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DOE OSTI2015

Anti-WASP intrabodies inhibit inflammatory responses induced by Toll-like receptors 3, 7, and 9, in macrophages

Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we showed that the WASP N-terminal domain interacted with the SH3 domain of Bruton's tyrosine kinase (Btk), and that the complex formed by WASP and Btk was important for TLR2 and TLR4 signaling in macrophages. Several other studies have shown that Btk played important roles in modulating innate immune responses through TLRs in immune cells. Here, we evaluated the significance of the interaction between WASP and Btk in TLR3, TLR7, and TLR9 signaling. We established bone marrow–derived macrophage cell lines from transgenic (Tg) mice that expressed intracellular antibodies (intrabodies) that specifically targeted the WASP N-terminal domain. One intrabody comprised the single-chain variable fragment and the other comprised the light-chain variable region single domain of an anti-WASP N-terminal monoclonal antibody. Both intrabodies inhibited the specific interaction between WASP and Btk, which impaired the expression of TNF-α, IL-6, and IL-1β in response to TLR3, TLR7, or TLR9 stimulation. Furthermore, the intrabodies inhibited the phosphorylation of both nuclear factor (NF)-κB and WASP in response to TLR3, TLR7, or TLR9 stimulation, in the Tg bone marrow-derived macrophages. These results suggested that WASP plays important roles in TLR3, TLR7, and TLR9 signaling by associating with Btk in macrophages. - Highlights: • The interaction between WASP and Btk is critical for TLR3, TLR7, and TLR9 signaling. • Anti-WASP intrabodies inhibited several TLR pathways that led to cytokine expression. • Phosphorylation of NF-κB via TLR signaling was inhibited by anti-WASP intrabodies. • WASP phosphorylation via several TLR ligands was inhibited by anti-WASP intrabodies.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2015

HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 {sup low} and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96{sup ®}Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 {sup low}, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases expression of NR4A1. • Core protein increases the expression of smad7 in hepatocytes. • Core protein inhibits HepG2 cells apoptosis induced by cisplatin.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

β-elemene inhibits tumor-promoting effect of M2 macrophages in lung cancer

Macrophages in tumor are mostly M2-polarized and have been reported to promote tumorigenesis, which are also defined as tumor-associated macrophages (TAMs). β-elemene has therapeutic effects against several cancers, however, it remains unknown whether β-elemene could inhibit cancer by targeting TAMs. Herein, we examined the effect of β-elemene on macrophages to elucidate a novel mechanism of β-elemene in tumor therapy. We showed that the conditioned medium of M2 macrophages promoted lung cancer cells to migration, invasion and epithelial mesenchymal transition, which could be inhibited by β-elemene. Moreover, β-elemene regulated the polarization of macrophages from M2 to M1. β-elemene also inhibited the proliferation, migration, invasion of lung cancer cells and enhanced its radiosensitivity. These results indicate β-elemene suppresses lung cancer by regulating both macrophages and lung cancer cells, it is a promising drug for combination with chemotherapy or radiotherapy. - Highlights: • β-elemene inhibits M2 macrophages induced migration and invasion of cancer cells. • β-elemene inhibits M2 macrophages induced EMT in lung cancer cells. • β-elemene transforms macrophages from M2 to M1 phenotype. • This study reveals a novel anti-tumor mechanism of β-elemene.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Eupatilin with PPARα agonistic effects inhibits TNFα-induced MMP signaling in HaCaT cells

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is a flavonoid compound exhibiting several beneficial biological activities, including neuroprotection, anti-cancer, antinociception, chondroprotection, anti-oxidation, and anti-inflammation. Our previous study demonstrated that eupatilin specifically activates peroxisome proliferator-activated receptor alpha (PPARα) through direct binding. The PPAR subfamily includes ligand-dependent transcription factors that consist of three isotypes: PPARα, PPARβ/δ, and PPARγ. All isotypes are involved in inflammation, epidermal proliferation/differentiation and skin barrier function. Among them, PPARα regulates lipid and glucose metabolism and skin homeostasis. In this study, we confirm that the ability of eupatilin as a PPARα activator significantly inhibited tumor necrosis factor-alpha (TNFα)-induced matrix metalloproteinase (MMP)-2/-9 expression and proteolytic activity in HaCaT human epidermal keratinocytes. Furthermore, we found that eupatilin subsequently suppressed IκBα phosphorylation, blocked NF-κB p65 nuclear translocation and down-regulated MAPK/AP-1 signaling via PPARα activation. Taken together, our data suggest that eupatilin inhibits TNFα-induced MMP-2/-9 expression by suppressing NF-κB and MAPK⁄AP-1 pathways via PPARα. Our findings suggest the usefulness of eupatilin for preventing skin aging. - Highlights: • Eupatilin Increases PPARα Transactivation and Expression. • Eupatilin Inhibits TNFα-induced MMP-2/-9 Expression. • Eupatilin Inhibits TNFα-induced p65 Translocation and IκBα Phosphorylation via PPARα. • Eupatilin Inhibits TNFα-induced AP-1 and MAPK Signaling via PPARα.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Inhibition of CIP2A attenuates tumor progression by inducing cell cycle arrest and promoting cellular senescence in hepatocellular carcinoma

Highlights: • High expression level of CIP2A was frequently observed in hepatocellular carcinoma (HCC). • CIP2A expression level was associated with unfavorable overall and recurrence-free survival in HCC patients. • Inhibition of CIP2A attenuated HCC progression via inducing cell cycle arrest and promoting cellular senescence. CIP2A is a recent identified oncogene that inhibits protein phosphatase 2A (PP2A) and stabilizes c-Myc in cancer cells. To investigate the potential oncogenic role and prognostic value of CIP2A, we comprehensively analyzed the CIP2A expression levels in pan-cancer and observed high expression level of CIP2A in majority cancer types, including hepatocellular carcinoma (HCC). Based on a validation cohort including 60 HCC and 20 non-tumorous tissue samples, we further confirmed the high mRNA and protein expression levels of CIP2A in HCC, and found high CIP2A mRNA expression level was associated with unfavorable overall and recurrence-free survival in patients with HCC. Mechanistic investigations revealed that inhibition of CIP2A significantly attenuated cellular proliferation in vitro and tumourigenicity in vivo. Bioinformatic analysis suggested that CIP2A might be involved in regulating cell cycle. Our experimental data further confirmed CIP2A knockdown induced cell cycle arrest at G1 phase. We found accumulated cellular senescence in HCC cells with CIP2A knockdown, companying expression changes of senescence associated proteins (p21, CDK2, CDK4, cyclin D1, MCM7 and FoxM1). Mechanistically, CIP2A knockdown repressed FoxM1 expression and induced FoxM1 dephosphorylation. Moreover, inhibition of PP2A by phosphatase inhibitor rescued the repression of FoxM1. Taken together, our results showed that CIP2A was highly expressed in HCC. Inhibition of CIP2A induced cell cycle arrest and promoted cellular senescence via repressing FoxM1 transcriptional activity, suggesting a potential anti-cancer target for patients with HCC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Cholesterol inhibits entotic cell-in-cell formation and actomyosin contraction

Highlights: • Lipofectamine 2000 inhibits entotic cell-in-cell formation. • Lipofectamine 2000 inhibits actomyosin contraction. • Cholesterol and phospholipids inhibits entotic cell-in-cell formation. • Cholesterol inhibits the phosphorylation of myosin light chain. Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced β-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didn't. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes' effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Accelerated dentinogenesis by inhibiting the mitochondrial fission factor, dynamin related protein 1

Highlights: • DRP1 inhibition increased dentin formation in mouse first molar cultures. • DRP1 inhibition enhanced mouse dental papilla cell odontoblastic differentiation. • The enhancement was due to increased mitochondrial elongation and ATP production. • Thus, DRP1 could be a therapeutic target for developmental disorders of teeth. Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Loquacious-PD removes phosphate inhibition of Dicer-2 processing of hairpin RNAs into siRNAs

Highlights: • Inorganic phosphate inhibits Dicer-2 processing of hairpin RNAs. • Phosphate does not inhibit Dicer-2 + Loqs-PD processing of blunt-end hairpin RNAs. • Loqs-PD removes phosphate inhibition of Dicer-2 processing of blunt hairpin RNAs. Drosophila Dicer-2 processes RNA substrates into short interfering RNAs (siRNAs). Loquacious-PD (Loqs-PD), a dsRNA-binding protein that associates with Dicer-2, is required for processing of a subset of RNA substrates including hairpin RNAs into siRNAs. Inorganic phosphate—a small molecule present in all cell types—inhibits Dicer-2 from processing precursor of microRNAs (pre-miRNAs), which are processed by Dicer-1. Whether or how Loqs-PD modulates the inhibitory effect of inorganic phosphate on Dicer-2 processing of RNA substrates is unknown. To address this question, I performed in vitro hairpin RNA processing assay with Dicer-2 in the presence or absence of Loqs-PD and/or inorganic phosphate. I found that inorganic phosphate inhibits Dicer-2 alone, but not Dicer-2 + Loqs-PD, from processing blunt-end hairpin RNAs into siRNAs. Thus, Loqs-PD removes the inhibitory effect of inorganic phosphate on Dicer-2 processing of blunt-end hairpin RNAs, allowing siRNA production in the presence of inorganic phosphate.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Citrus bioflavonoids dipeptidyl peptidase-4 inhibition compared with gliptin antidiabetic medications

Highlights: • The citrus bioflavonoids may possess dipeptidyl peptidase-4 (DPP-4) inhibition activity similar to gliptins. • Various citrus bioflavonoids may have a variable DPP-4 inhibition activity. • The measurement of the DPP-4 inhibition activity can be done using DPP-4 inhibition kit. • Commercial citrus bioflavonoid supplements contain various citrus bioflavonoids with different DPP-4 inhibition activities. This study compared dipeptidyl peptidase-4 (DPP-4) inhibitory activity of citrus bioflavonoid nutraceuticals compared with three gliptins. Citrus bioflavonoid standards and three commercially available citrus bioflavonoid supplements (Thompson's Super Bioflavonoid Complex{sup ®}(SB), Ethical Nutrients Bioflavonoids Plus Vitamin C{sup ®}(EN), and Country Life Citrus Bioflavonoids and Rutin{sup ®}(CB)) were considered in this study. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis was undertaken to identify and quantitate the citrus bioflavonoids present in each supplement. The DPP-4 inhibitory activity was determined by fluorometric assay. All of the tested individual citrus flavonoids demonstrated DPP-4 inhibitory activity, with IC{sub 50} values ranging from 485 μM (rutin) to 5700 μM (hesperitin and eriodictyol). Similarly, the flavonoid supplements had IC{sub 50} values of 16.9 mg/mL (EN), 3.44 mg/mL (SB) and 2.72 mg/mL (CB). These values compare with gliptin IC{sub 50} values of 0.684 μM (sitagliptin), 0.707 μM (saxagliptin) and 2.286 μM (vildagliptin). The supplement flavonoid content varied from 11.98% (CB) to 5.26% (EN) and 14.51% (SB) of tablet mass, corresponding to daily flavonoid doses of around 300, 150 and 400 mg, respectively, with CB and SB containing rutin at levels of 7.0% and 7.5% of tablet mass, respectively. While our data demonstrated that citrus bioflavonoid based supplements do possess DPP-4 inhibitory activity, they are several orders of magnitude less potent than gliptins. Further studies using higher concentrations of citrus bioflavonoids, as well as investigations into antioxidant properties which may add additional benefit are warranted.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

MicroRNA-139-5p suppresses myosin heavy chain I and IIa expression via inhibition of the calcineurin/NFAT signaling pathway

Highlights: • MiR-139-5p was higher in mouse slow-twitch than in fast-twitch muscle. • MiR-139-5p overexpression downregulated the expression of MyHC I and MyHC IIa. • CaN/NFAT signaling was notably inhibited by miR-139-5p overexpression. • MiR-139-5p suppressed MyHC I and IIa expression via inhibiting CaN/NFAT signaling. MicroRNAs (miRNAs) are a class of small non-coding RNAs that are widely involved in a variety of biological processes. Different skeletal muscle fiber type composition exhibits characteristic differences in functional properties and energy metabolism of skeletal muscle. However, the molecular mechanism by which miRNAs control the different type of muscle fiber formation is still not fully understood. In the present study, we characterized the role of microRNA-139-5p (miR-139-5p) in the regulation of myosin heavy chain (MyHC) isoform expression and its underlying mechanisms. Here we found that the expression of miR-139-5p was significantly higher in mouse slow-twitch muscle than in fast-twitch muscle. Overexpression of miR-139-5p downregulated the expression of MyHC I and MyHC IIa, whereas inhibition of miR-139-5p upregulated them. We also found that the levels of calcineurin (CaN), NFATc1, MEF2C and MCIP1.4, which are the components of CaN/NFAT signaling pathway that has shown to positively regulate slow fiber-selective gene expression, were notably inhibited by miR-139-5p overexpression. Furthermore, treatment of phenylephrine (PE), a α1-adrenoceptor agonist, abolished the inhibitory effect of miR-139-5p on MyHC I and MyHC IIa expression. Together, our findings indicated that the role of miR-139-5p in regulating the MyHC isoforms, especially MyHC I and MyHC IIa, may be achieved through inhibiting CaN/NFAT signaling pathway.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

HBV polymerase-derived peptide exerts an anti-HIV-1 effect by inhibiting the acetylation of viral integrase

Highlights: • A HBV derived 6-mer peptide, Poly6, can elicit an anti-HIV-1 effect. • The anti-HIV-1 effects of Poly6 is due to the inhibition of viral integration. • Poly6 can lead to downregulation of HIV-1 IN acetylation via p300 suppression. Here, we found that a 6-mer peptide, Poly6, derived from the hepatitis B virus (HBV), which overlaps with a polymerase corresponding to a preS1 deletion reported to contribute to liver disease progression, can elicit an antiviral effect against human immunodeficiency virus (HIV)-1 by inhibiting HIV-1 integrase (IN) activity of infected cells. Mechanistic studies revealed that the anti-HIV-1 effects of Poly6 occurred via the inhibition of integration, which resulted from the inhibition of acetylation of HIV-1 IN possibly by downregulation of p300 histone acetyltransferase. Our data suggest the potential therapeutic use of a 6-mer HBV-derived peptide, Poly6, as an anti-HIV-1 agent to suppress HIV-1 infection via inhibiting integrase activity.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

microRNA-29a inhibition induces Gab1 upregulation to protect OB-6 human osteoblasts from hydrogen peroxide

Highlights: • Gab1 is required for H{sub 2}O{sub 2}-induced Akt activation in OB-6 osteoblasts. • miR-29a inhibition upregulates Gab1 to protect OB-6 cells from H{sub 2}O{sub 2}. • Gab1 over-expression promotes Akt activation to inhibit H{sub 2}O{sub 2}-induced OB-6 cell death. • Gab1 is the direct and primary target of antagomiR-29a in mediating its anti-H{sub 2}O{sub 2} actions. The present study determines the role of the Gab1 in hydrogen peroxide (H{sub 2}O{sub 2})-induced death of human osteoblasts. We show that Gab1 is required for H{sub 2}O{sub 2}-induced Akt activation to promote osteoblast survival. In OB-6 human osteoblasts, Gab1 silencing (by targeted-shRNA) or complete knockout (by CRISPR-Cas9 KO plasmid) largely attenuated Akt activation by H{sub 2}O{sub 2}. Gab1-depleted OB-6 cells were more vulnerable to H{sub 2}O{sub 2}. Conversely, forced over-expression of Gab1 by an adenovirus vector increased Akt activation to protect OB-6 cells from H{sub 2}O{sub 2}. Significantly, the anti-sense of microRNA-29a (“antagomiR-29a”) induced Gab1 expression to facilitate H{sub 2}O{sub 2}-induced Akt activation, which protected OB-6 cells from apoptosis. AntagomiR-29a was however ineffective in Gab1-deficient and Akt-inhibited OB-6 cells. Forced over-expression of miR-29a induced Gab1 downregulation to inhibit H{sub 2}O{sub 2}-induced Akt activation, causing enhanced OB-6 cell death. miR-29a-induced actions were abolished by an adenovirus constitutively-active Akt1 (Ad-caAkt1) in OB-6 cells. Together, microRNA-29a inhibition induces Gab1 upregulation and Akt activation to protect OB-6 osteoblasts from H{sub 2}O{sub 2}.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Rubiadin-1-methyl ether from Morinda officinalis How. Inhibits osteoclastogenesis through blocking RANKL-induced NF-κB pathway

Highlights: • Rubiadin-1-methyl ether (RBM) inhibits osteoclastogenesis at early stage. • RBM inhibits bone resorption by reducing expression of osteoclastic c-Fos and NFATc1. • RBM is involved in RANKL-induced NF-κB signaling pathway in osteoclasts. Rubiadin-1-methyl ether (RBM) is a natural anthraquinone compound isolated from the root of Morinda officinalis How. In our previous study, RBM was found to have inhibitory effects on the TRAP activity of osteoclasts, which means that RBM may be a candidate for therapy of bone diseases characterized by enhanced bone resorption. However, the further effect of RBM on osteoclasts and the underlying mechanism remain unclear. In the present study, we investigated the effects of RBM isolated from Morinda officinalis How. on osteoclasts derived from bone marrow macrophages (BMMs) and the underlying mechanism in vitro. RBM at the dose that did not affect the viability of cells significantly inhibited RANKL-induced osteoclastogenesis and actin ring formation of osteoclast, while RBM performed a stronger effect at the early stage. In addition, RBM downregulated the expression of osteoclast-related proteins, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), cellular oncogene Fos (c-Fos), matrix metallopeptidase 9 (MMP-9) and cathepsin K (CtsK) as shown by Western blot. Furthermore, RBM inhibited the phosphorylation of NF-κB p65 and the degradation of IκBα as well as decreased the nuclear translocation of p65. Collectively, the results suggest that RBM inhibit osteoclastic bone resorption through blocking NF-κB pathway and may be a promising agent for the prevention and treatment of bone diseases characterized by excessive bone resorption.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2019

High-throughput screening and chemotype-enrichment analysis of ToxCast phase II chemicals evaluated for human sodium-iodide symporter (NIS) inhibition

In support of the Endocrine Disruptor Screening Program (EDSP), the U.S.EPA's Office of Research and Development (ORD) is developing high-throughput screening (HTS) approaches to identify chemicals that alter target sites in the thyroid hormone (TH) pathway. The sodium iodide symporter (NIS) is a transmembrane glycoprotein that mediates iodide uptake into the thyroid as the initial step of TH biosynthesis. Previously, we screened 293 ToxCast chemicals (ph1v2) using a HEK293T cell line expressing human NIS in parallel radioactive iodide uptake (RAIU) and cell viability assays to identify potential environmental NIS inhibitors. Here, we expanded NIS inhibitor screening for a set of 768 ToxCast Phase II (ph2) chemicals, and applied a novel computational toxicology approach based on the ToxPrint chemotype to identify chemical substructures associated with NIS inhibition. Following single-concentration screening (at 1 × 10 -4 M with a 20% inhibition cutoff), 235 samples (228 chemicals) were further tested in multipleconcentration (1 × 10 -9 - 1 × 10 -4 M) format in both RAIU and cell viability assays. The 167 chemicals that exhibited significant RAIU inhibition were then prioritized using combined RAIU and cell viability responses that were normalized relative to the known NIS inhibitor sodium perchlorate. Some of the highest ranked chemicals, such as PFOS, tributyltin chloride, and triclocarban, have been previously reported to be thyroid disruptors. In addition, several novel chemicals were identified as potent NIS inhibitors. The present results were combined with the previous ph1v2 screening results to produce two sets of binary hit-calls for 1028 unique chemicals, consisting of 273 positives exhibiting significant RAIU inhibition, and 63 positives following application of a cell viability filter. A ToxPrint chemotype-enrichment analysis identified > 20 distinct chemical substructural features, represented in > 60% of the active chemicals, as significantly enriched in each NIS inhibition hit-call space. A shared set of 9 chemotypes enriched in both hit-call sets indicates stable chemotype signals (insensitive to cytotoxicity filters) that can help guide structure-activity relationship (SAR) investigations and inform future research.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2012

Inhibiting the Aurora B Kinase Potently Suppresses Repopulation During Fractionated Irradiation of Human Lung Cancer Cell Lines

Purpose: The use of molecular-targeted agents during radiotherapy of non-small-cell lung cancer (NSCLC) is a promising strategy to inhibit repopulation, thereby improving therapeutic outcome. We assessed the combined effectiveness of inhibiting Aurora B kinase and irradiation on human NSCLC cell lines in vitro. Methods and Materials: NSCLC cell lines were exposed to concentrations of AZD1152-hydroxyquinazoline pyrazol anilide (AZD1152-HQPA) inhibiting colony formation by 50% (IC50{sub clone}) in combination with single dose irradiation or different fractionation schedules using multiple 2-Gy fractions per day up to total doses of 4-40 Gy. The total irradiation dose required to control growth of 50% of the plaque monolayers (TCD50) was determined. Apoptosis, G2/M progression, and polyploidization were also analyzed. Results: TCD50 values after single dose irradiation were similar for the H460 and H661 cell lines with 11.4 {+-} 0.2 Gy and 10.7 {+-} 0.3 Gy, respectively. Fractionated irradiation using 3 Multiplication-Sign 2 Gy/day, 2 Multiplication-Sign 2 Gy/day, and 1 Multiplication-Sign 2 Gy/day schedules significantly increased TCD50 values for both cell lines grown as plaque monolayers with increasing radiation treatment time. This could be explained by a repopulation effect per day that counteracts 75 {+-} 8% and 27 {+-} 6% of the effect of a 2-Gy fraction in H460 and H661 cells, respectively. AZD1152-HQPA treatment concomitant to radiotherapy significantly decreased the daily repopulation effect (H460: 28 {+-} 5%, H661: 10 {+-} 4% of a 2-Gy fraction per day). Treatment with IC50{sub clone} AZD1152-HPQA did not induce apoptosis, prolong radiation-induced G2 arrest, or delay cell cycle progression before the spindle check point. However, polyploidization was detected, especially in cell lines without functional p53. Conclusions: Inhibition of Aurora B kinase with low AZD1152-HQPA concentrations during irradiation of NSCLC cell lines affects repopulation during radiotherapy. Thus, concomitant Aurora B kinase inhibition and irradiation may be a promising strategy for fast repopulating tumors, which are difficult to cure by dose escalation based on conventional fractionation.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

DOE OSTI2011

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A{sub 2}-induced degranulation in mast cells

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of {beta}-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G{sub M1}), di-sialoganglioside (G{sub D1a}) and tri-sialoganglioside (G{sub T1b}). In contrast, honeybee venom-derived phospholipase A{sub 2} induced the net degranulation directly without cytotoxicity, which was not inhibited by G{sub M1}, G{sub D1a} and G{sub T1b}. For analysis of distribution of G{alpha}{sub q} and G{alpha}{sub i} protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of G{alpha}{sub q} and G{alpha}{sub i} at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A{sub 2}-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A{sub 2}-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2013

Mechanism of HERG potassium channel inhibition by tetra-n-octylammonium bromide and benzethonium chloride

Tetra-n-octylammonium bromide and benzethonium chloride are synthetic quaternary ammonium salts that are widely used in hospitals and industries for the disinfection and surface treatment and as the preservative agent. Recently, the activities of HERG channel inhibition by these compounds have been found to have potential risks to induce the long QT syndrome and cardiac arrhythmia, although the mechanism of action is still elusive. This study was conducted to investigate the mechanism of HERG channel inhibition by these compounds by using whole-cell patch clamp experiments in a CHO cell line stably expressing HERG channels. Tetra-n-octylammonium bromide and benzethonium chloride exhibited concentration-dependent inhibitions of HERG channel currents with IC{sub 50} values of 4 nM and 17 nM, respectively, which were also voltage-dependent and use-dependent. Both compounds shifted the channel activation I–V curves in a hyperpolarized direction for 10–15 mV and accelerated channel activation and inactivation processes by 2-fold. In addition, tetra-n-octylammonium bromide shifted the inactivation I–V curve in a hyperpolarized direction for 24.4 mV and slowed the rate of channel deactivation by 2-fold, whereas benzethonium chloride did not. The results indicate that tetra-n-octylammonium bromide and benzethonium chloride are open-channel blockers that inhibit HERG channels in the voltage-dependent, use-dependent and state-dependent manners. - Highlights: ► Tetra-n-octylammonium and benzethonium are potent HERG channel inhibitors. ► Channel activation and inactivation processes are accelerated by the two compounds. ► Both compounds are the open-channel blockers to HERG channels. ► HERG channel inhibition by both compounds is use-, voltage- and state dependent. ► The in vivo risk of QT prolongation needs to be studied for the two compounds.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2013

Nicotine-induced retardation of chondrogenesis through down-regulation of IGF-1 signaling pathway to inhibit matrix synthesis of growth plate chondrocytes in fetal rats

Previous studies have confirmed that maternal tobacco smoking causes intrauterine growth retardation (IUGR) and skeletal growth retardation. Among a multitude of chemicals associated with cigarette smoking, nicotine is one of the leading candidates for causing low birth weights. However, the possible mechanism of delayed chondrogenesis by prenatal nicotine exposure remains unclear. We investigated the effects of nicotine on fetal growth plate chondrocytes in vivo and in vitro. Rats were given 2.0 mg/kg·d of nicotine subcutaneously from gestational days 11 to 20. Prenatal nicotine exposure increased the levels of fetal blood corticosterone and resulted in fetal skeletal growth retardation. Moreover, nicotine exposure induced the inhibition of matrix synthesis and down-regulation of insulin-like growth factor 1 (IGF-1) signaling in fetal growth plates. The effects of nicotine on growth plates were studied in vitro by exposing fetal growth plate chondrocytes to 0, 1, 10, or 100 μM of nicotine for 10 days. Nicotine inhibited matrix synthesis and down-regulated IGF-1 signaling in chondrocytes in a concentration-dependent manner. These results suggest that prenatal nicotine exposure induces delayed chondrogenesis and that the mechanism may involve the down-regulation of IGF-1 signaling and the inhibition of matrix synthesis by growth plate chondrocytes. The present study aids in the characterization of delayed chondrogenesis caused by prenatal nicotine exposure, which might suggest a candidate mechanism for intrauterine origins of osteoporosis and osteoarthritis. - Highlights: ► Prenatal nicotine-exposure could induce delayed chondrogenesis in fetal rats. ► Nicotine inhibits matrix synthesis of fetal growth plate chondrocytes. ► Nicotine inhibits IGF-1 signaling pathway in fetal growth plate chondrocytes.

60 APPLIED LIFE SCIENCES↗