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This study introduces a facile method for synthesizing covalently bonded magnetic carbon nanoparticles (MCNs) in which carboxylic acid-functionalized activated carbon nanospheres (ACN-COOH) are connected with amine-terminated iron oxide nanoparticles (NPs) (Fe3O4-NH2) via a carbodiimide crosslinking reaction. The adsorption characteristics of the developed magnetic nanoparticles (ACN-Fe3O4) were investigated using a standard cationic dye (methylene blue, MB). Two additional MCNs (multi-core and core@shell structures) were also prepared, and their adsorption performances were extensively compared. The developed ACN-Fe3O4 material thoroughly utilizes the strengths of activated carbon and Fe3O4 themselves, exhibiting large specific surface areas (708.4 m2/g) and strong magnetic properties (40.3 emu/g), resulting in high adsorption capacity (349.5 mg/g) and recycling efficiency (76 % of adsorption performance after four cycles). In addition, a study of the mechanism reveals that pore-filling processes are dominant with minor contributions from electrostatic interactions, π–π interactions, and n–π interactions. The developed covalently bonded magnetic carbon nanoparticles (ACN-Fe3O4) can thus be considered as competent adsorbents with the potential to compensate for the drawbacks of contemporary MCNs, such as, low adsorption capacity, and weak magnetic properties.

Silicon particles have garnered attention as promising biomedical probes for hyperpolarized 29Si magnetic resonance imaging and spectroscopy. However, due to the limited levels of hyperpolarization for nanosized silicon particles, microscale silicon particles have primarily been the focus of dynamic nuclear polarization (DNP) applications, including in vivo magnetic resonance imaging (MRI). To address these current challenges, we developed a facile synthetic method for partially 29Si-enriched porous silicon nanoparticles (NPs) (160 nm) and examined their usability in hyperpolarized 29Si MRI agents with enhanced signals in spectroscopy and imaging. Hyperpolarization characteristics, such as the build-up constant, the depolarization time (T1), and the overall enhancement of the 29Si-enriched silicon NPs (10 and 15%), were thoroughly investigated and compared with those of a naturally abundant NP (4.7%). During optimal DNP conditions, the 15% enriched silicon NPs showed more than 16-fold higher enhancements─far beyond the enrichment ratio─than the naturally abundant sample, further improving the signal-to-noise ratio in in vivo29Si MRI. The 29Si-enriched porous silicon NPs used in this work are potentially capable to serve as drug-delivery vehicles in addition to hyperpolarized 29Si in vivo, further enabling their potential future applicability as a theragnostic platform.

Background Background: Hot springs have been traditionally used as an alternative treatment for a wide range of diseases, including rheumatoid arthritis, bronchial asthma, diabetes, hypertension, psoriasis and atopic dermatitis. However, the clinical effects and therapeutic mechanisms associated with hot springs remain poorly defined. Objective The purpose of this study was to demonstrate the different effects of hot springs on cellular viability and secretion of inflammatory cytokines on keratinocyte in two geographically representative types of hot springs: NaHCO3-type and NaCl-type, which are the most common types in South Korea. Methods We performed WST-1, BrdU measurements, human inflammatory cytokine arrays and enzyme-linked immunosorbent assay in HaCaT cells stimulated with toll-like receptor 3 by polyinosinicpolycytidylic acid. Results The interaction effects of cell viability and cell proliferation were not significantly different regardless of polyinosinic-polycytidylic acid stimulation and cultured hot springs type. Cytokine array and enzymelinked immunosorbent assay analysis showed increased expression of inflammatory cytokines such as interleukin6 and granulocyte-macrophage colony-stimulating factor by polyinosinic-polycytidylic acid stimulation, with expression levels differing according to hot springs hydrochemical composition. Cytokine reduction was not significant. Conclusion The effects and mechanisms of hot springs treatment in keratinocytes were partially elucidated.

This study introduces a robust synthetic method of fabricating co-assembled biodegradable microcapsules composed mainly of representative natural sugar polymers, hydroxyethylcellulose (HEC), alginate, and sorbitol. We propose a facile enzymatic method evaluating initial biodegradation processes of the microcapsules utilizing a linewidth analysis on 1H nuclear magnetic resonance (NMR) spectroscopy. Linewidth analysis on the 1H resonance signals represents that broad resonance signals of the sugar-based microcapsules are changed to relatively narrower signals as an association of cellulase enzyme. This phenomenon explains co-assembled and cross-linked microcapsule structures are destroyed or/and loosened by the enzymatic breakdowns of ester bonds in the microcapsules. Moreover, closer inspection indicates that co-assembled sorbitol molecules are released from the main networked microcapsules, additionally supporting the biodegradation properties of the microcapsules. Considering the unique features of this method, the proposed study provides valuable information about the initial biodegradation processes and mechanisms of various microcapsules.

This study introduces a temperature-responsive attractive nanoemulsion (ANE) system, which is characterized by the polymer chain conformation-driven dipolar interaction across different oil droplets in an aqueous medium. To achieve this, highly stable ANEs were produced by co-assembly of amphiphilic triblock copolymers (ATCs), poly(ethylene oxide)-b-poly(e-caprolactone)-b-poly(ethylene oxide) (PEO-b-PCL-b-PEO), with lecithin at the oil-water interface. The dipolar attraction of the methoxy terminated-PEO (mPEO) of ATCs on one drop surface with the lecithin head located on the other drop surface led to the drop-to-drop association. We showed that the efficiency of this interdrop association was dominantly influenced by the chain conformation of mPEO blocks. From dense suspension rheology studies, it was demonstrated that the ANEs formed a gel-like phase below the lower critical solution temperature (LCST) of the mPEO, but transformed to a liquid-like phase above the LCST, which occurred reversibly, thus enabling the development of temperature-responsive emulsion fluids.

The decarboxylation reaction of phenylglyoxylic acid with hydrogen peroxide is studied by real-time hyperpolarized carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy at room temperature. A non-observable reaction intermediate is identified using blind selective saturation pulses in the expected chemical shift range, thereby revealing information on the reaction mechanism.

Silicon particles in combination with the dynamic nuclear polarization (DNP) method have recently attracted attention as promising biomedical platforms for drug delivery and medical diagnostics in ...

Introduction: RX-3117 is an oral, small molecule cytidine analog anticancer agent with an improved pharmacological profile relative to gemcitabine and other nucleoside analogs. The agent has excellent activity against various cancer cell lines and xenografts including gemcitabine-resistant variants and it has excellent oral bioavailability; it is not a substrate for the degradation enzyme cytidine deaminase. RX-3117 is being evaluated at a daily oral schedule of 700 mg (5 days/week for 3 weeks) which results in plasma levels in the micromolar range that have been shown to be cytotoxic to cancer cells. It has shown clinical activity in refractory bladder cancer and pancreatic cancer. Areas covered: The review provides an overview of the relevant market and describes the mechanism of action, main pharmacokinetic/pharmacodynamic features and clinical development of this investigational small molecule. Expert opinion: RX-3117 is selectively activated by uridine-cytidine kinase 2 (UCK2), which is expressed only in tumors and has a dual mechanism of action: DNA damage and inhibition of DNA methyltransferase 1 (DNMT1). Because of its tumor selective activation, novel mechanism of action, excellent oral bioavailability and candidate biomarkers for patient selection, RX-3117 has the potential to replace gemcitabine in the treatment of a spectrum of cancer types.

In this study, we measured the force of electrostatic interactions between poly(methyl methacrylate) (PMMA) particles dispersed in organic solvent mixtures of cyclohexyl bromide (CHB) and n-decane. Optical laser tweezers were employed to directly measure interactive forces between paired PMMA particles in a CHB medium that contained n-decane in various volume ratios. CHB, having a moderate dielectric constant, provided an environment with a high charge storage capacity. The addition of n-decane lowered the effective refractive index of the medium, which increased the optical trapping efficiency. We also fabricated microscope flow cells with a commonly used UV-curable adhesive and quantified the effects of dissolved adhesive compounds through interactive force measurements and nuclear magnetic resonance analysis. In addition, we studied the impact of CHB dissociation into H+ and Br- ions, which could screen electrostatic interactions.

Obesity is a public health problem characterized by increased body weight due to abnormal adipose tissue expansion. Bioactive compound consumption from the diet or intake of dietary supplements is one of the possible ways to control obesity. Natural products with adipogenesis-regulating potential act as obesity treatments. We evaluated the synergistic antiangiogenesis, antiadipogenic and antilipogenic efficacy of standardized rebaudioside A, sativoside, and theasaponin E1 formulations (RASE1) in vitro in human umbilical vein endothelial cells (HUVECs), 3T3-L1 preadipocytes respectively, and in vivo using a high-fat and carbohydrate diet-induced obesity mouse model. Orlistat was used as a positive control, while untreated cells and animals were normal controls (NCs). Adipose tissue, liver, and blood were analyzed after dissection. Extracted stevia compounds and green tea seed saponin E1 exhibited pronounced antiobesity effects when combined. RASE1 inhibited HUVEC proliferation and tube formation by suppressing VEGFR2, NF-κB, PIK3, and-catenin beta-1 expression levels. RASE1 inhibited 3T3-L1 adipocyte differentiation and lipid accumulation by downregulating adipogenesis- and lipogenesis-promoting genes. RASE1 oral administration reduced mouse body and body fat pad weight and blood cholesterol, TG, ALT, AST, glucose, insulin, and adipokine levels. RASE1 suppressed adipogenic and lipid metabolism gene expression in mouse adipose and liver tissues and enhanced AMP-activated protein kinase levels in liver and adipose tissues and in serum adiponectin. RASE1 suppressed the NF-κB pathway and proinflammatory cytokines IL-10, IL-6, and TNF-α levels in mice which involve inflammation and progression of obesity. The overall results indicate RASE1 is a potential therapeutic formulation and functional food for treating or preventing obesity and inflammation.

(1) Background: RX-3117 (fluorocyclopentenyl-cytosine) is a cytidine analog that inhibits DNA methyltransferase 1 (DNMT1). We investigated the mechanism and potential of RX-3117 as a demethylating agent in several in vitro models. (2) Methods: we used western blotting to measure expression of several proteins known to be down-regulated by DNA methylation: O6-methylguanine-DNA methyltransferase (MGMT) and the tumor-suppressor genes, p16 and E-cadherin. Transport of methotrexate (MTX) mediated by the proton-coupled folate transporter (PCFT) was used as a functional assay. (3) Results: RX-3117 treatment decreased total DNA-cytosine-methylation in A549 non-small cell lung cancer (NSCLC) cells, and induced protein expression of MGMT, p16 and E-cadherin in A549 and SW1573 NSCLC cells. Leukemic CCRF-CEM cells and the MTX-resistant variant (CEM/MTX, with a deficient reduced folate carrier) have a very low expression of PCFT due to promoter hypermethylation. In CEM/MTX cells, pre-treatment with RX-3117 increased PCFT-mediated MTX uptake 8-fold, and in CEM cells 4-fold. With the reference hypomethylating agent 5-aza-2&prime, deoxycytidine similar values were obtained. RX-3117 also increased PCFT gene expression and PCFT protein. (4) Conclusion: RX-3117 down-regulates DNMT1, leading to hypomethylation of DNA. From the increased protein expression of tumor-suppressor genes and functional activation of PCFT, we concluded that RX-3117 might have induced hypomethylation of the promotor.

Background: RX-5902 (Supinoxin) is a novel anti-cancer compound that targets phosphorylated p68 RNA helicase, a member of the DEAD box family of helicases, affecting upstream and downstream molecules in the Wnt canonical pathway. As a single agent, RX-5902 exhibits strong growth inhibition in both in vitro and in vivo settings. Specifically, RX-5902 enhances survival and tumor growth inhibition in numerous xenograft models, including ovarian, renal and breast. We have previously shown RX-5902 inhibits cell growth in a dose-dependent fashion in the triple-negative breast cancer (TNBC) xenograft MDA-MB231. In the current study, we have expanded our investigation of the therapeutic potential of RX-5902 against TNBC using both in vitro and in vivo preclinical models. Methods: RX-5902 was provided by Rexahn, Inc. (Rockville, MD). Cell proliferation was measured using the Cell-Titer Glo luminescent cell viability assay (Promega). Apoptosis was assessed using Incucyte Caspase 3/7 Green apoptosis assay (Essenbioscience). Immunoblots of MDA-MB-231 cell line were probed for ß-catenin (Cell Signaling). Syngeneic 4T1 murine TNBC mice were obtained from Sippr-BK Laboratory Animal Co (Shanghai, China) and tumor volumes were measured twice a week. When the mean tumor volumes reached ˜90 mm3, mice were randomized and treated with vehicle or RX-5902 PO daily alone or in combination with anti-CTLA4 or anti-PD-1 BIW for 3 weeks. Tumor growth inhibition (TGI) was calculated at Day 25. Results: A panel of 18 TNBC cell lines were treated with RX-5902 and effects on cell proliferation were measured by the Cell Titer-Glo assay. Using 100nM as a cutoff, 14 sensitive lines and 4 resistant lines were identified, with an average IC50 of 56 nM in the sensitive lines. Of these, we chose 2 sensitive lines (MDA-MB-231, HCC1806) and 2 resistant lines (MDA-MB-436 and CAL-120) and assessed induction of apoptosis by the Incucyte caspase activity assay. Robust induction of apoptosis was observed in both sensitive lines (N=3). These lines were then subjected to cell cycle analysis by flow cytometry, which revealed a pronounced G2/M cell cycle arrest and aneuploidy following exposure to RX-5902. Western blot analysis of the MDA-MB-231 cell line showed decreases in the Wnt pathway-related protein nuclear ß-catenin in doses ranging from 20 nM to 200 nM. Finally, the therapeutic efficacy of RX-5902 was assessed as a single agent and in combination with two immune-oncology agents in the treatment of the TNBC 4T1 animal model. RX-5902 as a single agent showed dose dependency in the 4T1 model, and when given in combination with either anti-CTLA4 or anti-PD1 showed an additive effect (p Conclusions: RX-5902 showed efficacy against several in vitro and in vivo preclinical models of TNBC. RX-5902 resulted in G2/M arrest and induced apoptosis in sensitive TNBC cell lines and decreases in nuclear beta-catenin. In vivo, RX-5902 demonstrated additive anti-tumor effects when combined with either anti-CTLA4 or anti-PD1 immunotherapies. Together, these finding indicate that RX-5902 may have important clinical implications for the treatment of TNBC. A phase 2a clinical study in metastatic TNBC is ongoing..training_cert Citation Format: Tentler JJ, Frank JG, Kim DJ, George C, Lee YB, Ely B, Tan AC, Kim J, Pitts TM, Capasso A, Dailey KL, Eckhardt G, Diamond JR. Preclinical studies of RX-5902, a beta-catenin modulator in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-16.

Heavy metal pollution in soil has been concerned because of toxicity in ecosystem and adverse effect on human health. Main objective of this study was to examine reduction of bioavailable heavy metals and consequently, decrease of ecotoxicity to biota when chemical amendments were applied in soil. Three chemical amendments, acid mine drainage sludge (AMDS), lime stone (LS), and steel slag (SS) were applied with varied application ratio (1, 3, 5%) in heavy metal polluted soil and bioavailable fraction of heavy metal was monitored. In addition, ecotoxicity test using earthworm (Eisenia fetida) was conducted for 28 days examining mortality, weight increase, and bioaccumulation of heavy metal in the earthworm. Result showed that AMDS was the most efficient amendment for reducing bioavailable heavy metals in soil while SS showed the least efficiency. Reduction ratio of bioavailable-As, Cd, and Pb was ranged 39.0–92.0% depending on application ratio and heavy metal species for AMDS application. However, only bioavailable-Pb was reduced at the range between 39.1% and 56.5% when SS was applied in soil. In contrast, the lowest concentration of As, Cd, and Pb and ecotoxicity effect in the earthworm was observed in SS treatment indicating that exposure route of heavy metals or particle size of amendments might effect on uptake of heavy metals to the earthworm. Overall, ecotoxicity test in combination with chemical concentration monitoring is a useful tool for evaluating remediation efficiency of heavy metal polluted soil.

RX-5902 is a first-in-class anticancer agent targeting phosphorylated-p68 and attenuating nuclear shuttling of β-catenin. The purpose of this study was to evaluate the efficacy of RX-5902 in preclinical models of triple-negative breast cancer (TNBC) and to explore effects on β-catenin expression. A panel of 18 TNBC cell lines was exposed to RX-5902, and changes in proliferation, apoptosis, cellular ploidy, and effector protein expression were assessed. Gene expression profiling was used in sensitive and resistant cell lines with pathway analysis to explore pathways associated with sensitivity to RX-5902. The activity of RX-5902 was confirmed in vivo in cell line and patient-derived tumor xenograft (PDX) models. RX-5902 demonstrated potent antiproliferative activity in vitro against TNBC cell lines with an average IC50 of 56 nmol/L in sensitive cell lines. RX-5902 treatment resulted in the induction of apoptosis, G2–M cell-cycle arrest, and aneuploidy in a subset of cell lines. RX-5902 was active in vivo against TNBC PDX models, and treatment resulted in a decrease in nuclear β-catenin. RX-5902 exhibited dose-proportional pharmacokinetics and plasma and tumor tissue in nude mice. Pathway analysis demonstrated an increase in the epithelial-to-mesenchymal transformation (EMT), TGFβ, and Wnt/β-catenin pathways associated with sensitivity to RX-5902. RX-5902 is active against in vitro and in vivo preclinical models of TNBC. Target engagement was confirmed with decreases in nuclear β-catenin and MCL-1 observed, confirming the proposed mechanism of action. This study supports the continued investigation of RX-5902 in TNBC and combinations with immunotherapy.

We utilized dynamic nuclear polarization to hyperpolarize three metabolic imaging agents as single agents and together (co-polarization with two different organic radicals; OX63 and BDPA). In addition, we describe our initial two slice selective spectroscopy in vivo experiments in mice. These techniques along with advances by other laboratories will allow for multiple metabolic pathways to be imaged in real-time and in vivo .

1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl] piperazine (RX-5902) exhibits strong growth inhibition in various human cancer cell lines with IC50 values ranging between 10 and 20 nM. In this study, we demonstrate that p68 RNA helicase is a cellular target of RX-5902 by the drug affinity responsive target stability (DARTS) method, and confirmed the direct binding of 3H-labeled RX-5902 to Y593 phospho-p68 RNA helicase. We further demonstrated RX-5902 inhibited the β-catenin dependent ATPase activity of p68 RNA helicase in an in vitro system. Furthermore, we showed that treatment of cancer cells with RX-5902 resulted in the downregulation of the expression of certain genes, which are known to be regulated by the β-catenin pathway, such as c-Myc, cyclin D1 and p-c-Jun. Therefore, our study indicates that the inhibition of Y593 phospho-p68 helicase - β-catenin interaction by direct binding of RX-5902 to Y593 phospho-p68 RNA helicase may contribute to the anti-cancer activity of this compound. J. Cell. Biochem. 116: 1595–1601, 2015. © 2015 Wiley Periodicals, Inc.

RX-3117 is a novel cytidine analog showing encouraging results in ongoing Phase 2a studies with gemcitabine-resistant pancreatic ductal adenocarcinoma and advanced urothelial bladder cancer. Cellular uptake of RX-3117 is mediated by the equilibrative nucleoside transporter 1 (hENT1). RX-3117 is activated by uridine-cytidine kinase-2 (UCK2) and is not a substrate for cytidine deaminase (CDA), and shows a good oral bioavailability. In an earlier study accumulation of RX-3117 nucleotides was associated with sensitivity to the drug. RX-3117 is incorporated into RNA and DNA and inhibits DNA methyltransferase 1 (DNMT1). RX-3117 is active against gemcitabine-resistant non-small cell lung cancer (NSCLC) cell lines. In this study we aimed to elucidate potential resistance mechanisms against RX-3117. For that purpose the NSCLC cell lines H460, A549 and SW1573 (including its gemcitabine resistant variant SW-G) were exposed to increasing concentrations of RX-3117. For A549 and SW1573 two variants were obtained: A549/RX1, A549/RX2, SW1573/RX1 and SW1573/RX2. Resistance varied from 10 (H460; SW-G) to >100-fold for the other cell lines. Resistance was stable except for H460 cells. Cross-resistance in the same range was observed for the cytidine analogs ethynyl-cytidine (ETC), cyclopentenylcytosine (CPEC) and aza-cytidine (aza-CR), which are all activated by UCK2; no cross-resistance was observed for gemcitabine and aza-deoxycytidine which are activated by deoxycytidine kinase. No change was observed in the expression of DNMT1, while expression of ribonucleotide reductase 1 was decreased. However, analysis of UCK2 at the enzyme activity level, protein and gene expression did not reveal a decrease in UCK2 while for ETC, aza-CR and CPEC resistance was reported to be mediated by a UCK2 deficiency. No change was found in the other activating enzymes UMP-kinase, CMP-kinase and NME1/NDKA. Despite this lack of change, accumulation of RX-3117 and RX-3117 nucleotides (mono-, di- and triphosphate) was 2-5-fold lower in the resistant cells after incubation with 10 or 100 μM RX-3117 for 4-24 hr. No evidence for accumulation of RX-3117 deoxynucleotides was found in both the parent and the resistant cell lines. Since no change in hENT1 was found, cell lines were further investigated by using RNA-seq, and western blotting focusing on nucleoside/nucleotide degradation enzymes. CDA was decreased in resistant cells, but the cytosolic pyrimidine nucleotidase NT5C3 was increased in the resistant cell lines as well as DCTPP1, which can degrade deoxynucleoside triphosphates. Moreover several DNA repair enzymes SAMHD1, MTH1 and TDP1 were increased. However, inhibition of MTH1 and DCTPP1 did not increase sensitivity to RX-3117. In conclusion, induction of resistance to the novel cytidine analog RX-3117 led to a decreased accumulation of RX-3117 ribonucleotides, which might be associated with an increased degradation. Citation Format: Godefridus J. Peters, Dzjemma Sarkisjan, Btissame El Hassouni, Richard J. Honeywell, Joris R. Julsing, Beatrice Balboni, Daniel J. De Klerk, Safet Zekanovic, Kees Smid, Elisa Giovannetti, Young B. Lee, Deog J. Kim. Unusual mechanism of resistance to the novel cytidine analog fluorocyclopentenylcytosine (RX-3117) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1267.

Phosphorylated p68 (p-p68 or phospho-DDX5) has been shown to be associated with cell transformation, epithelial mesenchymal transition (EMT) and cell migration, and furthermore play a vital role in cell proliferation and tumor/cancer progression by promoting the nuclear translocation of β-catenin. RX-5902 is an oral, small molecule inhibitor of β-catenin nuclear translocation mediated by p-p68. Previous studies have shown that RX-5902 inhibits the growth of cancer cells at low nanomolar IC50 (10 to 24nM) and disrupts the p-p68-β-catenin signaling pathway through an interaction with p-p68 on Tyr 593. By interfering with the p-p68-β-catenin signaling pathway cancer cells undergo apoptosis. In this study, we expanded our investigation to study the effect of RX-5902 on nuclear β-catenin in several types of cancer cell lines that include colon, gastric, hepatic, head and neck, melanoma, breast, pancreatic, ovarian, and prostate. Western blot data, in the cell lines tested, showed a decrease of nuclear β-catenin protein as well as p-p68 and c-myc in a concentration-dependent manner. Furthermore, an in vivo study of the MDA-MD-231 xenograft model demonstrated the ability of RX-5902 to inhibit the tumor growth by decreasing p-p68, β-catenin, cyclin D1 and c-myc proteins. Collectively our findings in both in vitro and in vivo studies support the potential therapeutic effect of RX-5902 in multiple cancer indications through the disruption of the phospho-p68/nuclear β-catenin interaction and blocking the nuclear translocation of β-catenin. Citation Format: Young B. Lee, Christina George, Deog J. Kim. The anticancer effects of RX-5902 result from inhibition of phosphorylated p68-mediated β-catenin nuclear translocation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1298.

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 μM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 μM RX-3117 resulted in 90 % inhibition of RNA synthesis but 100 μM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 μM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.

Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression (r = 0.803 and 0.915, respectively), but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117.

BACKGROUND/AIMS An association between reduced pulmonary function and diabetes has been observed. Our aim was to evaluate the prevalence and risk factors associated with reduced pulmonary function in diabetic patients. METHODS We analyzed data from the Korea National Health and Nutrition Examination Survey 2011 to 2013. The study population included data from 8,784 participants (including 1,431 diabetics) aged ≥ 40 years. Reduced pulmonary function was defined as patients with restrictive (forced expiratory volume in 1 second/forced vital capacity [FEV1/FVC] ≥ 0.7 and FVC < 80% of predicted value) or obstructive (FEV1/FVC < 0.7) patterns. RESULTS Subjects with diabetes had a higher prevalence of restrictive (18.4% vs. 9.4%, p < 0.001) and obstructive impairments (20% vs. 12.6%, p < 0.001) than those without diabetes. The adjusted odds ratios (aORs) (95% confidence interval [CI]) for obstructive and restrictive pulmonary impairment were 0.91 (0.75 to 1.11) and 1.57 (1.30 to 1.89), respectively. In the diabetes population, age (aOR, 1.04; 95% CI, 1.02 to 1.06), male sex (aOR, 1.40; 95% CI, 1.04 to 1.88), and body mass index (aOR, 1.15; 95% CI, 1.10 to 1.21) were independently associated with restrictive pulmonary impairment. Age (aOR, 1.12; 95% CI, 1.09 to 1.14), male sex (aOR, 4.24; 95% CI, 2.42 to 7.44), and smoking at any point (ever-smoker: aOR, 1.96; 95% CI, 1.16 to 3.33) were independent risk factors for obstructive pulmonary impairment in diabetics. Diabetes duration or glycated hemoglobin had no association with pulmonary impairment in diabetes. CONCLUSIONS Subjects with diabetes had a higher risk of restrictive pulmonary impairment than those without diabetes after adjusting for confounding factors. Older age, male sex, body mass index, and smoking were associated with reduced lung function in diabetes.

RX-3117 (fluorocyclopentenyl-cytosine) is a novel cytidine analog currently being evaluated in a Phase Ib clinical trial in cancer patients with solid tumors. The radiosensitizing effect of RX-3117 was studied in A2780 ovarian cancer cells and non-small cell lung cancer cell lines and related to cell survival and the effect on cell cycle and cell cycle proteins. RX-3117 has a schedule-dependent radiosensitizing effect, but only at pre-incubation (dose modifying factors: 1.4–1.8), observed at pulse and fractionated irradiation. Radiosensitizion was also seen in a three-dimensional spheroid model. At the low radiosensitizing concentration, RX-3117 in combination with radiation led to an accumulation of cells in S-phase, which was accompanied with an increase of cell cycle proteins such as p-Chk2 and p-cdc25C. In addition, RX-3117 caused DNA damage and increased apoptosis. In conclusion, our in vitro experiments showed a radiosensitizing effect of RX-3117.

Clavulanic acid is a psychoactive compound that has been shown to modulate central nervous system activity. Importantly, in neurotoxin-induced animal models, clavulanic acid has been shown to improve motor function (Huh et al., 2010) suggesting that it can be neuroprotective; however, the mechanism as how clavulanic acid can induce neuroprotection is not known. We demonstrate here that clavulanic acid abrogates the effects of the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) which mimics Parkinson's disease (PD) by inducing neurodegeneration. To further establish the mechanism we identified that clavulanic acid inhibits neurotoxin-induced loss of mitochondrial membrane potential and ROS production. Consistent with these results, neurotoxin-induced increase in Bax levels was also decreased in clavulanic acid treated cells. Importantly, neurotoxin-induced release of cytochrome c levels as well as caspase activation was also inhibited in clavulanic acid treated cells. In addition, Bcl-xl levels were also restored and the Bcl-xl/Bax ratio that is critical for inducing apoptosis was increased in clavulanic acid treated cells. Overall, these results suggest that clavulanic acid is intimately involved in inhibiting neurotoxin-induced loss of mitochondrial function and induction of apoptosis that contributes towards neuronal survival.

On the basis of the potent biological activity of cyclopentenyl-pyrimidines, fluorocyclopentenyl-pyrimidines were designed and synthesized from D-ribose. Among these, the cytosine derivative 5a showed highly potent antigrowth effects in a broad range of tumor cell lines and very potent antitumor activity in a nude mouse tumor xenograft model implanted with A549 human lung cancer cells. However, its 2'-deoxycytidine derivative 5b did not show any antigrowth effects, indicating that 2'-hydroxyl group is essential for the biological activity.

This study introduces a new type of uniform liposome-analogous vesicle with a highly stable shell structure in which water-in-oil-in-water double emulsion drops fabricated in a capillary-based microfluidic device are used as templates. The vesicles developed in this work consist of a poly(ethylene glycol) hydrogel core surrounded by a polyurethane (PU) film between 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) layers. Subjecting the double emulsion templates to UV irradiation leads to the formation of a PU elastomer film between the DPPC layers. The presence of a thin PU film sandwiched between the DPPC layers is confirmed by confocal laser microscopy. The thicknesses of the PU films are measured to be approximately ⿼4 μm. Further study reveals the incorporation of the PU film between the DPPC layers remarkably improves the shell impermeability. Our vesicle system is expected to be useful for regulating the permeation of small molecules through lipid-based vesicular films.

Long-pulsed 755-nm alexandrite and long-pulsed 1064-nm neodymium:yttrium-aluminum-garnet (Nd:YAG) lasers have been used for photorejuvenation of the face. The aim of this study was to investigate the safety and efficacy of long-pulsed alexandrite and long-pulsed Nd:YAG lasers for photorejuvenation in Korea. One hundred and sixteen Korean patients with photo-aged facial skin were enrolled. Sixty-two patients with facial pigmentation underwent long-pulsed alexandrite laser treatment. Eleven patients that wanted to improve facial pigmentation with minimal pain had quasi-long-pulsed alexandrite laser treatment. Forty three patients had long-pulsed Nd:YAG laser therapy. Outcome assessments included standard photographs and global evaluation by blinded investigators. The self-assessment grade was provided in questionnaires. Forty-four percent of patients reported excellent or good improvement of their pigmentary lesions (>50% improvement) using a long-pulsed alexandrite laser. Of patients who underwent long-pulsed Nd:YAG laser treatment, 36% reported excellent or good improvement in skin tightening, 50% in facial flushing and 45% in pigmentary lesions. We conclude that long-pulsed alexandrite and long-pulsed Nd:YAG lasers are safe and effective for facial photorejuvenation in Koreans.

Summary Background Hair greying is an obvious sign of ageing in humans. White (nonpigmented) hair is thicker than black (pigmented) hair. The growth rate of white hair is also significantly higher than that of black hair. However, the mechanism underlying this is largely unknown. Objectives To examine the association between hair greying and hair growth patterns by evaluating expression of the genes or proteins related to hair growth in white and black hairs. Methods Morphological characteristics were observed in eyebrow and scalp hairs. The differential expression of genes was analysed in black and white hairs from human scalp by a microarray analysis. Reverse transcription–polymerase chain reaction (RT-PCR) and immunohistochemistry for genes and proteins related to hair growth were performed in black and white hairs. Results Keratin and keratin-associated protein (KRTAP) genes in white hair were upregulated at least two-fold in comparison with black hair in a microarray analysis. Upregulation of selected keratin genes and KRTAP4 isoform genes in white hair was validated by RT-PCR. Immunoreactivity for KRT6, KRT14/16 and KRT25 was increased in the hair follicle of white hair compared with black hair. Gene expression of fibroblast growth factor 5 (FGF5) was downregulated in white hair compared with black hair. However, gene expression of FGF7 was upregulated in white hair compared with black hair. Conclusions Expression of genes and proteins associated with active hair growth is upregulated in white (nonpigmented) hair compared with black (pigmented) hair. These results suggest that hair greying is associated with active hair growth.

RX-3117 is a novel, investigational, oral, small molecule nucleoside compound that shows promising antitumor activity in xenografts including patient-derived xenografts resistant to gemcitabine. RX-3117 is currently being evaluated in a Phase IIa multi-center, open-label clinical study in patients with relapsed or refractory pancreatic cancer and advanced bladder cancer. RX-3117 can downregulate DNMT1 and activate genes controlled by methylation, such as E-cadherin and p16. We aimed to elucidate the mechanism of RX-3117 mediated downregulation of DNMT1. Expression of DNMT1 was evaluated by western blotting. Binding of DNMT1 to DNA was measured after DNA isolation, DNAse treatment and gel electrophoresis. DNMT1 trapping in the nucleus was also studied with ImageStream FACS analysis. DNMT1 ubiquitination was measured by immunoprecipitation and quantification of the ubiquitin tag K48. Translocation of DNMT1 was studied by confocal microscopy. Modeling studies were performed with Quantum Mechanics (QM) and Molecular Dynamics (MD). DNMT1 acts on the DNA during the so-called base flipping necessary for methyl transfer. To determine whether RX-3117 treatment would lead to trapping of DNMT1, NSCLC A549 and PDAC SUIT-028 cells were treated for 24 hr with 1 µM RX-3117. Immunostaining of DNMT1-DNA and ImageStream FACS analysis demonstrated binding of DNMT1 to DNA. Treatment with RX-3117 also resulted in DNMT1 ubiquitination, as shown by an increase in the ubiquitin tag K48, reaching the highest level after 48 hr. Incubation of A549 and SUIT-028 cells with 80 nM of the specific proteasome inhibitor bortezomib prevented RX-3117 mediated degradation of DNMT1 (at 24 and 24 hr exposure to 1 µM RX-3117). This underlines the role of the proteasome in DNMT1 degradation. With confocal microscopy it was demonstrated that DNMT1 and the proteasome co-localized and translocated to the cytosol of A549 and SUIT-028 cells treated for 48 hours with 1 µM RX-3117. With molecular modelling using QM and MD, changes in nucleophilicity of C4-C5 double bound were observed indicating that DNMT1 would initiate methyl transfer but cannot proceed. In summary we conclude that RX-3117 induces epigenetic changes in cancer cells by trapping DNMT1 in the nucleus followed by translocation of the protein to the proteasome for degradation. Citation Format: Dzjemma Sarkisjan, Ferry Pronk, Rosan Kuin, Btissame el Hassouni, Young B. Lee, Deog J. Kim, Marc Dijk, Daan P. Geerke, G. J. Peters. RX-3117 promotes epigenetic effects in cancer cells through enhanced degradation of DNMT1 [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 5861.

Vitamin C is used as an anti-ageing agent because of its collagen enhancing effects. The precise cellular signalling mechanism of vitamin C is not well known. Here, we investigate the profibrotic mechanism of vitamin C against LL-37. Antimicrobial peptide LL-37 decreases collagen expression at mRNA and protein levels in human dermal fibroblasts (HDFs). The ability of LL-37 to inhibit collagen expression is dependent on phosphorylation of extracellular signal-regulated kinase (ERK). HDFs and human keloid fibroblasts were treated with vitamin C followed by 2 h of LL-37 treatment. Collagen mRNA expression and total soluble collagen production inhibited by LL-37 was enhanced by treatment with 0.5 mm vitamin C. Vitamin C also decreased intracellular reactive oxygen intermediates (ROI) levels that were increased by LL-37. Furthermore, the phosphorylation of ERK was analysed by Western blot following treatment with vitamin C and LL-37. Vitamin C turned off phosphorylation of ERK that was induced by LL-37. Ets-1 transcriptional factor, which is involved in the regulation of collagen expression by LL-37, was also inhibited by vitamin C. This study shows that vitamin C enhances collagen production by inhibiting the ERK pathway induced by LL-37.

In vitro culture of shoot tip of garlic (Allium sativium L. cv. Seosan) was carried out to find medium condition of the induction of multiple shoots and bulbing for muliproduction of virus-free seed bulbs. For this work, tank culture was introduced. In shoot tip culture on MS solid medium the induction of multiple shoots and bulbing were better by adding 3% sucrose than 8%. Supplementation with 2mg/L 2ip and 0.2 mg/L IAA in this medium was effective. Three point three shoots including 2.7 bulbs were formed from a shoot tip after cultivation for 30 days on this medium. Bulbing of garlic in liquid culture with plastic water tank of 20L supplied air at the side of the lower part was better by adding 3% sucrose than 8% by subculture for 45 days with shoots obtained from shoot tip culture for 30 days on soid MS medium. Shoot growth was vigorous at 3% sucrose however bulb growth was more effective on the medium of 8% sucrose. Because of the effectiveness on solid medium added 3% sucrose, 2 mg/L 2ip and 0.2 mg/L IAA for initial production of multi-shoot in stem tip culture and the effectiveness in liquid culture with water tank for growth of bulbs, the method of two-step culture could be introduced for the multiple production of seed bulb of high quality. It was more desirable by supply of 0.2 mg/L BA and 0.02 mg/L NAA at tank culture time. But growth of the bulbs became poor by increasing concentration of NAA of the medium.

Single-node stem pieces ca. 1 cm in length containing a axillary bud were obtained from in vitro plants of potato (Solanum tuberosum L.). The influences by a position of the node and the existence of a leaf at the node were observed in the single-node culture on the 8% sucrose MS medium. The effect of CCC was also investigated for the microtuberization. The apical part node was excellent in the tuberization not to mention shoot length, fresh weight, diameter, the number of node on the in vitro culture of a single-node than the lower part. The differences in the diameter of a tuber formed in the part of the axillary bud on all treatments including the cultivation of the apical part node were not recognized. However, the fresh weight of the tuber showed high value in the tuber formed at the axillary bud of shoot apex part. At 20 days after cultivation, tuberization was promoted in the new stolen that developed from the bud of node with a leaf under SD condition of 8 hours at . The tuberization from axillary bud of the single-node without leaf was inhibited at high temperature of regardless of daylength. Whereas, tuberization at and was similar without the difference under SD condition but the tuber formation ratio were low. CCC 500 mg/L promoted tuberization and the effect was also showed even under LD condition at . The inhibiton of tuberization under LD and high temperature condition could be solved by treatment with CCC.

Embryogenic calluses derived from shoot apical meristem explants of sweetpotato were subjected to particle bombardment to generate transgenic plants for antisense expression of cDNAs encoding two different AGPase small subunit (ibAGP1 and ibAGP2). Plants were generated via somatic embryogenesis. PCR and Southern analysis demonstrated that the incorporation of ibAGP1 and ibAGP2 into the genome in an antisense orientation. Immunoblot analysis confirmed reduced levels of AGPase small subunit in transgenic plant leaves. Plants with both ibAGP1 and ibAGP2 produced a lower level of the protein than plants with ibAGP1 alone. iodine test demonstrated that transgenic plant leaves and storage root accumulated reduced amounts of starch. Iodine staining of leaf tissues indicated that transgenic plants accumulated less amount of starch than control. In accordance with western blot analysis, plants with both ibAGP1 and ibAGP2 accumulated a lower amount of starch than plants with ibAGP1 alone. Both transgenic plants exhibited a severely retarded growth, resulting in bare survival. It is suggested that disrupted expression of the gene encoding AGPase small subunit is lethal to the growth of sweetpotato contrast to other species including potato.

One-node stem pieces ca. 1 cm in length containing a axillary bud and a fully expanded leaf were obtained from it in vitro plants of potato (Solanum tuberosum L.). Leaves were removed and the nodes were cultured on the MS medium to investigate the effects of temperature, day length, sucrose, and CCC in microtuber formation and development. The fresh weight of microtubers after 80 days increased significantly at 8% sucrose and compared with . The tuberization and development were reduced at except short-day treatment of 8 hours at 8% sucrose. The fresh weight and diameter were increased on the culture medium added CCC 500 mg/L. The potato tuberization was promoted under short daylength, and it showed great effect by treatment with the CCC. Though the tuberization was promoted at low temperature of in a histologic change of an axillary bud part cell of a potato, the cells were able to observe the swelling growth. Swelling growth of tissue was stimulated in the darkness and was more remarkable by addition of CCC. In particular, in the visual ratio of cell division for each position in the tissue, the cortex part showed larger ratio of cell expansion than that of the pith part. The effect of CCC was identified at 8% sucrose in the darkness. The effect of CCC was not showed in sucrose 3% under long daylength of 16 hours. As a result, the fact of a substance with AGPase important for starch composition was certified by the result with the inclose of AGPase activity on high concentration of sucrose, CCC, and dark treatment by which tuber formation and development are promoted.

Poly(vinyl alcohol) (PVA) films embedded with functional polydiacetylene (PDA) are efficiently prepared for color and fluorescence imaging. Intensely blue films are obtained by mixing and drying solutions containing PDA vesicles and PVA. A blue-to-red color transition is observed upon heating the polymer films. In addition, selective UV irradiation (through a photomask) of PVA films containing diacetylene monomer results in the generation of micropatterned color (without heating) and both color and fluorescent images (after heating the films at 120 °C for 10 s). Patterned two-color (blue and red) images in the polymer film are readily obtained by a sequential process of photomasked irradiation, heating, and unmasked irradiation.

In order to evaluate the antifungal activity of bacteria against plant pathogenic fungi, 8 bacteria were isolated from mushroom compost hot spring, seaweed, and forest soil and mutants from them were induced by gamma radiation(). Bacillus circulans K1, Burkholderia gladioli K4 and Bacillus subtilis YS1 showed wide antifungal spectrum against 12 kinds of plant pathogenic fungi. From the radiation sensitivity test, B. gladioli K4 was very sensitive to gamma radiation and its value was 0.11 kGy. Antifungal activities of B. circulans Kl-1004 and B. subtilis YS1-1009, which were induced by the radiation of increased against Botryosphaeria dothidea. The mutant strains, B. subtilis YS1-1006 and B. subtilis YS1-1009 were resistant to tebuconazole and copper hydroxide. SAR535, SAR5108, and SAR5118 mutated from Streptomyces sp. SAR01 were antifungal activity deficient mutants against 5 kinds of plant pathogenic fungi compared to wild strain, so that they could be supposed to be model strains far studying antifungal mechanism. It is suggested that various functional types of mutants could be induced by gamma radiation and applied usefully.

Nasolabial folds are a sign of aging and increasing number of people want filler injections in their nasolabial folds to look younger. Various dermal fillers are used for the correction of nasolabial folds. Recently, a novel injectible filler, polymethylmethacrylate (PMMA) and cross-linked dextran in hydroxypropyl methylcellulose, was introduced for facial contouring. This study was designed as a six-month, prospective, single-blinded, and open-label study in two centers located in Korea. Nineteen Korean patients received the novel filler injections on both nasolabial folds. At Weeks 4, 12, and 24, the efficacy and safety of the dermal filler were evaluated by blinded-investigators using clinical photographs. The mean Wrinkle Severity Rating Scale revealed significant decrease after dermal filler injections at each study visit. The decreased Wrinkle Severity Rating Scale was maintained for 6 months (p < 0.0001). The Global Aesthetic Improvement score showed an improvement greater than 2 in 95% of the per-proto col population 24 weeks after the injections. All patients (100%) experienced an improvement of their nasolabial folds at Week 24. There were no complications related to the novel filler injection. The novel dermal filler, PMMA, and cross-linked dextran in hydroxylpropyl methylcellulose, can be another safe and effective treatment option in the treatment of nasolabial folds.

Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nϵ-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His·Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His·Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd≤ 0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH 7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH 7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.

was added per literwith other growth supplements as follows; 30 mM glucose,0.2 µM thiamine hydrochloride, 0.8 mM L-arginine hydro-chloride and 0.2 mM uracil. Since strain SWM1 is, unlikethe wild type, resistant to chloramphenicol, this antibiotic(60 mg/mL in ethanol) was added just before inoculation toa final concentration of 60 µg/L. One liter cultures in L-broth were grown overnight and used to inoculate 25 litersof medium in a New Brunswick Scientific Pilot Fermenter.The cell were grown at 37

Deoxyhypusine synthase catalyzes the first step in the post-translational synthesis of hypusine [Ne-(4-amino-2-hydroxybutyl)lysine] in eukaryotic translation initiation factor 5A. We recently reported biochemical evidence for a covalent enzyme-substrate intermediate involving a specific lysine residue (Lys329) in human deoxyhypusine synthase (Wolff, E. C., Folk, J. E., and Park, M. H. (1997) J. Biol. Chem. 272, 15865–15871). In an effort to evaluate the role of this enzyme-substrate intermediate in catalysis, we carried out site-directed mutagenesis (Lys to Arg and/or Ala) of the conserved lysine residues in human deoxyhypusine synthase. A drastic reduction in enzyme intermediate formation and enzymatic activities was observed with mutant proteins with substitution at Lys287 but not with those with mutations at residues 141, 156, 205, 212, 226, 251, or 338. Lys to Ala or Lys to Arg substitution at Lys329 totally abolished covalent enzyme-substrate intermediate formation and deoxyhypusine synthesis activity, indicating that Lys329 is the unique site for the enzyme intermediate and that it is absolutely required for deoxyhypusine synthesis in the eukaryotic translation initiation factor 5A precursor. The K329A mutant showed spermidine cleavage activity (∼6% of the wild type enzyme) suggesting that in contrast to deoxyhypusine synthesis, spermidine cleavage can occur without enzyme intermediate formation.

Akt-1 is a protein kinase that contributes to cancer progression by promoting proliferation and inhibiting apoptosis of tumor cells. RX-0201 (Archexin®), a 20-mer phosphorothioate antisense oligonucleotide that inhibits the expression of Akt-1, is currently in a Phase IIa clinical trial for metastatic renal cancer. To further improve the efficacy of RX-0201, folate receptor-targeted lipid coated albumin nanoparticles containing RX-0201 (F-LCAN-RX-0201) were synthesized and evaluated for Akt-1 inhibition and antitumor activity. These nanoparticles are composed of DOTAP/soyPC/TPGS/folate-PEG-DSPE (25:70:4:1), a cationic human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate, and RX-0201. F-LCAN-RX-0201 had a particle size of 108.6±5.8 nm, zeta potential of 10.5±3.2 mV and 71.5% antisense loading efficiency. F-LCAN-RX-0201 in folate receptor-expressing KB cells inhibited cell growth at IC50 of 11.9 ± 4.0 μM while free RX-0201 did not show significant cytotoxicity at concentration up to 250 μM. Cellular uptake and Akt-1 inhibition of F-LCAN-RX-0201 was greater than the non-targeted LCAN-RX-0201 and was partially blocked by excess free folate. Pharmacokinetic studies showed that F-LCAN-RX-0201 had a longer half-life than that of free RX-0201 (30 ± 4 min vs 10 ± 2 min). In a KB xenograft tumor model, F-LCAN-RX-0201 exhibited higher tumor suppressive activity compared to the non-targeted LCAN-RX-0201 at 16 mg/kg (as a RX-0201 amount) without body weight changes. Moreover, the antitumor activity of F-LCAN-RX-0201 at a dose of 16mg/kg (as a RX-0201 amount) was similar to free RX-0201 at a dose of 90mg/kg. In conclusion, LCAN is a highly effective formulation for therapeutic delivery of antisense agents and F-LCAN-RX-0201 could be a potential therapeutic agent for tumors with increased folate-receptor expression. Citation Format: Hong Li, Xinwei Cheng, Yang Liu, Young Bok Lee, Deog Joong Kim, Chang-ho Ahn, Robert J. Lee. Folate receptor-targeted lipid coated albumin nanoparticles (F-LCAN) for therapeutic delivery of RX-0201 (Archexin®), an antisense oligonucleotide against Akt-1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1322.

Several studies have indicated that the DEAD box RNA helicase DDX5/p68 plays several important roles in cancer (1, 2). In particular, p68 that is phosphorylated on Tyr593 has been shown to be associated with cell transformaton, epithelial mesenchymal transition (EMT) and cell migration (3). Therefore, phosphorylated p68 may be a promising target for novel anti-cancer therapeutics. We previously reported that 1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl) aminocarbonyl] piperazine (RX-5902, Supinoxin®) inhibits the growth of cancer cells at low nanomolar concentrations by interacting with phosphorylated p68 on Tyr593, interfering with the phosphorylated p68-β-catenin signaling pathway (4). In this study, we sought to determine whether phosphorylated p68 on Tyr593 plays a key role in RX-5902's ability to inhibit cancer cell growth by knocking down p68. p68-siRNA efficiently down-regulated the expression of phosphorylated p68 on Tyr593 as well as p68 in the triple-negative (TN) breast cancer cell line, MDA-MB-231. Exposure of p68-siRNA-transfected cells to the IC50 concentration of RX-5902 protected MDA-MB-231 cells from the cytotoxic effects of RX-5902, indicating the phosphorylated p68 on Tyr593 is a key molecule for RX-5902 cytotoxic effects. We also examined the tumor growth inhibition (TGI) of RX-5902 in the human TN-breast tumor (MDA-MB-231) xenograft mouse model. Not only did RX-5902 demonstrate potent efficacy in this model but also oral administration with RX-5902 resulted in dose-dependent TGI and extended the overall survival of these animals. Oral administration of 160, 320 and 600 mg/kg of RX-5902 showed 54.4%, 84.4% and 100% TGI, respectively whereas 5 mg/kg of Abraxane (iv) showed only 48.2% TGI at day 29. Further studies demonstrated the inhibitory effects of RX-5902 on cellular motility in MDA-MB-231 in wound healing assays, suggesting the potential function of phosphorylated p68 on Tyr593 in cell migration (5). These data support the potential therapeutic activity of RX-5902 in triple negative breast cancers. A Phase 1 study of RX-5902 on relapse/refractory solid tumors is ongoing. References 1. Fuller-Pace, FV, RNA Biology 10, 121–132 (2013) 2. Dai et al. Journal of Experimental & Clinical Cancer Research, 33, 64-71 (2014 3. Yang et al., Cell, 127, 139–155 (2006) 4. Kost et al., Journal of Cellular Biochemistry ;online: 3 FEB 2015 05:14PM EST | DOI: 10.1002/jcb.25113) (2015). 5. Remenyi et al, presented at 2015 AACR (2015). Citation Format: Kim DJ, Yang MY, Lee YB, Remenyi J, Fuller-Pace F. The anticancer effects of Supinoxin® (RX-5902) in triple-negative breast cancer MDA-MB-231 through phosphorylated p68 on Tyr593. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-13.

Background: Although the therapeutic mechanism of balneotherapy for atopic dermatitis has not been clarified, many atopic patients who visit thermomineral springs have shown clinical improvements. Objective: This study was aimed to evaluate the immunomodulatory effect of thermomineral water balneotherapy on the atopic dermatitis murine model. Methods: The oxazolone-induced atopic dermatitis murine model was used to evaluate the therapeutic effect of balneotherapy with Deokgu thermomineral water compared with distilled water. Histologic evaluation and confocal microscopic imaging were performed to analyze the lesional expression of cluster-of-differentiation (CD)4 and forkhead box p3 (Foxp3). Lesional mRNA expression of interleukin (IL) 33, thymic stromal lymphopoietin (TSLP), and Foxp3 was evaluated by real-time reverse transcription polymerase chain reaction. Results: Compared with the distilled water bath group, confocal microscopic evaluation of CD4 and Foxp3 merged images showed increased expression of regulatory T cells in the thermomineral balneotherapy group. The lesional mRNA level of IL-33 showed a reduced trend in the thermomineral balneotherapy group, whereas the level of mRNA of Foxp3 was increased. TSLP showed a decreased trend in both distilled water and thermomineral water bath groups. There was a trend of reduced expression in lesional IL-33 mRNA but increased cell count of CD4 + Foxp3 + regulatory T cells in thermomineral balneotherapy compared with distilled water bath. Conclusion: Therefore, thermomineral balneotherapy can be an effective and safe adjuvant therapeutic option for atopic dermatitis. (Ann Dermatol 28(2) 192∼198, 2016)

Nonablative lasers are used for dermal collagen remodeling. Although clinical improvements have been reported using various laser devices, the mechanism of dermal collagen remodeling remains unknown.To investigate the effects of energy fluences of the long-pulsed neodymium-doped yttrium aluminum garnet (Nd:YAG) nonablative laser on dermal collagen remodeling and evaluate the dermal collagen remodeling mechanism.Hairless mice were pretreated with ultraviolet B irradiation to produce photo-damage. The laser treatment used a long-pulse 1,064-nm Nd:YAG laser at energy fluences of 20, 40, and 60 J/cm(2) . The amount of dermal collagen and expressions of transforming growth factor beta (TGF-β), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1) of laser treated skin were compared with those of nontreated control skin.The long-pulse Nd:YAG laser treatment increased dermal collagen and significantly increased TGF-β expression. The expression of MMP-1 decreased with low energy fluence. The expression of TIMP-1 was not significantly different.Long-pulsed 1,064-nm Nd:YAG laser increases the dermal collagen in association with the increased expression of TGF-β.

Certain guanyl diamines are effective inhibitors of deoxyhypusine synthase (Jakus, J., Wolff, E. C., Park, M. H., and Folk, J. E. (1993) J. Biol. Chem. 268, 13151-13159), the first enzyme involved in the biosynthesis of the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Evidence that hypusine is implicated in cell growth prompted this study of the cellular effects of these inhibitors. In Chinese hamster ovary (CHO) cells, inhibition of hypusine biosynthesis followed by progressive arrest in cellular proliferation was observed with both N-mono- and N,N'-bisguanyl derivatives of 1,6-diaminohexane, 1,7-diaminoheptane, and 1,8-diaminooctane. Cells treated with these compounds showed no significant change in polyamine distribution, suggesting that the observed growth inhibition is not mediated through an interference with polyamine metabolism. N1-guanyl-1,7-diaminoheptane, the most potent inhibitor of deoxyhypusine synthase both in vitro and in cells, exhibited the highest antiproliferative activity toward CHO cells. No early cytotoxic effects were observed with this inhibitor, and its antiproliferative activity appeared to be reversible. Transport studies showed that N1-guanyl-1,7-diaminoheptane is actively taken up by the polyamine transport system. Mutant CHO cells defective in polyamine transport were found to be resistant to growth inhibition by this compound. The findings suggest that the antiproliferative effect of N1-guanyl-1,7-diaminoheptane is exerted intracellularly through inhibition of hypusine synthesis.

Based on the anticancer activity of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives published in Bioorg. Med. Chem. 2010 , 18 , 7966, we further explored the synthesis of 7 or 8-substituted quinoxalinyl piperazine derivatives. From in vitro studies of the newly synthesized compounds using human cancer cell lines, we identified some of the 8-substituted compounds, for example 6p , 6q and 6r , which inhibited the proliferation of various human cancer cells at nanomolar concentrations. Compound 6r , in particular, showed the lowest IC 50 values, ranging from 6.1 to 17 nM, in inhibition of the growth of cancer cells, which is better than compound 6k (compound 25 in the reference cited above). In order to select and develop a leading compound among the quinoxaline compounds with substitutions on positions 5, 6, 7 or 8, the compounds comparable to compound 6k in in vitro cancer cell growth inhibition were chosen and their pharmacokinetic properties were evaluated in rats. In these studies, compound 6k showed the highest oral bioavailability of 83.4%, and compounds 6j and 6q followed, with 77.8% and 57.6%, respectively. From the results of in vitro growth inhibitory activities and the pharmacokinetic study, compound 6k is suggested for further development as an orally deliverable anticancer drug.