Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells

Abstract

We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 µM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells.

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

“…These results indicate the specificity of DFMO and CGP 48664 in inhibiting the appropriate biosynthetic enzymes. DFMO caused a 50-70% decrease in putrescine and an up to 90% decrease in spermidine levels in MCF-7 cells [34,39]. However, spermine levels were not significantly altered.…”

“…3) [37,38]. MCF-7 cells treated with 1 mM DFMO or 1 mM CGP 48664 showed comparable levels of growth inhibition [39]. If DFMO was added to the cells with spermidine, growth inhibition could be prevented [7].…”

“…However, spermine levels were not significantly altered. Treatment of MCF-7 cells with CGP 48664 resulted in an 50% decrease in spermidine and spermine, but a sixfold increase in putrescine level, compared with the control [38,39]. Inhibition of SAMDC by CGP 48664 in other cell types has also resulted in compensatory increases of putrescine levels [37].…”

“…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.…”

“…Alternatively, in the presence of polyamine analogues or metabolic inhibitors, decreased expression of growth-stimulatory genes or increased expression of genes in the cell death pathway can force cells to the choice of cell death. In addition, specific associations between transcription factors, coactivators and the transcription machinery appear to be sensitive to polyamine action [39]. Similarly, interactions between mediators of cell death, loss of the integrity of mitochondria [71] as well as activation of caspases [120] might be part of cell death induced by excessive accumulation or depletion of polyamines under specific cellular contexts.…”