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.     

Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)‐γ‐induced protein 10 (IP‐10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN‐γ depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN‐γ and TNF‐α have not been adequately elucidated in human monocytes. In this study, we showed that TNF‐α had more potential than IFN‐γ to induce CXCL10 production in THP‐1 monocytes. Furthermore, IFN‐γ synergistically enhanced the production of CXCL10 in parallel with the activation of NF‐κB in TNF‐α‐stimulated THP‐1 cells. Blockage of STAT1 or NF‐κB suppressed CXCL10 production. JAKs inhibitors suppressed IFN‐γ plus TNF‐α‐induced production of CXCL10 in parallel with activation of STAT1 and NF‐κB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF‐κB, but not that of STAT1. IFN‐γ‐induced phosphorylation of JAK1 and JAK2, whereas TNF‐α induced phosphorylation of ERK1/2. Interestingly, IFN‐γ alone had no effect on phosphorylation and degradation of IκB‐α, whereas it significantly promoted TNF‐α‐induced phosphorylation and degradation of IκB‐α. These results suggest that TNF‐α induces CXCL10 production by activating NF‐κB through ERK and that IFN‐γ induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF‐α‐induced NF‐κB may be the primary pathway contributing to CXCL10 production in THP‐1 cells. IFN‐γ potentiates TNF‐α‐induced CXCL10 production in THP‐1 cells by increasing the activation of STAT1 and NF‐κB through JAK1 and JAK2. J. Cell. Physiol. 220: 690–697, 2009. © 2009 Wiley‐Liss, Inc.     

NF023 binding to XIAP‐BIR1: Searching drugs for regulation of the NF‐κB pathway

Inhibitor of Apoptosis Proteins (IAPs) are the target of extensive research in the field of cancer therapy since they regulate apoptosis and cell survival. Smac‐mimetics, the most promising IAP‐targeting compounds specifically recognize the IAP‐BIR3 domain and promote apoptosis, competing with caspases for IAP binding. Furthermore, Smac‐mimetics interfere with the NF‐κB survival pathway, inducing cIAP1 and cIAP2 degradation through an auto‐ubiquitination process. It has been shown that the XIAP‐BIR1 (X‐BIR1) domain is involved in the interaction with TAB1, an upstream adaptor for TAK1 kinase activation, which in turn couples with the NF‐κB survival pathway. Preventing X‐BIR1 dimerization abolishes XIAP‐mediated NF‐κB activation, thus implicating a proximity‐induced mechanism for TAK1 activation. In this context, in a systematic search for a molecule capable of impairing X‐BIR1/TAB1 assembly, we identified the compound NF023. Here we report the crystal structure of the human X‐BIR1 domain in the absence and in the presence of NF023, as a starting concept for the design of novel BIR1‐specific compounds acting synergistically with existing pro‐apoptotic drugs in cancer therapy. Proteins 2015; 83:612–620. © 2015 Wiley Periodicals, Inc.     

Soluble CXCL16 promotes TNF‐α‐induced apoptosis in DLBCL via the AMAD10‐NF‐κB regulatory feedback loop

We had previously identified that the co‐expression of transmembrane CXCL16 (TM‐CXCL16) and its receptor CXCR6 is an independent risk factor for poor survival in patients with diffuse large B‐cell lymphoma (DLBCL). However, the impact of the soluble form of CXCL16 (sCXCL16) on the pathogenesis of DLBCL remains unknown. In the present study, the synergistic effect of sCXCL16 and tumor necrosis factor α (TNF‐α) on apoptosis in DLBCL cell lines (OCI‐LY8 and OCI‐LY10) was investigated in vitro. sCXCL16 reinforced TNF‐α‐mediated inhibition of DLBCL cell proliferation, as determined by the cell counting kit‐8 assay. The results of annexin V staining showed that sCXCL16 enhanced TNF‐α‐induced apoptosis in OCI‐LY8 and OCI‐LY10 cells through a death receptor‐caspase signaling pathway. The results of gene microarray suggested a significant upregulation of differentially expressed genes in the TNF signaling pathway. sCXCL16 increased the concentration of extracellular TNF‐α by binding to CXCR6 to activate the nuclear factor‐κB (NF‐κB) signaling pathway. TNF‐α also induced the secretion of sCXCL16 by increasing the expression of ADAM10, which is known to cleave TM‐CXCL16 to yield sCXCL16. Moreover, bioinformatics analysis revealed that elevated TNF‐α and ADAM10 expression levels in tumor tissues predicted better survival in patients with DLBCL. Thus, our study suggests that sCXCL16 enhances TNF‐α‐induced apoptosis of DLBCL cells, which may involve a positive feedback loop consisting of TNF‐α, ADAM10, sCXCL16, and members of the NF‐κB pathway. sCXCL16 and TNF‐α may be used as prognostic markers in the clinic, and their combinational use is a promising approach in the context of DLBCL therapy.     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

Chlorogenic acid: A potent molecule that protects cardiomyocytes from TNF‐α–induced injury via inhibiting NF‐κB and JNK signals

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti‐inflammatory, anti‐oxidant and anti‐apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)‐induced heart failure mouse model, through mitigating the TNF‐α–induced toxicity. We further used TNF‐α‐induced cardiac injury in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) to elucidate the underlying mechanisms. CGA pre‐treatment could reverse TNF‐α–induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF‐κB/p65 and major mitogen‐activated protein kinases (MAPKs) signalling pathways involved in TNF‐α–induced apoptosis of hiPSC‐CMs. Importantly, CGA can directly inhibit NF‐κB signal by suppressing the phosphorylation of NF‐κB/p65. As for the MAPKs, CGA suppressed the activity of only c‐Jun N‐terminal kinase (JNK), but enhanced extracellular signal‐regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF‐κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.     

Geraniin suppresses ovarian cancer growth through inhibition of NF‐κB activation and downregulation of Mcl‐1 expression

This study investigated the anticancer effects of geraniin on ovarian cancer cells and the signaling pathways involved. Ovarian cancer cells were treated with different concentrations of geraniin for 48 h and examined for viability, apoptosis, mitochondrial membrane depolarization, and gene expression. Xenograft tumor studies were performed to determine the anticancer activity of geraniin in vivo. Geraniin significantly decreased cancer cell viability in a concentration‐dependent fashion. Geraniin significantly triggered apoptosis, which was accompanied by loss of mitochondrial membrane potential and increased cytochrome c release and caspsase‐3 activity. Mechanistically, geraniin significantly downregulated Mcl‐1 and impaired NF‐κB p65 binding to the mcl‐1 promoter. Overexpression of Mcl‐1 significantly reversed geraniin‐induced apoptosis in OVCAR3 cells. In addition, geraniin retarded ovarian cancer growth and reduced expression of phospho‐p65 and Mcl‐1. Collectively, geraniin elicits growth suppression in ovarian cancer through inhibition of NF‐κB and Mcl‐1 and may provide therapeutic benefits for this malignancy.     

Blocking NF-κB and Akt by Hsp90 inhibition sensitizes Smac mimetic compound 3-induced extrinsic apoptosis pathway and results in synergistic cancer cell death

NF-κB and Akt are two main cell survival pathways that attenuate the anticancer efficacy of therapeutics. Our previous studies demonstrated that the Smac mimetic compound 3 (SMC3) specifically suppresses c-IAP1 and induces TNF-α autocrine to kill cancer cells. However, SMC3 also induces a cell survival signal through NF-κB activation. In this report, we further found that SMC3 potently activates Akt, which inhibits SMC3-induced cancer cell death. Strikingly, concurrent blocking NF-κB and Akt resulted in a significantly potentiated cytotoxicity. Because heat shock protein 90 (Hsp90) plays an important role in maintaining the integrity of both the NF-κB and Akt pathways in cancer cells, we examined if suppression of Hsp90 is able to potentiate SMC3-induced cancer cell death. The results show that targeting Hsp90 does not interfere with SMC3-induced c-IAP1 degradation and TNF-α autocrine, the key processes for SMC3-induced cancer cell apoptosis. However, Hsp90 inhibitors effectively blocked SMC3-induced NF-κB activation through degradation of RIP1 and IKKβ, two key components of the NF-κB activation pathway, and reduced both the constitutive and SMC3-induced Akt activity through degradation of the Akt protein. Consistently, with the co-treatment of SMC3 and Hsp90 inhibitors, apoptosis was markedly sensitized and a synergistic cytotoxicity was observed. The results suggest that concurrent targeting c-IAP1 and Hsp90 by combination of SMC3 and Hsp90 inhibitors is an effective approach for improving the anticancer value of SMC3.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

NF‐κB and estrogen receptor α interactions: Differential function in estrogen receptor‐negative and ‐positive hormone‐independent breast cancer cells

Estrogen receptor (ER)‐positive breast cancer cells have low levels of constitutive NF‐κB activity while ER negative (?) cells and hormone‐independent cells have relatively high constitutive levels of NF‐κB activity. In this study, we have examined the aspects of mutual repression between the ERα and NF‐κB proteins in ER+ and ER? hormone‐independent cells. Ectopic expression of the ERα reduced cell numbers in ER+ and ER? breast cancer cell lines while NF‐κB‐binding activity and the expression of several NF‐κB‐regulated proteins were reduced in ER? cells. ER overexpression in ER+/E2‐independent LCC1 cells only weakly inhibited the predominant p50 NF‐κB. GST‐ERα fusion protein pull downs and in vivo co‐immunoprecipitations of NF‐κB:ERα complexes showed that the ERα interacts with p50 and p65 in vitro and in vivo. Inhibition of NF‐κB increased the expression of diverse E2‐regulated proteins. p50 differentially associated directly with the ER:ERE complex in LCC1 and MCF‐7 cells by supershift analysis while p65 antibody reduced ERα:ERE complexes in the absence of a supershift. ChIP analysis demonstrated that NF‐κB proteins are present on an endogenous ERE. Together these results demonstrate that the ER and NF‐κB undergo mutual repression, which may explain, in part, why expression of the ERα in ER? cells does not confer growth signaling. Secondly, the acquisition of E2‐independence in ER+ cells is associated with predominantly p50:p50 NF‐κB, which may reflect alterations in the ER in these cells. Since the p50 homodimer is less sensitive to the presence of the ER, this may allow for the activation of both pathways in the same cell. J. Cell. Biochem. 107: 448–459, 2009. © 2009 Wiley‐Liss, Inc.     

TNF‐α regulates the early development of avascular necrosis of the femoral head by mediating osteoblast autophagy and apoptosis via the p38 MAPK/NF‐κB signaling pathway

Previous studies have shown that the tumor necrosis factor‐α (TNF‐α) levels in serum and bone tissues formed in avascular necrosis of femoral head (ANFH) patients were higher than those of normal individuals, indicating TNF‐α might play a role in the pathogenesis of ANFH. However, the underlying mechanisms remain unclear. Hematoxylin and eosin staining was performed to show the pathological changes of ANFH bone tissues. TNF‐α expression in normal and ANFH tissues was examined by quantitative real‐time polymerase chain reaction and western blot analyses. Osteoblast autophagy and apoptosis, as well as signaling pathways activation, were measured by their corresponding marker proteins. Osteoblast proliferation, autophagy, and apoptosis were evaluated using cell counting kit‐8, transmission electron microscopy, and flow cytometry. The structures of bone tissues of ANFH were obviously damaged. TNF‐α expression was significantly upregulated in ANFH bone tissues compared to normal tissues. Autophagy and apoptosis were remarkably promoted, and p38 mitogen‐activated protein kinase (MAPK)/nuclear factor‐κB (NF‐κB) signaling pathways were markedly activated in ANFH. Suppression of the p38 MAPK/NF‐κB pathway significantly attenuated the TNF‐α‐induced autophagy, however, enhanced the TNF‐α‐induced apoptosis in osteoblasts. Increased TNF‐α in ANFH regulated osteoblast autophagy and apoptosis by p38 MAPK/NF‐κB signaling pathways, blocking the pathway by inhibitors exacerbated TNF‐α‐induced apoptosis through impairing autophagy flux.     

TNF‐α increases αvβ3 integrin expression and migration in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumour with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Tumour necrosis factor (TNF)‐α is a key cytokine involved in inflammation, immunity, cellular homeostasis and tumour progression. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. However, the effects of TNF‐α in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that TNF‐α increased the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. Activations of MAPK kinase (MEK), extracellular signal‐regulating kinase (ERK) and nuclear factor‐κB (NF‐κB) pathways after TNF‐α treatment were demonstrated, and TNF‐α‐induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK and NF‐κB cascades. Taken together, our results indicated that TNF‐α enhances the migration of chondrosarcoma cells by increasing αvβ3 integrin expression through the MEK/ERK/NF‐κB signal transduction pathway. J. Cell. Physiol. 226: 792–799, 2011. © 2010 Wiley‐Liss, Inc.     

Interleukin‐12 P40 induces the expression of TNF‐α in microglia and macrophages

Recently, it has been found that overproduction of IL‐12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits – p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL‐12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF‐α in microglia. Interestingly, we have found that IL‐12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose‐dependently induced the production of TNF‐α in BV‐2 microglial cells. This induction of TNF‐α production was accompanied by an induction of TNF‐α mRNA. In addition to BV‐2 glial cells, p70, p402 and p40 also induced the production of TNF‐α in mouse primary microglia and peritoneal macrophages. Since the activation of both NF‐κB and C/EBPb is important for the expression of TNF‐α in microglial cells, we investigated the effect of p40 on the activation of NF‐κB as well as C/EBPb. Activation of NF‐κB as well as C/EBPb by p40 and inhibition of p40‐induced expression of TNF‐α by Dp65, a dominant‐negative mutant of p65, and DC/EBPb, a dominant‐negative mutant of C/EBPb, suggests that p40 induces the expression of TNF‐α through the activation of NF‐κB and C/EBPb. This study delineates a novel role of IL‐12 p40 in inducing the expression of TNF‐α in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases. Acknowledgements: This study was supported by NIH grants (NS39940 and AG19487).