Molecular correlates of the action of bis(ethyl)polyamines in breast cancer cell growth inhibition and apoptosis.

Polyamines are known to be involved in cell growth regulation in breast cancer. To evaluate the efficacy of bis(ethyl)polyamine analogs for breast cancer therapy and to understand their mechanism of action we measured the effects of a series of polyamine analogs on cell growth, activities of enzymes involved in polyamine metabolism, intracellular polyamine levels, and the uptake of putrescine and spermidine using MCF-7 breast cancer cells. The IC50 values for cell growth inhibition of three of the compounds, N1,N12-bis(ethyl)spermine, N1,N11-bis(ethyl)norspermine, and N1,N14-bis(ethyl)homospermine, were in the range of 1-2 microM. Another group of three compounds showed antiproliferative activity at about 5 microM level. These compounds are also capable of suppressing colony formation in soft agar assay and inducing apoptosis of MCF-7 cells. The highly effective growth inhibitory agents altered the activity of polyamine biosynthetic and catabolic enzymes and down-regulated the transport of natural polyamines, although each compound produced a unique pattern of alterations in these parameters. HPLC analysis showed that cellular uptake of bis(ethyl)polyamines was highest for bis(ethyl)spermine. We also analyzed polyamine analog conformations and their binding to DNA minor or major grooves by molecular modelling and molecular dynamics simulations. Results of these analyses indicate that tetramine analogs fit well in the minor groove of DNA whereas, larger compounds extend out of the minor groove. Although major groove binding was also possible for the short tetramine analogs, this interaction led to a predominantly bent conformation. Our studies show growth inhibitory activities of several potentially important analogs on breast cancer cells and indicate that multiple sites are involved in the mechanism of action of these analogs. While the activity of an analog may depend on the sum of these different effects, molecular modelling studies indicate a correlation between antiproliferative activity and stable interactions of the analogs with major or minor grooves of DNA.

Effects of genistein and structurally related phytoestrogens on cell cycle kinetics and apoptosis in MDA-MB-468 human breast cancer cells.

We have studied the effects of phytoestrogens (genistein, quercetin, daidzein, biochanin A and kaempferol) on proliferation, cell cycle kinetics, and apoptosis of MDA-MB-468 breast cancer cells. Genistein and quercetin inhibited cell growth with IC50 values of 8.8 and 18.1 muM, respectively, while the other phytoestrogens were less effective. Flow cytometric analysis showed G2/M cell cycle arrest with 25 muM and higher concentrations of genistein. At 100 muM, genistein, quercetin and kaempferol caused accumulation of 70, 60 and 35% of cells, respectively, in G2/M phase by 24 h. In contrast, biochanin A and daidzein were ineffective. APO-BRDU analysis revealed apoptosis with 10 muM genistein (19.5%), reaching 86% at 100 muM. Apoptosis by genistein was confirmed by Hoechst 33342 staining and fluorescence microscopy. With 100 muM quercetin, 47% of the cells were apoptotic, while the other bioflavonoids had little effect. Genistein treatment resulted in a biphasic response on cyclin B1: 70% increase in cyclin B1 level at 25 muM, and 50 and 70% decrease at 50 and 100 muM, respectively. In contrast, the action of quercetin involved an increase in cyclin B1 level. Genistein had no effect on cdc2 level up to 50 muM concentration; however, there was a decrease in the phosphorylated form of the protein at 100 muM. Quercetin had no effect on cdc2 levels. Our results suggest that the action of genistein and quercetin involves G2/M arrest and apoptosis in MDA-MB-468 cells. Biochanin A and daidzein, although structurally related to genistein, did not share this mechanism. Thus, structurally related phytoestrogens have discrete target sites and mechanisms in their growth inhibitory action on breast cancer cells.

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

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.