Dehydroxymethylepoxyquinomicin Research Articles

Antitumor effect of dehydroxymethylepoxyquinomicin, a small molecule inhibitor of nuclear factor- B, on glioblastoma

Glioblastoma is the most malignant type of brain tumor. Despite recent advances in therapeutic modalities, the prognosis of glioblastoma remains very poor. Recent studies have indicated that RelA/nuclear factor (NF)-κB is consistently activated in human glioblastoma. In this study, we searched for a new treatment modality for glioblastoma, by examining the effects of dehydroxymethylepoxyquinomicin (DHMEQ), a unique small molecule inhibitor of NF-κB. Addition of DHMEQ to cultured human glioblastoma cells inhibited the nuclear translocation of RelA. It also reduced the growth rate of human glioblastoma cells significantly in 6 cell lines and modestly in 3 among 10 cell lines examined. Then, we performed further analyses using 3 sensitive cell lines (U87, U251, and YKG-1). The growth retardation was accompanied by G2/M arrest in vitro. Increased apoptosis was observed in U87 and YKG-1, but not U251 cells after DHMEQ treatment. Then, we tested the efficacy of DHMEQ in chemoprevention through the use of a nude mouse model. Subcutaneous tumors formed by U87 or U251 cells were reduced by ∼40% in size by intraperitoneal administration of DHMEQ started immediately after implantation of the cells. DHMEQ treatment achieved statistically significant improvements in survival curves of mice intracranially implanted with U87 or U251 cells. Histological analysis revealed increased areas of necrosis, increased numbers of collapsed microvessels, decreased nuclear immunoreactivity of RelA, and decreased immunoreactivity of urokinase-type plasminogen activator in the DHMEQ-treated U87 tumor tissues. These results suggest that the targeting of NF-κB by DHMEQ may serve as a promising treatment modality in glioblastoma.

A novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates inflammatory colonic injury in mice

BackgroundIn inflammatory bowel disease (IBD), gut inflammation is associated with the activation of nuclear factor kappa B (NF-κB), a key pro-inflammatory transcription factor. AimTo investigate the therapeutic potential of a novel, specific NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), we examined its effect on IBD using murine experimental colitis models. MethodsThe in vitro effect of DHMEQ was evaluated by inflammatory cytokine production and p65 immunostaining using HT-29 and RAW264.7 cells. The in vivo therapeutic effect of DHMEQ was studied in colitis induced by dextran sulphate sodium (DSS) and trinitrobenzenesulphonic acid (TNBS). In these, progression and severity of colitis was mainly assessed by the disease activity index (DAI), histopathology, cellular infiltration, and mRNA expression levels of pro-inflammatory cytokines in the colonic tissues. ResultsIn RAW264.7 cells, DHMEQ significantly inhibited tumour necrosis factor (TNF)-α and interleukin (IL)-6 production induced by LPS in a dose-dependent manner by blocking the nuclear translocation of NF-κB. In addition, DHMEQ inhibited IL-8 production induced by LPS in HT-29 cells. DHMEQ significantly ameliorated DSS colitis as assessed by DAI scores, colonic oedema, and histological scores. Immunohistochemistry revealed that DHMEQ inhibited colonic infiltration of nuclear p65+ cells, CD4+ lymphocytes, and F4/80+ macrophages. mRNA expression levels of the pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, IL-12p40, IL-17, and MCP-1 were also suppressed by DHMEQ administration. Furthermore, DHMEQ significantly ameliorated TNBS colitis as assessed by body-weight changes and histological scores. ConclusionDHMEQ ameliorated experimental colitis in mice. These results indicate that DHMEQ appears to be an attractive therapeutic agent for IBD.

Design and synthesis of biotinylated DHMEQ for direct identification of its target NF-κB components

The design and synthesis of dehydroxymethylepoxyquinomicin (DHMEQ) derivatives were carried out to investigate the intracellular targets. The synthetic biotin probe exhibited membrane permeability and combined selectively with the target protein p65.

Bioimaging analysis of nuclear factor‐κB activity in Philadelphia chromosome‐positive acute lymphoblastic leukemia cells reveals its synergistic upregulation by tumor necrosis factor‐α‐stimulated changes to the microenvironment

To gain an insight into the microenvironmental regulation of nuclear factor (NF)-κB activity in the progression of leukemia, we established a bioluminescent imaging model of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) cells transduced with a NF-κB/luciferase (Luc) reporter and cocultured with murine stromal cells and cytokines. Stromal cells alone did not augment Luc activity, taken as an index of NF-κB, but Luc activity was synergistically upregulated by the combination of stromal cells and tumor necrosis factor (TNF)-α. Dehydroxymethylepoxyquinomicin (DHMEQ), a specific inhibitor of NF-κB DNA binding, rapidly induced the apoptosis of Ph+ALL cells, indicating that NF-κB is necessary for the growth and survival of these cells. However, the DHMEQ-induced suppression of NF-κB activity and the apoptosis of leukemia cells were attenuated by the presence of stromal cells and TNF-α. In NOD-SCID mice transplanted with NF-κB/Luc reporter-containing Ph+ALL cell lines and monitored periodically during the progression of the leukemia, murine TNF-α was significantly expressed in lesions in which the leukemia cells emitted a significant NF-κB signal. These results support the notion that TNF-α also triggers microenvironmental upregulation of NF-κB activity in vivo. Collectively, the results indicated that TNF-α-stimulated microenvironment may contribute to the survival and progression of Ph+ALL cells through the synergistic upregulation of NF-κB activity.

Anti-tumor activity of dehydroxymethylepoxyquinomicin against human oral squamous cell carcinoma cell lines in vitro and in vivo

Several reports have indicated that nuclear factor-kappa B (NF-κB) is constitutively activated in a variety of cancer cells including human oral squamous carcinoma cells, and play a key role in their growth and survival. Recent studies report that NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), inhibits proliferation and induces apoptosis in prostate cancer cell lines. However this anti-tumor effects are still unknown in end human oral squamous carcinoma cells. In the present study, we investigated the effects of DHMEQ on oral squamous carcinoma cell (OSCC) lines in vitro and in vivo. Human OSCC cell lines (HSC-3, SAS) were treated with DHMEQ and examined for cell viability by MTT assay, cell cycle distribution by flow-cytometry, apoptosis by TUNEL assay, and protein expression by western blotting, respectively. In vivo activities were also investigated in a mouse xenograft model. DHMEQ inhibited growth of two OSCC cell lines in a dose-dependent manner measured by MTT assay. A flow cytometric analysis demonstrated that treatment with DHMEQ induced accumulation in sub-G1 phase. TUNEL assay showed that DHMEQ induced DNA fragmentation. Protein expression by western blotting analysis revealed that DHMEQ induced nuclear down regulation of Survivin, cIAP-1, and cIAP-2. In nude mice, DHMEQ inhibited growth of OSCC without major toxic side effects. The present results demonstrated that administration of DHMEQ is suggested to be a novel anti-tumor approach to the treatment of OSCC.

Inhibition of nuclear factor‐kappaB suppresses peritoneal dissemination of gastric cancer by blocking cancer cell adhesion

Currently, patients with peritoneal dissemination of gastric cancer must accept a poor prognosis because there is no standard effective therapy. To inhibit peritoneal dissemination it is important to inhibit interactions between extracellular matrices (ECM) and cell surface integrins, which are important for cancer cell adhesion. Although nuclear factor-kappa B (NF-κB) is involved in various processes in cancer progression, its involvement in the expression of integrins has not been elucidated. We used a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), to study whether NF-κB blocks cancer cell adhesion via integrins in a gastric cancer dissemination model in mice and found that DHMEQ is a potent suppressor of cancer cell dissemination. Dehydroxymethylepoxyquinomicin suppressed the NF-κB activity of human gastric cancer cells NUGC-4 and 44As3Luc and blocked the adhesion of cancer cells to ECM when compared with the control. Dehydroxymethylepoxyquinomicin also inhibited expression of integrin (α2, α3, β1) in in vitro studies. In the in vivo model, we injected 44As3Luc cells pretreated with DHMEQ into the peritoneal cavity of mice and performed peritoneal lavage after the injection of cancer cells. Viable cancer cells in the peritoneal cavities were evaluated sequentially by in vivo imaging. In mice injected with DHMEQ-pretreated cells and lavaged, live cancer cells in the peritoneum were significantly reduced compared with the control, and these mice survived longer. These results indicate that DHMEQ could inhibit cancer cell adhesion to the peritoneum possibly by suppressing integrin expression. Nuclear factor-kappa B inhibition may be a new therapeutic option for suppressing postoperative cancer dissemination.

Chemoenzymatic synthesis of (2S,3S,4S)-form, the physiologically active stereoisomer of dehydroxymethylepoxyquinomicin (DHMEQ), a potent inhibitor on NF-κB

A new route for (2S,3S,4S)-form, the physiologically active stereoisomer of dehydroxymethylepoxyquinomicin (DHMEQ), a potent NF-κB inhibitor, was established by chemoenzymatic approach. Elaboration on the asymmetric epoxidation of a p-benzoquinone monoketal with benzylcinchonidinium tert-butylhydroperoxide yielded an epoxyenone, in 79.8% ee and 57% yield in reproducible manner. By way of the transformation of this key intermediate to enantiomerically pure (2S,3S,4S)-DHMEQ, the contaminating undesired enantiomer could be effectively removed by applying Burkholderia cepacia lipase-catalyzed hydrolysis of diacylated precursor. The above integrated combination of chemical asymmetric synthesis and enzyme-catalyzed kinetic resolution enabled us to prepare active DHMEQ in a large-scale.

Dehydroxymethylepoxyquinomicin, a novel nuclear factor‐κB inhibitor, prevents inflammatory injury induced by interferon‐γ and histamine in NCTC 2544 keratinocytes

1. The novel nuclear factor (NF)-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) is a derivative of the antibiotic epoxyquinomicin C from Amycolatopsis sp. that has been found to inhibit tumour necrosis factor (TNF)-alpha-induced activation of NF-kappaB by suppressing nuclear translocation of NF-kappaB. The aim of the present study was to determine the effects of DHMEQ on interferon (IFN)-gamma- and histamine-activated NCTC 2544 keratinocytes. 2. Keratinocytes were stimulated or not with 200 U/mL IFN-gamma and 10(-4) mol/L histamine in the absence or presence of different concentrations of DHMEQ (1, 5 and 10 microg/mL) or hydrocortisone (10(-5) mol/L), which was used as a reference anti-inflammatory drug. After 48 h, each sample was tested for the presence of intercellular adhesion molecule (ICAM)-1 by western blot analysis, as well as for the release of monocyte chemoattractant protein (MCP)-1, RANTES and interleukin (IL)-8 using specific sandwich ELISAs. To verify the effect of DHMEQ on cell viability of non-stimulated NCTC 2544 keratinocytes, the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used. 3. The results showed that 10 microg/mL DHMEQ potently inhibited ICAM-1 production (by 50%), as well as the release of MCP-1 (to 25% of control), RANTES (to 5% of control) and IL-8 (to 2% of control). The results of the MTT assay indicated that DHMEQ has no effect on cell viability. 4. In conclusion, DHMEQ inhibits the IFN-gamma- and histamine-induced activation of the keratinocyte cell line NCTC 2544. The anti-inflammatory effects of DHMEQ could be exploited by applying the drug topically alone or in combination with sub-toxic concentrations of anti-inflammatory drugs to producer a synergistic effect.

Regulation of human dendritic cells by a novel specific nuclear factor–κB inhibitor, dehydroxymethylepoxyquinomicin

Regulation of antigen-presenting cells (APC) is crucial in controlling allograft rejection. Dendritic cells (DC) are the most potent APC and must mature to present antigens to T-cell receptors. During DC maturation, nuclear factor–κB (NF-κB) is a key transcriptional factor. We synthesized dehydroxymethylepoxyquinomicin (DHMEQ), which specifically inhibits the final step of nuclear translocation of activated NF-κB proteins and examined its immunoregulatory effects on human monocyte–derived DC (Mo-DC). Regulatory Mo-DC were generated by pretreatment with DHMEQ before LPS stimulation, which were termed dl-DC. DHMEQ pretreatment (5 μg/ml) completely inhibited nuclear translocation of activated NF-κB. DHMEQ significantly inhibited DC production of proinflammatory cytokines (IL-6, TNF-α, and IL-12 p70) in a dose-dependent manner. IL-12 was most potently inhibited. However, IL-10 production by dl-DC was only moderately affected by DHMEQ. Although CD40 and the expression of HLA-DR (HLA-DR) expression on dl-DC was downregulated, CD80 and CD86 expression was moderately upregulated. Induction of T helper 1 cell responses was efficiently impaired by dl-DC. This confirmed that DHMEQ-treated Mo-DC exhibited immunoregulatory effects. These findings suggest that DHMEQ has potential as an immunosuppressive drug for human immune cells.

Donor Pretreatment with DHMEQ Improves Islet Transplantation

Currently, pancreatic islet transplantation to achieve normoglycemia in insulin-dependent diabetes mellitus (IDDM) requires two or more donors. This may be due to the inability to transplant functionally preserved and viable islets after isolation. Islets have already been subjected to various harmful stresses during the isolation process leading to apoptosis. One of the intracellular signaling pathways, the transcription factor nuclear factor-kappaB (NF-kappaB)-related pathway, is relevant to the mechanism of beta-cell apoptosis in isolated islets. We attempted to prevent islet apoptosis during isolation by a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). DHMEQ was injected intraperitoneally into donor mice 2 h prior to isolation. NF-kappaB activation, the functioning of isolated islets, apoptosis after isolation, and cytokine- and apoptosis-related genes were analyzed. After 160 equivalents of islets were transplanted into diabetic mice, graft survival and function were evaluated. Intra-islet NF-kappaB was activated immediately after isolation, and DHMEQ inhibited NF-kappaB activation without deterioration of islet function. DHMEQ significantly prevented apoptosis by inhibiting caspase 3/7 activities and down-regulated Bax, a pro-apoptotic gene. Donor pretreatment with DHMEQ significantly improved engraftment in syngeneic islet transplantation in mice, thus preserving insulin contents in the graft liver, as assessed by functional and histologic analyses. DHMEQ is a promising agent in islet transplantation because it protects islets from apoptosis during isolation stress. Donor pretreatment with DHMEQ can significantly affect the success of islet engraftment.

Abstract 4058: Possibility of clinical application of dehydroxymethylepoxyquinomicin in liver metastasis of pancreatic cancer

Abstract Activation of the nuclear transcription factor-kappaB (NF-κB) has been implicated in the progression and metastasis of pancreatic cancer. This study was designed to evaluate the effects of a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in the inhibition of liver metastasis of pancreatic cancer in a clinical liver metastasis-simulating mouse model in which cancer cells were intra-portal injected via small laparotomy. Moreover, synergistic effect of combination therapy with DHMEQ and gemcitabine was investigated. Male BALB/c nude mice were xenografted with intra-portal vein injection of AsPC-1. Mice were divided into four groups; 1) positive control (PC), without any treatment, 2) gemcitabine treated group (GEM), 3) DHMEQ treated group, and 4) combined treatment group (GEM+DHMEQ). Mice were fed for four weeks, and the number of liver metastases, induction of apoptosis and angiogenesis in metastasis foci were investigated after sacrifice. The number of liver metastases decreased significantly in the GEM group, DHMEQ group and GEM+DHMEQ group when compared with the PC group. Significant inductions of apoptosis were revealed in the GEM group and DHMEQ group compared to the PC group, in which apoptotic bodies in metastasis sections were counted by using the TUNEL method. Moreover, in the DHMEQ+GEM group, synergistic effect of combination therapy was observed in induction of apoptosis. Tumor vessels were indentified by immunohistochemical staining of tumor metastasis sections with antibody against CD31. There was a large reduction in the number of CD31-positive vessels in the tumor of DHMEQ or/and GEM-treated mice as compared with control group. Gene expression in metastasis sites was investigated by quantitative RT-PCR. DHMEQ down-regulated IL-8 and MMP-9 remarkably, while gemcitabine down-regulated VEGF moderately. The differences in down-regulated target genes and mechanisms of anti-tumor effects between DHMEQ and gemcitabine point towards the validity of combination therapy with DHMEQ and gemcitabine. In our intra-portal vein injection model, which mimics liver metastasis of pancreatic cancer very well, DHMEQ exhibited anti-tumor effect by inhibiting angiogenesis, invasiveness of tumor cells, and induction of apoptosis. Moreover, when DHMEQ and gemcitabine were administered simultaneously, our result suggested the possibility of a synergistic effect. DHMEQ is lipophilic agent and insoluble in common solvents. Thus, DHMEQ might be a potent drug for the treatment of pancreatic cancer, although in the case of clinical application, some difficulties remain to be solved, such as a dissolution method and an administration route. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4058.

Abstract 4218: Potential roles of NF-kappa B pathways in breast cancer-initiating cells

Abstract Accumulating evidence suggests that Tumor-initiating cells (TICs) or cancer stem cells_which make up only a small proportion of heterogeneous tumor cells_possess a greater ability to maintain tumor formation than other tumor cell types. In human breast cancers, a population characterized by the expression of cell surface markers, CD24−/low/CD44+, was reported to be highly enriched in TICs as compared to CD24+/CD44+ cell populations. However, the signaling pathways activated in TICs that contribute to tumorigenesis are not fully understood. We observed various expression levels of CD24 and CD44 among 8 human breast cancer cell lines. The CD24−/low/CD44+ cell populations of HCC1954 and MCF7, breast cancer cell lines, generated significantly larger tumors than the CD24+/CD44+ cell populations in a xenograft model, suggesting that the CD24−/low/CD44+ cell populations have the properties of TICs at some extent. To identify expressed genes that were highly enriched in CD24−/low/CD44+ cell populations, we next performed DNA microarray analysis using HCC1954, MCF7, and HCC70 cell lines that have small populations of CD24−/low/CD44+ cells. We then applied a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. GSEA revealed that the CD24−/low/CD44+ cell populations are enriched for genes involved in a tumor necrosis factor (TNF) or interferon (IFN) response pathway. TNF or IFN response pathway is involved in the expression of many inflammatory cytokines/chemokines, including IL8 and CCL5. We measured expression levels of IL8 and CCL5 by quantitative RT-PCR (qRT-PCR) and confirmed that they were expressed at significantly higher levels in CD24−/low/CD44+ cell populations compared with the CD24+/CD44+ cell populations. Moreover, we found the relatively high nuclear factor kappa B (NF-kappa B) activity in the CD24−/low/CD44+ cell populations. In addition, NF-kappa B inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) prevented tumorigenesis of CD24−/low/CD44+ cell populations in vivo. Our findings suggest that NF-kappa B play a crucial role for orchestration of transcription factors regulating breast cancer stem cell biology. Further studies will be presented about the mechanism of NF-kappa B activation and its role in breast TICs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4218.

Inhibition of active HIV-1 replication by NF-κB inhibitor DHMEQ

Previous reports indicate that nuclear factor (NF)-κB regulates induction of human immunodeficiency virus type 1 (HIV-1) gene expression in latently infected cells. However, the role of NF-κB in cells with active HIV-1 replication is not well understood. In this study, we examined the effect of a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on HIV-1 replication in a human T cell line and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMCs). We further explored the mechanism of DHMEQ-mediated inhibition of HIV-1 replication. DHMEQ inhibited HIV-1 replication in HIV-1-infected Molt-4 and PHA-PBMCs. DHMEQ inhibited constitutive NF-κB activity in HIV-1-infected PHA-PBMCs and HIV long terminal repeat promoter activity driven by tumor necrosis factor (TNF)-α and the trans-activator Tat. The single-round assay using vesicular stomatitis virus-pseudotyped virus in the human T cell line M8166 indicated that DHMEQ treatment resulted in decreased integration of HIV-1 provirus into the host genome and decreased HIV-1 expression. These results indicate that NF-κB regulates early events as well as the initial and accelerated expression of HIV-1 in its life cycle. Therefore, we conclude that NF-κB is a molecular target for controlling active HIV-1 replication.

Potent Cytotoxic Effect of a Novel Nuclear Factor-κB Inhibitor Dehydroxymethylepoxyquinomicin on Human Bladder Cancer Cells Producing Various Cytokines

To explore the potential therapeutic effects of the nuclear factor-kappaB (NF-kappaB) inhibitor dehydroxymethylepoxyquinomicin (DHMEQ). KU-19-19 cells, originally derived from a patient with invasive bladder cancer who exhibited marked leukocytosis, produce multiple cytokines. This model of clinically advanced bladder cancer, in which NF-kappaB is constitutively activated, was used in this study. Expression of p65 protein in fractionated KU-19-19 cells was determined by Western blotting analysis. DNA-binding activity of NF-kappaB was detected by electrophoretic mobility shift assay. The cytotoxic effects and induction of apoptosis by DHMEQ were analyzed, and cytokines in the supernatant of KU-19-19 cells cultured with or without DHMEQ were measured by enzyme-linked immunosorbent assay (ELISA). Athymic nude mice bearing KU-19-19 subcutaneous tumors were subjected to intraperitoneal administration of 2 mg/kg/d DHMEQ for 3 weeks. Tumor growth was monitored and microvessel density, vascular endothelial growth factor expression, and the apoptotic index of tumors were evaluated by tissue immunohistochemistry. NF-kappaB was constitutively activated in KU-19-19 cells. DHMEQ reversibly inhibited the DNA-binding activity of NF-kappaB by blocking its nuclear translocation. Both cell viability and production of cytokines were significantly and dose-dependently suppressed by DHMEQ, and significant apoptosis was also induced. In in vivo studies, the mean tumor volume in mice treated with DHMEQ was significantly smaller than in controls. Immunohistochemical analysis of tumors revealed marked reduction in microvessel density, vascular endothelial growth factor expression, and induction of apoptosis. Blockade of NF-kappaB function by DHMEQ may be a useful new molecular targeting treatment for highly aggressive bladder cancer.

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

The present research was carried out to determine the effects of a nuclear factor-kappaB (NF-ĸB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), derivative of the antibiotic epoxyquinomicin C, on normal human chondrocytes treated with interleukin-1β (IL-1β). 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-1β-induced chondrocytes. DHMEQ may become a therapeutic agent for treatment of chondro-degenerative diseases.

Gene set enrichment analysis provides insight into novel signalling pathways in breast cancer stem cells

Background:Tumour-initiating cells (TICs) or cancer stem cells can exist as a small population in malignant tissues. The signalling pathways activated in TICs that contribute to tumourigenesis are not fully understood.Methods:Several breast cancer cell lines were sorted with CD24 and CD44, known markers for enrichment of breast cancer TICs. Tumourigenesis was analysed using sorted cells and total RNA was subjected to gene expression profiling and gene set enrichment analysis (GSEA).Results:We showed that several breast cancer cell lines have a small population of CD24−/low/CD44+ cells in which TICs may be enriched, and confirmed the properties of TICs in a xenograft model. GSEA revealed that CD24−/low/CD44+ cell populations are enriched for genes involved in transforming growth factor-β, tumour necrosis factor, and interferon response pathways. Moreover, we found the presence of nuclear factor-κB (NF-κB) activity in CD24−/low/CD44+ cells, which was previously unrecognised. In addition, NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) prevented tumourigenesis of CD24−/low/CD44+ cells in vivo.Conclusion:Our findings suggest that signalling pathways identified using GSEA help to identify molecular targets and biomarkers for TIC-like cells.

Amelioration of Experimental Autoimmune Uveoretinitis with Nuclear Factor-κB Inhibitor Dehydroxy Methyl Epoxyquinomicin in Mice

Purpose. Experimental autoimmune uveoretinitis (EAU), a Th1/Th17 cell-mediated autoimmune disease induced in mice, serves as a model of human endogenous uveitis. In this model, proinflammatory cytokines and various stimuli activate the transcriptional factor, nuclear factor-kappaB (NF-kappaB), in the retina. The therapeutic effect of the NF-kappaB inhibitor, dehydroxy methyl epoxyquinomicin (DHMEQ), was examined on EAU. Methods. EAU was induced in B10.BR mice by K2 peptide immunization. DHMEQ (40 mg/kg/d) was administered daily by intraperitoneal injection. Clinical severity and histopathologic severity were assessed. Translocation of NF-kappaB p65 into the nucleus in EAU retina was assessed. T cells were collected from draining lymph nodes of the K2-immunized mice to examine antigen (Ag)-specific T-cell active responses and cytokine production in vitro. Results. Disease onset was significantly delayed in DHMEQ-treated mice (15.6 days) compared with untreated mice (12.6 days; P < 0.01). Histologic severity was significantly milder in DHMEQ-treated mice (score, 1.13) than in controls (score, 2.33; P < 0.05). DHMEQ suppressed the Ag-specific T-cell active responses and downregulated the productions of Th-1 type cytokines in vitro in a dose-dependent manner. Alternation was not observed in Th-2 type cytokines. Pretreatment of primed T cells or Ag-presenting cells with DHMEQ reduced T-cell activation and Th1/Th17 cytokine production. DHMEQ treatment suppressed the translocation of the NF-kappaB p65 subunit into the nuclei. Conclusions. Systemic administration of DHMEQ suppressed NF-kappaB translocation in the retina, which might have reduced the inflammation of ocular tissues. DHMEQ-mediated regulation of NF-kappaB p65 could be a therapeutic target for the control of endogenous ocular inflammatory diseases.

Induction of oncogene addiction shift to NF-κB by camptothecin in solid tumor cells

The biological basis of the resistance of solid tumor cells to chemotherapy is not well understood. While addressing this problem, we found that gastric cancer cell line St-4/CPT, lung cancer cell line A549/CPT, and colon cancer cell line HT-29/CPT, all of which are resistant to camptothecin (CPT), showed strong and constitutive nuclear factor (NF)-κB activity driven by IκB kinase compared with their parental cell lines St-4, A549, and HT-29. A new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), reduced viability and induced apoptosis in St-4/CPT, A549/CPT, and HT-29/CPT cell lines, while their parental cell lines were resistant to DHMEQ. The results in this study present an example of the shift in signals that support the survival of solid tumor cells to NF-κB during the acquisition of resistance to CPT. The results also indicate that solid tumor cells that become resistant to chemotherapy may be more easily treated by NF-κB inhibitors.

Antitumor Effects of Dehydroxymethylepoxyquinomicin, a Novel Nuclear Factor-κB Inhibitor, in Human Liver Cancer Cells Are Mediated through a Reactive Oxygen Species-Dependent Mechanism

Activation of the nuclear transcription factor-kappaB (NF-kappaB) has been implicated in liver tumorigenesis. We evaluated the effects of a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in two human liver cancer cell lines HA22T/VGH and HuH-6. DHMEQ treatment dose dependently decreased the DNA-binding capacity of the NF-kappaB p65 subunit, inhibited cell growth and proliferation, and increased apoptosis as shown by caspase activation, release of cytochrome c, poly(ADP-ribose) polymerase cleavage, and down-regulation of survivin. DHMEQ also induced a dose-dependent activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling, and inhibition of this pathway significantly reduced cell growth. It is noteworthy that we observed that DHMEQ stimulated reactive oxygen species (ROS) production in a dose-dependent manner and that pretreatment of the cells with the antioxidant N-acetyl-L-cysteine (NAC) significantly reduced DHMEQ-induced ROS generation. Accordingly, NAC completely reversed the DHMEQ-induced growth inhibition, caspase activation, and cell death. DHMEQ-treated cells exhibited DNA damage, as evaluated by accumulation in nuclear foci of phospho-H2AX, which was completely reversed by NAC. Moreover, DHMEQ induced the expression of genes involved in the endoplasmic reticulum stress response (GRP78, CHOP, TRB3) and promoted the splicing of XBP1 mRNA in a dose-dependent fashion in both cell lines, which was reversed in the presence of NAC. Knockdown of TRB3 mRNA expression by small interference RNA significantly decreased DHMEQ-induced cell growth inhibition. These data suggest that DHMEQ antitumor effects are primarily mediated through ROS generation. Thereby, considering that cancer cells are under increased ER stress and oxidative stress conditions, DHMEQ may greatly improve various anticancer strategies.

Transient inhibition of NF‐κB by DHMEQ induces cell death of primary effusion lymphoma without HHV‐8 reactivation

Primary effusion lymphoma (PEL) is a refractory malignancy caused by human herpes virus 8 (HHV-8) in immunocompromised individuals. The tumor cells of PEL are characterized by constitutive NF-kappaB activation. Dehydroxymethylepoxyquinomicin (DHMEQ) is a new NF-kappaB inhibitor and is effective on various tumor cells with constitutively activated NF-kappaB. Thus, in search for a new therapeutic modality of PEL, we examined the effect of DHMEQ on PEL cells. We confirmed constitutive activation of NF-kappaB with subcomponents of p50 and p65 in PEL cell lines. DHMEQ quickly and transiently abrogated NF-kappaB activation and reduced the cell viability in dose- and time-dependent manners, inducing apoptosis through activation of both mitochondrial and membrane pathways. Array analysis revealed that DHMEQ down-regulated expression levels of NF-kappaB target genes, such as interleukin-6 (IL6), Myc, chemokine (C-C motif) receptor 5 (CCR5) and NF-kappaB1, whereas it up-regulated expression levels of some genes involved in apoptosis, and cell cycle arrest. DHMEQ did not reactivate HHV-8 lytic genes, indicating that NF-kappaB inhibition by DHMEQ did not induce virus replication. DHEMQ rescued CB-17 SCID mice xenografted with PEL cells, reducing the gross appearance of effusion. Thus, DHMEQ transiently abrogated the NF-kappaB activation, irreversibly triggering the apoptosis cascade without HHV-8 reactivation. In addition, DHMEQ could rescue the PEL-xenograft mice. Therefore, we suggest DHMEQ as a promising candidate for molecular target therapy of the PEL.