Luteolin Attenuates Diabetic Nephropathy through Suppressing Inflammatory Response and Oxidative Stress by Inhibiting STAT3 Pathway.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). DN has many pathological changes, but tubular injury is considered to be a crucial pathological feature and plays a key role in the progression of DN. Accumulating studies have confirmed that Luteolin (3,4,5,7-tetrahydroxyflavone, Lut) possesses anti-inflammatory and antioxidant activities, which may play a role in kidney protection in DN. OBJECTIVES: This paper described the effects of Lut on appropriated tubular injury in the kidneys of db/db mice and searched the possible mechanisms underlying the kidney protection effect in DN. METHODS: Twelve-week-old male C57BL/6 J db/db and C57BL/6 J db/m mice were used for the animal experiments. They were organized into the following five groups for the animal experiments: a db/m group (control, n=6); a db/db group(n=8) ; a db/db group receiving Lut (10 mg/kg/day, n=8)treatment by oral gavage; a db/db group receiving stattic (a selective STAT3 inhibitor,50 mg/Kg/day, n=8) treatment by oral gavage and a db/db group receiving both stattic and Lut treatment by oral gavage. RESULTS: In this study, we found that Lut might ameliorate glomerular sclerosis and interstitial fibrosis in DN mouse models through inhibiting the inflammatory response and oxidative stress. And it might play its biological function mainly through repressing the STAT3 activation. CONCLUSIONS: Lut attenuates DN mainly via suppression of inflammatory response and oxidative response. STAT3 pathway is the potential target, which ultimately reduces renal fibrosis and delays the progress of DN.

Possible New Strategies for the Treatment of Congenital Hyperinsulinism.

Objective: Congenital hyperinsulinism (CHI) is a rare disease characterized by persistent hypoglycemia as a result of inappropriate insulin secretion, which can lead to irreversible neurological defects in infants. Poor efficacy and strong adverse effects of the current medications impede successful treatment. The aim of the study was to investigate new approaches to silence ß-cells and thus attenuate insulin secretion. Research Design and Methods: In the scope of our research, we tested substances more selective and more potent than the gold standard diazoxide that also interact with neuroendocrine ATP-sensitive K+ (KATP) channels. Additionally, KATP channel-independent targets as Ca2+-activated K+ channels of intermediate conductance (KCa3.1) and L-type Ca2+ channels were investigated. Experiments were performed using human islet cell clusters isolated from tissue of CHI patients (histologically classified as pathological) and islet cell clusters obtained from C57BL/6N (WT) or SUR1 knockout (SUR1-/-) mice. The cytosolic Ca2+ concentration ([Ca2+]c) was used as a parameter for the pathway regulated by electrical activity and was determined by fura-2 fluorescence. The mitochondrial membrane potential (ΔΨ) was determined by rhodamine 123 fluorescence and single channel currents were measured by the patch-clamp technique. Results: The selective KATP channel opener NN414 (5 µM) diminished [Ca2+]c in isolated human CHI islet cell clusters and WT mouse islet cell clusters stimulated with 10 mM glucose. In islet cell clusters lacking functional KATP channels (SUR1-/-) the drug was without effect. VU0071063 (30 µM), another KATP channel opener considered to be selective, lowered [Ca2+]c in human CHI islet cell clusters. The compound was also effective in islet cell clusters from SUR1-/- mice, showing that [Ca2+]c is influenced by additional effects besides KATP channels. Contrasting to NN414, the drug depolarized ΔΨ in murine islet cell clusters pointing to severe interference with mitochondrial metabolism. An opener of KCa3.1 channels, DCEBIO (100 µM), significantly decreased [Ca2+]c in SUR1-/- and human CHI islet cell clusters. To target L-type Ca2+ channels we tested two already approved drugs, dextromethorphan (DXM) and simvastatin. DXM (100 µM) efficiently diminished [Ca2+]c in stimulated human CHI islet cell clusters as well as in stimulated SUR1-/- islet cell clusters. Similar effects on [Ca2+]c were observed in experiments with simvastatin (7.2 µM). Conclusions: NN414 seems to provide a good alternative to the currently used KATP channel opener diazoxide. Targeting KCa3.1 channels by channel openers or L-type Ca2+ channels by DXM or simvastatin might be valuable approaches for treatment of CHI caused by mutations of KATP channels not sensitive to KATP channel openers.

Formulation of Stattic as STAT3 inhibitor in nanostructured lipid carriers (NLCs) enhances efficacy of doxorubicin in melanoma cancer cells.

Nowadays, nanoparticle-based combination therapy has been emerging as huge innovation in cancer treatment. Here, we studied the effect of Stattic (STAT3 inhibitor) loaded in nanostructured lipid carriers (NLCs) on enhancing the efficacy, cytotoxicity, and induction of apoptosis of doxorubicin in B16F10 mouse melanoma cancer cell. The evaluation of Stattic-loaded NLCs has been done in terms of zeta potential, particle size, scanning electron microscope (SEM), and cellular uptake. MTT assay was applied to evaluate the cell proliferation. Apoptotic cell death and identification of early and late apoptosis were assessed by DAPI staining and Annexin V/PI staining, respectively. Real-time RT-PCR was applied to measure the effects of doxorubicin and/or Stattic on key apoptotic genes such as Bad, Survivin, HIF1, and STAT3. The Stattic formulated into NLCs shown mean particle size of 56 ± 7 nm which was confirmed by SEM. The IC50 values for Stattic and doxorubicin were 2.95 ± 0.52 µM and 1.21 ± 0.36 µM, respectively. Stattic-loaded NLCs diminished percent of cell proliferation from 68 ± 6.8 to 54 ± 3.7% (p < 0.05). Combinational treatment of the cells with Stattic-loaded nanoparticles and doxorubicin give rise to a significant increase in the percentage of apoptosis (p < 0.05). The study of gene expression profile has shown a remarkable decrease in anti-apoptotic gene, Survivin, along with smooth decline in HIF1 as angiogenesis intermediator and increase in Bad mRNA levels. Our results recommend that NLCs as novel technology have potent strategy to augment efficacy of current chemotherapeutic agent in melanoma cancer cells.

The Novel Small-Molecule SR18662 Efficiently Inhibits the Growth of Colorectal Cancer In Vitro and In Vivo.

Krüppel-like factor 5 (KLF5), a member of the SP/KLF family of zinc finger transcription factors, is overexpressed in human colorectal cancer specimens, and this overabundance is associated with aggressive cancer development and progression. We demonstrated that mice haploinsufficient for Klf5 had reduced intestinal tumor burden in the background of germline mutation in Apc, a gatekeeper of intestinal tumorigenesis. Based on a high-throughput screening strategy, we developed ML264, a small-molecule compound that inhibits KLF5, and showed that it inhibits growth of colorectal cancer in vitro and in vivo Through optimization efforts based on the structure of ML264, we have now identified a new lead compound, SR18662. We find that treatment with SR18662 significantly reduces growth and proliferation of colorectal cancer cells as compared with treatment with vehicle control, ML264, or SR15006 (a less optimized analogue from SAR efforts leading to SR18662). SR18662 showed improved efficacy in reducing the viability of multiple colorectal cancer cell lines. Flow cytometry analysis following SR18662 treatment showed an increase in cells captured in either S or G2-M phases of the cell cycle and a significant increase in the number of apoptotic cells, the latter a unique property compared with ML264 or SR15006. SR18662 treatment also reduces the expression of cyclins and components of the MAPK and WNT signaling pathways. Importantly, we observed a significant dose-dependent inhibition of xenograft growth in mice following SR18662 treatment that exceeded the effect of ML264 at equivalent doses. These findings support further development of SR18662 and its analogues for colorectal cancer therapy.

Safety, Tolerability, and Pharmacokinetics of the ß-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor Verubecestat (MK-8931) in Healthy Elderly Male and Female Subjects.

ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is required for the production of ß-amyloid peptides, which are implicated in the etiology of Alzheimer's disease. The safety and pharmacokinetics of the BACE1 inhibitor verubecestat have previously been studied in young adults aged 19-45 years. In this randomized, placebo-controlled, phase I study (protocol MK-8931-006), we investigated the safety, tolerability, and pharmacokinetics of a single dose (100 mg) or multiple doses (30, 80, and 120 mg) once daily for 28 days of verubecestat in healthy elderly subjects. Safety end points were assessed at baseline and during the duration of the study period and indicated that verubecestat was generally well tolerated. Verubecestat pharmacokinetics were similar between healthy elderly male and female subjects and similar to those reported in healthy young males in previous studies. These data supported subsequent studies to assess the potential efficacy of verubecestat in subjects with Alzheimer's disease.

Pharmacokinetics and Pharmacodynamics of the BACE1 Inhibitor Verubecestat (MK-8931) in Healthy Japanese Adults: A Randomized, Placebo-Controlled Study.

ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of ß-amyloid (Aß) peptides and is considered a potential treatment target for Alzheimer's disease (AD). To support Japan's participation in the global clinical development program, we characterized the safety, pharmacokinetics (PKs), and pharmacodynamics of the BACE1 inhibitor verubecestat (MK-8931) in 24 healthy Japanese adults in a two-part, single-center, randomized, placebo-controlled phase I trial (protocol MK-8931-007) and compared the results with historical data from non-Japanese subjects. Both single (20, 100, and 450 mg) and multiple (80 and 150 mg once daily for 14 days) doses of verubecestat were well tolerated. Verubecestat's PK profile was similar in Japanese and non-Japanese subjects. Verubecestat also reduced mean cerebrospinal fluid concentrations of the Aß proteins Aß40, Aß42, and soluble ß fragment of amyloid precursor protein; the level of reduction was comparable between Japanese and non-Japanese subjects. These results support the continued global development of verubecestat as a potential disease-modifying agent for Japanese and non-Japanese subjects who are at risk for developing AD.

Pharmacokinetic characterization of 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone, a novel 11ß-hydroxysteroid dehydrogenase type 1 inhibitor in rats.

11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is associated with metabolic syndromes such as type 2 diabetes mellitus and obesity. A new 11ß-HSD1 inhibitor known as 2-(3-benzoyl)-4-hydroxy-1, 1-dioxo-2H-1, 2-benzothiazine-2-yl-1-phenylethanone (KR-66344) is being developed as a therapeutic agent for these metabolic diseases. The purpose of this study was to characterize the pharmacokinetics of KR-66344 to support further preclinical development. KR-66344 showed high liver microsomal stability with T1/2 values >3 h and high permeability with apparent permeability coefficients of 15.2-24.2 × 10(-6) cm/s in Caco-2 cell monolayers. KR-66344 was also strongly bound to plasma proteins (>98%). After intravenous dosing, KR-66344 exhibited low systemic clearance (0.27-0.37 L/h/kg) and a low to moderate volume of distribution at steady state (0.79-0.8 L/kg). The bioavailability and terminal half-lives of KR-66344 following oral administration were 25% and 1.7-3.3 h, respectively. In addition, KR-66344 showed dose-independent pharmacokinetics at 0.5-10 mg/kg in intravenous and oral pharmacokinetic studies.

STAT3 Inhibition Enhances the Therapeutic Efficacy of Immunogenic Chemotherapy by Stimulating Type 1 Interferon Production by Cancer Cells.

STAT3 is an oncogenic transcription factor with potent immunosuppressive functions. We found that pharmacologic inhibition of STAT3 or its selective knockout in cancer cells improved the tumor growth-inhibitory efficacy of anthracycline-based chemotherapies. This combined effect of STAT3 inhibition/depletion and anthracyclines was only found in tumors growing on immunocompetent (not in immunodeficient) mice. As compared with Stat3-sufficient control tumors, Stat3(-/-) cancer cells exhibited an increased infiltration by dendritic cells and cytotoxic T lymphocytes after chemotherapy. Anthracyclines are known to induce several stress pathways that enhance the immunogenicity of dying and dead cancer cells, thereby stimulating a dendritic cell-dependent and T lymphocyte-mediated anticancer immune response. Among these therapy-relevant stress pathways, Stat3(-/-) cancer cells manifested one significant improvement, namely an increase in the expression of multiple type-1 interferon-responsive genes, including that of the chemokines Cxcl9 and Cxcl10. This enhanced type-1 interferon response could be suppressed by reintroducing wild-type Stat3 (but not a transactivation-deficient mutant Stat3(Y705F)) into the tumor cells. This maneuver also abolished the improved chemotherapeutic response of Stat3(-/-) cancers. Finally, the neutralization of the common type-1 interferon receptor or that of the chemokine receptor CXCR3 (which binds CXCL9 and CXCL10) abolished the difference in the chemotherapeutic response between Stat3(-/-) and control tumors. Altogether, these results suggest that STAT3 inhibitors may improve the outcome of chemotherapy by enhancing the type-1 interferon response of cancer cells.

Discovery of 1-{4-[3-fluoro-4-((3s,6r)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (GNE-3500): a potent, selective, and orally bioavailable retinoic acid receptor-related orphan receptor C (RORc or RORγ) inverse agonist.

Retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) is a nuclear receptor that plays a major role in the production of interleukin (IL)-17. Considerable efforts have been directed toward the discovery of selective RORc inverse agonists as potential treatments of inflammatory diseases such as psoriasis and rheumatoid arthritis. Using the previously reported tertiary sulfonamide 1 as a starting point, we engineered structural modifications that significantly improved human and rat metabolic stabilities while maintaining a potent and highly selective RORc inverse agonist profile. The most advanced δ-sultam compound, GNE-3500 (27, 1-{4-[3-fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone), possessed favorable RORc cellular potency with 75-fold selectivity for RORc over other ROR family members and >200-fold selectivity over 25 additional nuclear receptors in a cell assay panel. The favorable potency, selectivity, in vitro ADME properties, in vivo PK, and dose-dependent inhibition of IL-17 in a PK/PD model support the evaluation of 27 in preclinical studies.

Effect of new oxicam derivatives on efflux pumps overexpressed in resistant a human colorectal adenocarcinoma cell line.

BACKGROUND: Oxicams are non-steroidal anti-inflammatory drugs (NSAIDs). Antitumor potential of NSAIDs has often been reported in literature. We studied antitumor activity of newly synthesized oxicam derivatives (PR17 and PR18) against doxorubicin-sensitive and resistant human colorectal adenocarcinoma cells (LoVo and LoVo/Dx). MATERIALS AND METHODS: The cytotoxicity of oxicam derivatives alone and in combination with doxorubicin was assessed. Inhibition of P-glycoprotein (ABCB1) transport activity was monitored by flow cytometry. Expression of ABCB1 gene was analyzed by semi-quantitative reverse transcription PCR, while ABCB1 protein expression was assessed by western blotting. RESULTS: Oxicam derivative PR18 was more cytotoxic to cancer cells than PR17. PR18 was observed to sensitize LoVo/Dx cells to doxorubicin and was identified as an effective multidrug resistance modulator. Additionally, ABCB1 expression was reduced in the presence of PR18. CONCLUSION: PR18 was identified as an effective modulator in LoVo/Dx resistant human colorectal adenocarcinoma cells which overexpressed ABCB1 efflux pump.

Relative contributions of presystemic and systemic peptidases to oral exposure of a novel metabotropic glutamate 2/3 receptor agonist (LY404039) after oral administration of prodrug pomaglumetad methionil (LY2140023).

Pomaglumetad methionil (LY2140023) is the prodrug of a novel metabotropic glutamate 2/3 receptor agonist (LY404039) being investigated for the treatment of schizophrenia. Using accelerator mass spectrometry (AMS) and an intravenous (i.v.) radiolabeled tracer approach, the absolute bioavailability of the prodrug and the extent of its conversion to active moiety (LY404039) were estimated at presystemic (intestinal/first pass) and systemic sites after simultaneous oral and i.v. dosing in healthy subjects. The mean absolute bioavailability of prodrug (80 mg oral) was 0.68. On the basis of these data and a previous radiolabeled mass balance study in which no prodrug was recovered in feces, we concluded that 0.32 of the dose is converted to active drug in the intestinal tract. The fraction of prodrug converted to active moiety was approximately 1, indicating complete conversion of the prodrug that reaches the systemic circulation to the active moiety. Prodrug (80 mg oral and 100 µg i.v.) and active moiety (100 µg i.v.) were well tolerated in healthy subjects. Thus, the absolute bioavailability of prodrug LY2140023 and the fraction converted presystemically and systemically to active moiety LY404039 were estimated simultaneously using radiolabeled tracer microdosing and AMS.

Determination of 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone by liquid chromatography-tandem mass spectrometry.

2-(3-Benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344), a 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitor, is newly developed for the control of type 2 diabetes mellitus (T2DM) and metabolic syndrome. A method for the determination of KR-66344 in rat plasma was developed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) to evaluate the pharmacokinetics of KR-66344. Plasma samples were processed by a liquid-liquid extraction method with ethyl acetate and introduced onto the LC-MS-MS system. The analyte and imipramine (internal standard) were analyzed by multiple reaction monitoring based on transitions at m/z 420.1 → 105.0 and 282.2 → 86.0, respectively. The calibration curve was linear (r = 0.9993) over the concentration range of 1.0-1,000 ng/mL. The mean recovery values for KR-66344 and imipramine were 83.8 and 86.2%, respectively. The mean inter-day and intra-day assay precision values were 3.9 and 2.4%, respectively. KR-66344 was stable under various handling and storage conditions. This developed method was applied to a pharmacokinetic study after the oral administration of KR-66344 in rats. The concentration of KR-66344 was readily measurable in rat plasma up to 24 h post-dose after an oral administration, suggesting that current assay is applicable to pharmacokinetic studies for KR-66344.

Gamma secretase inhibition promotes hypoxic necrosis in mouse pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease that is refractory to medical intervention. Notch pathway antagonism has been shown to prevent pancreatic preneoplasia progression in mouse models, but potential benefits in the setting of an established PDA tumor have not been established. We demonstrate that the gamma secretase inhibitor MRK003 effectively inhibits intratumoral Notch signaling in the KPC mouse model of advanced PDA. Although MRK003 monotherapy fails to extend the lifespan of KPC mice, the combination of MRK003 with the chemotherapeutic gemcitabine prolongs survival. Combination treatment kills tumor endothelial cells and synergistically promotes widespread hypoxic necrosis. These results indicate that the paucivascular nature of PDA can be exploited as a therapeutic vulnerability, and the dual targeting of the tumor endothelium and neoplastic cells by gamma secretase inhibition constitutes a rationale for clinical translation.

Effects of a novel mGlu2/3 receptor agonist prodrug, LY2140023 monohydrate, on central monoamine turnover as determined in human and rat cerebrospinal fluid.

RATIONALE: Accumulating evidence suggests that the primary symptoms of schizophrenia may be associated with altered central glutamate transmission. LY2140023 monohydrate is the methionine prodrug of the selective mGlu(2/3) receptor agonist LY404039 and is currently being assessed for the treatment of schizophrenia. OBJECTIVE: The objective of this study was to evaluate the central pharmacological activity of LY2140023 monohydrate in preclinical and clinical studies. METHODS: Effects on neurotransmitter/metabolite concentrations were assessed in male rats following single oral doses of LY2140023 monohydrate (microdiasylates from the prefrontal cortex), single intraperitoneal injection of LY404039 [cerebrospinal fluid (CSF)], or LY2140023 monohydrate dosed once daily for 7 days (CSF). A clinical study in 16 healthy subjects assessed the effects of LY2140023 monohydrate 40 mg orally twice daily for 14 days in lumbar CSF. RESULTS: Rat studies: Acute dosing with LY2140023 monohydrate resulted in significant dose-dependent increases in extracellular concentrations of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), but not 5-hydroxyindoleacetic acid (5-HIAA), in the prefrontal cortex. LY2140023 monohydrate dosing for 7 days elevated the concentrations of HVA in CSF, while acutely dosed LY404039 increased the concentrations of DOPAC, HVA, and methoxy-hydroxyphenylglycol (MHPG), but not 5-HIAA. Clinical study: Significant increases were seen for DOPAC, HVA, 5-HIAA, and MHPG in the CSF of subjects receiving LY2140023 monohydrate, but not placebo. CONCLUSIONS: LY2140023 monohydrate and/or LY404039 dosing potently affected dopamine turnover and also significantly affected serotonin turnover in the human and rat central nervous systems. The measurement of biogenic amine metabolites such as DOPAC and HVA may serve as useful biomarkers of LY2140023 monohydrate and/or LY404039 central pharmacodynamic activity.

Oral RKS262 reduces tumor burden in a neuroblastoma xenograft animal model and mediates cytotoxicity through SAPK/JNK and ROS activation in vitro.

Patients diagnosed with high-risk neuroblastoma (NB), an extracranial solid tumor in children, have metastases and low survival (30%) despite aggressive multi-modal therapy. Therefore new therapies are urgently needed. We show significant in vitro and in vivo antitumor efficacy of RKS262 in NB. RKS262 showed superior cytotoxicity (IC(50) = 6-25 µM) against six representative NB cell lines compared to its parent analog Nifurtimox (currently in phase 2). Pre-formulated RKS262 (150 mg/kg/daily) pellets administered orally, suppressed tumor growth (60%, p = 0.021) in NB xenograft mice within 28 days. RKS262-treated SMSKCNR cells showed TUNEL-positive DNA nicks and activation of ROS, MAPKs (SAPK/JNK), caspase-3, and p53, along with suppression of the IGF-1R/PI3K/PKC pathway and the Bcl2 family of proteins. RKS262 caused G(2)/M-phase arrest and suppressed cdc-2, cyclin B1, p21, and cyclin D1/D4 expression. N-acetyl-cysteine (NAC; 10 mM) pre-treatment rescued cell viability of RKS262 (23 µM)-treated SMSKCNR cells, and pre-treatment with ascorbic acid (100 µM) and a MAPK inhibitor SB203580 (20 µM) reversed SAPK/JNK, caspase-3 activation, PARP-1 cleavage, and suppression of IGF-1R, PI3K, and PKC phosphorylation. Further, treatment with exogenous BDNF (50 nM) did not suppress SAPK/JNK or ROS activation due to RKS262. Rather, BDNF (50 nM), EGF (100 nM) and IGF-1 (100 nM) co-treatment with RKS262 induced a remarkable S-phase arrest rather than a G(2)/M phase arrest when RKS262 was used alone. In summary, RKS262 shows oral efficacy in NB xenograft animals, and induces apoptosis in vitro in SMSKCNR cells via cell cycle arrest, MAPK and ROS activation, and suppression of IGF-1R/PI3K/PKC and Bcl2 family proteins in a growth factor (BDNF/EGF/IGF-1)-independent fashion.

Effects of cyclooxygenase-2 inhibitor and adenosine triphosphate-sensitive potassium channel opener in syngeneic mouse islet transplantation.

In the initial days after transplantation, islet grafts may be attacked by cytokines via cyclooxygenase-2 (COX-2), producing primary nonfunction. In addition, chronic overstimulation of ß-cells may impair insulin secretion. To enhance the function of transplanted islets, the present study investigated the effects of rofecoxib, a COX-2 inhibitor, and NN414 (6-chloro-3-[1-methylcyclopropyl]amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide), an adenosine triphosphate-sensitive potassium channel opener, on islet transplantation. Male inbred C57BL/6 mice were used as donors and recipients. One hundred fifty islets were isolated via collagenase digestion and density gradient, and syngeneically transplanted under the kidney capsule in mice with streptozotocin-induced diabetes. Recipients were treated with or without rofecoxib, 10 mg/kg/d orally, or with or without NN414, 3 mg/kg/d orally, for 4 weeks. After transplantation, recipient body weight, blood glucose concentration, and intraperitoneal glucose tolerance were measured. The grafted kidney was extracted for determination of insulin content at 4 weeks. In the rofecoxib-treated and NN414-treated groups and both control groups, body weight remained stable, and the blood glucose concentration decreased progressively. However, at 4 weeks after transplantation in the groups treated or not treated with rofecoxib or NN414, no significant difference was observed in recipient body weight, blood glucose concentration, and glucose tolerance or in insulin content of the graft. These data indicate that posttransplantation treatment with rofecoxib or NN414 has no beneficial effect on transplantation outcome in diabetic mouse recipients engrafted with a marginal islet mass.

The Notch target Hes1 directly modulates Gli1 expression and Hedgehog signaling: a potential mechanism of therapeutic resistance.

PURPOSE: Multiple developmental pathways including Notch, Hedgehog, and Wnt are active in malignant brain tumors such as medulloblastoma and glioblastoma (GBM). This raises the possibility that tumors might compensate for therapy directed against one pathway by upregulating a different one. We investigated whether brain tumors show resistance to therapies against Notch, and whether targeting multiple pathways simultaneously would kill brain tumor cells more effectively than monotherapy. EXPERIMENTAL DESIGN: We used GBM neurosphere lines to investigate the effects of a gamma-secretase inhibitor (MRK-003) on tumor growth, and chromatin immunoprecipitation to study the regulation of other genes by Notch targets. We also evaluated the effect of combined therapy with a Hedgehog inhibitor (cyclopamine) in GBM and medulloblastoma lines, and in primary human GBM cultures. RESULTS: GBM cells are at least partially resistant to long-term MRK-003 treatment, despite ongoing Notch pathway suppression, and show concomitant upregulation of Wnt and Hedgehog activity. The Notch target Hes1, a repressive transcription factor, bound the Gli1 first intron, and may inhibit its expression. Similar results were observed in a melanoma-derived cell line. Targeting Notch and Hedgehog simultaneously induced apoptosis, decreased cell growth, and inhibited colony-forming ability more dramatically than monotherapy. Low-passage neurospheres isolated from freshly resected human GBMs were also highly susceptible to coinhibition of the two pathways, indicating that targeting multiple developmental pathways can be more effective than monotherapy at eliminating GBM-derived cells. CONCLUSIONS: Notch may directly suppress Hedgehog via Hes1 mediated inhibition of Gli1 transcription, and targeting both pathways simultaneously may be more effective at eliminating GBMs cells.

Unphosphorylated STAT3 modulates alpha 7 nicotinic receptor signaling and cytokine production in sepsis.

The role of STAT3 in infectious diseases remains undetermined, in part because unphosphorylated STAT3 has been considered an inactive protein. Here, we report that unphosphorylated STAT3 contributes to cholinergic anti-inflammation, prevents systemic inflammation, and improves survival in sepsis. Bacterial endotoxin induced STAT3 tyrosine phosphorylation in macrophages. Both alpha 7 nicotinic receptor (alpha 7nAChR) activation and inhibition of JAK2 blunt STAT3 phosphorylation. Inhibition of STAT3 phosphorylation mimicked the alpha 7nAChR signaling, inhibiting NF-kappaB and cytokine production in macrophages. Transfection of macrophages with the dominant-negative mutant STAT3F, to prevent its tyrosine phosphorylation, reduced TNF production but did not prevent the alpha 7nAChR signaling. However, inhibition of STAT3 protein expression enhanced cytokine production and abrogated alpha 7nAChR signaling. Alpha 7nAChR controls TNF production in macrophages through a mechanism that requires STAT3 protein expression, but not its tyrosine phosphorylation. In vivo, inhibition of STAT3 tyrosine phosphorylation by stattic prevented systemic inflammation and improved survival in experimental sepsis. Stattic also prevented the production of late mediators of sepsis and improved survival in established sepsis. These results reveal the immunological implications of tyrosine-unphosphorylated STAT3 in infectious diseases.

In vivo effects of a combined 5-HT1B receptor/SERT antagonist in experimental pulmonary hypertension.

AIMS: A mechanism for co-operation between the serotonin (5-hydroxytryptamine, 5-HT) transporter and 5-HT1B receptor in mediating pulmonary artery vasoconstriction and proliferation of pulmonary artery smooth muscle cells has been demonstrated in vitro. Here we determine, for the first time, the in vivo effects of a combined 5-HT1B receptor/serotonin transporter antagonist (LY393558) with respect to the development of pulmonary arterial hypertension (PAH) and its in vitro effects in human pulmonary artery smooth muscle cells (hPASMCs) derived from idiopathic PAH (IPAH) patients. METHODS AND RESULTS: We determined the effects of LY393558 as well as a selective serotonin transporter inhibitor, citalopram, on right ventricular pressure, right ventricular hypertrophy, and pulmonary vascular remodelling in wildtype mice and mice over-expressing serotonin transporter (SERT+ mice) before and after hypoxic exposure. We also compared their effectiveness at reversing PAH in SERT+ mice and hypoxic mice. Further, we examined the proliferative response to serotonin in IPAH hPASMCs. We also clarified the pharmacology of serotonin-induced vasoconstriction and 5-HT1B receptor/serotonin transporter interactions in mouse isolated pulmonary artery. Citalopram had a moderate effect at preventing and reversing experimental PAH in vivo whereas LY393558 was more effective. LY393558 was more effective than citalopram at reversing serotonin-induced proliferation in IPAH hPASMCs. There is synergy between 5-HT1B receptor and serotonin transporter inhibitors against serotonin-induced vasoconstriction in mouse pulmonary arteries. CONCLUSION: 5-HT1B receptor and serotonin transporter inhibition are effective at preventing and reversing experimental PAH and serotonin-induced proliferation of PASMCs derived from IPAH patients. Targeting both the serotonin transporter and 5-HT1B receptor may be a novel therapeutic approach to PAH.