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One mechanism by which lymphoid malignancies resist standard apoptosis-intending (cytotoxic) treatments is genetic attenuation of the p53/p16-CDKN2A apoptosis axis. Depletion of the epigenetic protein DNA methyltransferase 1 (DNMT1) using the deoxycytidine analog decitabine is a validated approach to cytoreduce malignancy independent of p53/p16. In vivo decitabine activity, however, is restricted by rapid catabolism by cytidine deaminase (CDA). We, therefore, combined decitabine with the CDA-inhibitor tetrahydrouridine and conducted a pilot clinical trial in patients with relapsed lymphoid malignancies: the doses of tetrahydrouridine/decitabine used (∼10/0.2 mg/kg orally (PO) 2×/week) were selected for the molecular pharmacodynamic objective of non-cytotoxic, S-phase dependent, DNMT1-depletion, guided by previous Phase 1 studies. Patients with relapsed/refractory B- or T-cell malignancies (n = 7) were treated for up to 18 weeks. Neutropenia without concurrent thrombocytopenia is an expected toxicity of DNMT1-depletion and occurred in all patients (Grade 3/4). Subjective and objective clinical improvements occurred in 4 of 7 patients, but these responses were lost upon treatment interruptions and reductions to manage neutropenia. We thus performed parallel experiments in a preclinical in vivo model of lymphoma to identify regimen refinements that might sustain DNMT1-targeting in malignant cells but limit neutropenia. We found that timed-alternation of decitabine with the related molecule 5-azacytidine, and combination with inhibitors of CDA and de novo pyrimidine synthesis could leverage feedback responses of pyrimidine metabolism to substantially increase lymphoma cytoreduction but with less neutropenia. In sum, regimen innovations beyond incorporation of a CDA-inhibitor are needed to sustain decitabine DNMT1-targeting and efficacy against chemo-resistant lymphoid malignancy. Such potential solutions were explored in preclinical in vivo studies.

The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

Background/aim Chemoresistance is a major consequence of multicycle chemotherapy and can be attributed to constitutive activation of pro-survival signaling pathways. Nitric oxide is a ubiquitous signaling molecule which has been shown to inhibit several pathways involved with survival signaling in cancer cells. We have previously demonstrated the anti-tumor activity of a nitric oxide-donor, nitrosylcobalamin (NO-Cbl), mediated by increased expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. We also demonstrated that a functional Apo2L/TRAIL receptor is necessary for the induction of cell death by NO-Cbl and the Apo2L/TRAIL death receptor DR4 (TRAIL R1) is S-nitrosylated. The aim of the study was to examine the effects of nitric oxide (NO) on nuclear factor kappa B (NF-κB) and determine whether nitric oxide could sensitize drug-resistant melanomas to Apo2L/TRAIL via inhibition of NF-κB or inhibitor kappa B kinase (IKK). Materials and methods Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by the TUNEL assay. The activation status of NF-κB was established by assaying luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. Results NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL, but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα. Conclusion NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of nitric oxide to inhibit NF-κB and potentiate the effects of chemotherapeutic agents, such as Apo2L/TRAIL, represents a promising anti-cancer combination based on recent clinical investigations of anti-TRAIL antibodies for cancer treatment strategies.

Previously, we reported apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), has potent anti-melanoma activity. We used DNA microarray and bioinformatics to interrogate gene expression profiles of tumors from apoA-I expressing (A-I Tg+/–) versus apoA-I-null (A-I KO) animals to gain insights into mechanisms of apoA-I tumor protection. Differential expression analyses of 11 distinct tumors per group with > 1.2-fold cut-off and a false discovery rate adjusted p < 0.05, identified 176 significant transcripts (71 upregulated and 105 downregulated in A-I Tg+/– versus A-I KO group). Bioinformatic analyses identified the mevalonate and de novo serine/glycine synthesis pathways as potential targets for apoA-I anti-tumor activity. Relative to A-I KO, day 7 B16F10L melanoma tumor homografts from A-I Tg+/– exhibited reduced expression of mevalonate-5-pyrophosphate decarboxylase (Mvd), a key enzyme targeted in cancer therapy, along with a number of key genes in the sterol synthesis arm of the mevalonate pathway. Phosphoglycerate dehydrogenase (Phgdh), the first enzyme branching off glycolysis into the de novo serine synthesis pathway, was the most repressed transcript in tumors from A-I Tg+/–. We validated our mouse tumor studies by comparing the significant transcripts with adverse tumor markers previously identified in human melanoma and found 45% concordance. Our findings suggest apoA-I targets the mevalonate and serine synthesis pathways in melanoma cells in vivo, thus providing anti-tumor metabolic effects by inhibiting the flux of biomolecular building blocks for macromolecule synthesis that drive rapid tumor growth.

Myelodysplastic syndromes (MDS) ultimately will progress to a higher-risk form or acute myeloid leukemia. This evolution is accompanied by acquisition of genetic hits following ancestral mutations, with further subclonal diversification of the clonal architecture. Conceptually, therapeutic approaches targeting ancestral hits have the potential to eradicate MDS at early stages of ontogenesis. Founder SF3B1 mutations are frequent in MDS and therefore represent rational targets for drug development. During our drug discovery efforts we identified an existing drug that selectively inhibits the growth of SF3B1 mutant (SF3B1MT) cells. We consequently examined the effects of known compounds possibly arresting clonal expansion of SF3B1MT MDS cells. For our studies, we generated CRISPR/Cas9 knock-in human cells stably expressing the recurrent SF3B1 K700E mutation and a chromophore-tagged reporter (mRFP). Because of the high frequency of SF3B1 mutations and their effects on the splicing of a multitude of genes, we hypothesized that some of the corresponding downstream effects could be effectively targeted. Using luminescent cell viability assays and flow cytometry analysis, we screened a 3,000 compound library for selective sensitivity against isogenic SF3B1MT cells. A subset of this library contained 23 calcium2+ channel blockers (CCBs) of which one specific dihydropirimidine (DHP) showed the highest inhibitory activity against SF3B1MT cells. Several studies point towards a role of calcium signaling in MDS. Proteins active in calcium metabolism (GPR68, calpain, calpastatin) are involved in modulating sensitivity to drugs used in MDS (e.g., lenalidomide). Divalent substrates including Fe2+ may use CCs and it's plausible that our DHP can inhibit iron overload by blocking Fe2+ trafficking through L-type CCs. In this regard, CCBs have been shown to stimulate the activity of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters. Furthermore, it is known that the SF3B1 mutations reduce the expression of the iron transporter ABCB7, leading to increased iron accumulation. All of this led us to further evaluate the effects of DHP in vitro and in vivo. DHP had higher growth inhibitory activity against SF3B1MT cells when tested in vitro using 8 serial concentrations in half-log dilutions for a period of 3 days (20% and 60% growth inhibition at 1 and 3μM). Mixed competitive experiment of K700EmRFP and WTGFP cells treated with increased doses of DHP (1, 3, 5, 10, 20, 50 μM) reduced the competitiveness of K700EmRFP over the time inducing a 3-fold reduction at 50μM. K700EmRFP cells expressed half the amount of ABCB7 mRNA compared to WTGFP cells by RT-PCR. Therapeutic index provided by DHP was determined in vivo. Bone marrow cells of B6. Gt(ROSA)26Sortm1Sor/J (CD45.2) donors were transplanted in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients. Two weeks after transplantation, average engraftment (measured as percentage of CD45.2(+) donor cells) was 96% ± 0.02. DHP (10 mg/Kg) was orally administered. No decrease in the proportion of CD45.2(+) donor cells was seen post-treatment compared to pre-treatment (96±0.01 % vs. 96%±0.009). Similarly no change in the proportion of CD45.2(+) donor cells was observed in competitive repopulation experiments when B6. Gt(ROSA)26Sortm1Sor/J cells were mixed 1:1 with transgenic Sf3b1+/K700E cells (40%±0.13 vs. 44%±0.02). In contrast, preliminary experiments showed that DHP had an effect on reducing Sf3b1+/K700E allele burden in two chimeric mice (to 21% and 24%) compared to pre-treatment. DHP is structurally unique in comparison to other DHP-based CCBs; in that it possess a -CH20-CH2CH2NH2 moiety linked directly to the DHP scaffold that may in itself provide opportunities or modes for Fe2+ chelation as well as its L-type CCB activity. Our observations and structural analyses therefore provide impetus to explore this feature to possibly improve the drug's efficacy. In sum, we have demonstrated that the clonal growth of cells carrying SF3B1 mutations might be suppressed using the known L-type CCB DHP. This might represent a novel modulator of ATPase activity of ABC transporters in SF3B1MT expressing low ABCB7 levels. Disclosures Sekeres: Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

SF3B1 mutations disrupt normal pre-mRNA splicing to cause disease. Drugs inhibiting the interaction between the SF3b complex and RNA or agents degrading auxiliary splicing factors are being tested as new avenues for targeted therapy in myeloid neoplasia (MN) with SF3B1 mutations. Here we describe the ability of small molecules to restore altered RNA processes in SF3B1MT MN. We previously reported (Visconte, ASH 2018) the identification of the small molecule 4-pyridyl-2-anilinothiazole (PAT) which showed growth inhibition of CRISPR/Cas9 SF3B1+/K700E cells and primary SF3B1MT cells. PAT did not influence the growth of other cell models without (THP1, MOLM13FLT3, OCIAML3DNMT3A, SIGM5TET2/DNMT3A, K562PHF6) and with other splicing factor mutations (K562U2AF1, K562LUC7L2). We now describe data from medicinal chemistry, transcriptome, and in vivo studies to advance drug development for SF3B1MT MN. SAR studies focused on logical and systematic modifications of PAT, e.g., i) replacement of the 2,4-disubstituted thiazole spacer ring with other heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); ii) alternative linking groups for the NH linker of the aniline of the tail region (sulfonamide, amide, substituted amine linkers); iii) alternative substituted aromatic and aliphatic ring structures for the phenyl head region substituent, led us to identify permissive sites for further chemical optimization. For example, a 4-chlorophenyl analog demonstrated activity [IC50, 3μM] similar to PAT. Competitive repopulation assays of bone marrow (BM) cells from dual reporters (ACTBtdTomato; EGFP) B6.GtROSA26 mixed with BM cells from conditional knock-in Sf3b1+/K700E mice injected in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients (n=18) were used as a preclinical murine model. This model then allowed i) demonstration of drug efficacy in reducing the competitiveness of SF3B1MT cells and ii) evaluation of therapeutic index in normal hematopoiesis. Post-transplant recovery, recipients of B6.GtROSA26 cells underwent PAT treatment (10 mg/Kg/IP/5 days weekly) for a period of 6 weeks without showing any signs of distress or drug intolerance (drop in blood count, weight loss, abdominal swelling, liver or kidney toxicity). Two weeks after transplantation, donor Sf3b1+/K700E cells had an engraftment capability similar to that of donor B6.GtROSA26 cells (83.6 ± 4 vs. 86.4 ± 2.4) when transplanted as a sole graft in CD45.1 recipients. PAT reduced almost half the percentage of Sf3b1+/K700E donor cells at 6 weeks of treatment (47.4%) vs. pre-treatment (83.6%). In mixed (1:1) BM transplants, Sf3b1+/K700E cellshad a repopulative disadvantage against competitors B6.GtROSA26 contributing for 16% of the marrow reconstitution. Similar to single graft transplants, PAT decreased the percentage of Sf3b1+/K700E cells at 6 weeks vs. pre-treatment (average, 6% vs. 16%) in chimeras. Consistent with the lack of toxicity of PAT treatment B6.GtROSA26 cells in chimeras were not affected by PAT and gradually repopulated the host (post-treatment, 80% vs. pre-treatment, 64%). Subsequently, we focused our efforts identifying important genes known to be dysregulated in MDS that were mostly influenced by drug treatment and minimally affected in normal cells. Our approach was based on the analyses of genes linked to erythropoiesis (a key hallmark of low-risk MDS). In normal hematopoiesis TGF-β signaling inhibits terminal erythroid maturation. Out of 13,775 genes, 5% (664/13,775) were found differentially expressed between CRISPR/Cas9 SF3B1+/K700E and parental cells of which 60% of these genes were significantly up-regulated and 40% down-regulated. Pathway analysis showed that the expression levels of SMAD family of genes and GDF factors changed significantly upon drug treatment. SMAD7 mRNA levels are 3-fold lower in MDS CD34+ cells (n=159) compared to the ones of healthy subjects (n=17) (GEO accession GSE58831) leading to TGF-β over activation. PAT treatment normalized SMAD7 expression levels in CRISPR/Cas9 SF3B1+/K700E cells by 3-fold while reducing the levels of GDF11. In summary, we have identified new drug entities that are modulators of transcriptomic changes which decrease the competitiveness of SF3B1MT cells. These results suggest combination therapies with current TGF-β pathway inhibitors. Disclosures Advani: Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Kelly:Novartis, Bayer, Janssen, Pharmacyclics, Celgene, Astrazeneca, Seattle Genetics: Honoraria, Speakers Bureau; Takeda: Research Funding; Genentech, Verastem: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Pulmonary hypertension (PH) is a frequently under recognized complication of myelofibrosis (MF). The pathophysiology of PH in MF is unknown and no definitive therapies have been established. We studied 15 patients with MF-associated PH and compared their echocardiographic and PH relevant biomarkers (nitric oxide (NO), N-terminal pro-hormone of brain natriuretic peptide (NT-pro BNP), von Willebrand antigen (vWB), ristocetin-cofactor activity (RCA) and uric acid (UA)) pre- and post-ruxolitinib treatment. Ruxolitinib decreased the plasma levels of NT-pro BNP (73%; P=0.043), UA (60%), vWB (86%) and RCA (73%; P=0.036). Improvements in echocardiographic findings were also seen in 66% of patients (P=0.022). Furthermore, marked increase in NO compared with baseline (69.75 vs 40.1 picomolar (pM); P=0.001) was observed post-ruxolitinib therapy, whereas no changes were noted with conventional therapies. Treatment with ruxolitinib also resulted in the reduction of key cytokines (tumor necrosis factor alpha, interleukin-4 (IL-4), IL-6 and IL-8) and induction of interferon-gamma. Animal studies further supported the role of ruxolitinib in the induction of NO levels. In conclusion, aberrant Janus kinase (JAK)-signal transducer and activator of transcription signaling in MF may mediate PH through dysregulation of NO and cytokine levels, which can be restored by therapy with JAK inhibitors suggesting that inhibition of this pathway is a novel target for the management of patients with PH.

Microplasmas have become increasingly attractive for activating chemical reactions because of their advantages over conventional plasma technology and catalytic processes. Advantages of reduced power requirements, atmospheric pressure operation, portability, and the circumventing of catalyst issues have made microplasmas a fascinating alternative for hydrocarbon reforming. Through the course of previous work, these microplasma reactors have displayed process and plasma variability that make characterizing the microplasma reactor challenging. In the current study, 24 experiments were run using 16 different microplasma reactors, with carbon dioxide as the only reactant. Carbon dioxide decomposition was chosen because it provides a simple experimental reaction environment that eliminates the need for a carrier gas and reduces the number of undesirable products. Carbon dioxide decomposition also has potential application for greenhouse gas mitigation. For example, an array of microplasma devices on the exhaus...

Because of its tumor-suppressive effect, interferon-based therapy has been used for the treatment of melanoma. However, limited data are available regarding the antitumor effects of pegylated interferons, either alone or in combination with approved anticancer drugs. We report that treatment of human WM-266-4 melanoma cells with peginterferon beta-1a induced apoptotic markers. Additionally, peginterferon beta-1a significantly inhibited the growth of human SK-MEL-1, A-375, and WM-266-4 melanoma xenografts established in immunocompromised mice. Peginterferon beta-1a regressed large, established WM-266-4 xenografts in nude mice. Treatment of SK-MEL-1 tumor-bearing mice with a combination of peginterferon beta-1a and the MEK inhibitor PD325901 ((R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide) significantly improved tumor growth inhibition compared with either agent alone. Examination of the antitumor activity of peginterferon beta-1a in combination with approved anticancer drugs in breast and renal carcinomas revealed improved antitumor activity in these preclinical xenograft models, as did the combination of peginterferon beta-1a and bevacizumab in a colon carcinoma xenograft model.

On board reforming of hydrocarbons for fuel cell feed has become an attractive research topic due to the low energy densities of batteries. The implementation of a microplasma as a means for reforming the liquid fuel methanol is explored in this work. Hydrocarbon reforming is commonly accomplished through catalysis, but catalysts have a number of limitations such as poisoning, coking, coarsening, long start-up times and excessive costs. Published studies have shown the viability of plasma reforming but none have succeeded in achieving suitable system efficiencies for portable applications. Non-thermal microplasmas are particularly attractive for reforming due to their extremely high electron and power densities and the scale of microplasma devices make them well suited for portable applications. This study describes experimental microplasma reactors reforming methanol. The reactors are based on the microhollow cathode discharge (MHCD) structure fabricated with microelectromechanical systems (MEMS) fabrication techniques. Through modeling the reaction for all five experiments, conversions within the microchannel were found to be nearly 100%. Despite the variations in the five experiments due to input electrical power, flow rate and concentration, the model was validated in each test. The experiments discussed in this work show the promise of a portable, non-thermal microplasma reformer that generates hydrogen for fuel cells for portable power.

A novel approach to chemical process intensification based on microplasma reactor systems is presented. Microplasmas (plasmas generated in sub-mm volumes) possess a favorable reaction environment because of high densities of reactive ions, radicals, and electrons. Moreover, operation under atmospheric pressure and near-room temperature makes them convenient for implementation. Experiments utilized a microfabricated microplasma reactor to intensify the reforming of methanol into a hydrogen-rich product stream useful for energy applications such as combustion or fuel cells. A reaction model elaborates the key mechanisms and demonstrates that reaction rates in the microplasma environment surpass those of conventional plasma reactors. The outcome is that microplasma chemical processing is a viable approach for hydrocarbon reforming and merits consideration for other chemical reaction applications.

Drug resistance is a major obstacle in cancer treatments and diminishes the clinical efficacy of biological, cytotoxic, or targeted therapeutics. Being an antiapoptotic mediator of chemoresistance in breast and lung cancer cells, MKP1 phosphatase might be targeted for overcoming chemoresistance and improving therapeutic efficacy. In this work, tyrosine phosphatase inhibitor-3 (TPI-3) was identified as a novel small molecule inhibitor of MKP1 and was capable of sensitizing tumors to bio- and chemotherapeutics in mice as a tolerated oral agent. Effective against recombinant MKP1, TPI-3 selectively increased MKP1 phosphosubstrates in Jurkat cells and induced cell death via apoptosis at nanomolar concentrations. TPI-3 also increased MKP1 phosphosubstrates in WM9 human melanoma cells and synergized with biotherapeutic IFNα2b in the growth inhibition of melanoma cells in vitro (combination index

There is an increasing evidence that long-term alcohol intake can promote damage to most of the body's major organs. However, regular consumption of a small-moderate amount of alcohol is often recommended as being beneficial to health and of concern is that the effect of ingesting commercially available alcohol products on steroid hormone synthesis under variable nutritional conditions has not been thoroughly investigated. Many individuals consume alcohol alone prior to a meal and the aim of the present study was to assess the effect of consuming a small-moderate amount of commercially available alcohol on the level of salivary cortisol and salivary dehydroepiandrosterone sulfate (DHEAS) before and after a meal. A total of 24 males aged 19–22 years participated in the current investigation. The experimental procedure required participants to fast for 6 h before being asked to ingest either 40 g alcohol in the form of red wine ( n = 8), low alcohol and high beer ( n = 8), white wine ( n = 8) or the equivalent amount of placebo over a 135-min period before consuming food for 45-min. The level of blood alcohol, salivary cortisol and salivary DHEAS was assessed upon arrival and then at regular 45-min intervals during the 180-min experimental period. The results showed that the consumption of alcohol and placebo can significantly lower the level of salivary cortisol. However, the effect of consuming a small-moderate amount of commercially available alcohol on the level of salivary DHEAS was dependent on the nutritional content of the beverage with red wine promoting no change, white wine promoting a significant decrease, and beer having a variable effect on salivary DHEAS concentration when compared to placebo. It was concluded that the effect of commercially available alcohol on the HPA axis is not the same for all alcohol products and both the nutritional status of participants and the nutritional content of the alcoholic beverage being administered should be taken into consideration when investigating the effect of alcohol on the HPA axis.

Metal-organic chemical vapour deposition (MOCVD) of various phases in PrO x system has been studied in relation with deposition temperature (450–750 °C) and oxygen partial pressure (0.027–100 Pa or 0.2–750 mTorr). Depositions were carried out by pulsed liquid injection MOCVD using Pr(thd) 3 (thd = 2,2,6,6-tetramethyl-3,5-heptanedionate) precursor dissolved in toluene or monoglyme. By varying deposition temperature and oxygen partial pressure amorphous films or various crystalline PrO x phases (Pr 2 O 3 , Pr 7 O 12 , Pr 6 O 11 ) and their mixtures can be grown. The pure crystalline Pr 2 O 3 phase grows only in a narrow range of partial oxygen pressure and temperature, while high oxygen pressure (40–100 Pa) always leads to the most stable Pr 6 O 11 phase. The influence of annealing under vacuum at 750 °C on film phase composition was also studied. Near 90% step coverage conformity was achieved for PrO x films on structured silicon substrates with aspect ratio 1:10. In air degradation of Pr 2 O 3 films with transformation to Pr(OH) 3 was observed in contrast to Pr 6 O 11 films.

The removal of carbon dioxide (CO2) from industrial emissions has become essential in the fight against climate change. In this study, we employed Zeolite 13X for the capture and recovery of CO2 in a flow through system where the adsorbent was subjected to five adsorption-desorption cycles. The influent stream contained 1.5% CO2 at standard conditions. The adsorbent bed was 1 in. in length and 1 in. 3/8 in dia., and was packed with 10 g of the zeolite. Temperature swing adsorption (TSA) was employed as the regeneration method through heating to approximately 135 °C with helium as the purge gas. The adsorbent capacity at 90% saturation was found to decrease from 78 to 60gCO2/kgZeolite13X after the fifth cycle. The CO2 capture ratio or the mass of CO2 adsorbed to the total mass that entered the system decreased from 63% to only 61% after the fifth cycle. The CO2 recovery efficiency ranged from 82 to 93% during desorption, and the CO2 relative recovery, i.e., CO2 desorbed for the nth cycle to CO2 adsorbed for the first cycle, ranged from 88 to 68%. The service life of the adsorbent was determined to be equal to eleven cycles at a useful capacity of 40gCO2/kgZeolite13X. © 2007 American Institute of Chemical Engineers AIChE J, 2007

Background— Ultrasound can increase tissue blood flow, in part, through the intravascular shear produced by oscillatory pressure fluctuations. We hypothesized that ultrasound-mediated increases in perfusion can be augmented by microbubble contrast agents that undergo ultrasound-mediated cavitation and sought to characterize the biological mediators. Methods and Results— Contrast ultrasound perfusion imaging of hindlimb skeletal muscle and femoral artery diameter measurement were performed in nonischemic mice after unilateral 10-minute exposure to intermittent ultrasound alone (mechanical index, 0.6 or 1.3) or ultrasound with lipid microbubbles (2×10 8 IV). Studies were also performed after inhibiting shear- or pressure-dependent vasodilator pathways, and in mice with hindlimb ischemia. Ultrasound alone produced a 2-fold increase ( P P P 2a receptors and epoxyeicosatrienoic acids had minimal effect. Limb nitric oxide production and muscle phospho-endothelial nitric oxide synthase increased in a stepwise fashion by ultrasound and ultrasound with microbubbles. In mice with unilateral hindlimb ischemia (40%–50% reduction in flow), ultrasound (mechanical index, 1.3) with microbubbles increased perfusion by 2-fold to a degree that was greater than the control nonischemic limb. Conclusions— Increases in muscle blood flow during high-power ultrasound are markedly amplified by the intravascular presence of microbubbles and can reverse tissue ischemia. These effects are most likely mediated by cavitation-related increases in shear and activation of endothelial nitric oxide synthase.

Preclinical studies had determined that tamoxifen and interferon-alpha2b (IFN-alpha2b) synergistically inhibited growth of both estrogen-receptor positive and negative murine tumor xenografts and had combined antiangiogenic effects and that tamoxifen potentiated IFN-stimulated gene (ISG) expression. A phase I trial in 26 patients was conducted using the combination to define tolerance and potentiation of ISG expression. IFN- alpha2b at a dose of 3 x 10(6) units/m(2) daily was given subcutaneously (s.c.), and tamoxifen was initiated as a loading dose of 150 mg/m(2) and then 60 mg/m(2) twice daily on day 8. At this initial dose, reduction of dose of IFN- alpha2b was required in 4 of 11 patients, primarily because of fatigue. Another group of patients was treated with an identical tamoxifen dose but with IFN-alpha2b reduced to 2 x 10(6)/m(2) U; this was better tolerated. As the projected serum tamoxifen level to reduplicate preclinical effects was 300 mg/m(2), dose escalation in a third cohort was undertaken; it had to be discontinued secondary to grade III or IV toxicity in 2 of 2 patients. Increases in products of transcriptionally regulated ISGs, beta (2)-microglobulin, neopterin, and ISG15 were assessed. All ISGs increased after IFN-alpha2b, but only ISG15 had a further significant rise after initiation of tamoxifen. Because at doses not limited by unacceptable toxicities, no marked potentiation of ISGs by tamoxifen could be identified, clinical evaluation of the combination was terminated.

The remanent magnetization and the magnetic anisotropy energy of 5–16 monolayer (ML) epitaxial Fe films grown on GaAs(001) were quantitatively monitored in situ by superconducting quantum interference device magnetometry and ferromagnetic resonance as a function of oxygen exposure at 300 K. We find that at an oxygen dose of 10 L the perpendicular uniaxial anisotropy decreases by almost 50%, whereas the magnetization is decreased by 8% only in the case of a 5 ML Fe film. The fourfold anisotropy constant K4 and in-plane uniaxial anisotropy constant , however, remain unchanged. Low-energy electron diffraction spots of the Fe surface disappear after 6 L of oxygen, indicating a disordered Fe oxide growth. For exposures below 70 L we observe a reduction of the remanent magnetization irrespective of the film thickness. At exposures >70 L we find a faster decrease of the remanent magnetization for the thinner films which is attributed to the finite size effect and a thickness dependent surface roughness of the iron films, respectively. As evidenced by Auger electron spectroscopy and x-ray diffraction, the iron oxide is found to be FeO in the first stage of oxidation, transforming into γ-Fe2O3 at higher dosages (>1000 L).

IL-2 therapy results in 10–20% response rates in advanced renal cell carcinoma (RCC) via activating immune cells, in which the protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) is a key negative regulator. Based on finding that sodium stibogluconate (SSG) inhibited SHP-1, the anti-RCC potential and action mechanism of SSG and SSG/IL-2 in combination were investigated in a murine renal cancer model (Renca). Despite its failure to inhibit Renca cell proliferation in cultures, SSG induced 61% growth inhibition of Renca tumors in BALB/c mice coincident with an increase (2-fold) in tumor-infiltrating macrophages (Mφ). A combination of SSG and IL-2 was more effective in inhibiting tumor growth (91%) and inducing tumor-infiltrating Mφ (4-fold), whereas IL-2 alone had little effect. Mφ increases were also detected in the spleens of mice treated with SSG (3-fold) or SSG/IL-2 in combination (6-fold), suggesting a systemic Mφ expansion similar to those in SHP-deficient mice. T cell involvement in the anti-Renca tumor action of the combination was suggested by the observations that the treatment induced spleen IFN-γ T cells in BALB/c mice, but failed to inhibit Renca tumor growth in athymic nude mice and that SSG treatment of T cells in vitro increased production of IFN-γ capable of activating tumoricidal Mφ. The SSG and SSG/IL-2 combination treatments were tolerated in the mice. These results together demonstrate an anti-Renca tumor activity of SSG that was enhanced in combination with IL-2 and functions via a T cell-dependent mechanism with increased IFN-γ production and expansion/activation of Mφ. Our findings suggest that SSG might improve anti-RCC efficacy of IL-2 therapy by enhancing antitumor immunity.

Objective: The protocol was designed to examine the biological effects and clinical activity of interferon-β in patients with platinum/taxane-resistant ovarian cancer. Methods: Patients with resistant ovarian and fallopian tube cancers and primary peritoneal carcinoma were treated with recombinant human interferon-β (Rebif®, Serono International) at doses ranging from 6 to 24 million international units (MIU)/day, based on their tolerance to therapy. Levels of IP-10, an interferon-inducible protein, were measured in the serum to evaluate the biological effects of the drug. Also, the peripheral blood mononuclear cells and serum were examined for the induction of previously described novel regulators of interferon-induced death. Results: Eighteen patients were treated, of whom 9 (50%) could be treated at the highest dose level (24 MIU). The major toxicities were fever, chills and fatigue. The median duration of therapy was 6 weeks (range 1–22). No objective responses were observed. IP-10 levels were significantly increased, compared with baseline, at 2, 4, and 6 weeks after initiation of therapy (p < 0.01). Conclusions: Recombinant human interferon-β produced a definite biological effect in the serum of treated patients, but this outcome was not translated into any clinically observable or meaningful impact on the disease process.

Interferons (IFNs), pleiotropic cytokines that regulate antiviral, antitumor, apoptotic, and cellular immune responses, were the first endogenous antiangiogenic regulators identified. In a species-specific manner, IFNs inhibit secretion of such angiogenic factors as basic fibroblast growth factor from tumor cells. The antiangiogenic activity of IFNs is enhanced when they are combined with other antiangiogenic agents, such as tamoxifen and thalidomide.

Cancer cell resistance limits the efficacy of IFNs. In this study, we show that sodium stibogluconate (SSG) and IFN-α synergized to overcome IFN-α resistance in various human cancer cell lines in culture and eradicated IFN-α-refractory WM9 human melanoma tumors in nude mice with no obvious toxicity. SSG enhanced IFN-α-induced Stat1 tyrosine phosphorylation, inactivated intracellular SHP-1 and SHP-2 that negatively regulate IFN signaling, and induced cellular protein tyrosine phosphorylation in cancer cell lines. These effects are consistent with inactivation of phosphatases as the basis of SSG anticancer activity. Characterization of SSG by chromatography revealed that only selective compounds in SSG were effective protein tyrosine phosphatase inhibitors. These observations suggest the potential of SSG as a clinically usable protein tyrosine phosphatase inhibitor in cancer treatment and provide insights for developing phosphatase-targeted therapeutics.

The interferon (IFN)-β and all-trans-retinoic acid combination suppresses tumor growth by inducing apoptosis in several tumor cell lines. A genetic technique permitted the isolation of human thioredoxin reductase (TR) as a critical regulator of IFN/all-trans-retinoic acid-induced cell death. Our recent studies have shown that TR1:thioredoxin 1-regulated cell death is effected in part through the activation of p53-dependent responses. To understand its death regulatory function, we have performed a mutational analysis of TR. Human TR1 has three major structural domains, the FAD binding domain, the NADPH binding domain, and an interface domain (ID). Here, we show that the deletion of the C-terminal interface domain results in a constitutive activation of TR-dependent death responses and promotes p53-dependent gene expression. TR mutant without the ID still retains its dependence on thioredoxin for promoting these responses. Thus, our data suggest that TR-ID acts as a regulatory domain.

The nonamethylene bridge of the title compound shows high conformational mobility and an asymmetric structure in the crystal. Molecular mechanics (MM+) calculations reproduced well the crystal structure, which has the lowest energy conformation.

To investigate the function of mGBP-2, a member of the interferon (IFN)-induced guanylate-binding protein family of GTPases, NIH 3T3 fibroblasts were generated that constitutively expressed mGBP-2. mGBP-2 induced a faster growth rate, with the highest expressing clones showing approximately a 50% reduction in doubling time. mGBP-2-expressing cells also grew to higher density and exhibited partial loss of contact growth inhibition, as evidenced by the formation of foci in post-confluent cultures. In addition, mGBP-2-expressing cells showed decreased dependence on serum-derived growth factors. However, they did not lose the requirement for anchorage-dependent growth. Finally, NIH 3T3 cells expressing mGBP-2 formed tumors in athymic mice. An mGBP-2 protein carrying a point mutation (S52N) that reduced GTP binding failed to produce these phenotypes when expressed at the same levels as wild type. The additional finding that IFN-γ treatment of NIH 3T3 cells resulted in an increase in proliferation similar to that observed for mGBP-2 in the absence of other IFN-induced proteins suggests that mGBP-2 may indeed be important for these growth changes.

Platinum (Pt), ruthenium (Ru), and ruthenium dioxide (RuO 2 ) have been considered as possible candidates as electrodes for high permittivity and ferroelectric oxides. The films were grown by metal organic chemical vapor deposition (MOCVD). High purity Pt films with near bulk resistivities (11 w z cm) were deposited. The Pt, Ru and RuO 2 films were found to be continuous and smooth and exhibited excellent adhesion properties on the underlying substrates. The temperature dependent electrical conductivity of the Ru and RuO 2 films was metallic type. Room temperature resistivities as low as 18 and 30 w z cm were determined for Ru and RuO 2 , respectively.

Intratumoral Prostate cancer (PCa) heterogeneity is characterized by multiple intrinsic mechanisms including Androgen signaling, aerobic glycolysis, aberrant transcriptional activation of c-Myc and dysregulation of miRNAs. The ability of prostate cancer cells to alter growth and metabolism in a manner distinct from benign cells is a major concern in the treatment of castration-resistant prostate cancer. Therefore, targeting the diverse oncogenesis promoting pathways involved in PCa progression and resistance at the posttranscriptional level using micro RNAs (miRNAs) offers a novel therapeutic option. Using a miRNA mimic screen, we identified a human-specific miRNA miR-644a, which modulates expression of a diverse set of oncogenic pathways in PCa including androgen signaling, AKT/IGF-1R signaling, c-myc mediated signaling and EMT pathways. We have also demonstrated a potent tumor suppressive function of miR-644a in mouse prostate cancer xenograft model. miR-644a also sensitizes the PCa tumors to current therapeutic options including AR antagonist Enzalutamide by regulating the key modulators in development of drug resistance. In conclusion, our study demonstrates that miR-644a is a novel tumor suppressor miRNA regulating key prostate carcinogenesis promoting pathways. Citation Format: Jey Sabith Ebron, Jagjit Singh, Eswar Shankar, Crystal Weyman, Sanjay Gupta, Daniel J. Lindner, Girish C. Shukla. Fine-tuning the expression of heterogeneous network of genes involved in androgen signaling, aerobic glycolysis, apoptosis and epithelial-mesenchymal transition by microRNA-644a in prostate cancer potentiation of AR signaling therapy by miR-644a: Selective manipulation of the prostate cancer transcriptome by miR-644a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1470. doi:10.1158/1538-7445.AM2017-1470

Prostate cancer growth and proliferation depends on androgen signaling mediated by transactivation of Androgen Receptor (AR). Androgen ablation remains the mainstay therapy for treatment of the disease. However, despite androgen ablation, the disease relapses to more aggressive form known as castration-resistant prostate cancer (CRPC). Androgen Signaling Inhibitor such as Abiraterone Acetate and Enzalutamide are the most effective treatment methods currently being used to treat CRPC. However, more than one-third of CRPC patients develop resistance to these treatments, mostly due to the gain of function in the AR protein and increase in intratumoral dihydrotestosterone (DHT) synthesis. Intratumoral DHT synthesis from steroid precursors in tumors is augmented by up-regulation of enzymes of cholestrogenesis and steroidogenesis, such as HMG-CoA reductase, SCARB1, and 17βHSD respectively. Tumor-specific downregulation of microRNAs which regulate the AR and steroid biosynthesis has been implicated in tumor growth and resistance to therapeutics in CRPC. We are focusing on tumor suppressive role of miR-149-5p in PCa. We discovered that miR-149-5p expression was significantly lower in prostate cancer tissues compared to normal tissues. The mechanistic investigation revealed that miR-149-5p downregulates wild type and alternatively spliced variant of AR. It also down regulates the expression of SCARB1, HMGCS and HMG-CoA reductase, the proteins known to facilitate intratumoral DHT synthesis. Ectopic expression of miR-149-5p negatively regulated the expression of prostate-specific antigen, a downstream target of androgen signaling and it also inhibited invasion and proliferation of CRPC cells. Our findings indicate a significant role of miR-149-5p in regulating androgen signaling and possibly DHT synthesis in CRPC. This provides a strong rationale for further investigating the significance of miR-149-5p for generation of new therapeutic for CRPC. Citation Format: Savita Singh, Jey Sabith Ebron, Eswar Shankar, Sanjay Gupta, Daniel Lindner, Girish Shukla. Regulation of androgen signaling axis and tumor suppressive function of miR-149-5p in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2531. doi:10.1158/1538-7445.AM2017-2531

It was demonstrated by X-ray diffraction analysis that the reaction of 3,3-dimethyl-6-trifluoromethyl-2,3-dihydro-4-pyrone with hydroxylamine afforded 5-hydroxyamino-3-(2-hydroxy-1,1-dimethylethyl)-5-trifluoromethyl-Δ2-isoxaline.

MOCVD is a suitable technique for the synthesis of functional metal oxide thin films. In the present paper, injection-MOCVD was proved to be an efficient method, not only to grow single layers, but also heterostructures and superlattices of functional oxide materials. We report on the deposition of BaxSr1−xTiO3 (BST) films and BaTiO3/SrTiO3 superlattices [(BTO/STO)n] obtained by injection MOCVD. In a first step, BST films were deposited on various substrates such as LaAlO3(LAO)(012), SrTiO3:Nb(STO:Nb)(001) and Al2O3(0001), resulting in epitaxial films in the case of STO or LAO substrates and highly textured films in the case of Al2O3 substrates. The good crystallinity of the films obtained was evidenced using X-ray analysis. Concerning the dielectric properties of BST films on STO:Nb, the Tc values were found to be the same as those for bulk materials. In a next step the feasibility of (BTO/STO)n superlattices was studied. The multilayers obtained were epitaxially grown on LAO and STO:Nb substrates. XPS was used to check in detail the pattern repetition of the superlattice and proved the low interface roughness of 1 nm as well as the absence of carbon contamination in the film. The measured periodicity of these superlattices were found to be in good agreement with simulation experiments. Further superlattices have been obtained by combining ferroelectric, magnetic (CMR) and superconducting (HTS) oxides. Typical results of these superlattices will be reported.

New BaTiO 3 -SrTiO 3 (BTO-STO)-superlattices which may be interesting for future electronic applications have been investigated by X-ray photoelectron spectroscopy (XPS) depth profiling. At first XPS measuring conditions were optimized for that non-conducting and thin layer systems (21 nm double layer thickness) considering the practical instrumental limitations. Second a simulation of the sputtering process for the concrete experimental conditions were done by a dynamic T-DYN code. By comparison of experimental and simulated depth profiles the maximum sample roughness could be estimated to be in the range of 2 nm.

Monocyclic conjugated molecules have stabilities, bond lengths, and magnetic properties following expectations from Huckel's Rule. Two hydrocarbons which seem to deviate from these generalizations, s-indacene and cyclohepta(def)fluorene, were studied with density functional theory. The former has characteristics expected for both aromatic and antiaromatic molecules; the latter is a ground state triplet. Potentially aromatic transition states with 10 electrons were also investigated. The mechanism of the 5,5-sigmatropic shift is predicted to be stepwise involving diradical intermediates.

BaTiO 3 (BTO) and SrTiO 3 (STO) layers were deposited by injection MOCVD using both a mixed Ba 2 Ti 2 precursor (dissolved in hexane) and a Sr(thd) 2 , Ba(thd) 2 and Ti(OPr) 2 (thd) 2 precursor system (dissolved in monoglyme). Films have been deposited at different temperatures between 600°C and 850°C. The microstructural properties of the films obtained on different kinds of substrates MgO (100). LaAlO 3 (012), sapphire (1-102), Si (100), Pt on Si and YBa 2 Cu 3 O 7 (YBCO) on LaAlO 3 are compared.

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced β1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of β1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between β1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.—Sossey-Alaoui, K., Pluskota, E., Davuluri, G., Bialkowska, K., Das, M., Szpak, D., Lindner, D. J., Downs-Kelly, E., Thompson, C. L., Plow, E. F. Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis.

Inhibition of angiogenesis by anti-tumor agents may play a role in tumor growth arrest. Tamoxifen and interferon-alpha/beta (IFN-alpha/beta) exhibit potentiated anti-proliferative activity against tumor cells. However, additional host-mediated effects such as modulation of angiogenesis may also inhibit tumor growth in vivo. The effect of tamoxifen and IFN-beta on angiogenesis induced by 2 human tumors, MCF-7 breast carcinoma (estradiol dependent) and NIH-OVCAR-3 ovarian carcinoma (estradiol independent), was assessed. Treatment of nude mice bearing MCF-7 tumors with tamoxifen resulted in a 68% decrease in the number of vessels at the tumor periphery. Treatment with IFN-beta yielded a 33% reduction. Treatment of nude mice bearing NIH-OVCAR-3 tumors with tamoxifen resulted in a 73% decrease in the number of vessels. Treatment with IFN-beta yielded a 57% reduction. Combination treatment resulted in augmented anti-angiogenic effects. As single agents, both tamoxifen and IFN-beta inhibited xenograft tumor growth. Ten weeks of tamoxifen treatment resulted in growth inhibition of MCF-7 and NIH-OVCAR-3 carcinomas by 85% and 66%, respectively. Ten weeks of IFN-beta treatment resulted in inhibition of growth of MCF-7 and NIH-OVCAR-3 carcinomas by 67% and 88%, respectively. The combination of tamoxifen and IFN-beta completely prevented growth of MCF-7 and NIH-OVCAR-3 carcinomas. The anti-angiogenic effects of tamoxifen and IFN-beta were additive. Inhibition of angiogenesis was detectable before measurable effects on tumor volume in both MCF-7 and NIH-OVCAR-3 tumors. Potentiation of anti-angiogenic effects by tamoxifen and IFN-beta, possibly resulting from enhanced IFN-induced gene expression, may contribute to anti-tumor activity in both estradiol-dependent and estradiol-independent tumors in vivo.

Interferons (IFN) and retinoids failed to inhibit the growth of a number of breast tumor cell lines. However, a combination of these two biological response modifiers significantly suppressed the cell growth at pharmacologically achievable doses. The molecular basis for such enhancement was investigated in MCF-7, a breast tumor cell line resistant to growth inhibition by IFN-β. Pretreatment of cells with retinoic acid (RA) for 16 h followed by IFN-β, but not the converse, induced cytotoxic effects in the cells. Continuous presence of RA was not necessary, although it enhanced the degree of cell death when present. Further analyses revealed that IFN-β failed to activate IFN-stimulated gene transcription. However, IFN-β strongly up-regulated the gene expression in RA-pretreated cells. Both IFN-β- and IFN-γ-inducible gene expression were enhanced via a modulation of the transcriptional factor IFN-stimulated gene factors-3 and GAF binding to respective cognate regulatory elements. STAT1 was undetectable in these cells prior to RA treatment. RA increased the levels of this crucial regulator, thereby restoring IFN responses. Thus, RA augmentation of STAT1 may be an early step in the cooperative anti-tumor effects of IFN and RA.

A fully automated method for separation of L-[methyl-11C]Methionine from metabolites in patient plasma was developed. L-[methyl-11C]Methionine was isolated from plasma by solid-phase extraction (SPE). The radioactivity retained on the SPE column was eluted and injected onto the HPLC system for separation of in vivo formed L-[methyl-11C]methionine radiolabeled metabolites. The yield through the isolation procedure and HPLC analysis was greater than 95% with a precision better than 5% (R.S.D.). The calculated rate of L-[methyl-11C]methionine transport into tumor tissue was markedly different with and without compensation for radiolabeled metabolites in patient plasma.

We have successfully prepared sense expression libraries, characterized the size of their inserts which are fused to a green fluorescent protein tag, and packaged these libraries into lentivirus. Four different human MDS bone marrow isolates have been transfected with lentivirus at multiplicity of infection of 0.1. A collection of 252 puromycin resistant clones were isolated, genomic DNA was prepared, and clones were screened for ability to undergo myeloid differentiation in response to GM-CSF. Only 30% of the puromycin resistant clones (78) acquired this phenotype. Transfection of MDS cells with antisense libraries did not generate any clones that acquired the desired (differentiative) phenotype. PCR was used to identify presence of the trans gene in the 78 clones. DNA sequencing has identified the cDNA inserts. These inserts were expressed in na ve MDS cell pools. Eight of these cDNAs were validated; they induced myeloid colonies in vitro and engrafted in the marrow of SG3, but not NSG mice.

Breast cancer is the most commonly diagnosed cancer in women in the United States. There is a continued need for the development of selective and specific treatment options for all types of breast cancer, including hormone-dependent and triple-negative subtypes. Recombinant human Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (rhTRAIL), the optimized form of the endogenous death ligand, shows potential as an effective anti-cancer therapeutic due to its ability to induce apoptosis in cancer independent of wild-type p53 function, while displaying minimal toxicity to normal cells. However, a majority of breast cancer cell lines exhibit resistance to TRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors 4 and 5. To overcome TRAIL resistance, a cotreatment option has been explored utilizing the natural compound Quercetin (Q). Q is a flavonol found in certain fruits, vegetables, and teas. As a single agent, Q has been shown to have antiproliferative and pro-apoptotic effects on a variety of cancer cell lines. The aim of this study is to examine the capacity of Q to enhance TRAIL's pro-apoptotic and antiproliferative effects on breast cancer cells. Sulphorhodamine B (SRB) assays were performed on hormone dependent (MCF-7) and triple negative (BT-20) breast cancer cell lines to determine if the cotreatment of Q and TRAIL hinders cell growth. Growth for both MCF-7 and BT-20 cells was substantially inhibited by single agent Q treatments (12.5 μM; ∼20%, 25 μM; ∼40%, 50 μM; ∼60%) but not by single agent TRAIL treatment (100 ng/mL; Citation Format: Jasmine M. Manouchehri, Michael Kalafatis, Daniel Lindner. Evaluation of the efficacy of TRAIL plus quercetin as a potential breast carcinoma therapeutic. [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 1295.

Inositol hexakisphosphate kinase 2 (IP6K2), a member of the inositol hexakisphosphate kinase family, functions as a growth suppressive and apoptosis-enhancing kinase during cell stress. We created mice with a targeted deletion of IP6K2; these mice display normal embryogenesis, development, growth and fertility. Chronic exposure to the carcinogen 4-nitroquinoline 1-oxide (4-NQO, a UV-mimetic compound) in drinking water resulted in fourfold increased incidence of invasive squamous cell carcinoma (SCC) formation in the oral cavity and esophagus of the knockout (KO) mice compared to the wild-type (WT) littermates. Paradoxically, KO mice displayed relative resistance to ionizing radiation and exhibit enhanced survival following 8-10 Gy total body irradiation. Primary KO fibroblasts displayed resistance to antiproliferative effects of interferon-beta and increased colony forming units following ionizing radiation. Radioresistance of KO fibroblasts was associated with accelerated DNA repair measured by comet assay. Direct microinjection of 5-PP-Ins(1,2,3,4,6)P(5) (the enzymatic product of IP6K2), but not InsP(6) (the substrate of IP6K2) induced cell death in SCC22A squamous carcinoma cells.

Abnormal self-renewal and differentiation are defining features of acute myeloid leukemia (AML). Using models of hematopoiesis, we show that persistent self-renewal that ignores differentiation signals can be produced through two distinct pathways. One pathway prevents repression of stem cell genes, producing daughter cells that resemble parental stem cells despite differentiation inducing lineage-specifying factor or cytokines. Runx1 deficiency, a frequent abnormality in AML, demonstrates the second pathway, which allows cytokine/lineage-specifying factor mediated repression of stem cell genes but impairs subsequent activation of differentiation genes that terminate transit-amplification, producing self-renewal in daughter cells that do not resemble the parental stem cell. Use of this pathway by AML cells has therapeutic relevance: cell-fate regulation by the chromatin-modifying protein DNMT1 is differentiation-phase dependent. In the post lineage-commitment differentiation-phase at which AML cell self-renewal occurs, DNMT1 has an opposite role than in normal stem cells. Consequently, DNMT1 depletion, using a clinically applicable method, terminates AML cell self-renewal but increases normal stem cell self-renewal.

Direct separation of enantiomeric amino-alcohols using tartaric acid derivatives as chiral complexing agents in organic stationary or mobile phases is described. Among the tartaric acid derivatives studied, only tartrates having hydroxyl groups attached to the asymmetric carbon atoms (C2) and (C3) free, gave enantioselective retention for epherrine stereoisomers. (2R,3R)-di-n-butyl tartrate (DBT) dissolved inn-hexane as stationary liquid phase gave higher stereoselectivity than DBT in dichloromethane. Both hydrophilic (Nucleosil CN) and hydrophobic (porous graphitic carbon) solid supports were found to affect the retention and enantioselectivity when using a chiral liquid stationary phase. A retention model is presented and used qualitatively in the study of the effect of DBT concentration, organic solvent, addition of a competing amine and packing material on retention and stereoselectivity.

Model molybdenum disulfide-based solid state synthesis catalysts have been prepared and tested catalytically in the HDS of thiophene. Both promoted and sulfur-deficient samples were prepared and the kinetic analyses were done at temperatures ranging from 473–673 K and atmospheric pressure. Structural characterization was carried out using X-ray diffraction and analytical and high resolution electron microscopy. The results indicate that the active catalysts possess a high degree of disorder in the (1010), (1011), (1012) directions, which represent the edges of the MoS2 crystal. This work also establishes that both Group VIII promotion and sulfur deficiency lead to similar structural changes in the molybdenum sulfide lattice of this specific solid state system. These structural traits can be correlated with increased HDS activity.

Signal transducers and activators of transcription 3 (STAT3) was originally identified as a transcription factor that mediates cytokine-induced responses. In these pathways, Janus-activated kinase (JAK)–induced transient tyrosine phosphorylation of STAT3 promotes gene expression in response to a number of cytokines, which is inhibited by feedback mechanisms. A number of studies have shown that STAT3 is constitutively activated in human cancer cells, leading to cell proliferation. It is unclear, apart from a chronic tyrosyl phosphorylation of STAT3, what mechanisms contribute to the STAT3 deregulation in tumors. Earlier, we have isolated a novel growth inhibitory gene product, gene associated with retinoid-IFN–induced mortality 19 (GRIM-19), using a genetic approach. GRIM-19 is an IFN/retinoic acid–regulated growth suppressor. Subsequent analyses have shown that GRIM-19 binds to STAT3 and prevents interleukin-6–induced transcription of cellular genes. However, its effects on a constitutively active STAT3 and cellular transformation are unknown. In this study, we show that GRIM-19 suppresses constitutive STAT3-induced cellular transformation in vitro and in vivo by down-regulating the expression of a number of cellular genes involved in cell proliferation and apoptosis. [Cancer Res 2007;67(13):6212–20]

The incidence of malignant melanoma has been on the rise for the past 30 years with a lifetime risk of 1 in 75. In its early stage, melanoma is easily cured, but the prognosis associated with metastatic malignant melanoma remains very poor and is one of the most treatment-refractory malignancies. FDA-approved melanoma therapeutics, Dacarbazine (DTIC) and Interleukin-2 (IL-2) are associated with response rates of only 10-20% and do not yield long-lasting remissions. A direct and selective p53-independent cancer therapy is the application of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL). TRAIL can induce apoptosis in a broad range of transformed human cells while showing minimal toxicity towards non-malignant cells. However, some cancers are resistant to rhTRAIL-induced apoptosis as a direct result of overexpressed antiapoptotic proteins. One key driver mediating the expression of such antiapoptotic proteins is Casein Kinase 2 (CK2). Upregulated in the nucleus of all cancer cells, CK2 serves as an oncoprotein, promoting the expression of inhibitors of apoptosis resulting in resistance to cell death. By inhibiting CK2 and, therefore, downregulating its antiapoptotic products, TRAIL-resistant cells may become more sensitive to TRAIL-induced apoptosis. To date, Ellagic Acid (EA) is the most potent, naturally-occurring CK2 inhibitor, possessing a Ki of 20 nM. To test whether the addition of EA could sensitize cancer cells to the effects of TRAIL, we utilized cotreatment with TRAIL and EA against the V600E BRAF-mutated melanoma cell lines A375 and WM164, which have previously been reported as TRAIL-resistant. Our in vitro analyses showed that the cotreatment of TRAIL plus EA does not augment the ability of TRAIL to induce apoptosis in A375 as determined by Annexin-V Apoptosis assays and Western Blot analysis. TRAIL-alone was able to induce apoptosis in A375 characterized by the formation of Annexin-V positive cells, PARP fragmentation and the activation of caspase 3. TRAIL initiated both the extrinsic and intrinsic pathways of apoptosis marked by caspase 8-activation and the mitochondrial release of cytochrome c, respectively. However, EA treatment alone did not induce apoptosis marked by the absence of Annexin-V positive cells and PARP fragmentation. Additionally, both TRAIL and EA, as single-agents, inhibited the growth of A375 in a dose-dependent manner, and the combination treatment of TRAIL plus EA enhanced the cytostatic effects of the single-agent treatments as determined by Sulforhodamine-B (SRB) colorimetric assays. Cell cycle analysis through Propidium Iodide (PI) staining and FACS analysis revealed that EA impeded cell growth through an EA-mediated S-phase arrest in A375. Microarray analysis showed that EA downregulates antiapoptotic proteins survivin, XIAP, cIAP-2 and cIAP-1 and upregulates the death receptor DR5. In vivo, we showed that the combination treatment of TRAIL plus EA did not potentiate the ability of TRAIL to inhibit the growth of established A375 tumors. TRAIL alone inhibited tumor growth by ∼80% and H&E staining of TRAIL-treated tumors presented evidence of apoptotic cells. EA was not able to stop the growth of established A375 tumors and did not augment the in vivo antiproliferative effects of TRAIL. Lastly, we showed that TRAIL is cytotoxic towards WM164 cells and can reduce the viability of A375 as determined by Trypan Blue exclusion assays. Whereas EA, although it can inhibit the growth of WM164, does not reduce the viability of the cells. Additional analysis of the potential of EA to sensitize WM164 to the effects of TRAIL is ongoing. These data demonstrate that EA can enhance the cytostatic but not the cytotoxic or proapoptotic effects of TRAIL in vitro. In vivo, TRAIL can inhibit the growth of established tumors as a single-agent but is not augmented by cotreatment with EA. Citation Format: Katherine A. Turner, Daniel Lindner, Michael Kalafatis. Sensitization of TRAIL-resistant malignant melanomas by ellagic acid. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B34.

Ovarian cancer is a highly metastatic disease. Lysophosphatidic acid (LPA) levels are elevated in ascites from ovarian cancer patients, but its potential role in ovarian cancer metastasis has just begun to be revealed. In this work, we show that LPA stimulates invasion of primary ovarian cancer cells, but not ovarian epithelial or borderline ovarian tumor cells, although these benign cells indeed respond to LPA in cell migration. We have found that LPA downregulates tissue inhibitor of metalloproteinases (TIMPs). TIMP2 and TIMP3 play functional role in LPA-induced invasion as negative regulators. G(i) protein, phosphatidylinositol-3 kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), cytosolic phospholipase A(2) and urokinase type plasminogen activator (uPA) are required for LPA-induced cells invasion. TIMP3 may affect two independent downstream targets, vascular endothelial growth factor receptor and p38 MAPK. In vivo, LPA stimulates tumor metastasis in an orthotopic ovarian tumor model, which can be inhibited by a PI3K inhibitor, LY294002. In summary, LPA is likely a key component for promoting ovarian metastasis in vivo. LPA downregulates TIMP3, which may have targets other than metalloproteinases. Our in vivo metastasis mouse model is useful for studying the efficacy of therapeutic regimes of ovarian cancer.

Pre-clinical activity of SSG against melanoma and renal cancer has been identified recently although the drug's mechanism of action and activity against tumors of additional histological-types remain undefined.The effects of SSG and SSG combination with other agents on DU145 human prostate carcinoma xenograft tumors in mice and on DU145 cell subpopulations of differential SSG sensitivities were evaluated.DU145 tumor growth was inhibited by SSG (69%), IFNalpha2 (33%) or the combination (80%) that induced complete regression of WM9 human melanoma tumors. DU145 cells in culture were also partially growth inhibited by SSG at killing doses (200-800 mug/ml) for WM9 cells, indicating a correlation of SSG inhibition of cancer cell growth in vitro and in vivo. DU145 cells formed multiple micro tumors in mice treated with SSG or SSG/IFNalpha2 in contrast to the single large tumors in the control or IFNalpha2-treated mice, suggesting the existence of an SSG-resistant subpopulation in DU145 cells. Indeed, DU145 but not WM9 cells formed colonies (approximately 4% frequency) when cultured in the presence of SSG. Single cell clone (DU145-7) isolated from DU145 cells showed SSG-resistant growth in culture, unassociated with cross-resistance to IFNalpha2 and converted to SSG-responsive cells by BSO that inhibited intracellular glutathione levels.These results implicate a role for direct growth inhibition in SSG anti-tumor action, provide novel insights into the mechanism of tumor resistance to the drug and suggest a therapeutic potential for SSG and its combinations with IFNalpha2 or BSO for prostate cancer that warrants further investigation.