Abstract
The potent mitogenic activity of insulin-like growth factor I (IGF-I) on breast epithelium is inhibited by retinoic acid in oestrogen receptor-positive (ER+) breast cancer cell lines. We studied and compared the effects of N-(4-hydroxyphenyl)-retinamide (4-HPR) in terms of growth inhibition and modulation of the IGF-I system in ER+ (MCF-7) and oestrogen receptor-negative (ER-) (MDA-MB231) breast cancer cell lines. Treatment with 1-10 microM 4-HPR for up to 96 h induced a dose- and time-dependent inhibition of proliferation in both breast cancer cell lines. Induction of apoptosis was much more evident in MCF-7 than in MDA-MB231 cells (30-40% compared with 0-5% respectively at 5 microM for 48 h). Exogenous human recombinant IGF-I (hr-IGF-I)-stimulated cell proliferation was abolished by 1 microM 4-HPR in MCF-7 cells. Immunoreactive IGF-I-like protein concentration in conditioned medium was reduced by 38% in MCF-7 and by 90% in MDA-MB231 cell lines following treatment for 48 h with 5 microM 4-HPR. Western ligand blot analysis showed a reduction of IGF-binding protein 4 (BP4) and BP5 by 67% and 87%, respectively, in MCF-7, whereas IGF-BP4 and -BP1 were reduced by approximately 20% in MDA-MB231 cells. Exposure to 5 microM 4-HPR for 48 h inhibited [125I]IGF-I binding and Scatchard analysis revealed a decrease of more than 50% in maximum binding capacity (Bmax) and a reduced receptor number/cell in both cancer cell lines. Steady-state type I IGF-receptor mRNA levels were reduced by approximately 30% in both tumour cell lines. We conclude that 4-HPR induces a significant down-regulation of the IGF-I system in both ER+ (MCF-7) and ER- (MDA-MB231) breast cancer cell lines. These findings suggest that, in our model, interference with the ER signalling pathway is not the only mechanism of breast cancer growth inhibition by 4-HPR.
Highlights
(MCF-7) and ER- (MDA-MB231) breast cancer cell lines
Summary The potent mitogenic activity of insulin-like growth factor (IGF-I) on breast epithelium is inhibited by retinoic acid in oestrogen receptor-positive (ER+) breast cancer cell lines
We studied and compared the effects of N-(4-hydroxyphenyl)-retinamide (4-HPR) in terms of growth inhibition and modulation of the IGF-I system in ER+ (MCF-7) and oestrogen receptor-negative (ER-) (MDA-MB231) breast cancer cell lines
Summary
4-HPR was kindly provided by RW Johnson Pharmaceutical Research Institute, Spring House, PA, USA. Aliquots of concentrated CM from treated (5 gM 4-HPR for 48 h) and untreated cells were analysed to determine the concentration of IGF-I-like material using a specific radioimmunoassay (RIA), as previously described (Favoni et al, 1995). A separate assay for binding proteins was performed to ensure complete BP removal: heparintreated samples were dried and resuspended in 0.1I% BSA-PBS containing 30 000 counts per minute of radiolabelled IGF-I and incubated overnight at 4°C. Binding of IGF-I to its specific cell-surface type I IGF receptor (IGF-R) and its modulation by 4-HPR was studied by radioreceptor assay, using the procedure previously described (de Cupis et al, 1995). Statistical significance of the experimental results, reported as a mean percentage ± standard error, was evaluated by the nonparametric Wilcoxon test
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