NF-κB Inhibitor DHMEQ Research Articles

Causes and mechanisms of peritoneal fibrosis and possible application of NF-κB inhibitor for prevention and treatment

Peritoneal dialysis is an established form of the renal replacement therapy in patients with end stage renal failure. Continuous Ambulatory Peritoneal Dialysis developed by Moncrief and Popovich in 1975 was a revolutionary event, and contributed much to wide application of that form of treatment in uremic patients. On the other hand, the weak point of peritoneal dialysis is relatively short viability of the peritoneum as the dialysis membrane. Two main peritoneal pathologies are observed in patients treated with that form of renal replacement therapy: neovascularization of the membrane what causes increased peritoneal permeability to osmotic solutes and ultrafiltration failure, and fibrosis which results also in ultrafiltration failure due to its decreased hydraulic conductivity and reduced permeability to uremic toxins. Meanwhile, an NF-κB inhibitor DHMEQ was discovered in 2000 and has been successfully used to suppress various inflammatory and neoplastic disease models. NF-κB is likely to be involved in the mechanism of peritoneal inflammation and fibrosis. We have studied whether DHMEQ would inhibit cellular model of peritoneal inflammation and fibrosis. It inhibited inflammatory cytokine and collagen productions in primary culture of human peritoneal mesothelial cells, and intraperitoneal administration of NF-κB inhibitors would be useful to suppress peritoneal fibrosis.

Inhibition of Late and Early Phases of Cancer Metastasis by the NF-κB Inhibitor DHMEQ Derived from Microbial Bioactive Metabolite Epoxyquinomicin: A Review.

We previously designed and synthesized dehydroxyepoxyquinomicin (DHMEQ) as an inhibitor of NF-κB based on the structure of microbial secondary metabolite epoxyquinomicin C. DHMEQ showed anti-inflammatory and anticancer activity in various in vivo disease models without toxicity. On the other hand, the process of cancer metastasis consists of cell detachment from the primary tumor, invasion, transportation by blood or lymphatic vessels, invasion, attachment, and formation of secondary tumor. Cell detachment from the primary tumor and subsequent invasion are considered to be early phases of metastasis, while tumor cell attachment to the tissue and secondary tumor formation the late phases. The assay system for the latter phase was set up with intra-portal-vein injection of pancreatic cancer cells. Intraperitoneal administration of DHMEQ was found to inhibit liver metastasis possibly by decreasing the expression of MMP-9 and IL-8. Also, when the pancreatic cancer cells treated with DHMEQ were inoculated into the peritoneal cavity of mice, the metastatic foci formation was inhibited. These results indicate that DHMEQ is likely to inhibit the late phase of metastasis. Meanwhile, we have recently employed three-dimensional (3D) culture of breast cancer cells for the model of early phase metastasis, since the 3D invasion just includes cell detachment and invasion into the matrix. DHMEQ inhibited the 3D invasion of breast cancer cells at 3D-nontoxic concentrations. In this way, DHMEQ was shown to inhibit the late and early phases of metastasis. Thus, DHMEQ is likely to be useful for the suppression of cancer metastasis.

P3.01-064 The Overexpression and Cleavage of SASH1 by Caspase-3 Stimulates Cell Death in Lung Cancer Cells: Topic: Apoptosis in Lung Cancer

SASH1 (SAM and SH3 domain-containing protein 1) is a recently identified gene with tumor suppressor properties and has a role in induction of apoptosis. Previous work has shown that 90 % of lung cancer cell lines have a decrease in SASH1 mRNA levels (Zeller et al., 2003), however little characterization of SASH1 function in lung cancer has been undertaken. We evaluated SASH1 expression in transformed normal and malignant non-small cell lung cancer cell lines. We also utilized cell based assays to study the effects of altered SASH1 levels on cell survival and proliferation. Identification of a novel SASH1 targeting drug was performed through connectivity mapping. SASH1 protein expression was down regulated in two of the five lung cancer cell lines compared to immortalized normal bronchial epithelial cells. Prognoscan assessment identified decreased SASH1 mRNA expression reduced patient survival. The depletion of SASH1 in lung cells resulted in a significant increase in cellular proliferation in cancer lung cells. Connectivity mapping predicted the drug Chloropyramine would lead to an increase in SASH1 expression. We demonstrated that Chloropyramine upregulates SASH1 in malignant cell lines. In keeping with this we have demonstrated the Chloropyramine inhibited lung cancer proliferation in vitro. We also explored the role of SASH1 in apoptosis. Following ultraviolet light exposure SASH1 is cleaved by Caspase-3. The C-terminal fragment of SASH1 then translocates from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild type SASH1 or cleaved SASH1 amino acids 231-1247 leads to an increase in apoptosis, however loss of the SASH1 cleavage site and/or nuclear translocation prevents this initiation of apoptosis. Mechanistically SASH1 cleavage is required for the translocation of the transcription factor NF-κB to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a co-dependence between SASH1 and NF-κB for this process. We have shown that SASH1 contributes to apoptosis via a NF-κB-dependent mechanism. Agents that upregulate SASH1, such as chloropyramine or SASH1 gene therapy, are potential novel approaches to the management of NSCLC in the future.

Activation and cleavage of SASH1 by caspase-3 mediates an apoptotic response.

Apoptosis is a highly regulated cellular process that functions to remove undesired cells from multicellular organisms. This pathway is often disrupted in cancer, providing tumours with a mechanism to avoid cell death and promote growth and survival. The putative tumour suppressor, SASH1 (SAM and SH3 domain containing protein 1), has been previously implicated in the regulation of apoptosis; however, the molecular role of SASH1 in this process is still unclear. In this study, we demonstrate that SASH1 is cleaved by caspase-3 following UVC-induced apoptosis. Proteolysis of SASH1 enables the C-terminal fragment to translocate from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild-type SASH1 or a cleaved form of SASH1 representing amino acids 231–1247 leads to an increase in apoptosis. Conversely, mutation of the SASH1 cleavage site inhibits nuclear translocation and prevents the initiation of apoptosis. SASH1 cleavage is also required for the efficient translocation of the transcription factor nuclear factor-κB (NF-κB) to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a codependence between SASH1 and NF-κB for this process.

Inhibition of MMP-2-Mediated Mast Cell Invasion by NF-κB Inhibitor DHMEQ in Mast Cells

Background: Stimulation with antigen and IgE is known to activate NF-κB in mast cells. In the present research, we studied the role of NF-κB on cellular migration in mast cell-like RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) using the NF-κB inhibitor (-)-DHMEQ. Methods: A Matrigel invasion chamber was used to evaluate cell migration. A PCR array was used to screen the expression of 84 key genes involved in cell migration. Results: (-)-DHMEQ inhibited antigen/IgE-induced NF-κB activation and expressions of its target genes such as IL-6 and TNF-α. (-)-DHMEQ was found to inhibit in vitro invasion toward the antigen without any toxicity. We then looked for NF-κB-dependent genes that would be important for mast cell invasion using the PCR array. (-)-DHMEQ was found to lower the expression of matrix metalloproteinase (MMP)-2. The MMP inhibitor GM6001 also inhibited cellular invasion toward the antigen. These effects of (-)-DHMEQ were obtained in both RBL-2H3 cells and BMMCs. Conclusions: These findings indicate that (-)-DHMEQ suppressed mast cell migration via the inhibition of NF-κB-regulated MMP-2 expression.

Immunosuppression through constitutively activated NF-κB signalling in human ovarian cancer and its reversal by an NF-κB inhibitor.

Background:Although T-cell immunity is thought to be involved in the prognosis of epithelial ovarian cancer (EOC) patients, immunosuppressive conditions hamper antitumour immune responses. Thus, their mechanisms and overcoming strategies need to be investigated.Methods:The role of NF-κB in human EOC cells and macrophages was evaluated by in vitro production of immunosuppressive IL-6 and IL-8 by EOC cells and in vivo analysis of immune responses in nude mice implanted with human EOC cells using an NF-κB inhibitor DHMEQ.Results:In EOC patients, increased plasma IL-6, IL-8, and arginase were observed. The NF-κB inhibitor DHMEQ inhibited the production of IL-6 and IL-8 by EOC cell lines. Immunosuppression of human DCs and macrophages by culture supernatant of EOC cells was reversed with the pretreatment of DHMEQ. Administration of DHMEQ to nude mice implanted with human EOC resulted in the restoration of T-cell stimulatory activity of murine DCs along with the reduction of tumour accumulation and arginase expression of MDSCs. Nuclear factor-κB inhibition in tumour-bearing mice also enhanced antitumour effects of transferred murine naive T cells.Conclusions:NF-κB is involved in the immunosuppression induced by human EOC, and its inhibitor may restore antitumour immune responses, indicating that NF-κB is an attractive target for EOC treatment.

The NF-κB inhibitor DHMEQ decreases survival factors, overcomes the protective activity of microenvironment and synergizes with chemotherapy agents in classical Hodgkin lymphoma

The NF-κB inhibitor DHMEQ has shown preclinical activity in classical Hodgkin Lymphoma (cHL).Here we evaluated if DHMEQ could affect microenvironmental interactions and formation and improve the activity of drugs used in relapsed/refractory cHL. We demonstrated that DHMEQ down-regulated the NF-κB target genes IRF4 and CD40, the secretion of IL-6, CCL5, CCL17 and generated ROS. Cytotoxicity, CD30 down-modulation and CD30 shedding by DHMEQ were prevented by ROS scavenger NAC. DHMEQ overcame stimuli from CD40 engagement and fibroblasts and enhanced doxorubicin, cisplatin and gemcitabine activity. Our results suggest that DHMEQ may be a promising agent for future therapeutic strategies in cHL.

Abstract 3223: Active enhancement of anti-tumor effect by NF-κB inhibitor DHMEQ using PMBN conjugated with anti-EGFR antibody in human pancreatic adenocarcinoma cells in vivo and in vitro

Abstract Background and Objective: The NF-kB/Rel family of transcription factors is involved in the development of cancer. This family is constitutively activated in many human pancreatic cancers and pancreatic cell lines. DHMEQ inhibits NF-κB activity by preventing nuclear translocation and has been shown anti-tumor effect. However, because of its hydrophobic property, DHMEQ has to be administered intraperitoneally or subcutaneously every day. Therefore, it is necessary to develop effective drug delivery system (DDS) of DHMEQ for clinical application. In this study, we have used PMBN polymer, which forms a stable polymer aggregate in water that can enhance the solubility of hydrophobic substance such as DHMEQ. The active ester group of PMBN can be replaced by proteins such as anti-epidermal growth factor receptor (EGFR) antibody. It is expected that a selective increased accumulation of PMBN-DHMEQ antibody conjugate could lead to a reduction of its dose and/or numbers of administration. The aim of this study is that investigate active enhancement of anti-cancer effect by DHMEQ using PMBN. Methods: The human pancreas adenocarcinoma cell lines, AsPC-1, BxPC-3, MIAPaCa-2, which showed high expression level of EGFR were used in vitro study. DHMEQ, PMBN and mouse monoclonal anti-human EGFR antibody (528) were mixed at the weight ratio of 1:25:5 and sonicated to prepare PMBN-DHMEQ antibody conjugate in advance. Control DHMEQ was dissolved in 0.5% chrolomethylcellulose (CMC-DHMEQ). The effect of cell growth inhibition by each preparation was evaluated by WST assay in vitro. AsPC-1 cells which expressing Gaussia luciferase (AsPC-1-Luc) was used to investigate anti-tumor effect in vivo. 5×105 of AsPC-1-Luc was injected into nice portal vein to develop liver metastases model of pancreatic cancer. Mice were given PMBN-DHMEQ antibody conjugate (intravenously) or CMC-DHMEQ (intraperitoneally) every three days at a dose of 12mg/kg DHMEQ. Metastases level of each group was compared by measuring luciferase activity of mice whole liver homogenate using Renilla Luciferase Assay System (Promega). Result: PMBN-DHMEQ antibody conjugate showed higher cell growth inhibition than CMC-DHMEQ in vitro WST assay. Also it showed stronger growth inhibition effect than CMC-DHMEQ in vivo. Quantitative luciferase assay showed that PMBN-DHMEQ antibody conjugate have 49.2 times higher effect compare to CMC-DHMEQ. Conclusion: Our results demonstrate that the PMBN-DHMEQ-anti-EGFR antibody conjugate can efficiently decrease cancer development of cells overexpressing EGFR by an active enhancement effect. This result suggest that increased accumulation of PMBN-DHMEQ anti-EGFR antibody conjugate increased tumor concentration of DHMEQ and the therapeutic response. PMBN-mediated DDS, which in turn reduce its dose and/or numbers of administration of DHMEQ. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3223. doi:10.1158/1538-7445.AM2011-3223

Hydrogen andN-Acetyl-l-Cysteine Rescue Oxidative Stress-Induced Angiogenesis in a Mouse CornealAlkali-Burn Model

To investigate the role of reactive oxygen species (ROS) as the prime initiators of the angiogenic response after alkali injury of the cornea and observe the effects of antioxidants in preventing angiogenesis. The corneal epithelia of SOD-1-deficient mice or wild-type (WT) mice were removed after application of 0.15 N NaOH to establish the animal model of alkali burn. ROS production was semiquantitatively measured by dihydroethidium (DHE) fluorescence. Angiogenesis was visualized by CD31 immunohistochemistry. The effects of the specific NF-κB inhibitor DHMEQ, the antioxidant N-acetyl-L-cysteine (NAC), and hydrogen (H2) solution were observed. ROS production in the cornea was enhanced immediately after alkali injury, as shown by increased DHE fluorescence (P<0.01). NF-κB activation and the upregulation of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) were significantly enhanced (P<0.01), leading to a significantly larger area of angiogenesis. Angiogenesis in SOD-1-/- mice corneas were significantly higher in WT mice (P<0.01), confirming the role of ROS. Pretreatment with the specific NF-κB inhibitor DHMEQ or the antioxidant NAC significantly reduced corneal angiogenesis by downregulating the NF-κB pathway (P<0.01) in both WT and SOD-1-/- mice. Furthermore, we showed that irrigation of the cornea with hydrogen (H2) solution significantly reduced angiogenesis after alkali-burn injury (P<0.01). Immediate antioxidant therapy with H2-enriched irrigation solution is a new potent treatment of angiogenesis in cornea to prevent blindness caused by alkali burn.

A new NF-κB inhibitor based on the amino-epoxyquinol core of DHMEQ

The amino-epoxyquinols 6a and 6b were synthesized as soluble derivatives of an NF-κB inhibitor DHMEQ ( 1). In spite of the opposite configuration from 1, 6b rather than 6a affected the deactivation of NF-κB, based on NO secretion and MALDI-TOF MS analysis. It was indicated that 6b inhibited the activation by different manner from that of 1.

Abstract 664: Galiximab (anti-CD80 mAb) sensitizes TRAIL-resistant B-lymphoma to TRAIL-induced apoptosis via inhibition of the transcription repressor Yin Yang 1 (YY1)

Abstract Treatment of B-lymphomas with rituximab, alone or in combination with chemotherapy, resulted in significant clinical responses and prolongation of survival. However, a significant subset relapses or does not respond to initial treatments. Therefore, there is an urgent need to identify new targets and develop therapies to reverse resistance. CD80 is expressed on APCs and normal B cells and most B-lymphomas express CD80 on the surface. A primatized anti-CD80 mAb, galiximab, was developed and shown to inhibit tumor cell proliferation and mediates ADCC. A phase I/II clinical study with galiximab in patients with refractory B lymphomas demonstrated its safety TRAIL is a death-inducing ligand that is not toxic to normal cells but selectively cytotoxic to sensitive tumor cells and is currently in clinical trails in patients with a variety of cancers. The objective of this study was to investigate whether galiximab can sensitize TRAIL-resistant B lymphoma to TRAIL-induced apoptosis. We hypothesized that galiximab-induced inhibition of cell proliferation must have triggered the cells and inhibited constitutively activated survival pathways, such as NF-κB, and downstream anti-apoptotic gene products, thus, reducing the threshold of resistance and facilitating TRAIL to mediate apoptosis. This hypothesis was tested in vitro using CD80+ Raji cells as model and recombinant TRAIL. Galiximab was obtained from Biogen Idec. Treatment of Raji with galiximab (5-40 μg/ml) for 18h and followed by treatment with TRAIL (2.5-10 ng/ml) resulted in significant apoptosis as assessed by activation of Caspase 3. Galiximab treatment of Raji inhibited the NF-κB pathway and its DNA-binding activity as assessed by western and EMSA, respectively. The direct role of NF-κB-induced inhibition by galiximab in sensitization was corroborated by the use of the NF-κB inhibitor DHMEQ. We have reported that the TRAIL receptor DR5 transcription repressor YY1 regulates tumor-cell sensitivity to TRAIL. We have found that galiximab inhibits YY1 expression and DNA-binding activity in Raji in parallel with upregulation of DR5. The direct role of YY1-induced inhibition by galiximab in TRAIL sensitization was corroborated in cells transfected with siRNA YY1 and which were sensitive to TRAIL apoptosis. Sensitization by galiximab was due, in part; to activation of the type II mitochondrial pathway for apoptosis. Galiximab-induced inhibition of anti-apoptotic Bcl-2 family, such as Bcl-2 and Bcl-XL, in sensitization was verified by the use of the specific Bcl-2 inhibitor, 2 MAM-A3. Altogether, these findings established that galiximab is capable to sensitize TRAIL-resistant B lymphomas to TRAIL apoptosis. The findings also suggest the potential therapeutic application of the combination of galiximab and TRAIL or agonist DR4/DR5 antibodies in the treatment of resistant B lymphomas. 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 664.

Abstract 1466: The NO donor DETANONOate inhibits the EMT phenotype in human metastatic prostate carcinoma cell lines: pivotal roles of inhibition of NF-κB and Snail activities and induction of RKIP

Abstract Nitric oxide (NO) has been shown to mediate contrasting effects on tumorigenesis i.e. at low levels NO is pro-tumorigenic and at high levels NO is anti-tumorigenic. Recent studies revealed that treatment of tumor cell lines with NO donors at high levels significantly inhibited cell growth and survival pathways such as the constitutively activated NF-κB. Tumor cells with activated NF-κB were reported to have the capacity to initiate the metastatic cascade via induction of epithelial to mesenchymal transition (EMT). The EMT phenotype is characterized by decreased expression of epithelial gene products (e.g. E-cadherin, cytokeratin 18) and upregulation of mesenchymal gene products (e.g. vimentin, fibronectin). In addition, the tumor cells exhibit invasive and migratory properties. Based on these above findings, we hypothesized that treatment of metastatic prostate carcinoma cell lines, PC-3 and DU-145 with DETANONOate (500-1000μM) may reverse the EMT phenotype. This hypothesis was tested and treatment of the cells with DETANONOate resulted in inhibition of the EMT phenotype through inhibition of NF-κB activity. Inhibition of NF-κB was shown to be due, in part, to S-nitrosylation of p50. In addition to the inhibition of EMT phenotype, treatment with DETANONOate also inhibited invasive properties. The dual roles of DETANONOate-mediated inhibition of NF-κB and EMT were corroborated by the use of the NF-κB inhibitor DHMEQ. We examined the underlying mechanism by which NF-κB-induced inhibition by DETANONOate resulted in the inhibition of EMT. The EMT-induced transcription factor Snail was highly expressed in PC-3 and DU-145 cells. Treatment with DETANONOate inhibited Snail expression concomitant with inhibition of EMT. The direct role of Snail inhibition by DETANONOate and the inhibition of EMT was corroborated in cells transfected with Snail siRNA and such treatment reversed the EMT phenotype, mimicking DETANONOate. Inhibition of the repressor activity of Snail by DETANONOate resulted in the upregulation of the Raf-1 kinase inhibitory protein (RKIP). The role of RKIP induction in the reversal of EMT was corroborated in cells overexpressing RKIP. The in vitro findings with DETANONOate-induced inhibition of EMT were validated in mice bearing PC-3 xenografts. Treatment with DETANONOate and analysis by IHC of tumor biopsies revealed that the EMT phenotype was reversed. There was an inverse correlation between Snail inhibition and RKIP induction. The present findings demonstrate, for the first time, that NO donors (high concentrations) inhibit the EMT phenotype and suggest their potential therapeutic applications in vivo in patients. These studies also suggest that the NF-κB/Snail/RKIP circuitry is dysregulated in tumor cells and is responsible, in part, for the EMT phenotype. 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 1466.

Pivotal Roles of Snail Inhibition and RKIP Induction by the Proteasome Inhibitor NPI-0052 in Tumor Cell Chemoimmunosensitization

The novel proteasome inhibitor NPI-0052 has been shown to sensitize tumor cells to apoptosis by various chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although the mechanisms involved are not clear. We hypothesized that NPI-0052-mediated sensitization may result from NF-kappaB inhibition and downstream modulation of the metastasis inducer Snail and the metastasis suppressor/immunosurveillance cancer gene product Raf-1 kinase inhibitory protein (RKIP). Human prostate cancer cell lines were used as models, as they express different levels of these proteins. We show that NPI-0052 inhibits both NF-kappaB and Snail and induces RKIP expression, thus resulting in cell sensitization to CDDP and TRAIL. The direct role of NF-kappaB inhibition in sensitization was corroborated with the NF-kappaB inhibitor DHMEQ, which mimicked NPI-0052 in sensitization and inhibition of Snail and induction of RKIP. The direct role of Snail inhibition by NPI-0052 in sensitization was shown with Snail small interfering RNA, which reversed resistance and induced RKIP. Likewise, the direct role of RKIP induction in sensitization was revealed by both overexpression of RKIP (mimicking NPI-0052) and RKIP small interfering RNA that inhibited NPI-0052-mediated sensitization. These findings show that NPI-0052 modifies the NF-kappaB-Snail-RKIP circuitry in tumor cells and results in downstream inhibition of antiapoptotic gene products and chemoimmunosensitization. The findings also identified Snail and RKIP as targets for reversal of resistance.

Sensitization of Rituximab-Sensitive and Rituximab-Resistant B-NHL Cell Lines/Clones to TRAIL-Induced Apoptosis by Bortezomib and NF-κB Inhibitors.

Patients with B-NHL respond initially to conventional chemotherapy and/or to immunotherapy with rituximab (alone or in combination with chemotherapy). However, patients develop resistance to these modalities and novel approaches are needed. TRAIL is a cytotoxic molecule that exerts selective anti-tumor cytotoxic activity with minimal toxicity to normal tissues. Further, TRAIL or agonist monoclonal antibody (mAb) to TRAIL receptors, DR4 and DR5, are currently being tested clinically. The present study investigated the sensitivity of B-NHL cell lines to TRAIL-mediated apoptosis using the AIDS-related NHL (ARL) B-cell line, 2F7, and the B-NHL cell lines, Ramos and Daudi. Also, to recapitulate various aspects of acquired rituximab-resistance, we have generated rituximab-resistant (RR) clones from the parental wild type (wt) cells. Rituximab failed to chemo-sensitize the RR clones and the clones exhibited higher resistance to various drugs (e.g., CDDP, VP-16, ADR, Vincristine, Taxol) and to TRAIL (1–250 ng/ml-18 h) compared to the wt cells as analyzed by DNA fragment on assay. The findings demonstrate that the wild type and RR1 cells were resistant to TRAIL-mediated apoptosis at a wide range of TRAIL concentrations. We then examined means to reverse TRAIL resistance. We and others have reported that inhibition of NF-κB activity can sensitize TRAIL-resistant tumor cells to TRAIL-induced apoptosis. Hence, we examined the effect of the proteasome and NF-κB inhibitor, Bortezomib (Velcade), Bay 11–7085 and the specific NF-κB inhibitor DHMEQ (Kikuchi et. al, Cancer Research 2003; 63:107). Pretreatment of the NHL tumor cells with Bortezomib, Bay 11–7085 or DHMEQ for 6 h followed by treatment with TRAIL for 18h resulted in significant augmentation of apoptosis and synergy was achieved. Both the rituximab-sensitive and rituximab-resistant tumor cells were sensitized by these inhibitors, though higher concentrations were required for sensitization of the RR clones. Interestingly, detailed analysis of the signaling pathways in the RR clones revealed constitutive hyper-activation of the NF-κB survival pathway leading to over-expression of anti-apoptotic gene products Bcl-2, Bcl-xL and Mcl-1. Based on the findings, we postulate that patients with resistant B-NHL can be treated with combination of TRAIL/anti-DR4 or DR5 mAb and NF-κB inhibitors. Alternatively, these patients can be treated with agents that up-regulate TRAIL expression on host effectors (e.g., T cells, NK cells) in combination with NF-κB inhibitors.