Regulation of proliferation, survival and apoptosis by members of the TNF superfamily

Tumor necrosis factor (TNF) was first identified in 1984 as a cytokine with anti-tumor effects in vitro and in vivo. Extensive research since then has shown that there are at least 18 distinct members of the TNF super family and they exhibit 15-25% amino acid sequence homology with each other. These family members bind to distinct receptors, which are homologous in their extracellular domain. These cytokines have been implicated in a wide variety of diseases including tumorigenesis, septic shock, viral replication, bone resorption, rheumatoid arthritis, diabetes, and other inflammatory diseases. TNF blockers have been approved for human use in treating some of these conditions in the United States and other countries. Various members of the TNF super family mediate either proliferation, survival, or apoptosis of cells. Although distinct receptors, all members share a common cell signaling pathway that mediates the activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (e.g. c-jun N-terminal kinase). Regulation of cell growth and activation of NF-kappaB and of c-jun N-terminal kinase by the TNF super family is mediated through sequential activation/association of a set of cell signaling proteins named TNF receptor-associated factors, Fas-associated death domain and FADD-like ICE, caspases, receptor-interacting protein, NF-kappaB-inducing kinases, and IkappaBalpha kinases. Both apoptotic and antiapoptotic signals are activated simultaneously by the same cytokine in the same cell. Together these cytokines regulate cell growth/survival/apoptosis in a complex dance of changing partners and overlapping steps.     

Evidence that activation of nuclear factor-kappaB is essential for the cytotoxic effects of doxorubicin and its analogues

Several reports within the last 5 years have suggested that nuclear factor (NF)-kappaB activation suppresses apoptosis through expression of anti-apoptotic genes. In the present report, we provide evidence from four independent lines that NF-kappaB activation is required for the cytotoxic effects of doxorubicin. We used doxorubicin and its structural analogues WP631 and WP744, to demonstrate that anthracyclines activate NF-kappaB, and this activation is essential for apoptosis in myeloid (KBM-5) and lymphoid (Jurkat) cells. All three anthracyclines had cytotoxic effects against KBM-5 cells; analogue WP744, was most potent, with an IC(50) of 0.5 microM, and doxorubicin was least active, with an IC(50) of 2 microM. We observed maximum NF-kappaB activation at 1 microM with WP744 and at 50 microM with doxorubicin and WP631, and this activation correlated with the IkappaBalpha degradation. Because the anthracycline analogue (WP744), most active as a cytotoxic agent, was also most active in inducing NF-kappaB activation and the latter preceded the cytotoxic effects, suggests that NF-kappaB activation may mediate cytotoxicity. Second, receptor-interacting protein-deficient cells, which did not respond to doxorubicin-induced NF-kappaB activation, were also protected from the cytotoxic effects of all the three anthracyclines. Third, suppression of NF-kappaB activation by pyrrolidine dithiocarbamate, also suppressed the cytotoxic effects of anthracyclines. Fourth, suppression of NF-kappaB activation by NEMO-binding domain peptide, also suppressed the cytotoxic effects of the drug. Overall our results clearly demonstrate that NF-kappaB activation and IkappaBalpha degradation are early events activated by doxorubicin and its analogues and that they play a critical pro-apoptotic role.     

NF-kappaB and inflammation in genetic disease

By responding to pro-inflammatory cytokines, such as IL-1beta and TNF-alpha, and controlling itself the expression of numerous mediators of inflammation, NF-kappaB plays a pivotal role in controlling the proper sequence of events characterizing the inflammation process. Although excessive NF-kappaB activation is often associated with inflammatory signs in many different tissues, impaired NF-kappaB activation can also generate inflammation. This is the case in humans suffering from the genetic disease incontinentia pigmenti that exhibit severe skin inflammation. Identifying the molecular basis of this pathology, mutations affecting the gene coding for NEMO, has allowed production of mouse models for investigating the disease. Their characterization supports the view that a very tight positive and negative regulation of the NF-kappaB signaling pathway is required in vivo to ensure not only a fine-tuned response to injury or infection but also to maintain tissue homeostasis.     

Induction of apoptosis by phloroglucinol derivative from Ecklonia Cava in MCF-7 human breast cancer cells

Phloroglucinol derivatives, dioxinodehydroeckol (1) and 1-(3′,5′-dihydroxyphenoxy)-7-(2′′,4′′,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (2), were isolated from Ecklonia Cava. Their ability to inhibit the proliferation of human breast cancer cells were evaluated by measuring cell death via induction of apoptosis. Compound 1 exerted a higher anti-proliferative activity in human breast cancer cells compared with compound 2. Furthermore, compound 1 induced a significant proliferative inhibition and apoptosis in a dose-dependent manner on MCF-7 human cancer cells. Treatment with compound 1 also induced the increase in caspase (-3 and -9) activity, DNA repair enzyme poly-(ADP-ribose) polymerase (PARP) cleavage, and pro-apoptotic gene and the decrease in anti-apoptotic gene. In addition, NF-κB family and -dependent activated genes were down-regulated by compound 1. These results indicated that the potential inhibitory effect of compound 1 against growth of MCF-7 human breast cancer cells might be associated with induction of apoptosis through NF-κB family and NF-κB dependent pathway. The present results suggest that compound 1 has a promising potential to be used as a valuable chemopreventive agent.     

Role of the Fas/Fas ligand system in female reproductive organs: survival and apoptosis

For centuries, the question of "whether there is life after death" has intrigued the mind of philosophers and the same question fascinates researchers in the field of apoptosis today. The death of a cell is by no means the end of the story. On the contrary, growing evidence suggests that the clearance of apoptotic bodies by macrophages is an important regulatory component in tissue renewal. Without death by apoptosis, the life of reproductive tissues and their function would not be possible. The survival signals that counteract cell death also prepare the cells for apoptosis, and dead cells are important stimuli for tissue survival. The Fas/Fas ligand system is an important mediator of apoptosis and is an excellent example of this apparently contradictory phenomenon.     

Curcumin (diferuloylmethane) inhibits constitutive NF-kappaB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle cell lymphoma

Human mantle cell lymphoma (MCL), an aggressive B cell non-Hodgkin's lymphoma, is characterized by the overexpression of cyclin D1 which plays an essential role in the survival and proliferation of MCL. Because of MCL's resistance to current chemotherapy, novel approaches are needed. Since MCL cells are known to overexpress NF-kappaB regulated gene products (including cyclin D1), we used curcumin, a pharmacologically safe agent, to target NF-kappaB in a variety of MCL cell lines. All four MCL cell lines examined had overexpression of cyclin D1, constitutive active NF-kappaB and IkappaB kinase and phosphorylated forms of IkappaBalpha and p65. This correlated with expression of TNF, IkappaBalpha, Bcl-2, Bcl-xl, COX-2 and IL-6, all regulated by NF-kappaB. On treatment of cells with curcumin, however, downregulated constitutive active NF-kappaB and inhibited the consitutively active IkappaBalpha kinase (IKK), and phosphorylation of IkappaBalpha and p65. Curcumin also inhibited constitutive activation of Akt, needed for IKK activation. Consequently, the expression of all NF-kappaB-regulated gene products, were downregulated by the polyphenol leading to the suppression of proliferation, cell cycle arrest at the G1/S phase of the cell cycle and induction of apoptosis as indicated by caspase activation, PARP cleavage, and annexin V staining. That NF-kappaB activation is directly linked to the proliferation of cells, is also indicated by the observation that peptide derived from the IKK/NEMO-binding domain and p65 suppressed the constitutive active NF-kappaB complex and inhibited the proliferation of MCL cells. Constitutive NF-kappaB activation was found to be due to TNF, as anti-TNF antibodies inhibited both NF-kappaB activation and proliferation of cells. Overall, our results indicate that curcumin inhibits the constitutive NF-kappaB and IKK leading to suppression of expression of NF-kappaB-regulated gene products that results in the suppression of proliferation, cell cycle arrest, and induction of apoptosis in MCL.     

The regulation of the expression of inducible nitric oxide synthase by Src-family tyrosine kinases mediated through MyD88-independent signaling pathways of Toll-like receptor 4

Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) leading to the expression of inflammatory gene products. Src-family tyrosine kinases (STKs) are known to be activated by LPS in monocytes/macrophages. Therefore, we determined the role of STKs in TLR4 signaling pathways and target gene expression in macrophages. The activation of NFkappaB, and p38 MAPK, and the expression of inducible nitric oxide synthase (iNOS) induced by LPS were not affected in macrophages deficient in three STKs (Lyn, Hck, and Fgr). These results suggest that the deletion of the three STKs among possibly nine STKs is not sufficient to abolish total activity of STKs possibly due to the functional redundancy of other STKs present in macrophages. However, two structurally unrelated pan-inhibitors of STKs, PP1 and SU6656, suppressed LPS-induced iNOS expression in MyD88-knockout as well as wild-type macrophages. The suppression of iNOS expression by the inhibitors was correlated with the downregulation of IFNbeta (a MyD88-independent gene) expression and subsequent decrease in STAT1 phosphorylation. Moreover, PP1 suppressed the expression of IFNbeta and iNOS induced by TRIF, a MyD88-independent adaptor of TLR4. PP1 suppressed STAT1 phosphorylation induced by LPS, but not by IFNbeta suggesting that STKs are involved in the primary downstream signaling pathways of TLR4, but not the secondary signaling pathways downstream of IFNbeta receptor. Together, these results demonstrate that STKs play a positive regulatory role in TLR4-mediated iNOS expression in a MyD88-independent (TRIF-dependent) manner. These results provide new insight in understanding the role of STKs in TLR4 signaling pathways and inflammatory target gene expression.     

Mitigation of tumor necrosis factor alpha cytotoxicity by aurintricarboxylic acid in human peripheral B lymphocytes

The aims of this study were to ascertain whether aurintricarboxylic acid (ATA), an endonuclease inhibitor, known to interfere, with the actions of cytokines such as interferons, is able to antagonize the toxic effects produced by tumor necrosis factor alpha (TNF-alpha) in human healthy peripheral B lymphocytes and try to elucidate the molecular machinery through which this possible antagonism takes place. Results evidenced that the balance of survival signals of human B lymphocytes in the presence of TNF-alpha was altered by the interaction of TNF-alpha with a salicylate compound, ATA. Apoptosis effected by TNF-alpha alone was suppressed in the presence of ATA, and this effect appeared essentially characterized by: (i) phosphorylation of phosphatidylinositol-3 kinase (PI-3K), influencing in turn protein kinase B/Akt (Akt) and Bad phosphorylation; (ii) nuclear translocation of the nuclear factor kappa B (NF-kappaB) and (iii) nuclear translocation of protein kinase C zed (PKCzeta). Reversal of TNF-alpha/ATA effects occurred in the presence of the PI-3K specific inhibitors wortmannin or LY294002 in the culture medium and was coincident with inhibition of the translocation of PKCzeta in the nucleus, while NF-kappaB was less affected. These results indicate, therefore, that PI-3K-mediated activation and nuclear transfer of PKCzeta might be essential steps of ATA antagonism against TNF-alpha, suggesting that possible ATA pharmacological applications might be taken into account for staving off systemic or local toxic effects produced by TNF-alpha.     

Mechanisms of interferon-β effects on bone homeostasis

Restoration of dysregulated bone homeostasis is a therapeutic goal in many diseases including osteoporosis, rheumatoid arthritis and metastatic cancer. The molecular pathways regulating bone remodeling are major therapeutic targets, and studies continue to reveal endogenous factors that may be pathologically up- or down-regulated and lead to an uncoupling of bone formation and resorption. The purpose of this commentary is to highlight new mechanisms of bone homeostatic regulation mediated through the induction of endogenous interferon-β (IFN-β). The receptor activator of nuclear factor-κB (RANK) ligand (RANKL) is an important factor in the bone resorption cascade, and the RANK-RANKL interaction has been shown to induce IFN-β and osteoclastogenesis via induction of the c-fos gene. Subsequent binding of IFN-β to its biological receptor initiates a signal transduction cascade through the classic JAK/STAT pathway, causing an inhibition of c-fos protein production and osteoclast proliferation and differentiation (negative feedback). Another mechanism pertinent to the anti-resorptive effect of IFN-β is the induction of nitric oxide which has been shown to inhibit osteoclast formation. The role of IFN-β in bone metabolism could warrant its systematic evaluation as a potential adjunct to therapeutic regimens of osteolytic diseases. Here we also provide discussion of the potential challenges to optimizing IFN-β pharmacotherapy for such purposes.     

Brazilin selectively disrupts proximal IL-1 receptor signaling complex formation by targeting an IKK-upstream signaling components

The ligation of interleukin-1 receptor (IL-1R) or tumor necrosis factor receptor 1 (TNFR1) induces the recruitment of adaptor proteins and their concomitant ubiquitination to the proximal receptor signaling complex, respectively. Such are upstream signaling events of IKK that play essential roles in NF-κB activation. Thus, the discovery of a substance that would modulate the recruitment of key proximal signaling elements at the upstream level of IKK has been impending in this field of study. Here, we propose that brazilin, an active compound of Caesalpinia sappan L. (Leguminosae), is a potent NF-κB inhibitor that selectively disrupts the formation of the upstream IL-1R signaling complex. Analysis of upstream signaling events revealed that brazilin markedly abolished the IL-1β-induced polyubiquitination of IRAK1 and its interaction with IKK-γ counterpart. Notably, pretreatment of brazilin drastically interfered the recruitment of the receptor-proximal signaling components including IRAK1/4 and TRAF6 onto MyD88 in IL-1R-triggerd NF-κB activation. Interestingly, brazilin did not affect the TNF-induced RIP1 ubiquitination and the recruitment of RIP1 and TRAF2 to TNFR1, suggesting that brazilin is effective in selectively suppressing the proximal signaling complex formation of IL-1R, but not that of TNFR1. Moreover, our findings suggest that such a disruption of IL-1R-proximal complex formation by brazilin is not mediated by affecting the heterodimerization of IL-1R and IL-1RAcP. Taken together, the results suggest that the anti-IKK activity of brazilin is induced by targeting IKK upstream signaling components and subsequently disrupting proximal IL-1 receptor signaling complex formation.     

Inhibition of osteoclast differentiation and bone resorption by tanshinone IIA isolated from Salvia miltiorrhiza Bunge

Osteoclasts, multinuclear cells specialized for bone resorption, differentiate from the monocyte/macrophage lineage of hematopoietic cells. Intervention in osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone diseases involving osteoclasts. In this study, we found that tanshinone IIA, originating from Salvia miltiorrhiza Bunge, inhibited the differentiation of osteoclasts. Addition of tanshinone IIA to the osteoclast precursor culture caused a significant decrease in the level of calcitonin receptor, c-Src, and integrin beta3 mRNA, which are normally upregulated during the osteoclast differentiation dependent on RANKL (receptor activator of nuclear factor kappa B ligand). RANKL activated the ERK, Akt, and NF-kappaB signal transduction pathways in osteoclast precursor cells, and tanshinone IIA suppressed this activation. Tanshinone IIA also inhibited the bone resorptive activity of differentiated osteoclasts, which was accompanied with the disruption of the actin ring. Thus, tanshinone IIA has the potential to ameliorate bone-resorption diseases in vivo by reducing both the number and activity of osteoclasts.