NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.     

Nitric oxide induces apoptosis in cutaneous T cell lymphoma (HuT-78) by downregulating constitutive NF-κB

Constitutive active NF-κB have been shown to protect cutaneous T cell lymphoma (CTCL) cells from apoptosis. In the present study, we have studied the cytotoxic potential of nitric oxide generating compound, sodium nitroprusside (SNP) on CTCL cell line, HuT-78. Treatment of cells with SNP resulted in decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and poly (ADP ribose) polymerase cleavage. SNP treatment inhibited activation of NF-κB in a concentration dependent manner. SNP increased the expression of IκBα without affecting the phosphorylation of IκBα. Downregulation of NF-κB by SNP decreased p65 nuclear translocation as evident by confocal fluorescence microscopy. Further it was found that SNP treatment caused downregulation of Bcl-2 family member (Bcl-xl) in HuT-78 cells. Thus, we have provided evidence that SNP induces apoptosis in CTCL cell line, HuT-78 by downregulating constitutive NF-κB and thereby Bcl-xl expression.     

Suppression of uPA and uPAR blocks radiation-induced MCP-1 mediated recruitment of endothelial cells in meningioma

Chemokines play a vital role in recruiting various cell types in the process of tissue repair. Radiation, a major therapeutic modality in cancer treatment, has been described to induce inflammatory response that might lead to the expression of several chemokines. In the present study, we investigated the mechanism of monocyte chemoattractant protein-1 (MCP-1) induction by radiation in meningioma cell lines and the paracrine effect on human microvascular endothelial cells (HMEC). After radiation, meningioma cell lines (IOMM Lee and SF-3061) showed an increased expression of MCP-1. In addition, irradiated meningioma cancer cell conditioned medium (CM) showed an increased ability to attract HMEC and to stimulate MCP-1-induced protein (MCPIP), VEGF and angiogenin expression in HMEC. This chemotactic activity and angiogenic stimulator effect on HMEC were almost abrogated by depleting MCP-1 from the irradiated cancer cell CM. Further, inhibition of either ERK activation/expression or NF-κB nuclear translocation hindered radiation-induced MCP-1 expression in both meningioma cell lines. Further, supplementing cancer cells with exogenous ATF-uPA (with and without radiation) activated ERK phosphorylation, nuclear translocation of the NF-κB p65 sub-unit (Rel-A), and MCP-1 expression. Downregulation of uPA and uPAR, simultaneously by transfecting the cancer cells with bi-cistronic siRNA-expressing plasmid (pU) inhibited radiation-induced ERK activation, nuclear translocation of Rel-A, NF-κB DNA binding activity, and MCP-1 expression. In addition, pU-transfected cancer cells (with or without radiation) reduced radiation-induced MCP-1 and blocked the recruitment of other cell types during the inflammatory process induced by radiation both in in vitro and in vivo conditions.     

Thromboxane A2 increases endothelial permeability through upregulation of interleukin-8

Thromboxane A₂ (TXA₂), a major prostanoid formed from prostaglandin H₂ by thromboxane synthase, is involved in the pathogenesis of a variety of vascular diseases. In this study, we report that TXA₂ mimetic U46619 significantly increases the endothelial permeability both in vitro and in vivo. U46619 enhanced the expression and secretion of interleukin-8 (IL-8), a major inducer of vascular permeability, in endothelial cells. Promoter analysis showed that the U46619-induced expression of IL-8 was mainly regulated by nuclear factor-κB (NF-κB). U46619 induced the activation of NF-κB through IκB kinase (IKK) activation, IκB phosphorylation and NF-κB nuclear translocation. Furthermore, the inhibition of IL-8 or blockade of the IL-8 receptor attenuated the U46619-induced endothelial cell permeability by modulating the cell-cell junctions. Overall, these results suggest that U46619 promotes vascular permeability through the production of IL-8 via NF-κB activation in endothelial cells.     

Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50–60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88^negative population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88^negative A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88^positive TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.     

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.     

Hepatitis B virus X protein induces IKKα nuclear translocation via Akt-dependent phosphorylation to promote the motility of hepatocarcinoma cells

Hepatitis B virus (HBV) X protein (HBx) has been implicated in HBV-associated carcinogenesis through activation of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling pathway. Besides activating NF-κB in the cytoplasm, IKKα was found in the nucleus to regulate gene expression epigenetically in response to various stimuli. However, it is unknown whether nuclear IKKα plays a role in HBx-associated tumor progression. Moreover, the molecular mechanism underlying IKKα nuclear transport also remains to be elucidated. Here, we disclosed HBx as a new inducer of IKKα nuclear transport in hepatoma cells. HBx induced IKKα nuclear transport in an Akt-dependent manner. HBx-activated Akt promoted IKKα nuclear translocation via phosphorylating its threonine-23 (Thr23). In addition, IKKα ubiquitination enhanced by HBx and Akt also contributed to the IKKα accumulation in the nucleus, indicating the involvement of ubiquitination in Akt-increased IKKα nuclear transport in response to HBx. Furthermore, inhibition of IKKα nuclear translocation by mutation of its nuclear localization signal and Thr23 diminished IKKα-dependent cell migration. Taken together, our findings shed light on the molecular mechanism of IKKα nuclear translocation and provide a potential role of nuclear IKKα in HBx-mediated hepatocellular carcinoma (HCC) progression.     

The nucleoporin Nup88 is interacting with nuclear lamin A

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and mediate bidirectional nucleocytoplasmic transport. Their spatial distribution in the NE is organized by the nuclear lamina, a meshwork of nuclear intermediate filament proteins. Major constituents of the nuclear lamina are A- and B-type lamins. In this work we show that the nuclear pore protein Nup88 binds lamin A in vitro and in vivo. The interaction is mediated by the N-terminus of Nup88, and Nup88 specifically binds the tail domain of lamin A but not of lamins B1 and B2. Expression of green fluorescent protein-tagged lamin A in cells causes a masking of binding sites for Nup88 antibodies in immunofluorescence assays, supporting the interaction of lamin A with Nup88 in a cellular context. The epitope masking disappears in cells expressing mutants of lamin A that are associated with laminopathic diseases. Consistently, an interaction of Nup88 with these mutants is disrupted in vitro. Immunoelectron microscopy using Xenopus laevis oocyte nuclei further revealed that Nup88 localizes to the cytoplasmic and nuclear face of the NPC. Together our data suggest that a pool of Nup88 on the nuclear side of the NPC provides a novel, unexpected binding site for nuclear lamin A.     

IFN-γ Induces Gastric Cancer Cell Proliferation and Metastasis Through Upregulation of Integrin β3-Mediated NF-κB Signaling

Interferon γ (IFN-γ), a multifunctional cytokine, was upregulated in the resected gastric cancer tissue. However, whether IFN-γ is involved in the regulation of gastric cancer has not been well elucidated. Herein, we aimed to investigate the effects and mechanism of IFN-γ on gastric cancer. In this study, we found a vital role of IFN-γ in enhancing proliferation, inhibiting apoptosis, and promoting cell migration and invasion in gastric cancer cells SGC-7901 and MGC-803. Additionally, IFN-γ activated nuclear factor κB (NF-κB) signaling pathway by upregulating the phosphorylation expression of p65 and IκBα, and induced the expression of integrin β3 in vitro. Therefore, to further investigate the relationship between IFN-γ and integrin β3, SGC-7901 cells were transfected with integrin β3 siRNA. And then cells expressed lower cell viability, migration, and invasion rates, while cell apoptosis was significantly enhanced. Meanwhile, expression of integrin β3, MMP-2, MMP-9, and NF-κB, including p65 and IκBα, and the nuclear translocation of NF-κB/p65 were dramatically repressed, whereas IFN-γ significantly improved the effects. Moreover, in vivo, the experiment of xenograft model and pulmonary metastasis model also retarded in integrin β3 siRNA group. And the expression of integrin β3, MMP-2, MMP-9, and NF-κB was repressed. However, the treatment with IFN-γ improved tumor volume, lung/total weight, tumor nodules, and the protein expression described above compared with integrin β3 siRNA group. Overall, the results indicated that IFN-γ induces gastric cancer cell proliferation and metastasis partially through the upregulation of integrin β3-mediated NF-κB signaling. Hence, the inhibition of IFN-γ or integrin β3 may be the key for the treatment of gastric cancer.