstrated an absence of LMP1 + mitotic nuclei but enrichment of LMP1 + cells with small dense nuclei consistent with cells in a resting state, supporting a proliferation defect. Only those cells expressing high levels of STAT3 in addition to high levels of LMP1 progressed to G2/M phase of the cell cycle. Cultures with reduced LMP1 expression or reduced CD23hiCD58+ population also failed to establish transformed cell lines. We conclude that proliferation and subsequent transformation of EBV-infected B cells is dependent on the presence of activated STAT3 and high levels of total STAT3 as well as high levels of LMP1. These findings support a possible role for STAT3 modulation in the treatment of EBV-related cancers, particularly those arising in the context of immunocompromise. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A30.