All trans-retinoic acid acts synergistically with hydroxytamoxifen and transforming-growth factor beta to stimulate apoptosis in MCF-7 breast cancer cells

The anti-estrogen 4-hydroxytamoxifen (TAM) and vitamin A-related compounds, the retinoids, in combination act synergistically to inhibit growth of breast cancer cells in vitro and in vivo. To clarify the mechanism of this synergism, the effect of TAM and all trans-retinoic acid (AT) on proliferation of MCF-7 breast cancer cells was studied in vitro. TAM and AT acted synergistically to cause a time-dependent and dose-dependent inhibition of MCF-7 cell growth. In a temporally related manner, TAM+AT acted synergistically to downregulate Bcl-2 mRNA and Bcl-2 protein expression, and to stimulate apoptosis. TAM and AT each blocked cell cycle progression throughout 7 days of treatment but without any synergistic or additive effect on this process, indicating a selective synergism for apoptosis. The negative growth factor-transforming growth factor beta (TGFbeta) is secreted by these cells and was studied as a potential mediator of the synergistic effects of TAM+AT on apoptosis. TAM+AT acted synergistically to induce a fivefold increase in TGFbeta1 secretion over 72 h. TGFbeta1 alone had no apoptotic effects on these cells; however, TGFbeta1 in combination with AT acted synergistically to inhibit growth, to downregulate Bcl-2 mRNA and Bcl-2 protein expression, and to stimulate apoptosis of these cells in a manner comparable with that noted for TAM+AT. The synergism of both TAM+AT and TGFbeta1+AT for apoptosis was suppressed by estradiol. Co-incubation of TAM+AT with anti-TGFbeta antibody did not block down-regulation of Bcl-2 protein expression or stimulation of apoptosis. The synergistic effects of TAM+AT on apoptosis therefore occur independently of TGFbeta, although TGFbeta may interact with AT in a novel manner to provide another important anti-proliferative mechanism for breast cancer cells.