NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells

We previously demonstrated that indoxyl sulfate induces senescence and dysfunction of proximal tubular cells by activating p53 expression. However, little is known about the role of nuclear factor (NF)-κB in these processes. The present study examines whether activation (phosphorylation) of NF-κB by indoxyl sulfate promotes senescence and dysfunction in human proximal tubular cells (HK-2 cells). Indoxyl sulfate induced phosphorylation of NF-κB p65 on Ser-276, which was suppressed by N-acetylcysteine, an antioxidant. Furthermore, indoxyl sulfate induced NF-κB p65 expression. Inhibitors of NF-κB (pyrrolidine dithiocarbamate and isohelenin) and NF-κB p65 small interfering RNA (siRNA) suppressed indoxyl sulfate-induced senescence-associated β-galactosidase activity and expression of p53, transforming growth factor (TGF)-β1, and α-smoothe muscle actin (SMA). The induction of p53 expression and p53 promoter activity by indoxyl sulfate were inhibited by pifithrin-α, p-nitro, an inhibitor of p53, whereas p53-transfected cells showed enhanced p53 promoter activity. NF-κB inhibitors suppressed indoxyl sulfate-induced p21 expression, whereas NF-κB p65 siRNA enhanced its expression. NF-κB inhibitors partially alleviated indoxyl sulfate-induced inhibition of cellular proliferation. NF-κB p65 siRNA-transfected cells showed less proliferation in the presence of indoxyl sulfate than control cells. Phosphorylated NF-κB p65 was expressed and colocalized with p53, p21, β-galactosidase, TGF-β1, and α-SMA in the kidneys of chronic renal failure (CRF) rats. AST-120, which reduces serum indoxyl sulfate level, suppressed their expression in the CRF rat kidneys. Taken together, NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells. More notably, indoxyl sulfate accelerates proximal tubular cell senescence with progression of CRF through reactive oxygen species-NF-κB-p53 pathway.     

Profilin potentiates chemotherapeutic agents mediated cell death via suppression of NF-κB and upregulation of p53

The molecular mechanism by which Profilin acts as a tumor suppressor is still unclear. Several chemotherapeutic agents, used till date either have unfavorable side effects or acquired resistance in tumor cells. Our findings show that Profilin enhances cell death mediated by several chemotherapeutic-agents. The activation of NF-κB and its dependent genes, mediated by paclitaxel and vinblastine, was completely inhibited in Profilin overexpressing cells. This inhibition was due to the Profilin mediated attenuation of IκBα degradation, thereby preventing p65 nuclear translocation and low NF-κB DNA binding activity.Moreover, Profilin increases level of p53 in the presence of known inducers, such as doxorubicin, vinblastine, and benzofuran. This increased p53 level leads to enhanced cell death as indicated by activation of caspases 3, 8, 9, which results in cleavage of PARP.Furthermore, knocking down of p53 in Profilin overexpressing cells leads to decreased cell death. Ectopic expression of Profilin in HCT116 p53 knock out cells showed lesser cell death as compared to the HCT116 p53 wild type cells. For the first time, we provide evidences, which suggest that Profilin synergizes with chemotherapeutic drugs to induce tumor cell death by regulating NF-κB and p53. Thus, modulation of Profilin may be a useful strategy for effective combination therapy.     

P53 modulates hepatic insulin sensitivity through NF-κB and p38/ERK MAPK pathways

Besides its well-established oncosuppressor activity, the role of p53 in regulating metabolic pathways has been recently identified. Nevertheless, the function of p53 with respect to insulin resistance appears highly controversial. To address this issue, we investigated the expression of p53 in experimental model of insulin resistance. Then we used activator (nutlin-3α) and inhibitor (pifithrin-α, PFT-α) of p53 in HepG2 cell. Here we showed that p53 protein level was decreased in the hepatic tissue of high-fat diet-induced insulin resistance mice, genetically diabetic ob/ob mice and palmitate (PA) treated HepG2 cells. And high expression of phosphor-p38, ERK1/2 and nuclear factor kappa B (NF-κB) p65 accompanied with low expression of p53. But activation of p53 with nutlin-3α prevented PA-induced reduction of glucose consumption and suppression of insulin signaling pathways. At the same time, nutlin-3α downregulated the activation of NF-κB, p38 and ERK1/2 pathways upon stimulation with PA. In contrast, inhibition of p53 with PFT-α decreased glucose consumption and suppressed insulin signaling pathway. Furthermore, PFT-α activated NF-κB, p38 and ERK1/2 pathways in HepG2 cells. Overall, these results suggest that p53 is involved in improving insulin sensitivity of hepatic cells via inhibition of mitogen-activated protein kinases (MAPKs) and NF-κB pathways.     

PTEN inhibits the invasion and metastasis of gastric cancer via downregulation of FAK expression

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is essential in inhibiting tumor growth and metastasis. However, the mechanism by which PTEN restricts gastric cancer progression and metastasis remains largely elusive. Here we demonstrated that PTEN overexpression or knockdown in gastric cancer cells led to the downregulation or upregulation of focal adhesion kinase (FAK), and decreased or increased cell invasion, respectively. Moreover, FAK overexpression could rescue the inhibition of cell invasion by PTEN. These results were further confirmed in orthotropic gastric cancer nude mice model. In addition, in human gastric cancer tissues, PTEN protein level was conversely correlated with FAK protein level. Mechanistically, we found that PTEN inhibited PI3K/NF-κB pathway and inhibited the DNA binding of NF-κB on FAK promoter. Taken together, our data reveal a novel mechanism that PTEN inhibits the growth and invasion of gastric cancer via the downregulation of FAK expression and suggest that exploiting PTEN/PI3K/NF-κB/FAK axis is a promising approach to treat gastric cancer metastasis.     

Interaction between NF-κB signaling and Notch signaling in gliogenesis of mouse mesencephalic neural crest cells

In the present study, we elucidated that nuclear factor-κB (NF-κB) participates in the gliogenic specification of mouse mesencephalic neural crest cells. Whereas transfection of the NF-κB expression vector stimulated gliogenesis, treatment with the dominant negative NF-κB expression vector or NF-κB small interfering RNA suppressed the promotion of gliogenic specification by FGF treatment or Notch activation. This suppression was recovered by the treatment with the Deltex-1 expression vector or mammalian hairy and enhancer of split homologs expression vectors. Furthermore, transfection of the inhibitor of κB (IκB) expression vector inhibited gliogenesis. In addition, treatment with the NF-κB expression vector promoted the expression of Deltex-1. These data suggest that NF-κB signaling is implicated in the gliogenesis through the interaction with Notch signaling. Moreover, cells that contain Sox10 expressed NF-κB and Deltex-1 in the presumptive trigeminal ganglia of embryonic day 9.0-9.5 mouse embryos. This observation supports our notion that the interaction between NF-κB signaling and Notch signaling plays an important role in the gliogenic specification of mouse mesencephalic neural crest cells.     

IGF-I enhances α5β1 integrin expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. Insulin-like growth factor-I (IGF)-I plays an important role in regulating cell growth, proliferation, survival, and metabolism. However, the effects of IGF-I in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that IGF-I increased the migration and the expression of α5β1 integrin in human chondrosarcoma cells. Pretreatment of cells with IGF-I receptor antibody reduced IGF-I-induced cell migration and integrin expression. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and nuclear factor-κB (NF-κB) pathways after IGF-I treatment were demonstrated, and IGF-I-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and NF-κB cascades. Taken together, our results indicated that IGF-I enhances the migration of chondrosarcoma cells by increasing α5β1 integrin expression through the IGF-I receptor/PI3K/Akt/NF-κB signal transduction pathway.     

Control of p53 and NF-κB signaling by WIP1 and MIF: role in cellular senescence and organismal aging

The stress-activated signaling pathways, p53 and NF-κB, have a major role in the regulation of cellular senescence and organismal aging. These ancient signaling networks display functional antagonism via negative autoregulatory circuits. WIP1 (wildtype p53-induced phosphatase 1) and MIF (macrophage migration inhibitory factor) are signaling molecules which link together the p53 and NF-κB pathways via positive and negative feedback loops. It seems that the efficiency of the p53 signaling pathway declines during aging whereas that of NF-κB is clearly enhanced. Moreover, p53 is an important trigger of cellular senescence while NF-κB signaling seems to be involved in the induction of the senescence-associated secretory phenotype (SASP). MIF is a pro-inflammatory cytokine which inhibits the function of p53 signaling whereas it is linked to NF-κB signaling via a positive feedback loop. MIF knockout mice are healthier and live longer than their wild-type counterparts. An increased level of MIF can support inflammatory responses via enhancing NF-κB signaling and repressing the function of p53. p53 is an inducer of the expression of WIP1 which can subsequently inhibit NF-κB signaling. Several observations indicate that the activity of WIP1 decreases during the aging process, this being probably attributable to the decline in p53 function. Decreased WIP1 activity potentiates the activity of p38MAPK and NF-κB signaling leading to premature cellular senescence as well as low-level chronic inflammation. We will review the findings linking WIP1 and MIF to specific signaling responses of p53 and NF-κB and discuss their role in the regulation of cellular senescence and organismal aging.     

Identification of NME5 as a contributor to innate resistance to gemcitabine in pancreatic cancer cells

The limited therapeutic effect of gemcitabine on pancreatic cancer is largely attributed to pre-existing or acquired resistance of the tumor cells. This study was aimed at screening for candidate resistance-related gene(s) and elucidating the underlying mechanisms. NME5 was found to be highly expressed in an innate gemcitabine-resistant human pancreatic cancer sample and the cell line PAXC002 derived from the sample. Downregulation of NME5 significantly reversed gemcitabine resistance in PAXC002 cells, whereas NME5 overexpression induced gemcitabine resistance in the pancreatic cancer cell line BxPC-3. NME5 attenuated the induction of apoptosis and cell cycle arrest induced by gemcitabine, probably accounting for the blunted sensitivity to gemcitabine. Furthermore, NME5 was demonstrated to play its role in a nuclear factor kappaB (NF-κB)-dependent manner. NME5 was capable of directly binding NF-κB, and possibly regulated its expression level in PAXC002 cells. Our results also suggest that NF-κB is a key executor of NME5 in regulating apoptosis and cell cycle. All of these data suggest that NME5 is a promising target for relieving innate gemcitabine resistance in pancreatic cancer cells.     

Progesterone receptor activation of extranuclear signaling pathways in regulating p53 expression in vascular endothelial cells

We previously showed that progesterone (P4) inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) through a p53-dependent pathway. Now we investigated further the molecular mechanism underlying the hormone activity. In cultured HUVECs, P4 increased the protein levels of phosphorylated Src (p-Src), Raf-1, and ERK. The levels of p-Src and p-Src-progesterone receptor complex in HUVECs were increased by P4 treatment. These effects were blocked by pretreatment with a progesterone receptor antagonist, RU486. The P4-induced increase in p53 transactivity was abolished by pretreatment with Src kinase inhibitors. Moreover, administration with cSrc antisense oligonucleotide prevented the P4-induced increases of the levels of p53 mRNA and protein. These data suggest that P4-induced up-regulation of p53 might be mediated through activation of cSrc. Pretreatment with Src kinase inhibitors also prevented P4-induced membrane translocation of Kras and increases of the protein levels of phosphorylated Raf and phosphorylated ERK. Transfection with dominant-negative ERK2 prevented the P4-induced increases of protein level and promoter activity of p53 and a decrease of thymidine incorporation. P4 also increased nuclear factor-κB (NF-κB) nuclear translocation and NF-κB binding onto the p53 promoter. These effects were abolished by pretreatment with ERK inhibitors. The P4-induced up-regulation of the p53 promoter activity was prevented by preadministration with dominant-negative ERK2 or NF-κB inhibitors. Taken together, our data suggest that the cSrc/Kras/Raf-1/ERK2/NF-κB signaling pathway contributes to the P4-induced up-regulation of p53 in HUVECs. These findings highlight progesterone receptor activation of extranuclear signaling pathways in regulating p53 and cell cycle progression in HUVECs.     

Bone marrow mesenchymal stem cells-derived exosomal microRNA-16-5p restrains epithelial-mesenchymal transition in breast cancer cells via EPHA1/NF-κB signaling axis

ObjectiveThis study intends to conquer the mystery of microRNA-16-5p/erythropoietin-producing hepatocellular A1/nuclear factor-κB signaling (miR-16-5p/EPHA1/NF-κB signaling) in breast cancer.MethodsExpression of miR-16-5p, EPHA1 and NF-κB signaling-related proteins were detected. Gene overexpression or silencing was used to examine the biological roles of bone marrow mesenchymal stem cells (BMSCs)-derived exo-miR-16-5p in breast cancer. The effect of exo-miR-16-5p on tumorigenesis of breast cancer was confirmed by the xenograft nude mouse model.ResultsLow miR-16-5p and high EPHA1 expression were examined in breast cancer. BMSCs-derived exosomes, up-regulated miR-16-5p or down-regulated EPHA1 restrained epithelial-mesenchymal transition (EMT) of breast cancer cells and tumor growth in nude mice. Down-regulated miR-16-5p or up-regulated EPHA1 activated NF-κB signaling. Knockdown of EPHA1 or inhibition of NF-κB signaling reversed the effects of down-regulated miR-16-5p on breast cancer cells.ConclusionBMSCs-derived exosomal miR-16-5p hinders breast cancer cells progression via EPHA1/NF-κB signaling axis.     

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.     

Knockdown of hepatoma-derived growth factor-related protein-3 induces apoptosis of H1299 cells via ROS-dependent and p53-independent NF-κB activation

We previously identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistant biomarker in p53 wild-type A549 cells and found that p53-dependent induction of the PUMA pathway was a critical event in regulating the radioresistant phenotype. Here, we found that HRP-3 knockdown regulates the radioresistance of p53-null H1299 cells through a distinctly different molecular mechanism. HRP-3 depletion was sufficient to cause apoptosis of H1299 cells by generating substantial levels of reactive oxygen species (ROS) through inhibition of the Nrf2/HO-1 antioxidant pathway. Subsequent, ROS-dependent and p53-independent NF-κB activation stimulated expression of c-Myc and Noxa proteins, thereby inducing the apoptotic machinery. Our results thus extend the range of targets for the development of new drugs to treat both p53 wild-type or p53-null radioresistant lung cancer cells.     

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.