Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: Alterations in polyamine biosynthesis and the expression of p21/CIP1/WAF1

Abstract

We examined the effects of epidermal growth factor (EGF) on MDA-MB-468 cells to understand its mechanism of action in an EGF receptor-rich breast cancer cell line. EGF inhibited the growth of MDA-MB-468 cells with an IC50 of 1.5 +/- 0.5 nM, as determined by measurements of DNA content of cells in culture over a period of 4 to 6 days. This growth inhibition included apoptosis 24 h after EGF addition, as detected by an enzyme-linked immunosorbent assay (ELISA) and Hoechst 33342 staining. In EGF-treated cells, peak activities of two key enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC), were reduced by 57% and 83%, respectively. EGF treatment also caused a 30 to 50% decrease in cellular putrescine at all time points tested (12 to 48 h). EGF-induced inhibition of DNA synthesis was also partially reversed by the addition of putrescine or spermidine, but not by spermine. Western blot analysis of cell cycle regulatory proteins showed that EGF-mediated growth inhibition was associated with the induction of p21, an inhibitor of cyclin-dependent kinases. However, EGF had no significant effect on the expression of cyclin D1 or cyclin E. Furthermore, putrescine reversal of EGF effects was associated with the down-regulation of EGF-induced p21. These results suggest that the mechanism of growth inhibition by EGF in MDA-MB-468 cells include a down-regulation of polyamine biosynthesis and the induction of p21. Identification of growth regulatory pathways in breast cancer cells might be useful in the development of novel targets for therapeutic intervention.

Methionine Restriction and Cancer Biology

“…Polyamines are involved in the growth and proliferation of eukaryotic cells. Therefore, during polyamine depletion, there is a disruption in the cell cycle, inducing apoptosis [58,59]. Studies have shown that elevated levels of polyamines are associated with increased tumor growth [57].…”

“…Inhibitors of polyamine metabolism, such as alpha-difluoromethylornithine, lead to a reduction in polyamine production, and a disruption of the cell cycle and DNA synthesis in cancer cells [60,61]. In addition, other studies suggest that the mechanism of growth inhibition includes a downregulation of polyamine biosynthesis and the induction of the cyclin-dependent kinase inhibitor, p21 [59,62]. In cancer, there is often a disruption in polyamine metabolism leading to an increase in oxidative damage [56].…”

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

“…Interestingly, polyamine analogues are also active in p53-negative cell lines, indicating the interaction of polyamines with several targets in the cell death pathway. Our studies on the role of polyamines in epidermal growth factor (EGF)-induced apoptosis of MDA-MB-468 breast cancer cells also illustrate an inverse relationship between polyamine levels and induction of p21 [60]. Thus, the mechanism of the antiproliferative action of polyamine analogues and biosynthetic inhibitors includes disruption of the func-…”

Vascular smooth muscle cell‐specific regulation of cyclin‐dependent kinase inhibitor p21WAF1/Cip1 transcription by Sp1 is mediated via distinct cis‐acting positive and negative regulatory elements in the proximal p21WAF1/Cip1 promoter

“…p21 WAF1/Cip1 levels and expression can be influenced by a variety of agents such as FGF [Johnson et al, 1998; Kivinen and Laiho, 1999], EGF [Thomas et al, 1999], TGF‐beta; [Kivinen and Laiho, 1999; Yoo et al, 1999; Pardali et al, 2000], IL‐6 [Bellido et al, 1998], PMA [Shanmugam et al, 2001; Lai et al, 2002], and DNA damaging agents [Martinez et al, 2002]. The expression of p21 WAF1/Cip1 can also be modulated by transcription factors that bind to cis ‐regulatory elements located within the promoter [reviewed by Gartel and Tyner, 1999].…”