Additive Inhibitory Effect Research Articles

Elucidation of the Chemical Structure and Determination of the Production Conditions for a Bioactive Maillard Reaction Product, [5-(5,6-Dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]methanol, Isolated from a Glucose–Lysine Heated Mixture

We previously isolated a bioactive molecule, named F3-A, from an aqueous glucose (Glc) and lysine (Lys) Maillard reaction (MR) model system. Herein, F3-A was verified as [5-(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]methanol (5) and was subsequently synthesized for confirmation of bioactivity. Using Taguchi and factorial designs, we determined that the conditions which best increased the yield of F3-A were at pH 6 with a sugar:amino acid ratio of 2:1 and heating time of 12 h at 100 °C. The MR mixtures containing glucose produced highest yield, compared to fructose, lactose, and sucrose. Both the F3-A recovered from Glc-Lys MR mixture and the synthesized product exhibited significant (P < 0.05), dose dependent, nitric oxide (NO) inhibitory activity in Caco-2 cells that was comparable to aminoguanidine (AG) and pyrrolidine dithiocarbamate (PDTC), respectively. Finally, an additional inhibitory effect of F3-A was determined when coincubated with AG in cytokine-induced Caco-2 cells. This bioactivity points to a potential role in preventing intestinal inflammation.

Synergistic Ameliorative Effects of Resveratrol with Leflunomide on Serum Levels of Inflammatory Biomarkers and Joint Damage in Rats with Adjuvant Arthritis

Abstract Background: Rheumatoid arthritis (RA) is chronic irreversible inflammatory disorder and characterized by the thickening of synovial tissue with pannus formation and the destruction of joint structure; due to persistent overproduction of proinflammatory cytokines (TNF-α and IL-6), proinflammatory enzymes such as cyclooxygenase II (COX-2) and matrix metalloproteinases. Leflunomide, potent pyrimidine biosynthesis inhibitor, exhibiting anti-inflammatory and immunosuppressive actions and resveratrol is a potent antioxidant and anti-inflammatory agent; exerting potential antiarthritic effects and also can protect diverse tissue types as heart and colon in different experimental animal models. Objective: Evaluating outcomes of combinational use of both resveratrol and leflunomide on serum levels of proinflammatory biomarkers contributing to progression and severity of joint damage of adjuvant- induced arthritis in rats. Materials and Methods: 50 male Wistar rats divided into 5 equal groups, rheumatoid arthritis was induced by Complete Freund’s adjuvant. Arthritic rats were subdivided into 4 equal groups and taken the tested drugs daily for two weeks. Blood samples were taken for assay of serum levels of C-reactive protein, MDA, MMP-3, PGE2, IL-6, TNF-α and inflamed arthritic rat joints of hind paw from different rat groups were used for histological evaluation. Results: Arthritis rat group treated by concomitant resveratrol and leflunomide showed significant reduction in their serum levels C-reactive protein, MDA, MMP-3, PGE2, IL-6 , TNF-α, with histological improvement in comparison to others rat groups. Conclusion: Both drugs resveratrol and leflunomide exerted potent anti-inflammatory effects on serum levels of C-reactive protein, MDA, MMP-3, PGE2, IL-6, TNF-α and their concomitant use showed more significant ameliorative effects against joint damage reflected from histopathological analysis, with more additive inhibitory effects on serum levels of these cytokines and biomediators, that are strongly implicated in the pathogenesis and progression of RA and their combinational use is recommended for better management of RA in patients.

Vitamin D Potentiates the Inhibitory Effect of MicroRNA-130a in Hepatitis C Virus Replication Independent of Type I Interferon Signaling Pathway.

Calcitriol, the bioactive metabolite of vitamin D, was reported to inhibit HCV production in a synergistic fashion with interferon, a treatment in vitro. Our previous study established that miR-130a inhibits HCV replication by restoring the host innate immune response. We aimed to determine whether there is additive inhibitory effect of calcitriol and miR-130a on HCV replication. Here we showed that calcitriol potentiates the anti-HCV effect of miR-130a in both Con1b replicon and J6/JFH1 culture systems. Intriguingly, this potentiating effect of calcitriol on miR-130a was not through upregulating the expression of cellular miR-130a or through increasing the miR-130a-mediated IFNα/β production. All these findings may contribute to the development of novel anti-HCV therapeutic strategies although the antiviral mechanism needs to be further investigated.

X-Ray Structure of an Anticoagulant RNA Aptamer Bound to Factor Xa. Structural Basis for Its Ability to Disrupt Interactions Between Xa and Va within Prothrombinase

The prothrombinase complex assembles through high affinity interactions between factor Xa and factor Va on the surface of membranes exposing phosphatidylserine. Assembly of prothrombinase allows for the highly accelerated formation of thrombin localized at the injury site. Because the rate of thrombin formation by factor Xa alone is profoundly increased upon its incorporation into prothrombinase, disrupting the proteinase-cofactor interaction could represent an effective therapeutic strategy for the treatment of thrombosis. However, the extended interface between Xa and Va, likely involving shallow surfaces of the interacting proteins, limits the available strategies for the development of ligands that interfere with prothrombinase assembly with high affinity. The RNA aptamer, 11F7t, was selected in an unbiased SELEX screen using human factor Xa as a target and identified as a potent anticoagulant. Detailed functional characterization revealed that 11F7t binds both X and Xa with high affinity (~1 nM) and competes for the interaction between Xa and Va without occluding active site function of the proteinase. Competititive inhibition of the assembly of prothrombinase likely accounts for a major fraction of its anticoagulant effects. However, additional inhibitory effects of 11F7t on coagulation also probably arise from its ability to partially inhibit X activation by the intrinsic Xase and the inhibition of factor VIII activation by Xa. Here we report the crystal structure of 11F7t complexed with desGla-Xa containing the catalytic Ser195 replaced with Ala at 2.5 Å resolution and solved by molecular replacement. In the Xa model, the first 30 residues expected in the light chain, 5 residues in the autolysis loop and 7 residues at the C terminus were not defined by electron density. In 11F7t, 25 of 38 nucleotides could be unambiguously modeled. The tertiary fold of 11F7t presents an extended molecular surface for interactions with desGla-Xa and buries 1384 Å2 of solvent accessible surface area in the interacting species. The RNA aptamer exclusively binds the proteinase domain with contacts over a broad surface area that includes residues Leu59-Lys62, Val87-Arg93, Phe101, Lys236, Trp237 and Arg240 but not the catalytic site. Some of these residues have been implicated in heparin binding and have also been proposed to participate in Va binding based on modest changes in function upon mutagenesis. Even though 11F7t does not make any contacts in the 165 helix previously proposed to be a key region for the interaction with Va, aptamer binding is sufficient to disrupt the high affinity cofactor-proteinase interaction. The structure also reveals that most of the aptamer–protein contact involves nucleotides that constitute a base-paired stem (formed between G3-C6 and 31G-34C) and loop (C7-G11) of 11F7t with these interactions mostly specified by the nucleotide bases rather than the phosphate backbone which might be expected for a protein surface already implicated in binding a polyanion such as heparin or an acidic peptide from the C terminus of the A2 domain of Va. The regions of desGla-Xa occluded by the aptamer point to an extended surface that plays an important role in mediating interactions with factor Va within prothrombinase and suggests commonalities between this binding mode and the way that factor X might bind to factor VIIIa as a substrate within intrinsic Xase or the way that factor Xa might engage factor VIII as an enzyme. DisclosuresNo relevant conflicts of interest to declare.

Identification of coexistence of DNA methylation and H3K27me3 specifically in cancer cells as a promising target for epigenetic therapy.

Alterations of epigenetic modifications are promising targets for cancer therapy, and several epigenetic drugs are now being clinically utilized. At the same time, individual epigenetic modifications have physiological functions in normal cells, and cancer cell specificity is considered difficult to achieve using a drug against a single epigenetic modification. To overcome this limitation, a combination of epigenetic modifications specifically or preferentially present in cancer cells is a candidate target. In this study, we aimed to demonstrate (i) the presence of a cancer cell-specific combination of epigenetic modifications by focusing on DNA methylation and trimethylation of histone H3 lysine 27 (H3K27me3) and (ii) the therapeutic efficacy of a combination of DNA demethylation and EZH2 inhibition. Analyses of DNA methylation and H3K27me3 in human colon, breast and prostate cancer cell lines revealed that 24.7±4.1% of DNA methylated genes had both DNA methylation and H3K27me3 (dual modification) in cancer cells, while it was 11.8±7.1% in normal cells. Combined treatment with a DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC) and an EZH2 inhibitor, GSK126, induced marked re-expression of genes with the dual modification, including known tumor-suppressor genes such as IGFBP7 and SFRP1, and showed an additive inhibitory effect on growth of cancer cells in vitro. Finally, an in vivo combined treatment with 5-aza-dC and GSK126 inhibited growth of xenograft tumors more efficiently than a single treatment with 5-aza-dC. These results showed that the dual modification exists specifically in cancer cells and is a promising target for cancer cell-specific epigenetic therapy.

Antiplatelet activity of loureirin A by attenuating Akt phosphorylation: In vitro studies

Loureirin A is a flavonoid extracted from Dragon׳s Blood that has been used to promote blood circulation and remove stasis in Chinese traditional medicine. However, the mechanisms of these effects are not fully understood. We explored the anti-platelet activity and underlying mechanism of loureirin A in vitro. Our results indicated that loureirin A negatively affected agonist-induced platelet aggregation such as collagen, collagen-related peptide (CRP), ADP and thrombin. Loureirin A inhibited collagen-induced platelet ATP secretion and thrombin-stimulated P-selectin expression in a dose-dependent manner. Platelet spreading on immobilized fibrinogen was significantly impaired in the presence of loureirin A. Immunoblotting analysis indicated that 100μM of loureirin A almost completely eliminated collagen-induced Akt phosphorylation at Ser473. Interestingly, a submaximal dose (50μM) of loureirin A had an additive inhibitory effect with the phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 on collage-induced Akt phosphorylation in platelets. Taken together, loureirin A had an inhibitory effect on platelet activation, perhaps through an impairment of PI3K/Akt signaling.

Activation of formation of bacterial biofilms by azithromycin and prevention of this effect

Growth of members of most of the studied genera of gram-positive (Dietzia, Kocuria, and Rhodo- coccus) and gram-negative bacteria (Pseudomonas and Chromobacterium) in biofilms exhibited higher resistance to an translation inhibitor, azithromycin compared to the growth of planktonic cultures of the same strains. Low concentrations of azithromycin were found to stimulate biofilm formation by the studied saprotrophic strains. The rate of synthesis of the polysaccharide matrix component exceeded the rate of cell growth, indicating implementation of the biofilm phenotype under these conditions. It was found that an alkylhydroxybenzene (AHB) compound 4-hexylresorcinol was capable of almost uniform suppression of growth of both planktonic cultures and biofilms of the saprotrophic strains under study. In some cases, combined action ofazithromycin and AHB resulted in an additive inhibitory effect and prevented the stimulation of biofilm growth by subinhibitory azithromycin concentrations. Thus, AHB may be considered a promising antibiofilm agent.

Cadmium Telluride Quantum Dots (CdTe-QDs) and Enhanced Ultraviolet-B (UV-B) Radiation Trigger Antioxidant Enzyme Metabolism and Programmed Cell Death in Wheat Seedlings

Nanoparticles (NPs) are becoming increasingly widespread in the environment. Free cadmium ions released from commonly used NPs under ultraviolet-B (UV-B) radiation are potentially toxic to living organisms. With increasing levels of UV-B radiation at the Earth’s surface due to the depletion of the ozone layer, the potential additive effect of NPs and UV-B radiation on plants is of concern. In this study, we investigated the synergistic effect of CdTe quantum dots (CdTe-QDs), a common form of NP, and UV-B radiation on wheat seedlings. Graded doses of CdTe-QDs and UV-B radiation were tested, either alone or in combination, based on physical characteristics of 5-day-old seedlings. Treatments of wheat seedlings with either CdTe-QDs (200 mg/L) or UV-B radiation (10 KJ/m2/d) induced the activation of wheat antioxidant enzymes. CdTe-QDs accumulation in plant root cells resulted in programmed cell death as detected by DNA laddering. CdTe-QDs and UV-B radiation inhibited root and shoot growth, respectively. Additive inhibitory effects were observed in the combined treatment group. This research described the effects of UV-B and CdTe-QDs on plant growth. Furthermore, the finding that CdTe-QDs accumulate during the life cycle of plants highlights the need for sustained assessments of these interactions.

Сравнительное изучение влияния биогенных стимуляторов на образование радикалов кислорода фагоцитами крови пациентов с сердечной недостаточностью в условиях in vitro

Aim. To study the influence of biogenic stimulators Actovegine and Zifodine (deproteinized hemodrivates of calf blood) on reactive oxygen species formation by blood phagocytes in patients with chronic heart failure (CHF). Material and methods. Reactive oxygen species formation was registered by lucigenine-dependent chemiluminiscence on the luminomenter “Biotox-7” (Russia) in 20 patients with CHF of II-III functional classes by NYHA. The hemiluminiscences basal and stimulated by farbolic ether (RMA) were measured, as also inhibitory activity of Actovegine and Zifodine. Results. In CHF patients’ blood there was “spontaneous” secretion superoxide anion radicals, that is showing presence of preactivated phagocytes. In addition, a significant increase of oxygen radicals is shown as a response to RMA (1 mcM). Actovegine and Zifodine dose-dependently suppressed prominence of the response. While concomitantly applied Actovegine and Zifodine at the RMA background there was additive inhibitory effect found, which was not depended on consequence of additives. Conclusion. Actovegine and Zifodine decrease reactive oxygen species secretion by phagocytes, which are extracted from the blood of CHF patients. Addictiveness of such inhibitory effects of Actovegine and Zifodine shows absence of their identity by structure and of improper translation of this data into conclusions on effectiveness and safety, found in clinical practice while prescribing Actovegine and Zifodine.

Abstract 5505: Synergistic anticancer activity of clinical stage, non-genotoxic apoptosis inducing agents RG7388 (MDM2 antagonist) and ABT-199 (GDC-0199, BCL2 inhibitor) in p53 wild-type AML tumor models

Abstract MDM2, the most prominent natural inhibitor of the p53 tumor suppressor pathway, and BCL2, an anti-apoptotic regulator and important player in preventing induction of apoptosis in tumor cells, both play a pivotal role in leukemia cell survival and response to therapy in the clinic. The principal objectives of this study were to assess a synergistic antitumor effect by combining the clinical stage compounds RG7388, a MDM2 antagonist reactivating p53, and the BCL2 selective inhibitor GDC-0199 and to elucidate the underlying mechanism of action. Further insight into the biological rationale for this potential therapeutic strategy may pave the path for clinical combination studies in AML patients. We first investigated the effect of co-treatment with RG7388 and GDC-0199 on the viability and induction of apoptosis in AML tumor cell lines followed by high-resolution cell cycle kinetic analysis using BrdU-Hoechst quenching technology in-vitro. Cell viability assays demonstrated synergistic potency of combined RG7388 and GDC-0199 treatment with low nM IC50 values. Subsequent annexin binding assays confirmed the efficacy and revealed different kinetics of apoptosis induction. To further explain the mode of action, high-resolution cell cycle compartment analysis of consecutive cell cycles was performed. Mechanistically, RG7388 induces G1 arrest and preferentially causes nuclear fragmentation in G1 phase of the second cycle. The BCL2 inhibitor GDC-0199 mainly causes apoptosis in G1 compartments and hence cells transiently protected from apoptosis by RG7388 induced G1 arrest are hit by GDC-0199. In addition we analyzed changes in expression of pro- and anti-apoptotic genes and proteins upon combined treatment using qRT-PCR, Western Blot and Luminex technology. We identified down-modulation of MCL-1 levels, a known GDC-0199 resistance factor, as one potential mechanism for the synergistic efficacy and confirmed this finding by shRNA experiments in-vitro. Furthermore, the efficacy of the compounds was assessed in subcutaneous and orthotopic AML tumor cell line based xenograft models in-vivo, confirming the synergy observed in-vitro. Taken together, our study demonstrates that combined treatment with RG7388 and GDC-0199 shows a synergistic anticancer effect on AML cells. This indicates that activating the p53 tumor suppressor pathway and inhibiting a key anti-apoptosis target simultaneously induces a more than additive inhibitory effect by modulating the levels of key pro-survival proteins, such as Mcl-1, thereby further lowering the apoptotic threshold level for tumor cell death. Based on the observations from our study, the combined administration of RG7388 and GDC-0199 might offer a promising new therapeutic regimen for treating AML patients. Citation Format: Markus Dangl, Yuchen Chien, Christian Lehmann, Thomas Friess. Synergistic anticancer activity of clinical stage, non-genotoxic apoptosis inducing agents RG7388 (MDM2 antagonist) and ABT-199 (GDC-0199, BCL2 inhibitor) in p53 wild-type AML tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5505. doi:10.1158/1538-7445.AM2014-5505

Abstract 2314: The combination of DNA methylation and H3K27me3 is a cancer-specific dual epigenetic modification

Abstract Alterations of epigenetic modifications are promising targets of cancer therapy, and several epigenetic drugs are now used in the clinical field. At the same time, individual epigenetic modifications have physiological roles. Therefore, a cancer-specific combination of epigenetic modifications (dual modifications) is considered to be better as a therapeutic target. However, it is unclear whether or not such cancer-specific dual modification is present. In this study, we aimed to clarify whether cancer-specific dual modification is present by focusing on the combination of two major repressive modifications, DNA methylation and trimethylation of histone H3 lysine 27 (H3K27me3). DNA methylation and H3K27me3 analyses in human colon, breast, and prostate cancer cell lines by Infinium HumanMethylation450 and ChIP-on-chip, respectively, revealed that 24.7±4.1% of DNA methylated genes were co-localized with H3K27me3 in cancer cells. In contrast, only 11.8±7.1% of DNA methylated genes were co-localized with H3K27me3 in their normal counterpart cells. 29.9±17.4% of genes with the dual modifications had neither DNA methylation nor H3K27me3 in normal counterpart cells. Expression levels of genes with the dual modifications were as low as those of genes with only DNA methylation in cancer cells. Analysis of re-activation of genes with the dual modifications by a DNA demethylating reagent, 5-aza-2′-deoxycytidine (5-aza-dC), and/or an EZH2 inhibitor, GSK126, revealed that genes with the dual modifications, such as IGFBP7, SFRP1, and SLC6A15, were re-activated more efficiently by the combination treatment than by a single treatment with 5-aza-dC. In contrast, such a combination effect on gene re-activation was not observed for genes with only DNA methylation. Furthermore, the combination treatment by 5-aza-dC and GSK126 had an additive inhibitory effect on growth of cancer cells. These results showed that genes with the dual modifications, DNA methylation and H3K27me3, are increased in cancer cells, and the combination of a DNA demethylating reagent and an EZH2 inhibitor is useful for re-activation of these genes. Citation Format: Hideyuki Takeshima, Mika Wakabayashi, Naoko Hattori, Satoshi Yamashita, Toshikazu Ushijima. The combination of DNA methylation and H3K27me3 is a cancer-specific dual epigenetic modification. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2014-2314

Abstract 3363: Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3)

Abstract In human colon cancer cells, glycolytic activity shows a positive correlation with proliferation. PFKFB3 is an important enzyme regulating glycolysis in many tumor cells. We have studied the action of an established inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) as a single agent and in combination with other molecules that affect glycolysis and proliferation in four colon cancer cell lines (Caco-2, HCT116, HT29 and SW1116). Glucose uptake and cell proliferation were inhibited by 3PO and butyrate and additive effects were seen with 10 µM 3PO and 1 mM butyrate. Induction of alkaline phosphatase activity serves as a marker of differentiation in colon cancer cells. The large induction of alkaline phosphatase activity by butyrate seen in most colon cancer cell lines was not observed with 3PO. Similar contrasting effects on induction of alkaline phosphatase activity were seen with rat Morris hepatoma 7777 cells where 2-3 fold induction was seen with 1 µM vorinostat or 1 mM butyrate but no significant effect was seen with 3PO in the range of 5-30 µM. The biguanides, metformin and phenformin, increase glycolysis but inhibit proliferation of colon cancer cells. After combination treatment with 3PO, glycolysis was inhibited, as judged by glucose uptake and acidification of the medium, and additive inhibitory effects on cell proliferation could be observed. For example, after a 72 hour incubation of Caco-2 cells with 10 µM 3PO, 25 µM phenformin or 1 mM metformin, as single agents, proliferation relative to controls was decreased 20%, 17% and 34%, respectively. For combinations of 3PO and phenformin or 3PO and metformin the decreases relative to controls were 48% and 58%, respectively. In conclusion, 3PO was an inhibitor of proliferation of colon cancer cells, especially the rapidly growing HCT116 and HT29 cell lines. 3PO may be a useful agent in combination with other drugs that inhibit colon cancer cell proliferation. Citation Format: Michael A. Lea, Raymond Geherty, Rachelle David, Charles desBordes. Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3363. doi:10.1158/1538-7445.AM2014-3363

Abstract 2970: Differential effects of caloric and carbohydrate restriction on prostate cancer in a mouse model

Abstract Prostate cancer (PC) is the most common non-cutaneous malignancy in men in the United States. Animal studies have shown that dietary interventions, specifically dietary restriction, demonstrate an ability to inhibit prostate tumor growth and progression. Classically, dietary restriction has come in the form of global caloric restriction (CR). CR, without malnutrition, has been consistently shown to be effective in reducing age-related conditions as well as decreasing the spontaneous incidence and progression of multiple cancers. In contrast to the generalized reduction of calories seen in CR, an alternative manner of dietary restriction would be to restrict specific macronutrients. In prior studies, we have tested the theory that specifically restricting carbohydrates could slow tumor growth by providing mice a diet deplete of carbohydrates (i.e. a no-carbohydrate Ketogenic diet or NCKD). As such, we previously found that in a prostate cancer xenograft study, mice fed a NCKD, in the presence or absence of weight loss, had improved mouse survival as well as slowed tumor growth with respect to a diet complete of carbohydrates (i.e. Western diet). Moreover, NCKD, like CR, appeared to inhibit tumor growth via depression of the insulin/IGF-1 axis. These series of studies suggest that CR and NCKD may act via overlapping molecular mechanisms that inhibit tumor growth, however, no study to date has directly compared the effects of carbohydrate versus caloric restriction. Therefore we sought to investigate the role of global CR and NCKD without CR on tumor growth side by side. In addition, we sought to determine whether calorie restriction of NCKD would provide an additional inhibitory effect (i.e. CR + NCDK). This study consisted of four arms: 1) Western Diet (WD); 2) NCKD (pair fed with WD); 3) western diet 25% calorie restricted (WD-CR) and 4) NCKD 25% CR (NCKD-CR). By day 52 after randomization, when median tumor volumes for WD mice were ∼1000 mm3, NCKD, Western CR and NCKD CR arms had 32.2%, 41.6%, and 62.1% smaller tumor volumes, respectively, relative to WD mice (all p&amp;lt;0.01). At sacrifice, both calorically restricted groups had significantly lower serum glucose levels than the non-calorically restricted arms. Moreover, IGF-1:IGFBP-3 levels of all active experimental arms (NCKD, WD-CR, and NCKD-CR) were significantly lower than WD mice. Among the two CR arms, IGF-1: IGFBP-3 ratios were lower in the NCKD-CR mice. These data suggest that CR and NCKD have similar efficacy in reducing tumor growth and both act, at least in part, via reductions in IGF-1. Also, the combination of CR plus NCKD had an additive effect. Global gene expression analysis of xenograft tumors revealed genes altered in a similar fashion across the three diets in comparison to WD, but also genes uniquely altered in individual diets. Further analysis of these genes will allow us to better understand the molecular mechanisms through which these dietary regimens inhibit tumor growth. Citation Format: Everardo Macias, Jean A. Thomas, Elizabeth M. Masko, Alexis R. Gaines, Brian Whitley, Tanisha Coburn, Susan L. Poulton, Tameika E. Phillips, Stephen J. Freedland. Differential effects of caloric and carbohydrate restriction on prostate cancer in a mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2014-2970

Sulfasalazine and its metabolites inhibit platelet function in patients with inflammatory arthritis.

The purpose of this study is to assess the effect of sulfasalazine and its metabolites on platelet function in patients with inflammatory arthritis (IA). One hundred thirty-five consecutive patients with an established diagnosis of IA were screened. Those with a history of cardiovascular disease (CVD), taking anti-platelet agents or non-steroidal anti-inflammatory drugs (NSAIDs) were excluded. A total of 32 patients were investigated, 15 taking sulfasalazine and 17 taking other disease-modifying anti-rheumatic drugs (DMARDs) and no sulfasalazine. These two cohorts were compared to 15 patients with stable CVD on long-term aspirin. The effect of sulfasalazine and its metabolites on arachidonic acid (AA)-induced platelet aggregation was also tested in vitro in samples from healthy donors (n = 18). Demographics, CVD risk factors and disease activity indices were similar in the sulfasalazine and other DMARD groups. AA-induced platelet aggregation was significantly inhibited in the sulfasalazine group (9 ± 7 %) and comparable to that in the aspirin group (10 ± 6 %). In contrast, there was no effect on AA-induced platelet aggregation in the other DMARDs group (77 ± 12 %) (p < 0.001). Furthermore, sulfasalazine therapy had no effect on platelet aggregation in response to multiple other agonists. Sulfasalazine and its metabolites (5-aminosalicylic acid and sulfapyridine) exerted an additive and dose-dependent inhibitory effect on AA-induced platelet aggregation in vitro (p < 0.001). The inhibition of AA-induced platelet aggregation by sulfasalazine is comparable to that achieved by aspirin and is dependent on both sulfasalazine and its metabolites. This represents a potential mechanism that may contribute to the known cardioprotective effect of sulfasalazine in patients with IA.

The Inhibitory Effect of Pseudolaric Acid B on Gastric Cancer and Multidrug Resistance via Cox-2/PKC-α/P-gp Pathway

AimTo investigate the inhibitory effect of pseudolaric acid B on subcutaneous xenografts of human gastric adenocarcinoma and the underlying molecular mechanisms involved in its multidrug resistance.MethodsHuman gastric adenocarcinoma SGC7901 cells and drug-resistant SGC7901/ADR cells were injected into nude mice to establish a subcutaneous xenograft model. The effects of pseudolaric acid B with or without adriamycin treatment were compared by determining the tumor size and weight. Cyclo-oxygenase-2, protein kinaseC-α and P-glycoprotein expression levels were determined by immunohistochemistry and western blot.ResultsPseudolaric acid B significantly suppressed the tumor growth induced by SGC7901 cells and SGC7901/ADR cells. The combination of pseudolaric acid B and the traditional chemotherapy drug adriamycin exhibited more potent inhibitory effects on the growth of gastric cancer in vivo than treatment with either pseudolaric acid B or adriamycin alone. Protein expression levels of cyclo-oxygenase-2, protein kinaseC-α and P-glycoprotein were inhibited by pseudolaric acid B alone or in combination with adriamycin in SGC7901/ADR cell xenografts.ConclusionPseudolaric acid B has a significant inhibitory effect and an additive inhibitory effect in combination with adriamycin on the growth of gastric cancer in vivo, which reverses the multidrug resistance of gastric neoplasm to chemotherapy drugs by downregulating the Cox-2/PKC-α/P-gp/mdr1 signaling pathway.

Regulatory CD4+ T cells modulate the interaction between NK cells and hepatic stellate cells by acting on either cell type

NK cells regulate liver fibrosis by killing activated hepatic stellate cells (HSCs) and are controlled themselves by immune cells and/or soluble factors. Here, we analysed if CD4(+) regulatory T cells (Tregs) modify the interaction between NK cells and HSCs. The modification of NK cell activity against HSCs was studied in CD56(high)CD16(-) NK cells, using a flow cytometric CD107a degranulation assay and co-cultures with Tregs from healthy donors and patients with hepatitis C, respectively. We studied the underlying mechanisms in detail, applying Treg supernatants, Treg pretreated HSCs, and recombinant IL-8, TGF-ß1, and IL-10 as well as blocking experiments with neutralizing antibodies and analysed Treg-associated changes in the expression of NK cell receptor ligands on HSCs. Tregs suppressed NK cell activation during HSC co-culture in a cell-contact-dependent manner involving the cytotoxic T-lymphocyte antigen 4 (CTLA-4). NK cell degranulation was further reduced, when HSCs had been pretreated with Tregs (p=0.043), Treg supernatants (p=0.001) or recombinant IL-8 (R=0.630, p=0.001) and TGF-ß1 (R=0.608, p=0.002), respectively. This additional inhibitory effect corresponded to the IL-8/TGF-ß1-mediated downregulation of MIC-A/B and HLA class-I on HSCs. Tregs from hepatitis C likewise inhibited NK cell activity, which was reversed significantly in specific blocking experiments. Our data indicate that Tregs interfere with NK cell regulation of fibrogenesis via both direct cell-contact-dependent inhibition of NK cells and release of soluble factors, downregulating activating NK cell receptor ligands on HSCs. Our data may be particularly relevant for the intrahepatic accumulation of Tregs in chronic hepatitis C because downregulated NK cell activity against HSCs may blunt their control of fibrogenesis.

Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor.

Btk and Etk/BMX are Tec-family non-receptor tyrosine kinases. Btk has previously been reported to be expressed primarily in B cells and has an important role in immune responses and B-cell malignancies. Etk has been shown previously to provide a strong survival and metastasis signal in human prostate cancer cells, and to confer androgen independence and drug resistance. While the role of Etk in prostate carcinogenesis is well established, the functions of Btk in prostate cancer have never been investigated, likely due to the perception that Btk is a hematopoietic, but not epithelial, kinase. Herein, we found that Btk is overexpressed in prostate cancer tissues and prostate cancer cells. The level of Btk in prostate cancer tissues correlates with cancer grades. Knockdown of Btk expression selectively inhibits the growth of prostate cancer cells, but not that of the normal prostate epithelial cells, which express very little Btk. Dual inhibition of Btk and Etk has an additive inhibitory effect on prostate cancer cell growth. To explore Btk and Etk as targets for prostate cancer, we developed a small molecule dual inhibitor of Btk and Etk, CTN06. Treatment of PC3 and other prostate cancer cells, but not immortalized prostate epithelial cells with CTN06 resulted in effective cell killing, accompanied by the attenuation of Btk/Etk signals. The killing effect of CTN06 is more potent than that of commonly used inhibitors against Src, Raf/VEGFR and EGFR. CTN06 induces apoptosis as well as autophagy in human prostate cancer cells, and is a chemo-sensitizer for docetaxel (DTX), a standard of care for metastatic prostate cancer patients. CTN06 also impeded the migration of human prostate cancer cells based on a ‘wound healing' assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model.

The impact of S6K1 kinase on neuroblastoma cell proliferation is independent of GLI1 signaling

BackgroundThe crosstalk between Hedgehog (HH) signaling and other signal transduction cascades has been extensively studied in different cancers. In neuroblastoma, mTOR/S6K1 signaling is known to have a role in the development of this disease and recent evidence also implicates the HH pathway. Moreover, S6K1 kinase has been shown to phosphorylate GLI1, the effector of HH signaling, promoting GLI1 transcriptional activity and oncogenic function in esophageal adenocarcinoma. In this study, we examined the possible interplay of S6K1 and GLI1 signaling in neuroblastoma.MethodssiRNA knockdowns were used to suppress S6K1 and GLI1 expression, and the siRNA effects were validated by real-time PCR and Western blotting. Cell proliferation analysis was performed with the EdU incorporation assay. Cytotoxic analysis with increasing concentrations of PI3K/mTOR and GLI inhibitors, individually and in combination, was used to determine drug response.ResultsAlthough knockdown of either S6K1 or GLI1 reduces the cellular proliferation of neuroblastoma cells, there is little effect of S6K1 on the expression of GLI1 mRNA and protein and on the capacity of GLI1 to activate target genes. No detectable phosphorylation of GLI1 is observed prior or following S6K1 knockdown. GLI1 overexpression can not rescue the reduced proliferation elicited by S6K1 knockdown. Moreover, inhibitors of PI3K/mTOR and GLI signaling reduced neuroblastoma cell growth, but no additional growth inhibitory effects were detected when the two classes of drugs were combined.ConclusionOur results demonstrate that the impact of S6K1 kinase on neuroblastoma cells is not mediated through modulation of GLI1 expression/activity.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-600) contains supplementary material, which is available to authorized users.

PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors.

IntroductionTriple negative breast cancer (TNBC) is a heterogeneous collection of biologically diverse cancers, which contributes to variable clinical outcomes. Previously, we identified a TNBC subtype that has a luminal phenotype and expresses the androgen receptor (AR+). TNBC cells derived from these luminal AR + tumors have high frequency phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations. The purpose of this study was to determine if targeting phosphoinositide 3-kinase (PI3K) alone or in combination with an AR antagonist is effective in AR + TNBC.MethodsWe determined the frequency of activating PIK3CA mutations in AR + and AR- TNBC clinical cases. Using AR + TNBC cell line and xenograft models we evaluated the effectiveness of PI3K inhibitors, used alone or in combination with an AR antagonist, on tumor cell growth and viability.ResultsPIK3CA kinase mutations were highly clonal, more frequent in AR + vs. AR- TNBC (40% vs. 4%), and often associated with concurrent amplification of the PIK3CA locus. PI3K/mTOR inhibitors had an additive growth inhibitory effect when combined with genetic or pharmacological AR targeting in AR + TNBC cells. We also analyzed the combination of bicalutamide +/- the pan-PI3K inhibitor GDC-0941 or the dual PI3K/mTOR inhibitor GDC-0980 in xenograft tumor studies and observed additive effects.ConclusionsWhile approximately one third of TNBC patients respond to neoadjuvant/adjuvant chemotherapy, recent studies have shown that patients with AR + TNBC are far less likely to benefit from the current standard of care chemotherapy regimens and novel targeted approaches need to be investigated. In this study, we show that activating PIK3CA mutations are enriched in AR + TNBC; and, we show that the growth and viability of AR + TNBC cell line models is significantly reduced after treatment with PI3K inhibitors used in combination with an AR antagonist. These results provide rationale for pre-selection of TNBC patients with a biomarker (AR expression) to investigate the use of AR antagonists in combination with PI3K/mTOR inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0406-x) contains supplementary material, which is available to authorized users.

Treatment of poorly differentiated glioma using a combination of monoclonal antibodies to extracellular connexin-43 fragment, temozolomide, and radiotherapy.

Antitumor efficiencies of monoclonal antibodies to connexin-43 second extracellular loop (MAbE2Cx43), temozolomide, and fractionated γ-irradiation in the monotherapy mode and in several optimized combinations were studied in Wistar rats with induced C6 glioma. The survival of animals with glioma and the dynamics of intracerebral tumor development were evaluated by MRT. Temozolomide monotherapy (200 mg/m(2)) and isolated radiotherapy in a total dose of 36 Gy shifted the survival median from 28 days (no therapy) to 34 and 38 days, respectively; 100% animals died under conditions of temozolomide monotherapy and radiotherapy. Monotherapy with MAbE2Cx43 in a dose of 5 mg/kg led to significant regression of the tumor (according to MRT data), cure of 19.23% animals with glioma, and prolongation of the survival median to 39.5 days after tumor implantation. Combined therapy with MAbE2Cx43 and temozolomide completely abolished the antitumor effect (survival median 29 days). Treatment with MAbE2Cx43 in combination with radiotherapy was associated with mutual boosting of the therapeutic efficiencies, leading to a significant inhibition of tumor development and prolongation of the survival median to 60 days. The mechanism of tumorsuppressive activity of the antibodies could be due to connexon blockade in Cx43-positive glioma cells in the peritumor invasion zone. Higher efficiency of combined therapy was presumably due to the increase in blood-brain barrier permeability for antibodies after irradiation of the brain and to additional inhibitory effect of antibodies towards radioresistant migrating glioma cells. The results suggested that MAbE2Cx43 could be effective as the first-line drug in combined therapy for poorly differentiated gliomas.