Retinoid-related molecules require caspase 9 for the effective release of Smac and the rapid induction of apoptosis

Certain retinoid-related molecules (RRMs) with agonist or antagonist activities have been described to induce apoptosis in a variety of cancer cell lines and show promise for the treatment of cancer. Similar to other chemotherapeutic drugs, these retinoid analogs have been suggested to induce apoptosis through the intrinsic pathway, which requires the release of cytochrome c from the mitochondria for the effective activation of caspase 9. Expression of a catalytically inactive form of caspase 9, which functions as a dominant negative mutant, inhibits the induction of DEVDase activity and nuclear fragmentation by selective RRMs. Whereas the RRMs could induce the release of cytochrome c in the absence of caspase 9 activity, the later is necessary for the effective release of Smac/Diablo from the mitochondria. Furthermore, overexpression of Bcl-2 or Bcl-X(L) also inhibits RRM-induced apoptosis. We demonstrate that activation of caspase 2 by the agonist MX2870-1 requires caspase 9 activity and is inhibited by Bcl-2 overexpression. In contrast, the antagonist MX781 induces cleavage of procaspase 2 upstream of mitochondria and independently of caspase 9. Thus, two retinoid analogs with unique characteristics activate two distinct apical caspases (2 or 9) to initiate apoptosis. In addition to caspase-mediated cell death, sustained exposure to the RRMs can also lead to loss of cell viability in cells lacking caspase 9 activity or in cells stimulated in the presence of the caspase inhibitor Z-VAD-fmk. Moreover, MX2870-1 and MX781 produce cell cycle arrest independently of caspase activity and the retinoid receptors.     

Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-kappaB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (Ras(DN)), constitutively active MEK1 (MEK(CA)), dominant negative IkappaB kinase 2 (IKK(DN)), and constitutively active IKK2 (IKK(CA)). Inhibiting ERK activity by Ras(DN) overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEK(CA) remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-kappaB pathway with IKK(DN) virus suppressed the pit-forming activity of OCLs and NF-kappaB activation by IKK(CA) expression upregulated it without affecting their survival. Interleukin 1alpha (IL-1alpha) strongly induced ERK activation as well as NF-kappaB activation. Ras(DN) virus partially inhibited ERK activation, and OCL survival promoted by IL-1alpha. Inhibiting NF-kappaB activation by IKK(DN) virus significantly suppressed the pit-forming activity enhanced by IL-1alpha. These results indicate that ERK and NF-kappaB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-kappaB regulates osteoclast activation for bone resorption.     

Inhibition of IκB kinase-β and IκB kinase-α by heterocyclic adamantyl arotinoids

We recently reported on a series of retinoid-related molecules containing an adamantyl group, a.k.a. adamantyl arotinoids (AdArs), that showed significant cancer cell growth inhibitory activity and activated RXRα (NR2B1) in transient transfection assays while devoid of RAR transactivation capacity. We have now explored whether these AdArs could also bind and inhibit IKKβ, a known target that mediates the induction of apoptosis and cancer cell growth inhibition by related AdArs containing a chalcone functional group. In addition, we have prepared and evaluated novel AdArs that incorporate a central heterocyclic ring connecting the adamantyl-phenol and the carboxylic acid at the polar termini. Our results indicate that the majority of the RXRα activating compounds lacked IKKβ inhibitory activity. In contrast, the novel heterocyclic AdArs containing a thiazole or pyrazine ring linked to a benzoic acid motif were potent inhibitors of both IKKα and IKKβ, which in most cases paralleled significant growth inhibitory and apoptosis inducing activities.     

Mechanisms of fenretinide-induced apoptosis

Fenretinide, a synthetic retinoid, has emerged as a promising anticancer agent based on numerous in vitro and animal studies, as well as chemoprevention clinical trials. In vitro observations suggest that the anticancer activity of fenretinide may arise from its ability to induce apoptosis in tumor cells. Diverse signaling molecules including reactive oxygen species, ceramide, and ganglioside GD3 can mediate apoptosis induction by fenretinide in transformed, premalignant, and malignant cells. In many cell types, these signaling intermediates appear to be induced by mechanisms that are independent of retinoic acid receptor activation, and ultimately initiate the intrinsic or mitochondrial-mediated pathway of cell elimination. Numerous investigations conducted during the past 10 years have discovered a great deal about the apoptogenic activity of fenretinide. In this review we explore the mechanisms associated with fenretinide-induced apoptosis and highlight certain mechanistic underpinnings of fenretinide-induced cell death that remain poorly understood and thus warrant further characterization.     

Inhibition of IkappaB kinase activity by acetyl-boswellic acids promotes apoptosis in androgen-independent PC-3 prostate cancer cells in vitro and in vivo

Signaling through NF-kappaB has been implicated in the malignant phenotype as well as the chemoresistance of various cancers. Here we show that the natural compounds acetyl-beta-boswellic acid and acetyl-11-keto-beta-boswellic acid (AKbetaBA) inhibit proliferation and elicit cell death in chemoresistant androgen-independent PC-3 prostate cancer cells in vitro and in vivo. Induction of apoptosis was demonstrated in cultured PC-3 cells by several parameters including mitochondrial cytochrome c release and DNA fragmentation. At the molecular level these compounds inhibit constitutively activated NF-kappaB signaling by intercepting the IkappaB kinase (IKK) activity; signaling through the interferon-stimulated response element remained unaffected, suggesting specificity for IKK inhibition. The impaired phosphorylation of p65 and the reduced nuclear translocation of NF-kappaB proteins were associated with down-regulation of the constitutively overexpressed and NF-kappaB-dependent antiapoptotic proteins Bcl-2 and Bcl-x(L). In addition, expression of cyclin D1, a crucial cell cycle regulator, was reduced as well. Down-regulation of IKK by antisense oligodeoxynucleotides confirmed the essential role of IKK inhibition for the proliferation of the PC-3 cells. Both compounds tested were active in vivo, yet AKbetaBA proved to be far superior. Indeed, topical application of water-soluble AKbetaBA-gamma-cyclodextrin on PC-3 tumors xenografted onto chick chorioallantoic membranes induced concentration-dependent inhibition of proliferation as well as apoptosis. Similarly, in nude mice carrying PC-3 tumors, systemic application of AKbetaBA-gamma-cyclodextrin inhibited tumor growth and triggered apoptosis in the absence of detectable systemic toxicity. Thus, AKbetaBA and related compounds acting on IKK might provide a novel approach for the treatment of chemoresistant human tumors such as androgen-independent human prostate cancers.     

E1A sensitizes cells to tumor necrosis factor-induced apoptosis through inhibition of IkappaB kinases and nuclear factor kappaB activities.

The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor kappaB (NF-kappaB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-kappaB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-kappaB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IkappaB kinase (IKK) activity and IkappaB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IkappaB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-kappaB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.     

Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene diterpenes in macrophages. Specific effects on NF-kappa B-inducing kinase activity and on the coordinate activation of ERK and p38 MAPK

The anti-inflammatory action of most terpenes has been explained in terms of the inhibition of nuclear factor kappaB (NF-kappaB) activity. Ent-kaurene diterpenes are intermediates of the synthesis of gibberellins and inhibit the expression of NO synthase-2 and the release of tumor necrosis factor-alpha in J774 macrophages challenged with lipopolysaccharide. These diterpenes inhibit NF-kappaB and IkappaB kinase (IKK) activation in vivo but failed to affect in vitro the function of NF-kappaB, the phosphorylation and targeting of IkappaBalpha, and the activity of IKK-2. Transient expression of NF-kappaB-inducing kinase (NIK) activated the IKK complex and NF-kappaB, a process that was inhibited by kaurenes, indicating that the inhibition of NIK was one of the targets of these diterpenes. These results show that kaurenes impair the inflammatory signaling by inhibiting NIK, a member of the MAPK kinase superfamily that interacts with tumor necrosis factor receptor-associated factors, and mediate the activation of NF-kappaB by these receptors. Moreover, kaurenes delayed the phosphorylation of p38, ERK1, and ERK2 MAPKs, but not that of JNK, in response to lipopolysaccharide treatment of J774 cells. The absence of a coordinate activation of MAPK and IKK might contribute to a deficient activation of NF-kappaB that is involved in the anti-inflammatory activity of these molecules.     

NF-kappa B regulation by I kappa B kinase-2 in rheumatoid arthritis synoviocytes

IkappaB kinase-1 and IkappaB kinase-2 (IKK1 and IKK2; also called IKKalpha and IKKbeta, respectively) are part of the signal complex that regulates NF-kappaB activity in many cell types, including fibroblast-like synoviocytes (FLS). We determined which of these two kinases is responsible for cytokine-induced NF-kappaB activation in synoviocytes and assessed the functional consequences of IKK1 or IKK2 overexpression and inhibition. FLS were infected with adenovirus constructs encoding either wild-type (wt) IKK1 or IKK2, the dominant negative (dn) mutant of both kinases, or a control construct encoding green fluorescence protein. Analysis of the NF-kappaB pathway revealed that cytokine-induced IKK activation, IkappaB degradation, and NF-kappaB activation was prevented in cells expressing the IKK2 dn mutant, whereas baseline NF-kappaB activity was increased by IKK2 wt. In addition, synthesis of IL-6 and IL-8, as well as expression of ICAM-1 and collagenase, was only increased by IKK2 wt, and their cytokine-induced production was abrogated by IKK2 dn mutant. However, the IKK1 dn mutant did not inhibit cytokine-mediated activation of NF-kappaB or any of the functional assays. These data indicate that IKK2 is the key convergence pathway for cytokine-induced NF-kappaB activation. Furthermore, IKK2 regulates adhesion molecule, matrix metalloproteinase, and cytokine production in FLS.     

Cytokine-induced activation of nuclear factor-kappa B is inhibited by hydrogen peroxide through oxidative inactivation of IkappaB kinase

Rapid activation of the IkappaB kinase (IKK) complex is considered an obligatory step in the activation of nuclear factor-kappaB (NF-kappaB) in response to diverse stimuli. Since oxidants have been implicated in the regulation of NF-kappaB, the focus of the present study was the activation of IKK by tumor necrosis factor alpha (TNFalpha) in the presence or absence of hydrogen peroxide (H(2)O(2)). Exposure of mouse alveolar epithelial cells to H(2)O(2) was not sufficient to activate IKK, degrade IkappaBalpha, or activate NF-kappaB. In contrast, TNFalpha induced IKK activity rapidly and transiently resulting in IkappaBalpha degradation and NF-kappaB activation. Importantly, in the presence of H(2)O(2), the ability of TNFalpha to induce IKK activity was markedly decreased and resulted in prevention of IkappaBalpha degradation and NF-kappaB activation. Neither tyrosine kinases nor phosphatidylinositol 3-kinases, known regulators of NF-kappaB by oxidants, were involved in IKK inhibition by H(2)O(2). Direct addition of H(2)O(2) to the immunoprecipitated IKK complex inhibited enzyme activity. Inhibition of IKK activity by H(2)O(2) was associated with direct oxidation of cysteine residues present in the IKK complex and occurred only in enzymatically active IKK. In contrast to previously published observations, our findings demonstrate that the oxidant H(2)O(2) reduces NF-kappaB activation by inhibiting activated IKK activity.     

Signaling by the Mpl receptor involves IKK and NF-kappaB

Binding of tumor necrosis factor-alpha (TNF-alpha) to its receptor activates IKK complex, which leads to inducement of NF-kappaB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF-kappaB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand-responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKalpha and IKKbeta. Proliferating megakaryocytes displayed a constitutive DNA-binding activity of NF-kappaB p50 homodimers and of NF-kappaB p50-p65 heterodimers. As expected, reduced IKK activity in Mpl ligand-treated cells was associated with a significant reduction in NF-kappaB DNA binding activity and in the activity of a NF-kappaB-dependent promoter. Our study is thus the first to identify a constitutive NF-kappaB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF-kappaB activities. Since a variety of proliferation-promoting genes and anti-apoptotic mechanisms are activated by NF-kappaB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis.     

Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells

PS-341, also known as Velcade or Bortezomib, represents a new class of anticancer drugs which has been shown to potently inhibit the growth and/or progression of human cancers, including head and neck squamous cell carcinoma (HNSCC). Although it has been logically hypothesized that NF-kappaB is a major target of PS-341, the underlying mechanism by which PS-341 inhibits tumor cell growth is unclear. Here we found that PS-341 potently activated the caspase cascade and induced apoptosis in human HNSCC cell lines. Although PS-341 could inhibit NF-kappaB activation, the inhibition of NF-kappaB was not sufficient to initiate apoptosis in HNSCC cells. Using biochemical and microarray approaches, we found that proteasome inhibition by PS-341 induced endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in HNSCC cells. The inhibition of ROS significantly suppressed caspase activation and apoptosis induced by PS-341. Consistently, PS-341 could not induce the ER stress-ROS in PS-341-resistant HNSCC cells. Taken together, our results suggest that in addition to the abolishment of the prosurvival NF-kappaB, PS-341 might directly induce apoptosis by activating proapoptotic ER stress-ROS signaling cascades in HNSCC cells, providing novel insights into the PS-341-mediated antitumor activity.     

Acetyl-11-keto-beta-boswellic acid potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis by suppressing NF-kappa B and NF-kappa B-regulated gene expression

Acetyl-11-keto-beta-boswellic acid (AKBA), a component of an Ayurvedic therapeutic plant Boswellia serrata, is a pentacyclic terpenoid active against a large number of inflammatory diseases, including cancer, arthritis, chronic colitis, ulcerative colitis, Crohn's disease, and bronchial asthma, but the mechanism is poorly understood. We found that AKBA potentiated the apoptosis induced by TNF and chemotherapeutic agents, suppressed TNF-induced invasion, and inhibited receptor activator of NF-kappaB ligand-induced osteoclastogenesis, all of which are known to require NF-kappaB activation. These observations corresponded with the down-regulation of the expression of NF-kappaB-regulated antiapoptotic, proliferative, and angiogenic gene products. As examined by DNA binding, AKBA suppressed both inducible and constitutive NF-kappaB activation in tumor cells. It also abrogated NF-kappaB activation induced by TNF, IL-1beta, okadaic acid, doxorubicin, LPS, H2O2, PMA, and cigarette smoke. AKBA did not directly affect the binding of NF-kappaB to the DNA but inhibited sequentially the TNF-induced activation of IkappaBalpha kinase (IKK), IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation. AKBA also did not directly modulate IKK activity but suppressed the activation of IKK through inhibition of Akt. Furthermore, AKBA inhibited the NF-kappaB-dependent reporter gene expression activated by TNFR type 1, TNFR-associated death domain protein, TNFR-associated factor 2, NF-kappaB-inducing kinase, and IKK, but not that activated by the p65 subunit of NF-kappaB. Overall, our results indicated that AKBA enhances apoptosis induced by cytokines and chemotherapeutic agents, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-kappaB-regulated gene expression.     

IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therapy

Antiapoptotic activity of NF-kappaB in tumors contributes to acquisition of resistance to chemotherapy. Degradation of IkappaB is a seminal step in activation of NF-kappaB. The IkappaB kinases, IKK1 and IKK2, have been implicated in both IkappaB degradation and subsequent modifications of NFkappaB. Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes (IKK1/2(-/-)), we document a novel IkappaB degradation mechanism. We show that this degradation induced by a chemotherapeutic agent, doxorubicin (DoxR), does not require the classical serine 32 and 36 phosphorylation or the PEST domain of IkappaBalpha. Degradation of IkappaBalpha is partially blocked by phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the proteasome. Free NF-kappaB generated by DoxR-induced IkappaB degradation in IKK1/2(-/-) cells is able to activate chromatin based NF-kappaB reporter gene and expression of the endogenous target gene, IkappaBalpha. These results also imply that modification of NF-kappaB by IKK1 or IKK2 either prior or subsequent to its release from IkappaB is not essential for NF-kappaB-mediated gene expression at least in response to DNA damage. In addition, DoxR-induced cell death in IKK1/2(-/-) MEFs is enhanced by simultaneous inhibition of NF-kappaB activation by blocking the proteasome activity. These results reveal an additional pathway of activating NF-kappaB during the course of anticancer therapy and provide a mechanistic basis for the observation that proteasome inhibitors could be used as adjuvants in chemotherapy.     

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.     

NF-kappa B activation is not required for Chlamydia trachomatis inhibition of host epithelial cell apoptosis

Chlamydia trachomatis, an obligate intracellular bacterial species, is known to inhibit host cell apoptosis. However, the chlamydial antiapoptotic mechanism is still not clear. Because NF-kappaB activation is antiapoptotic, we tested the potential role of NF-kappaB activation in chlamydial antiapoptotic activity in the current study. First, no obvious NF-kappaB activation was detected in the chlamydia-infected cells when these cells were resistant to apoptosis induced via either the intrinsic or extrinsic apoptosis pathways. Second, inhibition of NF-kappaB activation with pharmacologic reagents failed to block the chlamydial antiapoptotic activity. Finally, NF-kappaB p65 gene deletion did not prevent chlamydia from inhibiting host cell apoptosis. These observations together have demonstrated that NF-kappaB activation is not required for the chlamydial antiapoptotic activity.