Simvastatin potentiates TNF-alpha-induced apoptosis through the down-regulation of NF-kappaB-dependent antiapoptotic gene products: role of IkappaBalpha kinase and TGF-beta-activated kinase-1

Numerous recent reports suggest that statins (hydroxy-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. Therefore, in this article, we investigated the effects of simvastatin on TNF-alpha-induced cell signaling. We found that simvastatin potentiated the apoptosis induced by TNF-alpha as indicated by intracellular esterase activity, caspase activation, TUNEL, and annexin V staining. This effect of simvastatin correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1 and cyclooxygenase-2), cell survival (Bcl-2, Bcl-x(L), cellular FLIP, inhibitor of apoptosis protein 1, inhibitor of apoptosis protein 2, and survivin), invasion (matrix mellatoproteinase-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor); all known to be regulated by the NF-kappaB. We found that simvastatin inhibited TNF-alpha-induced NF-kappaB activation, and l-mevalonate reversed the suppressive effect, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. Simvastatin suppressed not only the inducible but also the constitutive NF-kappaB activation. Simvastatin inhibited TNF-alpha-induced IkappaBalpha kinase activation, which led to inhibition of IkappaBalpha phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. NF-kappaB-dependent reporter gene expression induced by TNF-alpha, TNFR1, TNFR-associated death domain protein, TNFR-associated factor 2, TGF-beta-activated kinase 1, receptor-interacting protein, NF-kappaB-inducing kinase, and IkappaB kinase beta was abolished by simvastatin. Overall, our results provide novel insight into the role of simvastatin in potentially preventing and treating cancer through modulation of IkappaB kinase and NF-kappaB-regulated gene products.     

Differential pattern of inflammatory molecule regulation in intestinal epithelial cells stimulated with IL-1

To better predict the consequences of blocking signal transduction pathways as a means of controlling intestinal inflammation, we are characterizing the pathways up-regulated by IL-1 in intestinal epithelial cells (IEC). IL-1beta induced increased mRNA levels of MIP-2, MCP-1, RANTES, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) in the IEC-18 cell line. IL-1beta activated NF-kappaB but not ERK or p38. Infecting cells with adenovirus expressing a mutated gene for IkappaBalpha (IkappaBAA) blocked IL-1-induced mRNA increases in MIP-2, MCP-1, and iNOS but not COX-2 or RANTES. Expression of IkappaBAA attenuated the IL-1-induced increase in COX-2 protein. Unexpectedly, RANTES mRNA increased, and protein was secreted by cells expressing IkappaBAA in the absence of IL-1. Adenovirus-expressing IkappaBAA, blocking protein synthesis, and IL-1beta all resulted in activation of JNK. The JNK inhibitor SP600125 prevented the RANTES increases by all three stimuli. A human enterocyte line was similarly examined, and both NF-kappaB and JNK regulate IL-1-induced RANTES secretion. We conclude that in IEC-18, IL-1beta-induced increases in mRNA for MIP-2, MCP-1, and iNOS are NF-kappaB-dependent, whereas regulation of RANTES mRNA is independent of NF-kappaB but is positively regulated by JNK. IL-1beta-induced mRNA increases in COX-2 mRNA are both NF-kappaB- and MAPK-independent but the translation of COX-2 protein is NF-kappaB-dependent. This pattern of signaling due to a single stimulus exposed the complexities of regulating inflammatory genes in IEC.     

Short-term modulation of interleukin-1beta signaling by hyperoxia: uncoupling of IkappaB kinase activation and NF-kappaB-dependent gene expression

We have been interested in elucidating how simultaneous stimuli modulate inflammation-related signal transduction pathways in lung parenchymal cells. We previously demonstrated that exposing respiratory epithelial cells to 95% oxygen (hyperoxia) synergistically increased tumor necrosis factor-alpha (TNF-alpha)-mediated activation of NF-kappaB and NF-kappaB-dependent gene expression by a mechanism involving increased activation of IkappaB kinase (IKK). Because the signal transduction mechanisms induced by IL-1beta are distinct to that of TNF-alpha, herein we sought to determine whether hyperoxia modulates IL-1beta-dependent signal transduction. In A549 cells, simultaneous treatment with hyperoxia and IL-1beta caused increased activation of IKK, prolonged the degradation of IkappaBalpha, and prolonged the nuclear translocation and DNA binding of NF-kappaB compared with cells treated with IL-1beta alone in room air. Hyperoxia did not affect IL-1beta-dependent degradation of the interleukin receptor-associated kinase differently from treatment with IL-beta alone. In contrast to the effects on the IKK/IkappaBalpha/NF-kappaB pathway, simultaneous treatment with hyperoxia and IL-1beta did not augment NF-kappaB-dependent gene expression compared with treatment with IL-1beta alone. Similar observations were made in a different human respiratory epithelial cell line, BEAS-2B cells. In addition, simultaneous treatment with hyperoxia and IL-1beta caused hyperphosphorlyation of the NF-kappaB p65 subunit compared with treatment with IL-1beta alone. In summary, concomitant treatment of A549 cells with hyperoxia and IL-1beta augments activation of IKK, prolongs degradation of IkappaBalpha, and prolongs nuclear translocation and DNA binding of NF-kappaB. This activation, however, is not coupled to increased expression of NF-kappaB-dependent genes, and the mechanism of this decoupling is not related to decreased phosphorylation of p65.     

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.     

Artemisolide is a typical inhibitor of IkappaB kinase beta targeting cysteine-179 residue and down-regulates NF-kappaB-dependent TNF-alpha expression in LPS-activated macrophages

Nuclear factor (NF)-kappaB regulates a central common signaling for immunity and cell survival. Artemisolide (ATM) was previously isolated as a NF-kappaB inhibitor from a plant of Artemisia asiatica. However, molecular basis of ATM on NF-kappaB activation remains to be defined. Here, we demonstrate that ATM is a typical inhibitor of IkappaB kinase beta (IKKbeta), resulting in inhibition of lipopolysaccharide (LPS)-induced NF-kappaB activation in RAW 264.7 macrophages. ATM inhibited the kinase activity of highly purified IKKbeta and also LPS-induced IKK activity in the cells. Moreover, the effect of ATM on IKKbeta activity was completely abolished by substitution of Cys-179 residue of IKKbeta to Ala residue, indicating direct targeting site of ATM. ATM could inhibit IkappaBalpha phosphorylation in LPS-activated RAW 264.7 cells and subsequently prevent NF-kappaB activation. Further, we demonstrate that ATM down-regulates NF-kappaB-dependent TNF-alpha expression. Taken together, this study provides a pharmacological potential of ATM in NF-kappaB-dependent inflammatory disorders.     

Suberoylanilide hydroxamic acid potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis by suppressing nuclear factor-kappaB activation

Because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) is currently in clinical trials. How SAHA mediates its effects is poorly understood. We found that in several human cancer cell lines, SAHA potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents and inhibited TNF-induced invasion and 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 anti-apoptotic (IAP1, IAP2, X chromosome-linked IAP, Bcl-2, Bcl-x(L), TRAF1, FLIP, and survivin), proliferative (cyclin D1, cyclooxygenase 2, and c-Myc), and angiogenic (ICAM-1, matrix metalloproteinase-9, and vascular endothelial growth factor) gene products. Because several of these genes are regulated by NF-kappaB, we postulated that SAHA mediates its effects by modulating NF-kappaB and found that SAHA suppressed NF-kappaB activation induced by TNF, IL-1beta, okadaic acid, doxorubicin, lipopolysaccharide, H(2)O(2), phorbol myristate acetate, and cigarette smoke; the suppression was not cell type-specific because both inducible and constitutive NF-kappaB activation was inhibited. We also found that SAHA had no effect on direct binding of NF-kappaB to the DNA but inhibited sequentially the TNF-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation. Furthermore, SAHA inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRADD, TRAF2, NF-kappaB-inducing kinase, IkappaBalpha kinase, and the p65 subunit of NF-kappaB. Overall, our results indicated that NF-kappaB and NF-kappaB-regulated gene expression inhibited by SAHA can enhance apoptosis and inhibit invasion and osteoclastogenesis.     

Tetrandrine suppresses LPS-induced astrocyte activation via modulating IKKs-IκBα-NF-κB signaling pathway

Astrocyte activation has been implicated in the pathogenesis of many neurological diseases. These reactive astrocytes are capable of producing a variety of proinflammatory mediators and potentially neurotoxic compounds, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1beta (IL-1beta). In this study, we examined the suppressive effects of Tetrandrine (TET) on astrocyte activation induced by lipopolysaccharide (LPS) in vitro. We found that TET decreased the release of NO, TNF-alpha, IL-6 and IL-1beta in LPS-activated astrocytes. Also mRNA expression levels of inducible nitric oxide synthase (iNOS), macrophage inflammatory protein-1alpha (MIP-1alpha) and vascular cell adhesion molecule-1 (VCAM-1) were inhibited in TET pretreated astrocytes. Such suppressive effects might be resulted from the inhibition of nuclear factor kappa B (NF-kappaB) activation through downregulating IkappaB kinases (IKKs) phosphoration, which decreased inhibitor of nuclear factor-kappaB-alpha (IkappaBalpha) phosphoration and degradation. Our results suggest that TET acted to regulate astrocyte activation through inhibiting IKKs-IkappaBalpha-NF-kappaB signaling pathway.     

Propapoptotic effects of NF-kappaB in LNCaP prostate cancer cells lead to serine protease activation

LNCaP prostate cancer cells are resistant to induction of apoptosis by gamma-irradiation and partially sensitive to TNF-alpha or FAS antibody, irradiation sensitizes cells to apoptosis induced by FAS antibody or TNF-alpha. LNCaP cell clones stably expressing IkappaBalpha super repressor were resistant to apoptosis induced by death ligands in the presence or absence of irradiation. IkappaBalpha super repressor expression also increased clonogenic survival after exposure to TNF-alpha+irradiation, but had no effect on survival after irradiation alone. IkappaBalpha super repressor expression blocked the increase of whole cell and cell surface FAS expression induced by TNF-alpha, but did not effect induction of FAS expression and cell surface FAS expression that resulted from irradiation. In cells expressing IkappaBalpha super repressor there was diminished activation of caspases-8 and -7 and diminished production of proscaspases-8 and -7, usually required for death induction in LNCaP cells. Peptide inhibitors of caspase activation complemented the IkappaBalpha super repressor inhibition of apoptosis, but peptide inhibitors of serine proteases had no effect on LNCaP cells expressing IkappaBalpha super repressor. Moreover, cleavage of a serine protease substrate was induced by treatment of LNCaP cells with TNF-alpha and irradiation. The data suggest that in LNCaP cells NF-kappaB mediates a proapoptotic pathway that leads to activation of proapoptotic serine proteases.