Involvement of nuclear factor κB in platelet CD40 signaling

CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.     

Testes-specific protease 50 (TSP50) promotes cell proliferation through the activation of the nuclear factor κB (NF-κB) signalling pathway

TSP50 (testes-specific protease 50) is a testis-specific expression protein, which is expressed abnormally at high levels in breast cancer tissues. This makes it an attractive molecular marker and a potential target for diagnosis and therapy; however, the biological function of TSP50 is still unclear. In the present study, we show that overexpression of TSP50 in CHO (Chinese-hamster ovary) cells markedly increased cell proliferation and colony formation. Mechanistic studies have revealed that TSP50 can enhance the level of TNFα (tumour necrosis factor α)- and PMA-induced NF-κB (nuclear factor κB)-responsive reporter activity, IκB (inhibitor of NF-κB) α degradation and p65 nuclear translocation. In addition, the knockdown of endogenous TSP50 in MDA-MB-231 cells greatly inhibited NF-κB activity. Co-immunoprecipitation studies demonstrated an interaction of TSP50 with the NF-κB-IκBα complex, but not with the IKK (IκB kinase) α/β-IKKγ complex, which suggested that TSP50, as a novel type of protease, promoted the degradation of IκBα proteins by binding to the NF-κB-IκBα complex. Our results also revealed that TSP50 can enhance the expression of NF-κB target genes involved in cell proliferation. Furthermore, overexpression of a dominant-negative IκB mutant that is resistant to proteasome-mediated degradation significantly reversed TSP50-induced cell proliferation, colony formation and tumour formation in nude mice. Taken together, the results of the present study suggest that TSP50 promotes cell proliferation, at least partially, through activation of the NF-κB signalling pathway.     

Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation

Palmitate induces PTP1B expression in skeletal muscle cells. The purpose of this study was to investigate the mechanisms responsible for palmitate-induced PTP1B expression in mouse skeletal muscle cells. Three truncated fragments of PTP1B promoter were cloned into PGL3-basic vector and the promoter activity of PTP1B was assessed in C2C12 cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. EMSA was performed to examine binding of NF-κB to NF-κB consensus sequence and PTP1B oligonucelotides in the cells treated with palmitate. Lentiviral PTP1B-shRNA was used to knockdown PTP1B in myotubes. The phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. 0.5mM palmitate induced PTP1B promoter activity in fragment -1715/+59 by 50% (p<0.01). Palmitate increased NF-κB binding to both NF-κB consensus sequence and one NF-κB sequence (-920 to -935) in PTP1B promoter. Incubation of C2C12 cells with different concentrations of C2-ceramide enhanced PTP1B promoter activity dose-dependently. Inhibitors of de novo ceramide synthesis prevented palmitate-induced PTP1B promoter activity in myotubes. In addition, inhibitor of JNK pathway prevented ceramide-induced PTP1B promoter activity in myotubes. Knockdown of PTP1B also prevented ceramide-reduced IRS-1 and Akt phosphorylations in the myotubes. Exposure of the cells to PMA and calphostin C, an inhibitor of PKC, did not affect the activity of PTP1B promoter. Our data provide the evidence that the mechanism by which palmitate increased the expression of PTP1B seems to be through a mechanism involving the activation of ceramide-JNK and NF-κB pathways.     

The dichotomous role of TGF-β in controlling liver cancer cell survival and proliferation

Hepatocellular carcinoma (HCC) is the major form of primary liver cancer and one of the most prevalent and life-threatening malignancies globally. One of the hallmarks in HCC is the sustained cell survival and proliferative signals, which are determined by the balance between oncogenes and tumor suppressors. Transforming growth factor beta (TGF-β) is an effective growth inhibitor of epithelial cells including hepatocytes, through induction of cell cycle arrest, apoptosis, cellular senescence, or autophagy. The antitumorigenic effects of TGF-β are bypassed during liver tumorigenesis via multiple mechanisms. Furthermore, along with malignant progression, TGF-β switches to promote cancer cell survival and proliferation. This dichotomous nature of TGF-β is one of the barriers to therapeutic targeting in liver cancer. Thereafter, understanding the underlying molecular mechanisms is a prerequisite for discovering novel antitumor drugs that may specifically disable the growth-promoting branch of TGF-β signaling or restore its tumor-suppressive arm. This review summarizes how TGF-β inhibits or promotes liver cancer cell survival and proliferation, highlighting the functional switch mechanisms during the process.     

Curcumin inhibits cell proliferation of MDA-MB-231 and BT-483 breast cancer cells mediated by down-regulation of NFκB,cyclinD and MMP-1 transcription

Curcumin, an active constituent of turmeric, has been shown to possess inhibitory effect of cell proliferation and induction of apoptosis towards a board range of tumors. Cell inhibition activities of curcumin are behaved differently in various cell types. To investigate the mechanism basis for the cell inhibition of curcumin on breast cancer cell lines, we examine curcumin effect on NFκB, cell cycle regulatory proteins and matrix metalloproteinases (MMPs) in two breast cancer cell lines (MDA-MB-231 and BT-483). Cell proliferation was performed by water soluble tetrazolium WST-1 assay. The effect of curcumin's on the activity of matrix metalloproteinase-1, 3, 9 were analyzed by RT-PCR. Cell cycle regulatory protein including cyclin D1, CDK4 and p21 were examined by immunochemistry. The expressions of NFκB in breast cancer cells treated with curcumin were studied by immunochemistry and western blot. The results from WST-1 cell proliferation assay showed that curcumin exhibited the anti-proliferation effect on MDA-MB-231 and BT-483 cells in a time- and dose-dependent manner. In response to the treatment, while, the expression of cyclin D1 had declined in MDA-MB-231 and the expression of CDK4 in BT-483 had declined. MMP1 mRNA expression in BT-483 and MDA-MB-231 had significantly decreased in curcumin treatment group compared with control group. Our finding extrapolates the antitumor activity of curcumin in mediating the breast cancer cell proliferative rate and invasion by down-regulating the NFκB inducing genes.     

Distinct effect of forskolin and interferon-γ on cell proliferation and regulation of histocompatibility antigen expression in hematopoietic cells

The adenylate cyclase activator, forskolin, was found to induce expression of class I and class II major histocompatibility complex antigens in a B precursor cell line, Reh, as well as in a B lymphoid cell line, Raji. No such effect was, however, observed when the promyelocytic cells line HL-60 was treated with either forskolin or the cAMP analogue 8-bromoadenosine cyclic monophosphate. As expected, all three cell lines showed reduced proliferation upon forskolin treatment. Forskolin induced expression of class I and class II major histocompatibility complex antigens in cell lines not affected by interferon-γ and vice versa, indicating that cAMP is not involved in the regulation of histocompatibility antigens by interferon-γ. We also compared the effect of interferon-γ and 12-O-tetradecanoylphorbol 13-acetate on major histocompatibility complex class I and class II expression, and despite differences in the response on the tested cell lines, we can not at this point exclude the possibility that protein kinase C is involved in the action of interferon-γ.     

Retigeric acid B exhibits antitumor activity through suppression of nuclear factor-κB signaling in prostate cancer cells in vitro and in vivo

Previously, we reported that retigeric acid B (RB), a natural pentacyclic triterpenic acid isolated from lichen, inhibited cell growth and induced apoptosis in androgen-independent prostate cancer (PCa) cells. However, the mechanism of action of RB remains unclear. In this study, we found that using PC3 and DU145 cells as models, RB inhibited phosphorylation levels of IκBα and p65 subunit of NF-κB in a time- and dosage-dependent manner. Detailed study revealed that RB blocked the nuclear translocation of p65 and its DNA binding activity, which correlated with suppression of NF-κB-regulated proteins including Bcl-2, Bcl-x(L), cyclin D1 and survivin. NF-κB reporter assay suggested that RB was able to inhibit both constitutive activated-NF-κB and LPS (lipopolysaccharide)-induced activation of NF-κB. Overexpression of RelA/p65 rescued RB-induced cell death, while knockdown of RelA/p65 significantly promoted RB-mediated inhibitory effect on cell proliferation, suggesting the crucial involvement of NF-κB pathway in this event. We further analyzed antitumor activity of RB in in vivo study. In C57BL/6 mice carrying RM-1 homografts, RB inhibited tumor growth and triggered apoptosis mainly through suppressing NF-κB activity in tumor tissues. Additionally, DNA microarray data revealed global changes in the gene expression associated with cell proliferation, apoptosis, invasion and metastasis in response to RB treatment. Therefore, our findings suggested that RB exerted its anti-tumor effect by targeting the NF-κB pathway in PCa cells, and this could be a general mechanism for the anti-tumor effect of RB in other types of cancers as well.     

Simultaneous inhibition of the constitutively activated nuclear factor κB and of the Interleukin-6 pathways is necessary and sufficient to completely overcome apoptosis resistance of human U266 myeloma cells

Elevated Nuclear Factor κB (NF-κB) levels have been reported in multiple myeloma cells derived from patients relapsing after chemotherapy. In the search of an in vitro a model with molecular features similar to relapsing lesions, we focused our attention on an IL-6 autocrine human myeloma cell line (U266), characterized by apoptosis resistance due to up-regulation of two constitutive signaling pathways: NF-κB and STAT-3. NF-κB activity was inhibited with proteasome inhibitory agents, such as PS-341 and Withaferin A, with an IKK inhibitor (Wedelolactone) or with the adenoviral vector HD IκBαmut-IRES-EGFP encoding a mutant IκBα protein, resistant to proteasomal degradation. We observed that the NF-κB intracellular dislocation at the beginning of the treatment affected therapeutic effectiveness of PS-341, Withaferin A and Wedelolactone; interestingly, the adenoviral vector was highly effective in inducing apopotosis even with NF-κB being predominantly nuclear at the time of infection. We also observed that U266 treated with the Interleukin-6 antagonist Sant7 exhibited reduced STAT3 activity and preferential cytoplasmic NF-κB location; moreover they became capable of undergoing apoptosis mainly from the G1 phase. Adenoviral vector treated U266 have NF-κB localized completely in the cytoplasm and also showed down-regulation of nuclear phospho STAT-3. Finally, combined targeting of NF-κB and STAT3 signaling pathways was the most effective treatment in inducing apoptosis. These findings suggest that combined NF-κB κB and STAT3 targeting warrants further investigations in other apoptosis resistant MM cell lines as well as in suitable MM animal models.     

Caffeine inhibits cell proliferation and regulates PKA/GSK3β pathways in U87MG human glioma cells

Caffeine is the most commonly ingested methylxanthine and has anti-cancer effects in several types of cancer. In this study, we examined the anti-cancer effects of caffeine on gliomas, both in vitro and in vivo. In vitro, caffeine treatment reduced glioma cell proliferation through G(0)/G(1)-phase cell cycle arrest by suppressing Rb phosphorylation. In addition, caffeine induced apoptosis through caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. Caffeine also phosphorylated serine 9 of glycogen synthase kinase 3 beta (GSK3β). Pretreatment with H89, a pharmacological inhibitor of protein kinase A (PKA), was able to antagonize caffeine-induced GSK3β(ser9) phosphorylation, suggesting that the mechanism might involve a cAMP-dependent PKA-dependent pathway. In vivo, caffeine-treated tumors exhibited reduced proliferation and increased apoptosis compared with vehicle-treated tumors. These results suggest that caffeine induces cell cycle arrest and caspase-dependent cell death in glioma cells, supporting its potential use in chemotherapeutic options for malignant gliomas.     

Effects of tumor necrosis factor and interferon-β on proliferation and epidermal growth factor binding in young and senescent WI-38 cells

Summary Tumor necrosis factor-α (TNF) and various interferons (IFN) have potent cytostatic or cytotoxic effects on a variety of human tumor-derived cell lines. Their effects on normal cells are more controversial. We have examined the effects of TNF and IFN-β on the proliferation of WI-38 cells in a serum-free, growth factor-supplemented medium and in serum-containing medium. These cells respond to the combination of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and dexamethasone by DNA synthesis at a rate and extent equivalent to serum-stimulated cells. TNF has no effect on this growth factor-stimulated proliferation. However, it is stimulatory in serum-containing medium. IFN-β inhibits DNA synthesis 60 to 70% in both young and senescent cells. TNF and IFN-β together have a synergistic effect and completely inhibit growth factor-stimulated DNA synthesis in young cells. No synergism was observed with senescent cells. TNF stimulated an increase in the number of EGF specific binding sites two- to threefold in 24 h in both young and senescent cells. This seems to result from a proportional increase in a very high affinity binding site. IFN-β has little or no effect on EGF binding either alone or in combination with TNF.