Effect of a small molecule inhibitor of nuclear factor-κB nuclear translocation in a murine model of arthritis and cultured human synovial cells

A small cell-permeable compound, dehydroxymethylepoxyquinomicin (DHMEQ), does not inhibit phosphorylation and degradation of IκB (inhibitor of nuclear factor-κB [NF-κB]) but selectively inhibits nuclear translocation of activated NF-κB. This study aimed to demonstrate the antiarthritic effect of this novel inhibitor of the NF-κB pathway in vivo in a murine arthritis model and in vitro in human synovial cells. Collagen-induced arthritis was induced in mice, and after onset of arthritis the mice were treated with DHMEQ (5 mg/kg body weight per day). Using fibroblast-like synoviocyte (FLS) cell lines established from patients with rheumatoid arthritis (RA), NF-κB activity was examined by electrophoretic mobility shift assays. The expression of molecules involved in RA pathogenesis was determined by RT-PCR, ELISA, and flow cytometry. The proliferative activity of the cells was estimated with tritiated thymidine incorporation. After 14 days of treatment with DHMEQ, mice with collagen-induced arthritis exhibited decreased severity of arthritis, based on the degree of paw swelling, the number of swollen joints, and radiographic and histopathologic scores, compared with the control mice treated with vehicle alone. In RA FLS stimulated with tumor necrosis factor-α, activities of NF-κB components p65 and p50 were inhibited by DHMEQ, leading to suppressed expression of the key inflammatory cytokine IL-6, CC chemokine ligand-2 and -5, matrix metalloproteinase-3, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. The proliferative activity of the cells was also suppressed. This is the first demonstration of an inhibitor of NF-κB nuclear translocation exhibiting a therapeutic effect on established murine arthritis, and suppression of inflammatory mediators in FLS was thought to be among the mechanisms underlying such an effect

MOLECULAR DESIGN OF SUGAR-FREE MIGRACIN ANALOG MIGRACINAL THAT INHIBITS OVARIAN CANCER CELL MIGRATION AND INVASION

Introduction. Cancer metastasis consists of several steps including detachment from the primary tumor, migration, invasion, transport in the blood or lymphatic vessels, attachment at the secondary site, and growth of secondary tumor. Migration and invasion areinvolved in the mechanism of all types of cancer metastasis. We previously isolated novel cellular migration inhibitor migracin A and B from a culture filtrate of Streptomyces sp. Migracin A was shown to inhibit IGF-1-mediated cellular migration and invasion in ovarian carcinoma cells. However, it is difficult to prepare large amount of migracin A. Migracin A consists of substituted benzene and an alkylated sugar moiety. In the present research, we have designed and synthesized a simplified dialdehydederivative of migracin called migracinal having no sugar moiety. Material and methods. Migracinal was purchased from Techno Chem Co., Ltd., Tokyo, Japan. Migracinal was prepared from 2,4-dihydroxybenzaldehyde (2,4-DHBA). The structure was confirmed by proton and carbon NMR spectra and ESI mass spectroscopy. The antitumor activity of the new derivative was studied by standard tests under conditions in vitro. Results. Migracinal inhibited cellular migration and invasion in ovarian clear cell carcinoma ES-2 cells. It also inhibited IGF-1 expression as migracin A. Moreover, it induced anoikis rather than apoptosis in ES-2 cells.Conclusions. Migracinal is easier to prepare than migracins, and it may be useful for the mechanistic study and suppression of metastasis. Введение. Процесс метастазирования рака состоит из нескольких этапов: отсоединение клеток от первичной опухоли, миграцию, инвазию, перемещение в крови или лимфатических сосудах, присоединение и рост вторичной опухоли. Механизмы миграции и инвазии универсальны для всех видов рака. Ранее из культуры Streptomyces SP мы выделили Migracin А и В - новые ингибиторы клеточной миграции. Было продемонстрировано как Migracin А ингибирует IGF-1-опосредованную миграцию и инвазию клеток рака яичников. Однако большое количество Migracin А, состоящего из замещенного бензола и алкилированного углеводного фрагмента, синтезировать трудоемко. В настоящем исследовании мы разработали и синтезировали упрощенное производное диальдегида Migracin, не имеющего углеводного компонента, названное Migracinal. Материалы и методы. Migracin приобретался у компании «ТехноХим Co., Лтд» (Токио, Япония). Производное Migracinal получали взаимодействием Migracin с 2,4-дигидроксибензалдегидом. Структура была подтверждена спектрами ЯМР и масс-спектроскопией. Противоопухолевая активность нового производного изучалась стандартными тестами в условиях in vitro. Результаты. Установлено, что Migracinal ингибирует клеточную миграцию и инвазию клеток ES-2 рака яичника и аналогично Migracin A ингибирует IGF-1 экспрессию. Кроме того, он индуцировал аноикис, а не апоптоз в клетках ES-2.Заключение. Синтез Migracinal легче в сравнении с Migracin, а спектр противоопухолевой активности идентичен, что может быть использовано для подавления процессов метастазирования.

Перспективы ингибирования NF-kappa B внутрибрюшинным введением DHMEQ

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Dehydroxymethylepoxyquinomicin Inhibits Expression and Production of Inflammatory Mediators in Interleukin-1β β β β β-induced Human Chondrocytes

The present research was carried out to determine the effects of a nuclear factor-kappaB (NF-kappaB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), derivative of the antibiotic epoxyquinomicin C, on normal human chondrocytes treated with interleukin-1beta (IL-1beta). This is a cell model particularly useful to reproduce the mechanisms involved in degenerative arthropathies, where oxidative-inflammatory stress determines a progressive destruction of the articular cartilaginous tissue. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inter-cellular adhesion molecule (ICAM)-1 was evaluated through Western blot analysis. The release of chemokines like monocyte chemoattractant protein-1 (MCP-1), regulated upon normal activation T-cell expressed and secreted (RANTES), and interleukin-8 (IL-8) were determined by ELISA assays. DHMEQ acts as a potent inhibitor of iNOS and COX-2 gene expression while also suppressing the production of nitrite in human chondrocytes. In addition, DHMEQ induces a significant dose-dependent decrease in ICAM expression, MCP-1, RANTES, and IL-8 release. DHMEQ helps to decrease the expression and production of pro-inflammatory mediators in IL-1beta-induced chondrocytes. DHMEQ may become a therapeutic agent for treatment of chondro-degenerative diseases

Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors

AbstractDamnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis

Inhibition of NF-κB and Akt pathways by an antibody-avidin fusion protein sensitizes malignant B-cells to cisplatin-induced apoptosis

Multiple myeloma (MM) is an incurable disease of malignant plasma cells. Recent therapeutic advancements have resulted in improved response rates, however, there is no improvement in overall survival, therefore, new therapeutics are needed. Since the transferrin receptor is upregulated on the surface of MM cells, we previously developed an antibody fusion protein consisting of an IgG3 specific for the human transferrin receptor 1 (TfR1, CD71) genetically fused to avidin at its carboxy-terminus (ch128.1Av). We have previously shown that ch128.1Av exhibits intrinsic cytotoxicity against certain malignant B-cells by disrupting the cycling of the TfR and decreasing TfR cell surface expression resulting in lethal iron starvation. In addition, ch128.1Av can sensitize malignant cells to apoptosis induced by gambogic acid, a herbal drug used in Chinese medicine. In this study, we hypothesized that ch128.1Av may also sensitize drug-resistant malignant B-cells to chemotherapeutic agents by inhibiting key survival pathways. In this study we show that ch128.1Av sensitizes malignant B-cells to apoptosis induced by cisplatin (CDDP). The sensitization by ch128.1Av resulted in the inhibition of the constitutively activated Akt and NF-κB survival/antiapoptotic pathways and downstream decreased expression of antiapoptotic gene products such as BclxL and survivin. The direct role of the inhibition of the Akt and NF-κB pathways by ch128.1Av in CDDP-mediated cytotoxicity was demonstrated by the use of specific chemical inhibitors and siRNA which mimicked the effects of ch128.1Av. Overall, this study provides evidence of the therapeutic potential of ch128.1Av as a chemo-sensitizing agent in drug-resistant tumor cells.Fil: Suzuki, Eriko. Keio University; JapónFil: Daniels, Tracy R.. University of California at Los Angeles; Estados UnidosFil: Helguera, Gustavo Fernando. University of California at Los Angeles; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Penichet, Manuel L.. University of California at Los Angeles; Estados UnidosFil: Umezawa, Kazuo. Keio University; JapónFil: Bonavida, Benjamín. University of California at Los Angeles; Estados Unido

NF-κB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor

Inhibition of NF-κB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-κB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-κB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-κB inhibitors to rheumatoid arthritis therapy

Ligand-dependent EGFR activation induces the co-expression of IL-6 and PAI-1 via the NFkB pathway in advanced-stage epithelial ovarian cancer.

The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, is expressed in up to 70% of epithelial ovarian cancers (EOCs), where it correlates with poor prognosis. The majority of EOCs are diagnosed at an advanced stage, and at least 50% present malignant ascites. High levels of IL-6 have been found in the ascites of EOC patients and correlate with shorter survival. Herein, we investigated the signaling cascade led by EGFR activation in EOC and assessed whether EGFR activation could induce an EOC microenvironment characterized by pro-inflammatory molecules. In vitro analysis of EOC cell lines revealed that ligand-stimulated EGFR activated NFkB-dependent transcription and induced secretion of IL-6 and plasminogen activator inhibitor (PAI-1). IL-6/PAI-1 expression and secretion were strongly inhibited by the tyrosine kinase inhibitor AG1478 and EGFR silencing. A significant reduction of EGF-stimulated IL-6/PAI-1 secretion was also obtained with the NFkB inhibitor dehydroxymethylepoxyquinomicin. Of 23 primary EOC tumors from advanced-stage patients with malignant ascites at surgery, 12 co-expressed membrane EGFR, IL-6 and PAI-1 by immunohistochemistry; both IL-6 and PAI-1 were present in 83% of the corresponding ascites. Analysis of a publicly available gene-expression data set from 204 EOCs confirmed a significant correlation between IL-6 and PAI-1 expression, and patients with the highest IL-6 and PAI-1 co-expression showed a significantly shorter progression-free survival time (P=0.028). This suggests that EGFR/NFkB/IL-6-PAI-1 may have a significant impact on the therapy of a particular subset of EOC, and that IL-6/PAI-1 co-expression may be a novel prognostic marker

Determination of topological structure of ARL6ip1 in cells: Identification of the essential binding region of ARL6ip1 for conophylline

AbstractConophylline (CNP) has various biological activities, such as insulin production. A recent study identified ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) as a direct target protein of CNP. In this study, we revealed that ARL6ip1 is a three-spanning transmembrane protein and determined the CNP-binding domain of ARL6ip1 by deletion mutation analysis of ARL6ip1 with biotinyl-amino-CNP. These results suggest that CNP is expected to be useful for future investigation of ARL6ip1 function in cells. Because of the anti-apoptotic function of ARL6ip1, CNP may be an effective therapeutic drug and/or a novel chemosensitizer for human cancers and other diseases

Is the combination therapy of IKr-channel blocker and left stellate ganglion block effective for intractable ventricular arrhythmia in a cardiopulmonary arrest patient?

Background: We have previously reported that the defibrillation success rate of intravenous nifekalant hydrochloride (NIF), a pure IKr-channel (IKr: the rapid components of the delayed rectifier potassium current) blocker, was more than 75% for lidocaine-resistant ventricular tachycardia and fibrillation (VT/VF) in patients with out-of-hospital cardiopulmonary arrest (CPA). However, there was no effective treatment for the remaining 25% of patients in whom defibrillation was unsuccessful. We hypothesised that the combination therapy of NIF and left stellate ganglion block (LSGB) was useful for defibrillation in NIF-resistant VT/VF and investigated its efficacy in a retrospective study. Methods and results: We investigated sequentially 272 out-of-hospital CPA patients treated at Tokai University between April and December 2006. VT/VF occurred in 55 patients on arrival or during cardiopulmonary resuscitation (CPR). On the basis of our CPR algorithm, NIF was administered (0.15-0.3 mg/kg, i.v.) after the first direct-current cardioversion. NIF-resistant VT/VFs were observed in 15 out of 55 patients and LSGB was performed on 11 of these with administration of NIF. Sinus rhythm was restored in 7 patients following LSGB (64%) and complete recovery was achieved in 2 patients. In the non-LSGB group, however, all the patients died. Conclusions: The combination therapy of intravenous NIF and LSGB was useful for defibrillation in intractable VT/VF. It is a potential and innovative treatment strategy for IKr-channel blocker resistant VT/VF. (Cardiol J 2007; 14: 355-365