Overexpression of Notch1 has been associated with breast cancer. We recently showed that visfatin stimulates breast cancer cell proliferation and invasion. The present study was undertaken to determine whether Notch1 signaling is affected by visfatin and to characterize the functional role of the visfatin-Notch1 axis in breast cancer. Visfatin and Notch1 were expressed at higher levels in breast tumors than in matched control tissues. Visfatin induced Notch1 expression in MDA-MB-231 breast cancer cell line and in nontransformed MCF10A mammary epithelial cells, whereas visfatin depletion reduced Notch1 mRNA and protein levels. Depletion of Notch1 in MDA-MB-231 cells attenuated cell growth in vitro and in vivo; visfatin depletion produced similar effects, but was less potent. Additionally, Notch1 depletion inhibited cell proliferation induced by visfatin. Analysis of the signaling pathways underlying visfatin-mediated Notch1 upregulation revealed that visfatin activated NF-κB p65. Blockade of NF-κB signaling suppressed the effects of visfatin on Notch1 upregulation and breast cancer cell proliferation. Breast tumors expressing high levels of NF-κB p65 exhibited increased expression of Notch1. Our results demonstrate that the visfatin-Notch1 axis contributes to breast tumor growth through the activation of the NF-κB pathway. Study of the visfatin-Notch1 axis may offer new therapeutic directions for breast cancer.

Obesity is frequently associated with breast cancer. Such associations are possibly mediated by adipokines. Visfatin, an adipokine, has recently been shown to be related to the development and progression of breast cancer. Therefore, the down-regulation of visfatin may be a novel strategy for breast cancer therapy. Curcumin has anticancer activities by modulating multiple signaling pathways and genes. The purpose of this study was to investigate whether visfatin gene expression is affected by curcumin in human breast cancer cells and to characterize the functional role of visfatin in breast cancer. We found that the mRNA and protein levels of visfatin were down-regulated by curcumin in MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cells, along with decreased activity of constitutive nuclear factor (NF)-κB. We confirmed the repressive effect of curcumin on visfatin transcription by performing a visfatin promoter-driven reporter assay and identified two putative NF-κB-binding sites on visfatin promoter that are important for this effect. EMSA and chromatin immunoprecipitation analysis indicated the binding of p65 to the visfatin promoter, which was effectively blocked by curcumin. Enforced expression of p65 protein increased visfatin promoter activity, whereas blocking NF-κB signaling suppressed visfatin gene expression. Visfatin could enhance the invasion of MDA-MB-231 cells and also attenuate curcumin-induced inhibition of cell invasion; on the other hand, visfatin knockdown by small interfering RNA led to the reduction of cell invasion. Our data demonstrate, for the first time, that curcumin down-regulates visfatin gene expression in human breast cancer cells by a mechanism that is, at least in part, NF-κB dependent and suggest that visfatin may contribute to breast cancer cell invasion and link obesity to breast cancer development and progression.

Our aims were to determine the role of Notch1 in mediating visfatin-induced angiogenesis and to explore potential target genes involved. Inhibition of Notch signalling attenuated visfatin-induced angiogenesis in vitro, ex vivo, and in vivo. Visfatin increased γ-secretase activity, Notch1 cleavage and activation, and Hes1 gene induction. Visfatin also stimulated fibroblast growth factor-2 (FGF-2) gene expression in a Notch1-dependent manner. Enforced expression of active Notch1 intracellular domain increased FGF-2 protein levels and stimulated endothelial tube formation, whereas blocking Notch1 signalling or knockdown of Notch1 by small interfering RNA suppressed visfatin-induced FGF-2 up-regulation and angiogenesis. Reporter analysis of FGF-2 promoter revealed the presence of CSL (CBF-1, suppressor of hairless, LAG-1)-binding site, and chromatin immunoprecipitation analysis demonstrated the binding of Notch1–CSL complex to this site in response to visfatin. Our data provide the first example of Notch1-dependent endothelial FGF-2 induction by visfatin and of Notch1 activation in visfatin-stimulated endothelial angiogenesis, suggesting that the signalling axis of visfatin/Notch1/angiogenic factors like FGF-2 might be a valuable target for pathological angiogenesis.

Dental pulp plays an important role in the health of teeth. The aging of teeth is strongly related to the senescence of dental pulp cells. A novel adipokine, visfatin, is closely associated with cellular senescence. However, little is known about the effect of visfatin on the senescence of human dental pulp cells (hDPCs). Here, it was found that in vivo visfatin levels in human dental pulp tissues increase with age and are upregulated in vitro in hDPCs during premature senescence activated by H₂O₂, suggesting a correlation between visfatin and senescence. In addition, visfatin knockdown by small interfering RNA led to the reduction in hDPC senescence; however, treatment with exogenous visfatin protein induced the senescence of hDPCs along with increased NADPH consumption, which was reversed by FK866, a chemical inhibitor of visfatin. Furthermore, visfatin-induced senescence was associated with both the induction of telomere damage and the upregulation of senescence-associated secretory phenotype (SASP) factors as well as NF-κB activation, which were all inhibited by FK866. Taken together, these results demonstrate, for the first time, that visfatin plays a pivotal role in hDPC senescence in association with telomere dysfunction and the induction of SASP factors.