Selective Inhibition Of NF-κB Research Articles

Abstract 889: NF-κB-mediated mitophagy contributes to the maintenance of cancer stem cells in ovarian cancer

Abstract Tumors contain phenotypically and functionally heterogeneous cancer cells. Among them, a minority of cell subpopulation, termed cancer stem cells (CSCs), which possess the self-renewal capacity and are able to generate the heterogeneous lineage of cancer cells, has been believed to be responsible for the initiation and progression of the tumor. However, it is still unclear how the stemness properties of CSCs are maintained in the tumor. Mitophagy is a process by which damaged mitochondria are cleared via autophagy. The significance of mitophagy in the maintenance of CSC has been investigated in multiple CSC types. Our recent study shows that ovarian CSCs also exhibit enhanced mitophagy, which is mainly attributed to elevated expression of mitophagy receptors BNIP3 and BNIP3L. Downregulation of BNIP3 or BNIP3L significantly diminished mitophagy in ovarian CSCs and compromised their self-renewal capability. Mechanistic investigation further revealed that enhanced NF-κB signaling contributes to the increased expression of BNIP3 and BNIP3L. Inhibition of NF-κB signaling pathway via p65 knockdown or specific inhibitors is able to reduce mitophagy in ovarian CSCs. In summary, our data suggest that selective inhibition of NF-κB is able to deplete CSCs by interfering with mitochondria quality control in these cells and has potential to prevent tumor relapse and metastasis. Citation Format: Na Li, Tejinder Pal, Shurui Cai, Ananya Banerjee, Xuetao Bai, Kousalya Lavudi, Qi-En Wang. NF-κB-mediated mitophagy contributes to the maintenance of cancer stem cells in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 889.

NF-κB and neutrophil extracellular traps cooperate to promote breast cancer progression and metastasis

Aberrant NF-κB activation and neutrophil extracellular traps (NETs) are associated with breast cancer progression. How NF-κB and NETs modulate each other in breast cancer development remains unclear. Here, we found that NETs induced by phorbol 12-myristate 13-acetate promote breast cancer cell progression. In turn, cancer cells–derived factors, such as IL-8 and granulocyte colony-stimulating factor, stimulate neutrophils to form NETs. Mechanistically, NETs increased the interaction of NF-κB essential modifier (NEMO) with IκB kinase (IKK)α/β and enhanced NF-κB activation. We then employed a cell-permeable peptide corresponding to the NEMO-binding domain (NBD) of IKKα/β, termed NBD peptide, which disrupts NETs-mediated NEMO interaction with IKKα/β and abolished NF-κB activation in vitro. NBD peptide also reduced IL-8 level and NETs formation, and suppressed primary tumor growth and/or lung metastasis in human breast cancer mouse xenograft models and mouse spontaneous breast cancer model. Blockade of NET formation using a peptidylarginine deiminase 4 (PAD4) pharmacologic inhibitor decreased NF-κB activation and tumor metastasis. Collectively, these data suggest that NF-κB associates with NETs to form a positive loop facilitating breast tumor progression and metastasis, and that selective inhibition of NF-κB and PAD4-dependent NETs provides an effective therapeutic approach for treating breast cancer.

The Role of NF-κB in Uterine Spiral Arteries Remodeling, Insight into the Cornerstone of Preeclampsia.

Preeclampsia is one of the three leading causes of maternal morbidity and mortality worldwide. It afflicts 2–8% of pregnancies and is the most common cause of gestational hypertension. This article is focused on nuclear factor kappa B (NF-κB), its role in normal and pathological spiral arteries remodelling and development of preeclampsia, with evaluation if it is a promising therapeutic target. NF-κB is a key mediator of placentation. Since insemination, it stimulates production of proinflammatory cytokines by the uterine epithelium, which leads to activation of macrophages, uterine natural killer cells (uNKs), and other leukocytes. The trophoblast/uNK/macrophage crosstalk is crucial for implantation and spiral arteries remodeling, and NF-κB regulates that process through modification of cytokine expression, as well as cell phenotype and function. In the course of preeclampsia, the remodeling processes is disturbed by excessive inflammation and increased NF-κB activation. The pathological remodeling leads to uteroplacental dysfunction, release of proinflammatory cytokines into the maternal circulation, endothelial stress, and development of preeclampsia. The analysis of genetic and environmental inductors of NF-κB helps to distinguish preeclampsia risk groups. Furthermore, a selective inhibition of NF-κB or NF-κB activating pathways alleviates symptoms of preeclampsia in rat models; therefore, this could be an efficient therapeutic option.

Prophylactic inhibition of NF-\u03baB expression in microglia leads to attenuation of hypoxic ischemic injury of the immature brain

BackgroundPeriventricular leukomalacia (PVL), a devastating brain injury affecting premature infants, is the most common cause of cerebral palsy. PVL is caused by hypoxia ischemia (HI) and is characterized by white matter necrotic lesions, microglial activation, upregulation of NF-κB, and neuronal death. The microglia is the main cell involved in PVL pathogenesis. The goal of this study was to investigate the role of microglial NF-κB activity and its prophylactic inhibition in a neonate mouse model of HI.MethodsTransgenic mice with specific knockout NF-κB in microglia and colony stimulating factor 1 receptor Cre with floxed IKKβ (CSF-1R Cre + IKKβflox/wt ) were used. Postnatal day 5 (P5) mice underwent sham or bilateral temporary carotid artery ligation followed by hypoxia. After HI insult, inflammatory cytokines, volumetric MRI, histopathology, and immunohistochemistry for oligodendroglia and microglial activation markers were analyzed. Long-term neurobehavioral assessment, including grip strength, rotarod, and open field testing, was performed at P60.ResultsWe demonstrate that selective inhibition of NF-κB in microglia decreases HI-induced brain injury by decreasing microglial activation, proinflammatory cytokines, and nitrative stress. Rescue of oligodendroglia is evidenced by immunohistochemistry, decreased ventriculomegaly on MRI, and histopathology. This selective inhibition leads to attenuation of paresis, incoordination, and improved grip strength, gait, and locomotion.ConclusionWe conclude that NF-κb activation in microglia plays a major role in the pathogenesis of hypoxic ischemic injury of the immature brain, and its prophylactic inhibition offers significant neuroprotection. Using a specific inhibitor of microglial NF-κB may offer a new prophylactic or therapeutic alternative in preterm infants affected by HI and possibly other neurological diseases in which microglial activation plays a role.

NF-κB targeting for overcoming tumor resistance and normal tissues toxicity.

Radiotherapy andchemotherapy are two famous modalities in tumor-targeted therapy that lead to systemic and local toxicities for normal tissues. Moreover, several studies have confirmed that exposure of the tumor to radiation or chemotherapy drugs stimulate some signaling pathways in the tumor microenvironment (TME), leading to resistance of cancer cells to apoptosis, as well as promoting angiogenesis and tumor growth. Nuclear factor kappa B (NF-κB) plays a central role in the regulation of inflammatory responses in both normal tissues and tumors via the release of several cytokines, regulation of prostaglandins, reduction/oxidation (redox) reactions, angiogenesis, and cell death. Upregulation of NF-κB in normal tissues causes an appearance of inflammatory reactions and oxidative stress, whereas it regulates angiogenesis and suppresses apoptosis, leading to resistance to subsequent doses of radiation or chemotherapy. Selective inhibition of NF-κB in experimental studies has shown promising results for tumor sensitization via apoptosis induction, inhibition of angiogenesis, and increasing delay of tumor growth. The use of some agents for NF-κB inhibition has been shown to alleviate radiation/chemotherapy toxicities in normal cells/ tissues. In this current review, we explained the pivotal role of NF-κB in both normal tissue toxicity and tumor resistance. We also discussed the promising strategies for overcoming these problems with regard to chemotherapy and radiotherapy.

Stealing Tregs’ Identity (by Deleting NF-κB)

Stealing Tregs’ Identity (by Deleting NF-κB)

Selective inhibition of NF-kappaB and surfactant therapy in experimental meconium-induced lung injury.

Meconium aspiration syndrome (MAS) in newborns is characterized mainly by respiratory failure due to surfactant dysfunction and inflammation. Previous meta-analyses did not prove any effect of exogenous surfactant treatment nor glucocorticoid administration on final outcome of children with MAS despite oxygenation improvement. As we supposed there is the need to intervene in both these fields simultaneously, we evaluated therapeutic effect of combination of exogenous surfactant and selective inhibitor of NF-kappaB (IKK-NBD peptide). Young New Zealand rabbits were instilled by meconium suspension and treated by surfactant alone or surfactant in combination with IKK-NBD, and oxygen-ventilated for 5 h. PaO(2)/FiO(2), oxygenation index, oxygen saturation and ventilation efficiency index were evaluated every hour; post mortem, total and differential leukocyte counts were investigated in bronchoalveolar lavage fluid (BALF) and inflammatory, oxidative and apoptotic markers were assessed in lung tissue homogenates. Exogenous surfactant combined with IKK-NBD improved oxygenation, reduced neutrophil count in BALF and levels of IL-1beta, IL-6, p38 MAPK and caspase 3 in comparison with surfactant-only therapy. It seems that inhibition of inflammation may be strong supporting factor in surfactant treatment of MAS.

Microglia Induce Motor Neuron Death via the Classical NF-κB Pathway in Amyotrophic Lateral Sclerosis

Neuroinflammation is one of the most striking hallmarks of amyotrophic lateral sclerosis (ALS). Nuclear factor-kappa B (NF-κB), a master regulator of inflammation, is upregulated in spinal cords of ALS patients and SOD1-G93A mice. In this study, we show that selective NF-κB inhibition in ALS astrocytes is not sufficient to rescue motor neuron (MN) death. However, the localization of NF-κB activity and subsequent deletion of NF-κB signaling in microglia rescued MNs from microglial-mediated death invitro and extended survival in ALS mice by impairing proinflammatory microglial activation. Conversely, constitutive activation of NF-κB selectively in wild-type microglia induced gliosis and MN death invitro and invivo. Taken together, these data provide a mechanism by which microglia induce MN death in ALS and suggest a novel therapeutic target that can be modulated to slow the progression of ALS and possibly other neurodegenerative diseases by which microglial activation plays a role.

NF-κB Inhibition after Cecal Ligation and Puncture Reduces Sepsis-Associated Lung Injury without Altering Bacterial Host Defense

Introduction. Since the NF-κB pathway regulates both inflammation and host defense, it is uncertain whether interventions targeting NF-κB would be beneficial in sepsis. Based on the kinetics of the innate immune response, we postulated that selective NF-κB inhibition during a defined time period after the onset of sepsis would reduce acute lung injury without compromising bacterial host defense. Methods. Mice underwent cecal ligation and puncture (CLP). An NF-κB inhibitor, BMS-345541 (50 µg/g mice), was administered by peroral gavage beginning 2 hours after CLP and repeated at 6 hour intervals for 2 additional doses. Results. Mice treated with BMS-345541 after CLP showed reduced neutrophilic alveolitis and lower levels of KC in bronchoalveolar lavage fluid compared to mice treated with CLP+vehicle. In addition, mice treated with CLP+BMS had minimal histological evidence of lung injury and normal wet-dry ratios, indicating protection from acute lung injury. Treatment with the NF-κB inhibitor did not affect the ability of cultured macrophages to phagocytose bacteria and did not alter bacterial colony counts in blood, lung tissue, or peritoneal fluid at 24 hours after CLP. While BMS-345541 treatment did not alter mortality after CLP, our results showed a trend towards improved survival. Conclusion. Transiently blocking NF-κB activity after the onset of CLP-induced sepsis can effectively reduce acute lung injury in mice without compromising bacterial host defense or survival after CLP.

Inhibition of NF-κB prevents high glucose-induced proliferation and plasminogen activator inhibitor-1 expression in vascular smooth muscle cells

Recent epidemiologic studies clearly showed that early intensive glucose control has a legacy effect for preventing diabetic macrovascular complications. However, the cellular and molecular processes by which high glucose leads to macrovascular complications are poorly understood. Vascular smooth muscle cell (VSMC) dysfunction due to high glucose is a characteristic of diabetic vascular complications. Activation of nuclear factor-κB (NF-κB) may play a key role in the regulation of inflammation and proliferation of VSMCs. We examined whether VSMC proliferation and plasminogen activator inhibitor-1 (PAI-1) expression induced by high glucose were mediated by NF-κB activation. Also, we determined whether selective inhibition of NF-κB would inhibit proliferation and PAI-1 expression in VSMCs. VSMCs of the aorta of male SD rats were treated with various concentrations of glucose (5.6, 11.1, 16.7, and 22.2 mM) with or without an inhibitor of NF-κB or expression of a recombinant adenovirus vector encoding an IκB-α mutant (Ad-IκBαM). VSMC proliferation was examined using an MTT assay. PAI-1 expression was assayed by real-time PCR and PAI-1 protein in the media was measured by ELISA. NF-κB activation was determined by immunohistochemical staining, NF-κB reporter assay, and immunoblotting. We found that glucose stimulated VSMC proliferation and PAI-1 expression in a dose-dependent manner up to 22.2 mM. High glucose (22.2 mM) alone induced an increase in NF-κB activity. Treatment with inhibitors of NF-κB such as MG132, PDTC or expression of Ad-IκB-αM in VSMCs prevented VSMC proliferation and PAI-1 expression induced by high glucose. In conclusion, inhibition of NF-κB activity prevented high glucose-induced VSMC proliferation and PAI-1 expression.

Activation of AMP-activated Protein Kinase α1 Alleviates Endothelial Cell Apoptosis by Increasing the Expression of Anti-apoptotic Proteins Bcl-2 and Survivin

Accumulating evidence suggests that AMP-activated protein kinase (AMPK) activation exerts anti-apoptotic effects in multiple types of cells. However, the underlying mechanisms remain poorly defined. The aim of the present study was to determine how AMPK suppresses apoptosis in endothelial cells exposed to hypoxia and glucose deprivation (OGD). AMPK activity, NF-kappaB activation, and endothelial cell apoptosis were assayed in cultured endothelial cells and mouse common carotid artery with or without OGD treatment. OGD markedly activated AMPK as early as 30 min, and AMPK activity reached maximal at 2 h of OGD. Endothelial apoptosis was not detected until 2 h of OGD but became markedly elevated at 6 h of OGD treatment. Furthermore, AMPK inhibition by Compound C or overexpression of dominant negative AMPK (AMPK-DN) exacerbated, whereas AMPK activation by pharmacologic (aminoimidazole carboxamide ribonucleotide (AICAR)) or genetic means (overexpression of constitutively active AMPK) suppressed endothelial cell apoptosis caused by OGD. Concomitantly, AMPK activation increased the expression of both Bcl-2 and Survivin, two potent anti-apoptotic proteins. Furthermore, AMPK activation significantly enhanced IkappaBalpha kinase activation, NF-kappaB nuclear translocation, and DNA binding activity of NF-kappaB. Consistently, selective inhibition of NF-kappaB, which abolished OGD-enhanced expression of Bcl-2 and Survivin, accentuated endothelial apoptosis caused by OGD. Finally, we found that genetic deletion of the AMPKalpha1, but not AMPKalpha2, suppressed OGD-enhanced NF-kappaB activation, the expression of Bcl-2 and Survivin, and endothelial apoptosis. Overall, our results suggest that AMPKalpha1, but not AMPKalpha2 activation, promotes cell survival by increasing NF-kappaB-mediated expression of anti-apoptotic proteins (Bcl-2 and Survivin) and intracellular ATP contents.

Selective NF-κB inhibition, but not dexamethasone, decreases acute lung injury in a newborn piglet airway inflammation model

Acute respiratory failure in neonates (e.g. ARDS, meconium aspiration pneumonitis, pneumonia) is characterized by an excessive inflammatory response, governing the migration of polymorpho-nuclear leukocytes (PMNLs) into lung tissue and causing consecutive impairment of gas exchange and lung function. Critical to this inflammatory response is the activation of nuclear factor-κB (NF-κB) that is required for transcription of the genes for many pro-inflammatory mediators. We asked whether the inhibition of NF-κB activity using either a selective inhibitor (IKK-NBD peptide) or dexamethasone would be more effective in decreasing NF-κB activity and chemokine expression in pulmonary cells. Changes in lung function were repeatedly assessed for 24 h following induction of acute respiratory failure and therapeutic intervention. We conducted a randomized, controlled, prospective animal study with mechanically ventilated newborn piglets which underwent repeated airway lavage (20 ± 2 [SEM]) to remove surfactant and to induce lung inflammation. Admixed to 100 mg kg −1 surfactant, piglets then received either IKK-NBD peptide (S+IKK), a selective inhibitor of NF-κB activation, its control peptide without intrinsic activity, dexamethasone (S+Dexa), its solvent aqua, or an air bolus only (all groups n = 8). After 24 h of mechanical ventilation, the following differences were measured: PaO 2/FiO 2 (S+IKK 230 ± 9 mmHg vs. S+Dexa 188 ± 14, p < 0.05); ventilation efficiency index (0.18 ± 0.01 [3800/(PIP-PEEP) *f *PaCO 2] vs. 0.14 ± 0.01, p < 0.05); extravascular lung water (24 ± 1 ml kg −1 vs. 29 ± 2, p < 0.05); PMNL in BAL fluid (112 ± 21 cells μl −1 vs. 208 ± 34, p < 0.05), IL-8 (351 ± 117 pg ml −1 vs. 491 ± 144, p = ns) and leukotriene B 4 (23 ± 7 pg ml −1 vs. 71 ± 11, p < 0.01) in BAL fluid. NF-κB activity in the nucleus of pulmonary cells differed by 32 ± 5% vs. 55 ± 3, p < 0.001. Differences between these two intervention groups were more pronounced in the second half of the observation period (hours 12–24). At 24 h of mechanical ventilation, inhibition of NF-κB activity by IKK-NBD peptide admixed to surfactant as a carrier caused improved gas exchange, lung function and reduced pulmonary inflammation, as evidenced by reduction in PMNL migration into lung tissue due to reduced nuclear NF-κB activity. We conclude that IKK-NBD admixture to surfactant in acute neonatal respiratory failure is superior to dexamethasone administration within the first 24 h.

Targeted NF-κB inhibition of asthmatic serum-mediated human monocyte-derived dendritic cell differentiation in a transendothelial trafficking model

Transendothelial trafficking model mimics in vivo differentiation of monocytes into dendritic cells (DC). The serum from patients with systemic lupus erythematosus promotes the differentiation of monocytes into mature DC. We have shown that selective inhibition of NF-κB by adenoviral gene transfer of a novel mutated IκBα (AdIκBαM) in DC contributes to T cell tolerance. Here we demonstrated for the first time that asthmatic serum facilitated human monocyte-derived DC (MDDC) maturation associated with increased NF-κB activation in this model. Furthermore, selective blockade of NF-κB by AdIκBαM in MDDC led to increased apoptosis, and decreased levels of CD80, CD83, CD86, and IL-12 p70 but not IL-10 in asthmatic serum-stimulated MDDC, accompanied by reduced proliferation of T cells. These results suggest that AdIκBαM-transferred MDDC are at a more immature stage which is beneficial to augment the immune tolerance in asthma.

Abstract 2841: Endothelial Selective NF-κB Inhibition Improves Survival and Preserves Host Defense Capacity in Bacterial Model of Sepsis

NF-κB plays a predominant role in septic pathology and is a desired target for developing therapies for septic shock. However, NF-κB blockade impairs bacterial clearance capability (BCC), which is detrimental. We have demonstrated that endothelial-restricted NF-κB blockade effectively prevents endotoxemic multiple organ injury (Circulation, 114: II-257). In this study, we examined whether endothelial-selective NF-κB blockade preserves BCC and improves survival in bacterial model of sepsis. Wild type (WT) and transgenic mice (TG) that conditionally overexpress a degradation-resistant I-κBα (I-κBαmt) selectively on endothelial cells were intravenously injected with saline (1 ml/kg) or pathogenic bacteria. Bacterial colonies formed in blood and tissue homogenate cultures of lungs, liver, spleen and kidney were counted. TG mice were induced to express I-κBαmt by feeding with doxycycline in drinking water. No bacteria grew in blood and any tissue homogenate culture from WT and TG mice injected with saline. Bacterial colony counts in blood and the 4 tissue homogenate cultures were similar between TW and TG mice 12 hrs after 10 7 CFU S. pneumoniae or 10 8 CFU S. enterica injection or 24 hrs after 10 8 CFU L. monocytogenes injection (Table ). TG mice showed an improved survival in the cecal ligation and puncture model of sepsis. The survival rate was 33% and 67% for WT and TG mice. Our results demonstrate that endothelial-selective blockade of NF-κB activation improves survival and preserves bacterial clearance capacity in bacterial model of sepsis. Bacterial Colony Counts (CFU per ml blood or gram tissue)

Regulation of the G2–M cell cycle progression by the ERK5–NFκB signaling pathway

Elucidation of mechanisms regulating cell cycle progression is of fundamental importance for cell and cancer biology. Although several genes and signaling pathways are implicated in G1–S regulation, less is known regarding the mechanisms controlling cell cycle progression through G2 and M phases. We report that extracellular signal–regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinases, is activated at G2–M and required for timely mitotic entry. Stimulation of ERK5 activated nuclear factor κB (NFκB) through ribosomal S6 kinase 2 (RSK2)-mediated phosphorylation and degradation of IκB. Furthermore, selective inhibition of NFκB at G2–M phases substantially delayed mitotic entry and inhibited transcription of G2–M–specific genes, including cyclin B1, cyclin B2, Plk-1, and cdc25B. Moreover, inhibition of NFκB at G2–M diminished mitosis induced by constitutive activation of ERK5, providing a direct link between ERK5, NFκB, and regulation of G2–M progression. We conclude that a novel ERK5–NFκB signaling pathway plays a key role in regulation of the G2–M progression.

House dust mite allergen Der p 1 elevates the release of inflammatory cytokines and expression of adhesion molecules in co-culture of human eosinophils and bronchial epithelial cells

House dust mite (HDM) is a common allergen of allergic asthma. Eosinophils are principal effector cells of allergic inflammation and their adhesion onto human bronchial epithelial cells is mediated by a CD18-intracellular adhesion molecule-1 (ICAM-1)-dependent interaction. We studied the effects of HDM Dermatophagoides pteronyssinus (Der p) 1 on the activation of eosinophils and bronchial epithelial BEAS-2B cells. Cytokines and adhesion molecules were measured using flow cytometry. Transcription factor nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) and signaling molecule p38 mitogen-activated protein kinase (MAPK) were analyzed using electromobility shift assay and western blot, respectively. Der p 1 protein was found to potently induce the release of IL-1beta, IL-6, IL-10, tumor necrosis factor (TNF)-alpha and granulocyte macrophage colony-stimulating factor from eosinophils. Such induction was further up-regulated for IL-6 and IL-10, and down-regulated for TNF-alpha and IL-1beta in eosinophil-BEAS-2B cells co-culture. Surface expression of CD18 and ICAM-1 on eosinophils was greatly increased by Der p 1; such inductive effect on ICAM-1 was also found to be more prominent on BEAS-2B cells from the co-culture than BEAS-2B cells alone. Der p 1 was found to activate NF-kappaB and AP-1 activity in eosinophils alone and in co-culture and BEAS-2B cells in co-culture. Moreover, Der p 1 could activate p38 MAPK in BEAS-2B cells and eosinophils alone and in co-culture. Selective inhibition of NF-kappaB, AP-1 and p38 MAPK resulted in differential suppression of the Der p 1-induced cytokine release and adhesion molecule expression. As an allergen, HDM could therefore induce the release of inflammatory cytokines and expression of adhesion molecules from the interaction of human eosinophils and bronchial epithelial cells.

Down-regulating constitutive activation of the NF-κB canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis

AbstractConstitutive activation of the nuclear factor-kappaB (NF-κB) pathway has been shown to be involved in the resistance of tumor cells to apoptosis in several human malignancies of the hematopoietic lineage. By using electrophoretic mobility shift assay (EMSA) and confocal microscopic analysis, we demonstrate that NF-κB is constitutively activated in cutaneous T-cell lymphoma (CTCL) cell lines HuT-78, MyLa, and SeAx and in peripheral blood lymphocytes (PBLs) from patients with Sézary syndrome (SS) presenting a high ratio of tumor cells, with evidence of p50 and RelA/p65 in DNA-linked complexes. Transfection of SeAx line with a κB/luciferase reporter plasmid showed that translocated NF-κB complexes were functional. Selective inhibition of NF-κB, by transfecting CTCL cell lines with a super-repressor form of IκBα, led to apoptosis. We evidenced down-regulation of NF-κB activation and induction of CTCL cell apoptosis in the presence of proteasome 26S inhibitors ALLN, MG132, and bortezomib. Bortezomib at nanomolar concentrations inhibited constitutive activation of NF-κB and induced apoptosis of CTCL cells, with evidence of an upregulation of Bax expression. These results demonstrate the key role played by NF-κB in the resistance of CTCL to apoptosis and suggest that bortezomib might be useful for the treatment of patients with advanced stages of CTCL refractory to standard antineoplastic chemotherapy.

Targeting NF-Kβ for the Prevention of Graft Versus Host Disease.

NF-κB is a transcription factor that controls expression of a number of genes that are important for mediating immune and inflammatory responses, such as IL-1 and TNF-α , which are both overproduced during graft versus host disease (GVHD). To determine whether the NF-κB signaling pathway plays a role in the pathophysiology of GVHD, we examined the affect of bortezomib (PS-341, Velcade®), a proteasome inhibitor whose mechansism of action includes but is not limited to inhibition of NF-κB, and PS-1145, a IKB kinase (IKK) inhibitor that selectively inhibits NF-κB, on the induction of GVHD. Lethally irradiated C57BL/6 (H-2b) mice that were transplanted with MHC-incompatible B10.BR (H-2k) bone marrow (BM) and spleen cells and treated with a single dose of bortezomib (1 mg/kg) on the day of BMT had significantly prolonged survival compared to untreated GVHD controls (50% versus 0% survival at 60 days). In contrast, extended or delayed administration of this agent on a twice weekly schedule that recapitulated current clinical practice guidelines resulted in a significant increase in mortality as all animals died within ten days of transplantation. Histological analysis of bortezomib-treated mice demonstrated that mortality was attributable to severe pathological damage in the colon, indicating that there was a narrow therapeutic window within which bortezomib could be administered. Toxicity was GVHD-dependent as syngeneic marrow transplant recipients treated with a similar schedule for four weeks (8 doses) all survived for 60 days and had no evidence of pathological damage when compared to vehicle-treated controls. Given the narrow therapeutic window observed with bortezomib, we examined whether more selective inhibition of NF-κB with PS-1145 could prevent GVHD without inducing toxicity and early mortality. In vitro studies demonstrated that PS-1145 induced apoptosis in alloactivated T cells, similar to bortezomib, although a higher concentration was required for equivalent results (8 μM versus 0.32 μM). To achieve a serum concentration of PS-1145 necessary for the induction of T cell apoptosis in vivo, PS-1145 was administered at a dose of 50 mg/kg on days 0–2 post transplantation to recipients of MHC-incompatible marrow grafts. Treatment with PS-1145 resulted in similar GVHD protection when compared to animals administered a single dose of bortezomib. Prevention of GVHD by bortezomib and PS-1145 was associated with equivalent reductions of serum cytokine levels of IL-2, IL-10 and TNF-α when compared to GVHD controls. To determine whether the selective inhibition of NF-κB with PS-1145 caused less toxicity in BMT recipients, mice were administered this agent for 10 consecutive days beginning on the day of transplant. In contrast to results observed with bortezomib, PS-1145-treated mice had no early mortality and had superior survival when compared to GVHD controls (100% versus 0% survival at 60 days). GVHD protection in these mice was associated with significantly reduced expansion of alloreactive donor T cells in the spleen without compromise of donor engraftment. These results confirm a critical role for NF-κB in the pathophysiology of GVHD and indicate that selective inhibition of NF-κB may have a superior therapeutic index and constitute a viable approach to reduce GVHD.

NF-κB is Required for Survival of Immature Auditory Hair Cells In Vitro

Damage to auditory hair cells in the inner ear as a consequence of aging, disease, acoustic trauma, or exposure to ototoxins underlies most cases of hearing impairment. Because the mammalian ear cannot replace damaged hair cells, loss of hearing is irreversible and progressive throughout life. One of the current goals of inner ear biology is to develop therapeutic strategies to prevent hair cell degeneration. Although important progress has been made in discovering factors that mediate hair cell death, very little is known about the molecular pathway(s) that signal survival. Here we considered the role of NF-kappaB, a ubiquitous transcription factor that plays a major role in the regulation of many apoptosis- and stress-related genes, in mediating hair cell survival. NF-kappaB was detected in a constitutively active form in the organ of Corti of 5-day-old rats. Selective inhibition of NF-kappaB through use of a cell-permeable inhibitory peptide in vitro caused massive degeneration of hair cells within 24 h of inhibitor application. Hair cell death occurred through an apoptotic pathway through activation of caspase-3 and may involve transcriptional down-regulation of the gadd45beta gene, an anti-apoptotic NF-kappaB target. In view of our results, it seems likely that NF-kappaB may participate in normal hair cell function.

Evidence for radiosensitizing by gliotoxin in HL-60 cells: implications for a role of NF-kappaB independent mechanisms.

Radioresistance markedly impairs the efficacy of tumor radiotherapy and may involve antiapoptotic signal transduction pathways that prevent radiation-induced cell death. A common cellular response to genotoxic stress induced by radiation is the activation of the nuclear factor kappa B (NF-kappaB). NF-kappaB activation in turn can lead to an inhibition of radiation-induced apoptotic cell death. Thus, inhibition of NF-kappaB activation is commonly regarded as an important strategy to abolish radioresistance. Among other compounds, the fungal metabolite gliotoxin (GT) has been reported to be a highly selective inhibitor of NF-kappaB activation. Indeed, low doses of GT were sufficient to significantly enhance radiation-induced apoptosis in HL-60 cells. However, this effect turned out to be largely independent of NF-kappaB activation since radiation of HL-60 cells with clinically relevant doses of radiation induced only a marginal increase in NF-kappaB activity, and selective inhibition of NF-kappaB by SN50 did not result in a marked enhancement of GT-induced apoptosis. GT induced activation of JNKs, cytochrome c release from the mitochondria and potently stimulated the caspase cascade inducing cleavage of caspases -9, -8, -7 and -3. Furthermore, cleavage of the antiapoptotic protein X-linked IAP and downregulation of the G2/M-specific IAP-family member survivin were observed during GT-induced apoptosis. Finally, the radiation-induced G2/M arrest was markedly reduced in GT-treated cells most likely due to the rapid induction of apoptosis. Our data demonstrate that various other pathways apart from the NF-kappaB signaling complex can sensitize tumor cells to radiation and propose a novel mechanism for radiosensitization by GT, the interference with the G2/M checkpoint that is important for repair of radiation-induced DNA damage in p53-deficient tumor cells.