Role of c-myc in tamoxifen-induced apoptosis in estrogen-independent breast cancer cells

Background: The antiestrogen tamoxifen (TAM) is effective in the treatment of estrogen receptor (ER)-positive as well as some ER-negative breast cancers. However, the precise mechanism of action of TAM, especially in estrogen-independent cells, remains unclear. Previous work by our laboratory has demonstrated that TAM induces the morphologic and biochemical changes that are characteristic of apoptosis in both PR-positive and PR-negative cells. Purpose: We compared the effect of TAM at a clinically achievable concentration on cell growth and apoptosis with the effect of TAM on c-myc (also known as C-MYC) messenger RNA (mRNA) and protein expression in PR-negative MDA-231 cells. Methods: MDA-231 cells were treated for up to 72 hours with 1.0 mu M TAM alone or in the presence of 50 mu M c-myc antisense or nonsense oligonucleotides, c-myc mRNA expression was determined by northern blot analysis, protein expression by western blot analysis, cell growth inhibition by cell counts, and DNA cleavage by agarose gel electrophoretic analysis. Differences between the mean values from different treatment groups were compared with the use of the two-sided Wilcoxon rank-sum test. Results: TAM treatment for 72 hours increased c-myc mRNA fivefold (from a relative radiolabeled hybridization signal intensity of 17 +/- 4 up to 93 +/- 10; P<.05) and c-Myc protein threefold (from a relative immunofluorescence signal intensity of 28 +/- 7 up to 83 +/- 21; P<.05). The induction of c-myc by TAM was accompanied by internucleosomal DNA cleavage characteristic of apoptotic cell death, Addition of c-myc antisense oligonucleotide (5'-CACGTTGAGGGGCAT-3') to MDA-231 cells resulted in a nearly twofold decrease of basal c-myc mRNA (P<.05) and a sevenfold decrease of basal c-Myc protein (P<.05) expression. Addition of c-myc antisense oligomer also antagonized the TAM-induced increase in c-myc mRNA (P<.05) and protein expression (P<.05) and inhibited TAM-induced cytostasis (P<.01) and apoptosis. In parallel experiments, addition of the nonsense oligomer had no effect on any of the measured parameters. Conclusions: These results indicate that the effects of TAM on PR-negative MDA-231 cells may be mediated through c-myc overexpression, c-myc may play a critical role in the growth and progression of MDA-231 breast cancer cells.