Constitutive NF-kappaB Activation Research Articles

Blockage of interleukin-6 signaling with 6-amino-4-quinazoline synergistically induces the inhibitory effect of bortezomib in human U266 cells

The transcription factor nuclear factor-kappa B (NF-kappaB) regulates the transcription of a number of genes involved in a variety of cellular responses, including cell survival, inflammation, and differentiation. NF-kappaB is activated by a variety of stimuli, proinflammatory cytokines, mitogens, growth factors, and stress-inducing agents. Aberrant NF-kappaB expression is considered to be one of the oncogenic factors of cancer and the constitutive activation of NF-kappaB is observed in several hematologic disorders [classic Hodgkin's lymphoma, diffuse large B cell lymphoma, and multiple myeloma (MM)], and the modulation of NF-kappaB activation is emerging as a promising novel anticancer therapeutic strategy.Therefore, we focused on the regulation of NF-kappaB activation in MM. When U266 cells were treated with 6-amino-4-quinazoline, an NF-kappaB activation inhibitor, we determined that it most effectively blocked the interleukin (IL)-6-induced activation of MAPK and JAK/STAT pathways among different signaling inhibitors. The results of the luciferase assay indicated that 6-amino-4-quinazoline inhibited NF-kappaB activation with diminished NF-kappaB protein bound to NF-kappaB DNA binding sites. In addition, 6-amino-4-quinazoline suppressed the production of IL-6, which affected MM cell proliferation. Furthermore, combined treatment with bortezomib and 6-amino-4-quinazoline effectively inhibited the IL-6 and soluble IL-6R-induced activation of STAT3 and extracellular signal-regulated kinase phosphorylation. Our data showed that the inhibition of NF-kappaB activation abrogated MM cell proliferation induced by the IL-6 pathway, and might represent a promising therapeutic strategy for the treatment of MM.

DMNQ-S17 inhibits constitutive NF-kappaB activation leading to induction of apoptosis through the activation of caspase-3 in human myeloid leukemia U937 cells

Through cytotoxicity screening with naphthoquinone derivatives, a novel compound 6-(1-oxoallkyl)-5,8-dimethoxy-1,4-naphthoquinone-S17 (DMNQ-S17) showed its potency against human myeloid leukemia U937 cells. Thus, to elucidate the apoptotic mechanism of DMNQ-S17, this study was performed in myeloid leukemia U937 cells by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay, eletrophoretic mobility shift assay (EMSA), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay, 4′,6-diamidino-2-phenylindole (DAPI) staining, and Western blotting. In the present study, DMNQ-S17 inhibited constitutive NF κB activation and its transcriptional activity in U937 cells. In addition, DMNQ-S17 induced apoptotic features such as apoptotic bodies, cell shrinkage and chromatin condensation in U937 cells. Consistently, flow cytometric analysis showed that DMNQ-S17 increased sub-G1 portion and TUNEL positive cells in a concentration-dependent manner. Furthermore, DMNQ-S17 effectively attenuated mitochondrial membrane potential, released cytochrome C, activated caspase-3 expression, and cleaved poly (ADP-ribose) polymerase (PARP). Reversely, caspase-3 and -9 inhibitors also blocked the DMNQ-S17 induced caspase-3 activation and PARP cleavage in U937 cells. Taken together, these findings suggest that DMNQ-S17 can be a potent anticancer candidate for myeloid leukemias by the suppression of NF-κB activation leading to the activation of caspase-3 in human myeloid leukemia U937 cells.

ABINs inhibit EGF receptor-mediated NF-κB activation and growth of EGF receptor-overexpressing tumour cells

The epidermal growth factor receptor (EGFR) is frequently overexpressed in various tumours of epidermal origin and is held responsible for tumourigenicity and tumour persistence. Increased nuclear factor (NF)-kappaB activity has been suggested to be involved in the malignant behaviour of EGFR-overexpressing cells. However, the mechanisms that regulate EGF-induced NF-kappaB activation are still largely unknown. Here we show that EGF can induce NF-kappaB-dependent gene expression independently from IkappaBalpha degradation or p100 processing in EGFR-overexpressing HEK293T cells. Moreover, EGF-induced NF-kappaB activation could be inhibited by overexpression of ABINs, which were previously identified as intracellular inhibitors of tumour necrosis factor, interleukin-1 and lipopolysaccharide-induced NF-kappaB activation. Knockdown of ABIN-1 by RNA interference boosted the NF-kappaB response upon EGF stimulation. The C-terminal ubiquitin-binding domain containing region of ABINs was crucial and sufficient for NF-kappaB inhibition. Adenoviral gene transfer of ABINs reduced constitutive NF-kappaB activity as well as the proliferation of EGFR-overexpressing A431 and DU145 human carcinoma cells. Altogether, these results demonstrate an important role for an ABIN-sensitive non-classical NF-kappaB signalling pathway in the proliferation of EGFR-overexpressing tumour cells, and indicate a potential use for ABIN gene therapy in the treatment of cancer.

Expression of TNF-α leader sequence renders MCF-7 tumor cells resistant to the cytotoxicity of soluble TNF-α

Transmembrane TNF-alpha (tmTNF-alpha) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-alpha (tmTNF-alpha) at high level was resistant to soluble TNF-alpha (sTNF-alpha)-induced cytotoxicity, accompanied by constitutive NF-kappaB activation. In contrast, MCF-7 cells expressing tmTNF-alpha at very low level were sensitive to sTNF-alpha-induced cell death and had no detectable NF-kappaB activation. Consistently, siRNA-mediated tmTNF-alpha knockdown blocked NF-kappaB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-alpha, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-alpha containing LS constitutively activated NF-kappaB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-alpha lacking the cytoplasmic segment of LS neither activated NF-kappaB nor affected the sensitivity. However, NF-kappaB inhibitor PDTC suppressed NF-kappaB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-kappaB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)(2)-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-kappaB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-kappaB and resistance to sTNF-alpha-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-alpha-induced resistance in the breast cancer cells.

Inhibition of activation of dsRNA-dependent protein kinase and tumour growth inhibition

Inhibition of dsRNA-activated protein kinase (PKR), not only attenuates muscle atrophy in a murine model of cancer cachexia (MAC16), but it also inhibits tumour growth. In vitro the PKR inhibitor maximally inhibited growth of MAC16 tumour cells at a concentration of 200 nM, which was also maximally effective in attenuating phosphorylation of PKR and of eukaryotic initiation factor (eIF)2 on the alpha-subunit. There was no effect on the growth of the MAC13 tumour, which does not induce cachexia, even at concentrations up to 1,000 nM. There was constitutive phosphorylation of PKR and eIF2alpha in the MAC16, but not in the MAC13 tumour, while levels of total PKR and eIF2alpha were similar. There was constitutive upregulation of nuclear factor-kappaB (NF-kappaB) in the MAC16 tumour only, and this was attenuated by the PKR inhibitor, suggesting that it arose from activation of PKR. In MAC16 alone the PKR inhibitor also attenuated expression of the 20S proteasome. The PKR inhibitor potentiated the cytotoxicity of both 5-fluorouracil and gemcitabine to MAC16 cells in vitro. These results suggest that inhibitors of PKR may be useful therapeutic agents against tumours showing increased expression of PKR and constitutive activation of NF-kappaB, and may also prove useful in sensitising tumours to standard chemotherapeutic agents.

Expression of microRNA-146 suppresses NF-κB activity with reduction of metastatic potential in breast cancer cells

Cancer cells often acquire a constitutively active nuclear factor-kappaB (NF-kappaB) program to promote survival, proliferation and metastatic potential by mechanisms that remain largely unknown. Extending observations from an immunologic setting, we demonstrate that microRNA-146a and microRNA-146b (miR-146a/b) when expressed in the highly metastatic human breast cancer cell line MDA-MB-231 function to negatively regulate NF-kappaB activity. Lentiviral-mediated expression of miR-146a/b significantly downregulated interleukin (IL)-1 receptor-associated kinase and TNF receptor-associated factor 6, two key adaptor/scaffold proteins in the IL-1 and Toll-like receptor signaling pathway, known to positively regulate NF-kappaB activity. Impaired NF-kappaB activity was evident from reduced phosphorylation of the NF-kappaB inhibitor IkappaBalpha, reduced NF-kappaB DNA-binding activity and suppressed expression of the NF-kappaB target genes IL-8, IL-6 and matrix metalloproteinase-9. Functionally, miR-146a/b-expressing MDA-MB-231 cells showed markedly impaired invasion and migration capacity relative to control cells. These findings implicate miR-146a/b as a negative regulator of constitutive NF-kappaB activity in a breast cancer setting and suggest that modulating miR-146a/b levels has therapeutic potential to suppress breast cancer metastases.

Prevalence of bortezomib-resistant constitutive NF-kappaB activity in mantle cell lymphoma

BackgroundThe proteasome inhibitor bortezomib can inhibit activation of the transcription factor NF-κB, a mechanism implicated in its anti-neoplastic effects observed in mantle cell lymphoma (MCL). However, NF-κB can be activated through many distinct mechanisms, including proteasome independent pathways. While MCL cells have been shown to harbor constitutive NF-κB activity, what fraction of this activity in primary MCL samples is sensitive or resistant to inhibition by bortezomib remains unclear.ResultsProteasome activity in the EBV-negative MCL cell lines Jeko-1 and Rec-1 is inhibited by greater than 80% after exposure to 20 nM bortezomib for 4 hours. This treatment decreased NF-κB activity in Jeko-1 cells, but failed to do so in Rec-1 cells when assessed by electrophoretic mobility shift assay (EMSA). Concurrently, Rec-1 cells were more resistant to the cytotoxic effects of bortezomib than Jeko-1 cells. Consistent with a proteasome inhibitor resistant pathway of activation described in mouse B-lymphoma cells (WEHI231) and a breast carcinoma cell line (MDA-MB-468), the bortezomib-resistant NF-κB activity in Rec-1 cells is inhibited by calcium chelators, calmodulin inhibitors, and perillyl alcohol, a monoterpene capable of blocking L-type calcium channels. Importantly, the combination of perillyl alcohol and bortezomib is synergistic in eliciting Rec-1 cell cytotoxicity. The relevance of these results is illuminated by the additional finding that a considerable fraction of primary MCL samples (8 out of 10) displayed bortezomib-resistant constitutive NF-κB activity.ConclusionOur findings show that bortezomib-resistant NF-κB activity is frequently observed in MCL samples and suggest that this activity may be relevant to MCL biology as well as serve as a potential therapeutic target.

The Lymphotoxin-β Receptor Is an Upstream Activator of NF-κB-mediated Transcription in Melanoma Cells

The pleiotropic transcription factor nuclear factor-kappaB (NF-kappaB (p50/p65)) regulates the transcription of genes involved in the modulation of cell proliferation, apoptosis, and oncogenesis. Furthermore, a host of solid and hematopoietic tumor types exhibit constitutive activation of NF-kappaB (Basseres, D. S., and Baldwin, A. S. (2006) 25, 6817-6830). However, the mechanism for this constitutive activation of NF-kappaB has not been elucidated in the tumors. We have previously shown that NF-kappaB-inducing kinase (NIK) protein and its association with Inhibitor of kappaB kinase alphabeta are elevated in melanoma cells compared with their normal counterpart, leading to constitutive activation of NF-kappaB. Moreover, expression of dominant negative NIK blocked this base-line NF-kappaB activity in melanoma cells. Of the three receptors that require NIK for activation of NF-kappaB, only the lymphotoxin-beta receptor (LTbeta-R) is expressed in melanoma. We show in this manuscript that for melanoma there is a strong relationship between expression of the LTbeta-R and constitutive NF-kappaB transcriptional activity. Moreover, we show that activation of the LTbeta-R can drive NF-kappaB activity to regulate gene expression that leads to enhanced cell growth. The inhibition by LTbeta-R shRNA resulted in decreased NF-kappaB promoter activity, decreased growth, and decreased invasiveness as compared with control. These results indicate that the LTbeta-R constitutively induces NF-kappaB activation, and this event may be associated with autonomous growth of melanoma cells.

INHIBITION OF NFκB REDUCES CELLULAR VIABILITY IN GH3 PITUITARY ADENOMA CELLS

Adenomas of the pituitary gland are among the most common types of tumors of the adult brain. Although adenomas are histologically benign, they may be associated with significant morbidity and mortality, mostly because of their invasive growth pattern and hormone hypersecretion. Current medical therapies are suppressive, acting at a receptor level. Thus, there is a need to identify novel cellular and molecular targets for pituitary tumors. We investigated the possible role of the NFkappaB transcription factor in pituitary tumor cell growth. The effect of NFkappaB pathway inhibition on cellular viability was studied in the GH3 pituitary adenoma cell line, a well-characterized rat cell line that secretes growth hormone and prolactin. Cells were treated with mechanistically diverse pharmacological NFkappaB pathway inhibitors or with molecular inhibitors that were overexpressed in tumor cells before the assessment of cellular viability. NFkappaB activity was also assessed in GH3 cells using deoxyribonucleic acid binding assays. GH3 cells exhibited constitutive NFkappaB activity, which contributed to increased cellular proliferation. Treatment with wedelolactone, an IkappaB kinase inhibitor, or overexpression of an IkappaB super-repressor reduced cell viability, further implicating NFkappaB in pituitary tumor cell growth. Pharmacological or molecular inhibition of Akt similarly reduced GH3 viability and NFkappaB binding, suggesting that constitutive activation of NFkappaB may be, at least in part, mediated by Akt. Directed targeting of the Akt and NFkappaB signaling pathways may be a useful adjunct in the clinical management of pituitary tumors. Further elucidation of this pathway may yield novel information regarding the behavior of pituitary tumors in humans.

In Vitro Activation of the IκB Kinase Complex by Human T-cell Leukemia Virus Type-1 Tax

Human T-cell leukemia virus type-I expresses Tax, a 40-kDa oncoprotein that activates IkappaB kinase (IKK), resulting in constitutive activation of NFkappaB. Herein, we have developed an in vitro signaling assay to analyze IKK complex activation by recombinant Tax. Using this assay in combination with reporter assays, we demonstrate that Tax-mediated activation of IKK is independent of phosphatases. We show that sustained activation of the Tax-mediated activation of the NFkappaB pathway is dependent on an intact Hsp90-IKK complex. By acetylating and thereby preventing activation of the IKK complex by the Yersinia effector YopJ, we demonstrate that Tax-mediated activation of the IKK complex requires a phosphorylation step. Our characterization of an in vitro signaling assay system for the mechanism of Tax-mediated activation of the IKK complex with a variety of mutants and inhibitors results in a working model for the biochemical mechanism of Tax-induced activation.

Mycoplasma infection suppresses p53, activates NF-κB and cooperates with oncogenic Ras in rodent fibroblast transformation

Prokaryotes of the genus Mycoplasma are the smallest cellular organisms that persist as obligate extracellular parasites. Although mycoplasma infection is known to be associated with chromosomal instability and can promote malignant transformation, the mechanisms underlying these phenomena remain unknown. Since persistence of many cellular parasites requires suppression of apoptosis in host cells, we tested the effect of mycoplasma infection on the activity of the p53 and nuclear factor (NF)-kappaB pathways, major mechanisms controlling programmed cell death. To monitor the activity of p53 and NF-kappaB in mycoplasma-infected cells, we used a panel of reporter cell lines expressing the bacterial beta-galactosidase gene under the control of p53- or NF-kappaB-responsive promoters. Cells incubated with media conditioned with different species of mycoplasma showed constitutive activation of NF-kappaB and reduced activation of p53, common characteristics of the majority of human tumor cells, with M. arginini having the strongest effect among the species tested. Moreover, mycoplasma infection reduced the expression level and inducibility of an endogenous p53-responsive gene, p21(waf1), and inhibited apoptosis induced by genotoxic stress. Infection with M. arginini made rat and mouse embryo fibroblasts susceptible to transformation with oncogenic H-Ras, whereas mycoplasma-free cells underwent irreversible p53-dependent growth arrest. Mycoplasma infection was as effective as shRNA-mediated knockdown of p53 expression in making rodent fibroblasts permissive to Ras-induced transformation. These observations indicate that mycoplasma infection plays the role of a p53-suppressing oncogene that cooperates with Ras in cell transformation and suggest that the carcinogenic and mutagenic effects of mycoplasma might be due to inhibition of p53 tumor suppressor function by this common human parasite.

S2002 Impact of Vitamin C Supplementation On Regulators of Esophageal Inflammation and Carcinogenesis

Introduction Antioxidants such as vitamin C may have a role in chemoprevention and in modulating cancer treatment responses. The transcription factor NFkappaB is central in the regulation of genes encoding for mediators of inflammation, and Vitamin C modulates its responses In Vitro. The aim of this study was twofold: first, to assess the effect of vitamin C on the NFkappaB and associated cytokines in patients with Barrett's esophagus; and second, in a randomised study, to assess the effect of Vitamin C on the response of esophageal adenocarcinoma to neoadjuvant chemoradiation.. Methods In study 1, 25 patients with long segment Barretts received Vitamin C (1000mg/day) orally for four weeks, and had pre and post-VitaminC endoscopic biopsies. In study 2, 20 patients undergoingmultimodal treatment for esophageal adenocarcinoma were randomized to receive Vitamin C or no supplementation and standard chemoradiation. NFkappaB activity (activated p50 and p65 subunits) of nuclear extracts was assessed using the Active Motif NFkappaB TransAm assay. Cytokine analysis was performed using the Evidence Investigator biochip array. IkB expression was examined by Western blotting. Results NFkappaB (p50 & p65), along with proinflammatory (IL-8, VEGF, IL-1a, IL-1b) and anti-inflammatory cytokines (IL-4) were activated in the Barrett's tissue of all patients pre-treatment. Downregulation in activated NFkappaB (p50 subunit) and related cytokines was observed in 8/25 patients (32%). In study 2, NFkappaB (p50 & p65), along with proinflammatory (IL-8, VEGF, IL-1a, IL-1b) and anti-inflammatory cytokines (IL-4) were activated in the cancer tissue of all patients pre-treatment. Downregulation in NFkappaB and downstream cytokines was observed in five patients (25%), 2 from the Vitamin C arm. Conclusion Constitutive activation of NFkappaB and target cytokines was observed in all Barrett's esophagus and adenocarcinoma patients. Vitamin C supplementation however, in contrast to In Vitro studies, had little effect in modulating these responses, and the studies failed to support its value in chemoprevention or modulation of chemoradiation responses.

Rearranged NF‐κB2 gene in an adult T‐cell leukemia cell line

Adult T-cell leukemia (ATL) is an aggressive type of leukemia, originating from T-cells infected with human T-cell leukemia virus type 1. Accumulating evidence suggests the aberrant activation of NF-kappaB to be a causative factor mediating the abnormal proliferation of leukemic cells, thus resulting in the development of ATL. A rearranged NF-kappa B2/p100 gene was isolated from an ATL-derived cell line, which was generated by a chromosomal translocation. The isolated NF-kappa B2 mutant is fused with the with no (lysine) deficient protein kinase 1 gene, coding for a 58 kDa protein that retains the DNA binding Rel homology domain, but it lacks the entire ankyrin repeat inhibitory domain, thus suggesting its constitutive activation. This rearranged NF-kappa B2 gene product (p58) was localized in the nucleus, and formed a complex with NF-kappaB p65 or RelB. Moreover, a T-cell line expressing p58 increased the amount of an NF-kappa B2-inducible gene, NF-kappa B2/p100 by itself. These results suggest that such NF-kappa B2 gene rearrangement may therefore be a factor in the constitutive activation of NF-kappaB in ATL, and thereby playing a role in the ATL pathogenesis.

Flt3 Receptor Inhibition Abolishes Constitutive NFκB Activation in High-Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia.

High-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are characterized by the constitutive activation of the anti-apoptotic transcription factor NF-kappaB, via the activation of the IKK complex. We show that constitutive activation of the receptor tyrosine kinase Flt3 is responsible for IKK activation and this activation of the NF-kappaB pathway was found to involve a not yet described phosphorylation of the IKK and IkBa complex involving tyrosine residues compared to serine residues in the classical NF-kappaB pathway. Chemical inhibition or knockdown of Flt3 with small interfering RNAs abolished NF-kappaB activation in MDS and AML cell lines, as well as in primary CD34+ bone marrow cells from patients, causing mitochondrial apoptosis. Epistatic analysis involving the simultaneous inhibition of Flt3 and IKK indicated that both kinases act via the same anti-apoptotic pathway. An IKK2 mutant with a constitutive kinase activity and a plasma membrane-tethered mutant of NEMO that activates IKK1/2 prevented the cytocidal action of Flt3 inhibition. IKK2 and Flt3 physically associated in MDS and AML cells and Flt3 inhibition caused the release of IKK2 from a preferential association with the plasma membrane. Flt3 inhibition only killed CD34+ bone marrow cells from high-risk MDS and AML patients, in correlation with the blast numbers and the NF-kappaB activity, yet had no lethal effect on healthy CD34+ cells or cells from low-risk MDS. These results suggest that Flt3 inhibitors might exert an anti-neoplastic effect in high-risk MDS and AML through inhibition of constitutive NF kappaB activation.

Nuclear Factor-Kappa B: From Clone to Clinic

Nuclear transcription factor kappaB (NF-kappaB) was first discovered in 1986 in the nucleus of the B cell as an enhancer in the kappa immunoglobulin chain. However, this factor has identified in the cytoplasm in the resting state. When activated in response to inflammatory stimuli, carcinogens, stress, ionizing radiation, and growth factors; NF-kappaB translocates to the nucleus where it upregulates the expression of over 400 different gene products linked with inflammation, cell survival, proliferation, invasion, and angiogenesis. The activation of NF-kappaB has now been linked with a variety of inflammatory diseases, including cancer and pulmonary, autoimmune, skin, neurodegenerative, and cardiovascular disorders. Indeed, constitutive NF-kappaB activation frequently correlates with the proliferation, survival, chemoresistance, radioresistance, and progression of various cancers. Hence, NF-kappaB has both diagnostic and prognostic applications. In addition, pharmaceutical companies are aggressively pursuing development of inhibitors of NF-kappaB with therapeutic potential. Thus within last decades this transcription factor, discovered serendipitously, has moved from "clone to clinic".

Constitutive association of MyD88 to IRAK in HTLV-I–transformed T cells

Constitutive activation of nuclear factor (NF)-kappaB is a common feature of human T-cell leukemia virus type I (HTLV-I)-transformed T cells. Inhibition of NF-kappaB activity reduces cell growth and induces apoptosis of HTLV-I-transformed T cells, suggesting a central role of NF-kappaB in their proliferation and survival. In this study, we investigated whether MyD88, an adaptor protein of Toll-like receptor (TLR) signaling, contributes to constitutive NF-kappaB activation in HTLV-I-transformed T cells. Activation status of MyD88 and interleukin (IL)-1R-associated kinase 1 (IRAK1) in HTLV-I-transformed human T cells, MT2, MT4, and HUT102 was examined by using Western blot and immunoprecipitation. TLR expression was evaluated with reverse transcription polymerase chain reaction. An expression vector encoding a dominant negative MyD88 with a deletion of its death domain (MyD88dn) was transfected into MT2 cells to evaluate roles of MyD88 in spontaneous activation of cytokine gene promoters and transcription factors, proliferation, and apoptosis in HTLV-I-transformed T cells. Constitutive association of MyD88 with IRAK1 was observed in all three of HTLV-I-transformed T cells, but not in HTLV-I-negative T cells, such as Jurkat, HUT78, and MOLT4. MT2 cells showed expression of TLR-1, -6, and -10 mRNAs. Constitutive activation of NF-kappaB and NF-IL-6 and cytokine gene promoters, such as IL-1alpha, interferon-gamma, and tumor necrosis factor-alpha in MT2 cells was inhibited by MyD88dn expression. MyD88dn reduced proliferation and induced apoptosis of MT2 cells. HTLV-I Tax enhanced TLR expression and synergistically activated NF-kappaB with wild-type MyD88. Our results show a novel pathway in NF-kappaB activation in HTLV-I-transformed T cells and further demonstrate a critical role of MyD88 in their dysregulated gene activation, survival, and proliferation.

Curcumin downregulates the constitutive activity of NF-κB and induces apoptosis in novel mouse melanoma cells

Melanoma, the most deadly form of skin cancer, is very aggressive and resistant to present therapies. The transcription factor nuclear factor-kappa B (NF-kappaB) has been reported to be constitutively active in many types of cancer. Constitutively active NF-kappaB seen in melanoma likely plays a central role in cell survival and growth. We have established and characterized novel cell lines from our murine melanoma model. Here we report the constitutive activity of NF-kappaB in these melanoma-derived cells, as shown by electrophoretic mobility shift assay and reporter assays. We hypothesized that agents that inhibit NF-kappaB may also inhibit cell proliferation and may induce apoptosis in such melanoma cells. Curcumin has been shown to inhibit NF-kappaB activity in several cell types. In our system, curcumin selectively inhibited growth of melanoma cells, but not normal melanocytes. Curcumin induced melanoma cells to undergo apoptosis, as shown by caspase-3 activation, inversion of membrane phosphatidyl serine, and increases in cells in the sub-G1 phase. A curcumin dose-dependent inhibition of NF-kappaB-driven reporter activity correlated with decreased levels of phospho-IkappaBalpha, and decreased expression of NF-kappaB-target genes COX-2 and cyclin D1. This study demonstrates that the use of cells from our model system can facilitate studies of signaling pathways in melanoma. We furthermore conclude that curcumin, a natural and safe compound, inhibits NF-kappaB activity and the expression of its downstream target genes, and also selectively induces apoptosis of melanoma cells but not normal melanocytes. These encouraging in-vitro results support further investigation of curcumin for treatment of melanoma in vivo.

Induction of heme oxygenase-1 by cobalt protoporphyrin enhances the antitumour effect of bortezomib in adult T-cell leukaemia cells

Adult T-cell leukaemia (ATL) is a lethal neoplasia derived from HTLV-1-infected T lymphocytes frequently exhibiting nuclear factor-κB (NF-κB) activation. Despite the use of various treatment regimens, the prognosis of ATL is poor, and new treatment strategies are urgently needed. We therefore explored the effect and the molecular mechanism of a proteasome inhibitor, bortezomib, in ATL cells. We found bortezomib-induced cell death, and bortezomib suppressed constitutive NF-κB activation via I-κB stabilisation in three ATL cell lines (TaY, MT-2 and MT-4). An oligonucleotide DNA microarray analysis of TaY cells revealed upregulation of genes encoding heat shock proteins (HSPA1A, STIP1, HSPA1B, and HSPCA), genes related to protein folding (CDC37 and ANAPC5), Fas-associated factor 1(FAF1) and an oxidative stress-related gene, heme oxygenase-1(HMOX-1), known to be a target gene of hypoxia-inducible gene-1 alpha (HIF-1 alpha). Cobalt protoporphyrin induced HMOX-1, instead of HIF-1 alpha expression and increased bortezomib-induced apoptosis in the presence of pharmacologically effective doses of bortezomib. In contrast, zinc protoporphyrin downregulated HMOX-1 expression, thereby partially inhibiting bortezomib-induced cell death. This indicates that HMOX-1 may modulate anticancer effects of bortezomib in ATL cells, and could be a molecular target in treating ATL patients.

Transglutaminase-Mediated Activation of Nuclear Transcription Factor-κB in Cancer Cells: A New Therapeutic Opportunity

The nuclear factor kappaB (NF-kappaB) plays an important role in tumorigenesis by affecting processes such as tumor initiation, promotion, growth, and metastasis. NF-kappaB induces the expression of genes that are known to confer resistance to apoptosis. Therefore, its activation has been associated with the development of chemo- and radiation resistance in cancer cells. NF-kappaB is constitutively activated in many types of tumor cells by mechanisms that are not well understood. Like NF-kappaB, tissue-type transglutaminase (TG2), the most diverse and ubiquitous member of the calcium-dependent transglutaminase family of enzymes, is also aberrantly overexpressed in many human cancer types, blocks apoptosis, and promotes drug resistance and metastatic phenotypes. In this review, we will discuss the current understanding of the mechanisms thought to participate in constitutive activation of NF-kappaB. Particular focus is given to the implications of increased TG2 expression in NF-kappaB activation and its contributions to the development of drug resistance and metastatic phenotypes in cancer cells.

Multiple Myeloma: Lusting for NF-κB

Multiple myeloma (MM) is a late-stage B cell malignancy that has received much attention recently because of its therapeutic susceptibility to proteasome inhibitors. Two papers in this issue of Cancer Cell show that primary MM samples and MM cell lines frequently have mutations in genes encoding regulators and effectors of NF-kappaB signaling, and that these mutations lead to chronic NF-kappaB target gene expression, which is required for the viability of these MM tumor cells. These results reveal the molecular basis for constitutive NF-kappaB activity in many MMs and further validate the NF-kappaB signaling pathway as an appropriate target for MM therapy.