Activation of nuclear factor-kappaB (NFkappaB) identifies a high-risk subset of hormone-dependent breast cancers

Activation of nuclear factor-kappaB (NFkappaB) has been linked to the development of hormone-independent, estrogen receptor (ER)-negative human breast cancers. To explore the possibility that activated NFkappaB marks a subset of clinically more aggressive ER-positive breast cancers, NFkappaB DNA-binding was measured in ER-positive breast cancer cell lines and primary breast cancer extracts by electrophoretic mobility shift assay and ELISA-based quantification of specific NFkappaB p50 and p65 DNA-binding subunits. Oxidant (menadione 100 microMx30 min) activation of NFkappaB was prevented by pretreatment with various NFkappaB inhibitors, including the specific IkappaB kinase (IKK) inhibitor, parthenolide (PA), which was found to sensitize MCF-7/HER2 and BT474 but not MCF-7 cells to the antiestrogen tamoxifen. Early stage primary breast cancers selected a priori for lower ER content (21-87 fmol/mg; n=59) and known clinical outcome showed two- to four-fold increased p50 and p65 NFkappaB DNA-binding over a second set of primary breast cancers with higher ER content (>100 fmol/mg; n=22). Breast cancers destined to relapse (13/59) showed significantly higher NFkappaB p50 (but not p65) DNA-binding over those not destined to relapse (46/59; p=0.04). NFkappaB p50 DNA-binding correlated positively with several prognostic biomarkers; however, only NFkappaB p50 DNA-binding (p=0.04), Activator Protein-1 DNA-binding (AP-1; p相似文献   

The regulation of inducible nitric oxide synthase gene expression induced by lipopolysaccharide and tumor necrosis factor-alpha in C6 cells: involvement of AP-1 and NFkappaB

The roles of AP-1 and NFkappaB in the regulation of inducible nitric oxide synthase (iNOS) mRNA expression induced by the combination of lipopolysaccharide and tumor necrosis factor-alpha (LT) in C6 cells were examined in the present study. The iNOS mRNA level and NO release were increased by several cytokines alone or combination treatments at 24 hr. LT-induced iNOS mRNA level was maximally increased at 6 hr and maintained at higher level at least up to 24 hr. At 6 hr, iNOS protein level and NO release were also increased by LT. By western blot analysis, AP-1, such as Fra-1, Jun B, and phospho-CREB protein levels were increased by LT and translocation of NFkappaB p52 from the cytoplasm to the nucleus was increased. In addition, phosphorylations of MAPKs (ERK 1/2, p38, JNK 1/2) were increased by LT. LT-induced iNOS mRNA level was inhibited by PD98059 (MEK 1/2 inhibitor), SB203580 (p38 inhibitor), and cycloheximide (a protein synthesis blocker), indicating that the phosphorylation of ERK 1/2 and p38, and on-going protein synthesis are necessary for LT-induced iNOS expression. Electrophoretic mobility shift assay (EMSA) showed that AP-1 and NFkappaB DNA binding activities were increased at 6 hr and these AP-1 and NFkappaB DNA bands increased by LT were super-shifted when Fra-1, Jun B, or NFkappaB p50 antibody was coincubated. These findings strongly suggest that, in C6 cells, Fra-1, Jun B, NFkappaB p50, and NFkappaB p52 appear to be involved in the regulation of iNOS mRNA induced by LT.