KiSS1 suppresses TNFα‐induced breast cancer cell invasion via an inhibition of RhoA‐Mediated NF‐κB activation

Tumor necrosis factor‐alpha (TNFα) induces cancer development and metastasis, which is prominently achieved by nuclear factor‐kappa B (NF‐κB) activation. TNFα‐induced NF‐κB activation enhances cellular mechanisms including proliferation, migration, and invasion. KiSS1, a key regulator of puberty, was initially discovered as a tumor metastasis suppressor. The expression of KiSS1 was lost or down‐regulated in different metastatic tumors. However, it is unclear whether KiSS1 regulates TNFα‐induced NF‐κB activation and further tumor cell migration. In this study, we demonstrate that KiSS1 suppresses the migration of breast cancer cells by inhibiting TNFα‐induced NF‐κB pathway and RhoA activation. Both KiSS1 overexpression and KP10 (kisspeptin‐10) stimulation inhibited TNFα‐induced NF‐κB activity, suppressed TNFα‐induced cell migration and cell attachment to fibronectin in breast cancer cells while KP10 has little effect on cancer cell proliferation. Furthermore, KP10 inhibited TNFα‐induced cell migration and RhoA GTPase activation. Therefore, our data demonstrate that KiSS1 inhibits TNFα‐induced NF‐κB activation via downregulation of RhoA activation and suppression of breast cancer cell migration and invasion. J. Cell. Biochem. 107: 1139–1149, 2009. © 2009 Wiley‐Liss, Inc.     

The essential role of PKCalpha in the protective effect of heat-shock pretreatment on TNFalpha-induced apoptosis in hepatic epithelial cell line

During sepsis, hepatic apoptosis occurred, which is associated with inactivation of PKCalpha and elevation of tumor necrosis factor-alpha (TNFalpha), an apoptosis trigger. Heat shock, accompanied by the increase of heat-shock protein (Hsp72), has been shown to exhibit a protective role on cell survival. However, Hsp72 was unable to express during sepsis when the apoptosis was markedly increased. We hypothesized that hepatic apoptosis during sepsis may be due to the failure to induce expression of Hsp72, which is activated by PKC-phosphorylated HSF. This study was designed to examine the role of PKCalpha in Hsp72 expression and the anti-apoptotic effect of Hsp72 on hepatic epithelial cells by analyzing a TNFalpha-induced apoptosis system. The following results were observed: (1) Hsp72 was highly expressed at 8 h after heat-shock treatment in a clone 9 hepatic epithelial cell line; (2) the protein expression of PKCalpha in membrane-associated fraction was decreased by TNFalpha treatment; (3) the TNFalpha-induced cell death, especially apoptosis, was diminished by heat-shock pretreatment; (4) in the presence of PKCalpha antisense, which blocks the PKCalpha resynthesis, no protective effect of heat-shock pretreatment was observed, and the protein expression of Hsp72 was significantly suppressed. These results suggest that PKCalpha plays a critical role in the expression of Hsp72, which subsequently protects against TNFalpha-induced hepatic apoptosis.     

Mice lacking both TNFalpha receptors show increased constitutive expression of IFNgamma: a possible reason for lack of protection from fumonisin B1 hepatotoxicity

Fumonisin B1 is a mycotoxin prevalent in corn that produces species-, gender-, and organ-specific diseases. Mice lacking TNFalpha receptor (TNFR) 1 or 2 exhibited a diminished hepatotoxic response to fumonisin B1; however, the protection was lost when both TNFRs were deleted. We therefore investigated the constitutive expression of selected apoptotic factors and their response to fumonisin B1 in the liver from mice lacking both TNFRs (DRKO). Compared to their wild-type (WT) counterparts the DRKO strain had a higher constitutive mRNA expression of interferon (IFN)gamma, Fas, and interleukin (IL)-18. The mRNA expression of Bcl-2 was also higher in DRKO than in WT mice. The mRNA expression of IL-1 receptor antagonist (IL-1Ra) was decreased; that of TNF-related apoptosis-inducing ligand (TRAIL) was dramatically reduced. Induction of most apoptotic genes in response to fumonisin B1 was similar in both WT and DRKO strains; except in DRKO mice it was greater for Max and lesser for IL-1Ra than that in WT strain. Fumonisin B1 hepatotoxicity in DRKO mice was reduced by pretreatment with anti-IFNgamma antibody. It appears that in the absence of TNFalpha signaling other apoptotic pathways become operative; particularly the increase of IFNgamma, Fas and IL-18 may compensate for the loss of TNFalpha effects. Fumonisin B1 toxicity therefore appears to be a complex phenomenon that may utilize more than one cytotoxic pathway consequent to sphingoid deregulation; a higher expression of IFNgamma and other apoptotic factors in DRKO may be responsible for the observed fumonisin hepatotoxicity.     

Comparative analyses of complex formation and binding sites between human tumor necrosis factor-alpha and its three antagonists elucidate their different neutralizing mechanisms

Tumor necrosis factor-alpha (TNFα)-blocking therapy, using biologic TNFα antagonists, has been approved for the treatment of several diseases including rheumatoid arthritis, psoriasis and Crohn's disease. There have been few detailed studies of binding characterizations for the complex formation by TNFα and clinically relevant antagonists, particularly Infliximab (Remicade®) and Etanercept (Enbrel®). Here we characterized the binding stoichiometry and size of soluble TNFα–antagonist complexes and identified energetically important binding sites on TNFα for the three antagonists, Etanercept, Infliximab, and the recently developed humanized TNFα neutralizing monoclonal antibody, YHB1411-2. Size-exclusion chromatography and dynamic light scattering analyses revealed that the three antagonists formed distinct thermodynamically stable TNFα–antagonist complexes that exhibited differences in their size and composition. Energetically important binding residues on TNFα were identified for each antagonist by a sequence of experiments that consisted of competition binding assays, fragmentations, loop mutations, and single-point mutations using yeast surface-displayed TNFα, which was further confirmed for solubly purified TNFα mutants by surface plasmon resonance technique. Analyses of the binding geometry based on binding site location, spatial constraints, and valency satisfaction allowed us to interpret the thermodynamically stable complexes as follows: one molecule of Etanercept and one molecule of trimeric TNFα (Etanercept₁––TNFα₁), Infliximab₆–TNFα₃, and YHB1411–2₄-TNFα₂. The distinct features of the soluble antagonist–TNFα complex formation among the antagonists may give further insights into their different neutralizing mechanisms and pharmacokinetic profiles.     

LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

High serum levels of TNF-α after its administration for isolation perfusion of the limb

In a phase II study, 18 patients with locally spreading melanoma or sarcoma of lower limb were treated by isolation perfusion (ILP) with hyperthermia and local infusion of high dose of recombinant human tumor necrosis factor α (rHuTNF-α) (4 mg). Bioactive TNF-α and interleukin 6 (IL-6) serum levels were measured serially, In the limb, TNF-α rapidly reached a plateau at 2 μ/ml, while IL-6 appeared later and progressively increased until the end of ILP. In the systemic circulation TNF-α rose up to a median concentration of 31 ng/ml after 1 hour, then decreased and became negligible after 6 hours. IL-6 peaked only after 5 hours after start of ILP (median: 36.7 ng/ml). In patients with substantial leakage towards systemic circulation, both cytokines peaked higher and earlier as compared with patients with minimal leakage. No correlation was found between cytokine levels and severity of side effects which in all cases were reversible. We conclude that high dose TNF-α infusion in ILP results in extremely high levels of bioactive TNF-α in the systemic circulation without irreversible side effect, and provokes a delayed blood release of large amounts of IL-6; there was a correlation between leakage from the limb during procedure and the magnitude of systemic cytokines levels.     

NDR1/STK38 potentiates NF‐κB activation by its kinase activity

Human NDR1/STK38 belongs to the nuclear‐Dbf2‐related (NDR) family of Ser/Thr kinases. It has been implicated to function in centrosome duplication, control of cell cycle and apoptosis. However, the mechanism of NDR1 signaling pathway remains largely elusive. Here, we report a novel role of NDR1 in NF‐κB activation. By overexpression, NDR1 potentiates NF‐κB activation induced by TNFα, whereas knockdown of NDR1 expression inhibits NF‐κB activation induced by TNFα. Coimmunoprecipitation shows that NDR1 interacts with multiple signal components except p65 in NF‐κB signaling pathway. Furthermore, both phosphorylation and kinase dead mutants of NDR1 lose their synergistic effects on TNFα‐induced NF‐κB activation. siRNA oligo against NDR1 and kinase dead mutant as well mainly block the NF‐κB activation induced by TRAF2 but not RIP1. Furthermore, kinase dead mutant of NDR1 fails to interact with TRAF2. Taken together, our findings suggest an unknown function of NDR1, which may regulate NF‐κB activation by its kinase activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Rationale for using TNFα and chemotherapy in regional therapy of melanoma

Recombinant tumor necrosis factor-alpha (rTNFα) has potent antitumor activity in experimental studies on human tumor xenografts. However, in humans, the administration of rTNFα is hampered by severe systemic side-effects. The maximum tolerated dose range from 350 to 500 mg/m², which is at least 10-fold less than the efficient dose in animals. Isolation perfusion of the limbs (ILP) allows the delivery of high dose rTNFα in a closed system with acceptable side-effects. A protocol with a triple-drug regimen was based on the reported synergism of rTNFα with chemotherapy, with interferon-y, and with hydperthermia. In melanoma-in-transit metastases (stage IIIA or AB) we obtained a 91% complete response, compared with 52% after ILP with melphalan alone. Release of nanograms levels of TNFα in the systemic circulation was evident but control of this leakage and appropriate intensive care resulted in acceptable toxicity. Angiographic, immunohistological, and immunological studies suggest that the efficacy of this prtocol is due to a dual targeting: rTNFα activates and electively lyses the tumor endothelial cells while melphalan is mainly cytoxic to the tumor cells. ILP with rTNFα appears to be a useful model for studying the biochemotherapy of cancer in man.     

Possible involvement of ceramide in the regulation of rat Leydig cell function

In the present study, a possible role of a ceramide-dependent pathway in the regulation of Leydig cell function was investigated. Intracellular ceramide levels were increased by: (a) adding ceramide analogs; (b) inhibiting ceramidase activity; and (c) adding sphingomyelinase (SMase). The cell-permeable ceramide analogs N-acetyl-, N-hexanoyl- and N-octanoylsphingosine (C2, C6 and C8) were used. As inhibitor of ceramidase activity 1S,2R-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (MAPP) was used. Sphingomyelinase from S. aureus origin was utilized. Leydig cells were cultured for 3 or 24 h with or without the different drugs (10 microM) and SMase (0.3 U/ml) in the presence or absence of hCG (10 ng/ml). Basal testosterone production was not modified under any of the experimental conditions. A decrease in hCG-stimulated testosterone production was observed at 3 and 24 h in all cases. The inactive analog (N-hexanoyl dihydrosphingosine) did not produce inhibition in hCG-stimulated testosterone production. TNFalpha and IL1beta, two possible inducers of sphingomyelin hydrolysis, produced similar effects on hCG-stimulated testosterone production. In experiments performed in the presence of C6, inhibition in hCG-stimulated cAMP production was observed. The inhibitory effect of ceramide was also observed in dbcAMP-stimulated cultures indicating that this pathway inhibits post-cAMP formation events. To study possible loci for the action of ceramide on the steroidogenic pathway, cells were incubated with C6 and MAPP in the presence of different testosterone precursors. The drugs inhibited testosterone produced from 22(R)-hydroxycholesterol (22R-OHChol), pregnenolone and 17alpha-hydroxyprogesterone (17OHP4) but not from androstenedione (Delta4). These results suggest that a ceramide-dependent pathway regulates hCG-stimulated Leydig cell steroidogenesis at the level of cAMP production and at post-cAMP events.     

High Levels of sTNFR p75 and TNFα in Dengue-Infected Patients

Soluble forms of the two molecular species of the cell surface TNF receptors (sTNFR p55 and sTNFR p75) can reduce the activity of TNFα but they may also enhance its function by stabilizing the active TNFα oligomer. Considering the pathophysiological importance of sTNFR p75 for the regulation of the bioavailability of TNFα in the body, we determined the serum levels of sTNFR p75 and TNFα in 45 children and 28 adults with laboratory-confirmed dengue infection by using immunoassays. The serum samples were obtained from day 1 to day 15 after the onset of the disease during the 1989–1990 outbreak of dengue-3 in Tahiti, French Polynesia. The patients were clinically classified as having dengue hemorrhagic fever (DHF) and graded according to the criteria of the World Health Organization (WHO) into four grades from less severe (grade I) to severe (grade IV). The sera of both children and adult patients of all severity grades contained higher levels of sTNFR p75 than the sera of control subjects. Although high levels of TNFα were also detected in children and adults among grade I, II, III and IV patients, we found no correlation between sTNFR p75 and TNFα. We observed in adults a moderate elevation of sTNFR p75 and TNFα in sera compared with that observed in children. The raised levels of immunoreactive sTNFR p75 and TNFα in all clinical groups of dengue-infected patients strongly indicate activation of the TNFα system during dengue infection. The balance between sTNFR p75 and TNFα may be altered in dengue infection. Further investigations are needed to understand the role of sTNFR p75 and TNFα in the pathogenesis of DHF and to improve the management of dengue infection.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.