Resistance of Fetal Guinea Pig Ventricular Myocardium to Hypoxia: Maintained Intracellular ATP Prevents the Opening of ATP-Sensitive Potassium Channels.

The presence and function of the ATP-sensitive potassium channel current (IKATP) were examined in the guinea pig myocardium to clarify the mechanisms for the resistance of the fetal myocardium to hypoxia. Experimental hypoxia markedly reduced the action potential duration and contractile force in isolated ventricular myocardium from the adult, but only moderately in those from the fetus. In isolated ventricular cardiomyocytes, the density of the IKATP activated by cromakalim, as well as their sensitivity to intracellular ATP concentration, were not different between the fetus and adult. The tissue ATP content was similar between the fetal and adult myocardium under normal condition, but the hypoxia-induced decrease was smaller in the fetus. Confocal microscopic analysis revealed that the mitochondria in the fetal cardiomyocyte is less in quantity than that in the adult and is more localized to the cell center. These results indicate that IKATP in the fetal guinea pig myocardium has a current density and ATP sensitivity similar to those of the adult, but is not activated under hypoxic conditions because the energy metabolism of the fetal myocardium is less dependent on oxidative phosphorylation.

Low disease activity for up to 3 years after adalimumab discontinuation in patients with early rheumatoid arthritis: 2-year results of the HOPEFUL-3 Study.

BACKGROUND: This study was conducted to evaluate the feasibility of long-term adalimumab (ADA) discontinuation after achievement of low disease activity (LDA) in Japanese patients with early rheumatoid arthritis (RA) and to identify predictors of LDA maintenance. METHODS: In the HOPEFUL-1 study, patients received initial therapy with either ADA plus methotrexate (MTX; intensive therapy) or MTX alone (standard therapy) for 26 weeks, followed by ADA + MTX for 26 weeks. In the HOPEFUL-2 study, patients received ADA + MTX (ADA continuation) or MTX alone (ADA discontinuation) for 52 weeks. HOPEFUL-3 was an observational study that enrolled patients who had completed HOPEFUL-2; these patients were followed for an additional 104 weeks. RESULTS: Of the 172 patients enrolled in the HOPEFUL-3 study, 135 (ADA continuation, n = 61; ADA discontinuation, n = 74) with 28-joint Disease Activity Score using C-reactive protein (DAS28-CRP) values at both week 52 (start of HOPEFUL-2) and week 208 (end of HOPEFUL-3) were included in the effectiveness analysis. At week 208, 58 (95.1%) of 61 patients and 59 (79.7%) of 74 patients who continued or discontinued ADA, respectively, had LDA (DAS28-CRP <3.2). Initial intensive therapy was associated with a better outcome than standard therapy in terms of change in modified total Sharp score from week 0 to week 208, which was ≤0.5 (64% vs. 30%). The incidence of adverse events was significantly lower in the ADA discontinuation group than in the ADA continuation group (9.7% vs. 32.9%; p < 0.001). CONCLUSIONS: Approximately 80% of patients who discontinued ADA for 3 years after achieving LDA with ADA + MTX were still in LDA, with a lower incidence of adverse events than patients who continued ADA. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01346501. Registered 29 April 2011.

Impact of Adalimumab on Work Productivity and Activity Impairment in Japanese Patients with Rheumatoid Arthritis: Large-Scale, Prospective, Single-Cohort ANOUVEAU Study.

INTRODUCTION: The Adalimumab Non-interventional Trial for Up-verified Effects and Utility (ANOUVEAU) was a large-scale, multicenter, prospective, observational, single-cohort study that evaluated the effects of adalimumab (ADA) on rheumatoid arthritis (RA)-related work productivity and activity impairment (RA-related WPAI) and disease activity in routine rheumatology care in Japan. METHODS: Patients with RA were categorized as paid workers (PWs, ≥35 h/week), part-time workers (PTWs, <35 h/week), or homemakers (HMs, unemployed) and were administered the WPAI for RA (WPAI/RA) questionnaire. All patients who received ADA were followed for 48 weeks to evaluate safety and effectiveness. RESULTS: Of the 1808 patients analyzed, 825, 243, and 740 patients were PWs, PTWs, and HMs, respectively. WPAI/RA domain scores significantly improved at weeks 12, 24, and 48 in all groups, with maximum improvement observed for PWs (p < 0.05). Additionally, remission rates (according to Disease Activity Score 28, erythrocyte sedimentation rate, Simplified Disease Activity Index, or Health Assessment Questionnaire-Disability Index scores) and EuroQol 5-Dimension 3-Level scores significantly increased from baseline to 48 weeks in all groups (p < 0.0001). Analysis of patient subgroups revealed better WPAI/RA outcomes for patients who were biologic-naïve, treated with concomitant methotrexate, or with RA duration of ≤2 years (p < 0.05). The rate of serious adverse events over 48 weeks of ADA treatment was 5.23%. CONCLUSIONS: Treatment with ADA provided sustained improvement in WPAI and had an acceptable safety profile in patients with RA. FUNDING: AbbVie GK and Eisai Co., Ltd. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT01346488.

H2receptor-mediated positive inotropic effect of histamine in neonatal guinea-pig left atria.

The receptor type mediating the positive inotropic effect of histamine was examined in left atria from neonatal guinea pigs. The positive inotropic effect of histamine, as well as its action potential prolonging effect, was antagonized by ranitidine, but not by chlorpheniramine or thiperamide. The positive inotropic effect was enhanced by isobutylmethylxantine. Receptor binding studies revealed the presence of both H1 and H2 receptor types. These results suggest that the positive inotropic effect of histamine in the neonatal guinea-pig atrium is mediated by H2 receptors.

Histone deacetylase inhibitors valproic acid and depsipeptide sensitize retinoblastoma cells to radiotherapy by increasing H2AX phosphorylation and p53 acetylation-phosphorylation.

Although p53 is intact in most cases of retinoblastoma, it is largely inactivated by the ubiqutin-proteasome system through interaction with murine double minute 2 (MDM2) and murine double minute X (MDMX). The present study showed that the histone deacetylase (HDAC) inhibitors valproic acid (VPA) and depsipeptide (FK228) synergistically enhanced ionizing radiation (IR)-induced apoptosis, associated with activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Y79 and WER1-Rb1 human retinoblastoma cells. Both VPA and FK228 enhanced IR-induced phosphorylation of histone H2AX on Ser139 preceding apoptosis. Exposure of cells to IR in the presence of VPA or FK228 induced the accumulation of p53 acetylated at Lys382 and phosphorylated at Ser46 through the reduction of binding affinity with MDM2 and MDMX. These results suggest that acetylation of p53 by HDAC inhibitors is a promising new therapeutic target in refractory retinoblastoma.

Overall survival and updated results from a phase II study of sunitinib in Japanese patients with metastatic renal cell carcinoma.

BACKGROUND: In a phase II, open-label, multicentre Japanese study, sunitinib demonstrated antitumour activity and acceptable tolerability in metastatic renal cell carcinoma patients. Final survival analyses and updated results are reported. METHODS: Fifty-one Japanese patients with a clear-cell component of metastatic renal cell carcinoma (25 treatment-naïve; 26 cytokine-refractory) received sunitinib 50 mg orally, once daily (Schedule 4/2). Overall and progression-free survivals were estimated by the Kaplan-Meier method. Objective response rate (per Response Evaluation Criteria in Solid Tumours) and safety were assessed with an updated follow-up. RESULTS: First-line and pretreated patients received a median 6.0 and 9.5 treatment cycles, respectively. Investigator-assessed, end-of-study objective response rate was 52.0, 53.8 and 52.9% in first-line, pretreated and overall intent-to-treat populations, respectively. The median progression-free survival was 12.2 and 10.6 months in first-line and pretreated patients, respectively. Fourteen patients per group died (56 and 54%), and the median overall survival was 33.1 and 32.5 months, respectively. The most common treatment-related Grade 3 or 4 adverse events and laboratory abnormalities were fatigue (24%), hand-foot syndrome (18%), decreased platelet count (55%), decreased neutrophil count (53%) and increased lipase (49%). No Grade 5 treatment-related adverse events occurred. Forty patients (78%) required dose reduction, and 13 (25%) discontinued, due to treatment-related adverse events. CONCLUSIONS: With the median overall survival benefit exceeding 2.5 years, and acceptable tolerability, in first-line and pretreated Japanese metastatic renal cell carcinoma patients with Eastern Cooperative Oncology Group performance status 0/1, sunitinib showed a favourable risk/benefit profile, similar to Western studies. However, there was a trend towards greater efficacy and more haematological adverse events in Japanese patients.

A phase II study of sunitinib in Japanese patients with metastatic renal cell carcinoma: insights into the treatment, efficacy and safety.

OBJECTIVE: This study aims to assess the efficacy and safety of sunitinib in Japanese patients with metastatic renal cell carcinoma (RCC). METHODS: Fifty-one Japanese patients with prior nephrectomy, 25 treatment-naive patients (first-line group) and 26 cytokine-refractory patients (pretreated group) were enrolled in this phase II trial. Patients received sunitinib 50 mg orally, once daily, in repeated 6-week cycles (4 weeks on treatment, 2 weeks off). The primary endpoint was RECIST-defined objective response rate (ORR) with tumour assessments every 6 weeks via computed tomography or magnetic resonance imaging. Toxicity was assessed regularly. In the primary efficacy analysis of the intent-to-treat (ITT) population, ORR and 95% confidence interval were calculated based on independent review. Secondary time-to-event endpoints, such as progression-free survival (PFS), were estimated using the Kaplan-Meier method. RESULTS: In the ITT population, ORR was 48.0% in the first-line group (after a median 4 cycles), 46.2% in the pretreated group (5 cycles) and 47.1% overall, with median times to tumour response of 7.1, 10.7 and 10.0 weeks, respectively. Median PFS was 46.0, 33.6 and 46.0 weeks, respectively. The most common treatment-related grade 3/4 adverse events and laboratory abnormalities were fatigue (20%), hand-foot syndrome (14%) and hypertension (12%), decreased platelet count (55%), decreased neutrophil count (51%), increased lipase (39%) and decreased lymphocyte count (33%). CONCLUSIONS: In Japanese patients with RCC, sunitinib is consistently effective and tolerable with similar risk/benefit as that in Western patients, though there was a trend toward greater antitumour efficacy and higher incidence of haematological adverse events in Japanese patients.

Bone marrow and tumor cell colony-forming units and human tumor xenograft efficacy of noncamptothecin and camptothecin topoisomerase I inhibitors.

Topoisomerase I (TopoI), an established anticancer target, is an enzyme producing a single-strand DNA break during transcription. Several noncamptothecin TopoI inhibitors have been identified. One of these, ARC-111, was compared with two clinically used camptothecins, topotecan and irinotecan/SN-38. In mouse and human bone marrow colony formation [colony-forming units granulocyte-macrophage (CFU-GM)] assays, the IC(90) values were 519 and 331 nmol/L for topotecan and SN-38 mouse CFU-GM and were 19 and 26 nmol/L for human CFU-GM, giving mouse to human differentials of 28- and 13-fold. ARC-111 produced IC(90) values of 28 nmol/L in mouse and 6.2 nmol/L in human CFU-GM, thus only a 4.5-fold differential between species. Human bone marrow CFU-GM was more sensitive to topotecan than were several human cancer cell lines, but ARC-111 cytotoxicity was similar for human bone marrow CFU-GM and the seven human tumor cell lines tested. In HCT-116 xenografts, tumor growth delays (TGD) were 17 days for irinotecan and 20 days for ARC-111. In HT-29 xenografts, the TGD was 9 days for both irinotecan and ARC-111. Both ARC-111 and docetaxel had a TGD of 21 days in NCI-H460 xenografts, and both ARC-111 and gemcitabine had a TGD of 7 days in MiaPaCa2 xenograft. Current TopoI inhibitors have broad antitumor activity in human tumor xenografts that is not achieved in the clinic. This may be due to greater sensitivity of human bone marrow than mouse to the cytotoxicity of these agents. It may be possible to achieve similar levels of ARC-111 in patients as in mice allowing improved antitumor activity.

MUC1 oncoprotein blocks death receptor-mediated apoptosis by inhibiting recruitment of caspase-8.

Stimulation of the death receptor superfamily induces the activation of caspase-8 and thereby the apoptotic response. The MUC1 oncoprotein is aberrantly overexpressed by diverse human malignancies and inhibits stress-induced apoptosis. The present results show that MUC1 blocks activation of caspase-8 and apoptosis in the response of malignant cells to tumor necrosis factor alpha, tumor necrosis factor-related apoptosis-inducing ligand, and Fas ligand. The results show that MUC1 associates constitutively with caspase-8. The MUC1 cytoplasmic domain (MUC1-CD) binds directly to the caspase-8 p18 fragment upstream to the catalytic Cys(360) site. The results also show that MUC1-CD binds to Fas-associated death domain (FADD) at the death effector domain. In nonmalignant epithelial cells, MUC1 interacts with caspase-8 and FADD as an induced response to death receptor stimulation. The functional significance of these interactions is supported by the demonstration that MUC1 competes with caspase-8 for binding to FADD and blocks recruitment of caspase-8 to the death-inducing signaling complex. These findings indicate that MUC1 is of importance to the physiologic regulation of caspase-8 activity and that overexpression of MUC1, as found in human malignancies, could contribute to constitutive inhibition of death receptor signaling pathways.

MUC1 oncoprotein promotes growth and survival of human multiple myeloma cells.

The MUC1 oncoprotein is aberrantly expressed in human multiple myeloma cells by mechanisms that are not understood. Moreover, the functional role of MUC1 in multiple myeloma is not known. The present studies demonstrate that the MUC1 gene locus is amplified in multiple myeloma cell lines and in primary cells from patients. The KMS28PE multiple myeloma cell line, which was found to have MUC1 gene amplification, was stably silenced for MUC1 using different siRNAs. Silencing MUC1 was associated with a decrease in nuclear beta-catenin levels, consistent with the function of MUC1 in stabilizing beta-catenin. MUC1 is also known to activate the IKKbeta-->NF-kappaB pathway and KMS28PE cells silenced for MUC1 were found to have downregulation of IKKbeta and IkappaBalpha phosphorylation, and decreased nuclear targeting of NF-kappaB p65. The results also demonstrate that MUC1: i) contributes to KMS28PE cell proliferation, and ii) protects against apoptosis and loss of self-renewal in the response to melphalan and dexamethasone. These findings indicate that MUC1 activates the beta-catenin and NF-kappaB pathways in multiple myeloma cells and contributes to their growth and survival.

Increased myeloid cell responses to M-CSF and RANKL cause bone loss and inflammation in SH3BP2 "cherubism" mice.

While studies of the adaptor SH3BP2 have implicated a role in receptor-mediated signaling in mast cells and lymphocytes, they have failed to identify its function or explain why SH3BP2 missense mutations cause bone loss and inflammation in patients with cherubism. We demonstrate that Sh3bp2 "cherubism" mice exhibit trabecular bone loss, TNF-alpha-dependent systemic inflammation, and cortical bone erosion. The mutant phenotype is lymphocyte independent and can be transferred to mice carrying wild-type Sh3bp2 alleles through mutant fetal liver cells. Mutant myeloid cells show increased responses to M-CSF and RANKL stimulation, and, through mechanisms of increased ERK 1/2 and SYK phosphorylation/activation, they form macrophages that express high levels of TNF-alpha and osteoclasts that are unusually large. M-CSF and RANKL stimulation of myeloid cells that overexpress wild-type SH3BP2 results in similar large osteoclasts. This indicates that the mutant phenotype reflects gain of SH3BP2 function and suggests that SH3BP2 is a critical regulator of myeloid cell responses to M-CSF and RANKL stimulation.

A novel isocoumarin derivative induces mitotic phase arrest and apoptosis of human multiple myeloma cells.

PURPOSE: The isocoumarin NM-3 reverses resistance of human multiple myeloma (MM) cells to dexamethasone and is in clinical trials. In the present work, the NM-3 analog, 185322, has been studied for activity against MM cells. METHODS: Human U266, RPMI8226 and primary MM cells were analyzed for the effects of 185322 on cell cycle distribution, tubulin polymerization and induction of apoptosis. RESULTS: We show that, in contrast to NM-3, treatment with 185322 is associated with a marked arrest of MM cells in M phase. The results also demonstrate that treatment with 185322 is associated with a rapid decrease in tubulin assembly and an increase in Bcl-2 phosphorylation, consistent with disruption of mitosis. Our results further demonstrate that mitotic failure induced by 185322 results in activation of an apoptotic response in MM cell lines and primary MM cells. By contrast, 185322 had little if any effect on growth and survival of human carcinoma cells. CONCLUSION: These findings identify a novel inhibitor of microtubule assembly that induces mitotic arrest and apoptosis of MM cells.

2-(8-hydroxy-6-methoxy-1-oxo-1h-2-benzopyran-3-yl) propionic acid, an inhibitor of angiogenesis, ameliorates renal alterations in obese type 2 diabetic mice.

One of the mechanisms involved in the progression of diabetic nephropathy, the most common cause of end-stage renal failure, is angiogenic phenomenon associated with the increase of angiogenic factors such as vascular endothelial growth factor (VEGF)-A and angiopoietin (Ang)-2, an antagonist of Ang-1. In the present study, we examined the therapeutic efficacy of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3), a small molecule isocoumarin with antiangiogenic activity, using diabetic db/db mice, a model of obese type 2 diabetes. Increases in kidney weight, glomerular volume, creatinine clearance, urinary albumin excretion, total mesangial fraction, glomerular type IV collagen, glomerular endothelial area (CD31(+)), and monocyte/macrophage accumulation (F4/80(+)) observed in control db/db mice were significantly suppressed by daily intraperitoneal injection of NM-3 (100 mg/kg, for 8 weeks). Increases in renal expression of VEGF-A, Ang-2, fibrogenic factor transforming growth factor (TGF)-beta1, and chemokine monocyte chemoattractant protein-1 but not tumor necrosis factor-alpha were also inhibited by NM-3 in db/db mice. Furthermore, decreases of nephrin mRNA and protein levels in db/db mice were recovered by NM-3. In addition, treatment of db/db mice with NM-3 did not affect body weight, blood glucose, serum insulin, or food consumption. NM-3 significantly suppressed the increase of VEGF induced by high glucose in cultured podocytes and also suppressed the increase of VEGF and TGF-beta induced by high glucose in cultured mesangial cells. Taken together, these results demonstrate the potential use of NM-3 as a novel therapeutic agent for renal alterations in type 2 diabetes.

The angiogenesis inhibitor NM-3 is active against human NSCLC xenografts alone and in combination with docetaxel.

The novel isocoumarin 2-(8-hydroxy-6-methoxy-1-oxo-1 H-2-benzopyran-3-yl) propionic acid (NM-3) has completed phase I clinical evaluation as an orally bioavailable angiogenesis inhibitor. NM-3 directly kills both endothelial and tumor cells in vitro at low mM concentrations and is effective in the treatment of diverse human tumor xenografts in mice. The present work has assessed the activity of NM-3 against human non-small-cell lung cancer (NSCLC) cells when used alone and in combination with docetaxel. The results demonstrate that NM-3 decreases clonogenic survival of NSCLC cells at clinically achievable concentrations. The results also demonstrate that NM-3 is effective in the treatment of NSCLC (A549, NCI-H460) tumor xenografts in mice. Moreover, NM-3 potentiated the antitumor activity of docetaxel against NSCLC xenografts without increasing toxicity. Our findings indicate that NM-3 may be effective alone or in combination with docetaxel in the treatment of patients with NSCLC.

Effect of cytogenin, a novel immunomodulator, on streptozotocin-induced diabetes in mice.

The anti-diabetic effect of cytogenin was examined using streptozotocin-induced diabetes in mice. Cytogenin suppressed not only the increase of plasma glucose level but also the body weight reduction in diabetic mice. Histological examination of the pancreas taken from diabetic mice given cytogenin showed that cytogenin decreased the number of macrophages infiltrated into islet of pancreas. Further, cytogenin suppressed the nitric oxide generation by macrophages treated with lipopolysaccharide through decreasing of inducible nitric oxide synthase expression. Cytogenin suppressed interleukin-6 expression by macrophage treated with LPS, suggesting that the anti-diabetic activity of cytogenin might be partly attributed to the suppressive activity against nitric oxide generation.

2-(8-Hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl)propionic acid, a small molecule isocoumarin, potentiates dexamethasone-induced apoptosis of human multiple myeloma cells.

2-(8-Hydroxy-6-methoxy-1-oxo-1Eta-2-benzopyran-3-yl)propionic acid (NM-3) is a small molecule isocoumarin derivative that has recently entered clinical trials as an orally bioavailable anticancer agent. NM-3 induces lethality of human carcinoma cells by both apoptotic and nonapoptotic mechanisms and potentiates the effects of cytotoxic chemotherapeutic agents. The present studies have evaluated the effects of NM-3 on human multiple myeloma (MM) cells. The results demonstrate that NM-3 potentiates dexamethasone-induced killing of both dexamethasone-sensitive MM1.S and dexamethasone-resistant RPMI8226 and U266 MM cells. We show that NM-3 enhances dexamethasone-induced release of the mitochondrial apoptogenic factors cytochrome c and Smac/DIABLO. The results also demonstrate that NM-3 enhances dexamethasone-induced activation of the intrinsic caspase-9->caspase-3 apoptotic pathway. In concert with these results, NM-3 potentiates dexamethasone-induced apoptosis of MM1.S cells. Moreover, NM-3 acts synergistically with dexamethasone in inducing apoptosis of the dexamethasone-resistant RPMI8226 and U266 MM cells. These findings indicate that NM-3 may be effective in combination with dexamethasone in the treatment of MM.

Human MUC1 carcinoma-associated protein confers resistance to genotoxic anticancer agents.

The MUC1 transforming protein is overexpressed by most human carcinomas. The present studies demonstrate that the MUC1 C-terminal subunit (MUC1 C-ter) localizes to mitochondria in HCT116/MUC1 colon carcinoma cells and that heregulin stimulates mitochondrial targeting of MUC1 C-ter. We also show that MUC1 attenuates cisplatin-induced (1) release of mitochondrial apoptogenic factors, (2) activation of caspase-3, and (3) induction of apoptosis. Moreover, knockdown of MUC1 expression in A549 lung and ZR-75-1 breast carcinoma cells by MUC1siRNA was associated with increased sensitivity to genotoxic drugs in vitro and in vivo. These findings indicate that MUC1 attenuates the apoptotic response to DNA damage and that this oncoprotein confers resistance to genotoxic anticancer agents.

Antineoplastic effects of chemotherapeutic agents are potentiated by NM-3, an inhibitor of angiogenesis.

Antiangiogenic therapy, although effective in shrinking tumors, has not yet been established as a standalone treatment for cancer. This therapeutic limitation can be overcome by combining angiogenesis inhibitors with chemotherapeutic agents. NM-3, a small molecule isocoumarin, is a recently discovered angiogenesis inhibitor. Here we demonstrate that NM-3 inhibits the proliferation of human umbilical vein endothelial cells in vitro, at concentrations 10-fold less than those required to inhibit normal fibroblasts or tumor cells (HT29, MKN28, and MCF-7). NM-3 alone inhibits endothelial sprouting and tube formation in vitro. The results also show that synergistic antiproliferative activity is observed when human umbilical vein endothelial cells are treated with NM-3 in combination with 5-fluorouracil. The effects of treatment with NM-3 and various chemotherapeutic agents were also evaluated in tumor xenografts. The results demonstrate that combined treatment with NM-3 and chemotherapeutic agents significantly reduced mean tumor volume compared with either treatment alone, with no effects on body weight changes. Taken together, these findings demonstrate that NM-3 is a well-tolerated angiogenesis inhibitor that significantly increases the efficacy of existing antineoplastic agents.