Activation of Nuclear Factor κB by Polyamines in Breast Cancer Cells

Abstract

Polyamines-putrescine, spermidine, and spermine-are involved in the growth of breast cancer cells. A possible target of polyamine action is at the site of interaction of transcription factors with their response elements. NF-kappaB is a member of the rel family of transcription factors that regulate transcription of genes in the proliferative/anti-apoptotic pathways. We performed electrophoretic mobility shift assays to study the role of polyamines in NF-kappaB binding to NF-kappaB response elements (NREs), the consensus sequence of which is GGGGAATTCCCC. Using cellular extract from MCF-7 breast cancer cells, we found very little binding of NF-kappaB to NRE in the absence of polyamines. Addition of 1 mM spermidine or spermine caused a 4- and 6-fold increase in NF-kappaB-NRE binding, respectively. Putrescine induced a 2-fold increase in the binding at 2 mM concentration. Using antibody supershift assays, we identified the p50 subunit of NF-kappaB to be a major component in NF-kappaB-NRE complex formation in the presence of polyamines. However, the decreased intensity of the band corresponding to NF-kappaB-NRE complex in the presence of anti-p65, c-rel, relB and p52 antibodies suggested the participation of these subunits also. Spermine also stimulated NF-kappaB-NRE binding using cellular extracts from other breast cancer cell lines and a normal breast epithelial cell line. A differential effect of spermine analogues on NF-kappaB-NRE binding was observed, with spermine exerting the maximal effect. CD spectra of NRE containing oligonucleotides was asymmetric and distinct from that of a typical B-DNA CD spectrum. A concentration-dependent increase in T(m) of the duplex NRE was seen in the presence of polyamines. In transient transfection experiments using an NF-kappaB driven secreted alkaline phosphatase (SEAP) reporter, spermine induced NF-kappaB activity by approximately 2-fold as compared to controls. Spermine induced activation of NF-kappaB was also confirmed using an NF-kappaB-EGFP (enhanced green fluorescent protein) vector in transient transfections in which expression of the green fluorescent protein was visualized by fluorescence microscopy. These data show a gene regulatory function of polyamines involving enhanced binding of NF-kappaB to NRE and a possible mechanism for the action of polyamines in breast cancer cell proliferation.

Polyamine Depletion Induces Rapid NF-κB Activation in IEC-6 Cells

“…The polyamine spermine activates NF-B in human breast cancer cells (21,22). These findings appear to be in conflict with the results reported herein.…”

Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications

“…Results of these studies and recent results from a spermidine/spermine acetyl transferase (SSAT) overproducing transgenic mice [25] indicate that polyamines and/or their analogues do not need to be oxidized for the induction of apoptosis, at least in some cell types. Excessive accumulation or depletion of polyamines may disrupt many cellular functions, including DNA-protein interactions [70], protein-protein interactions [39] and mitochondrial integrity [71], leading to apoptosis. In addition, cell types that undergo apoptosis in the presence of ODC overexpression might be highly sensitive to apoptosis.…”

Polyamine Analogues Bind Human Serum Albumin

“…Association of polyamines with nucleic acids and protein is included in their mechanism of action. The aim of this study was to examine the interaction of human serum albumin (HSA) with several polyamine analogues such as 1,3,7,11,7,11,15, in aqueous solution at physiological conditions, using a constant protein concentration and various polyamine contents (μM to mM). FTIR, UVvisible and CD spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure.…”