Currently, tamoxifen is the standard endocrine therapy for breast cancer in premenopausal women (27
) and also being widely used to treat breast cancer in postmenopausal women, either as monotherapy or combined with aromatase inhibitors (11
). Due to their reduced estrogen levels, menopausal women experience symptoms such as hot flashes, sleeping disorders and mood swings, which are exacerbated by antiestrogen treatment. Traditionally, HRT has been used to relieve these symptoms, but many oncologists do not recommend HRT to breast cancer patients due to the concern that it may increase the risk of tumor recurrence, which, directly or indirectly, results in 28–91% of breast cancer patients using one or more alternative therapies without the knowledge of their physicians (30
). This indicates that many breast cancer patients, possibly also being treated with tamoxifen, self-medicate with complementary treatments, including soy isoflavones, without proper guidance. Genistein, the major soy isoflavone, has been reported to bind to ER and exhibit weak but substantial estrogenic effects (6
). Thus, if genistein negates the effect of tamoxifen, the simultaneous consumption of genistein and tamoxifen may lead to an undesirable outcome for breast cancer patients.
We have demonstrated that estrogen silastic tubing implanted in athymic mice was able to produce blood estrogen at similar levels with those observed in postmenopausal women and thus is an appropriate model for postmenopausal breast cancer (32
). Using this postmenopausal animal model, we reported that 1000 p.p.m. genistein negated the inhibitory effect of tamoxifen on MCF-7 breast tumor growth in athymic mice implanted with a 0.25 mg E silastic tubing (18
). In the present study, we evaluated the effect of the interaction between tamoxifen and various doses of dietary genistein on mammary tumor growth using the previously utilized model but with a lower dose of estrogen silastic implant (0.08 mg) to better represent blood levels of estrogen in postmenopausal women. Our results revealed that the 0.08 mg E implant produced a circulating estrogen level of 119 ± 16 pg/ml (0.4 nM), whereas the implantation of a 1–2 mg estrogen pellet generated circulating estrogen levels of 2–3 nM in mice that were similar to levels observed in premenopausal women (32
). The low dose of estrogen presented here was sufficient to stimulate MCF-7 tumor growth and this stimulation effect was significantly inhibited by tamoxifen, indicating a successful establishment of a postmenopausal ER+ breast cancer model.
Previous reports have demonstrated that genistein can act as an estrogen agonist and stimulate ER+ cancer cell growth both in vitro
and in vivo
). Our laboratory has reported that 250, 500 and 1000 p.p.m. genistein increased MCF-7 tumor growth in a dose-dependent manner in the absence of exogenous estrogen and tamoxifen (14
). These doses of dietary genistein produced plasma levels of total genistein in the range of 1.4–3.3 μM (14
), which were similar to those in humans consuming high soy isoflavones-containing diets (34
). Thus, to evaluate the interaction between genistein and tamoxifen, the same doses of genistein were utilized and we hypothesized that genistein would abrogate the inhibitory effect of tamoxifen on tumor growth induced by estrogen in a dose-dependent manner. In the present study, we observed that the two lower doses of genistein (250 and 500 p.p.m.) abrogated the inhibitory effect of tamoxifen on tumor prevalence and tumor size of MCF-7 tumors in a dose-dependent manner, whereas the high dose of genistein (1000 p.p.m.) did not interfere with the antitumor action of tamoxifen. Our results were similar to an earlier study conducted by Liu et al.
) showing that a diet containing 211 μg/g total isoflavone abrogated the preventative effect of tamoxifen on MCF-7 tumor growth in MMTV-wt-erbB-2/neu transgenic mice. The percentage of tumor-free mice in this group was reduced to 46.8% when compared with 87.5% in the tamoxifen-treated group, whereas a diet containing 491 μg/g total isoflavone had no impact. These results suggested that low doses of dietary genistein may interfere with tamoxifen, thereby decreasing its efficacy on breast cancer treatment. However, there were some reports showing that genistein synergistically enhanced the preventive effect of tamoxifen on the growth of MCF-7 tumors in mice (22
). This inconsistency to our result was probably caused by the interaction of genistein with different estrogen and tamoxifen levels. In the present study, we used a lower E silastic implant that caused a more relevant tumor growth rate. We also used lower tamoxifen levels (3 mg in a slow releasing silastic implant), whereas Mai et al.
) used 25 mg in a more rapidly releasing pellet. Thus, the interaction of genistein with lower E2
and tamoxifen levels was the most likely reason for differences in the two studies.
The differential effect of dietary genistein at low lose or high dose observed here on tumor growth in mice bearing MCF-7 tumors in the presence of both estrogen and tamoxifen was probably due to the ability of genistein to exhibit an estrogenic effect at low doses through estrogen receptor-mediated pathways (15
) and an inhibitory effect at high doses via ER-independent mechanisms, such as induction of cell apoptosis and cell cycle arrest, protein tyrosine kinase inhibition and topo-isomerase II inhibition (39
). Although no apparent interaction with tamoxifen was observed on tumor growth in the current study, we previously showed that 1000 p.p.m. genistein counteracted the effect of tamoxifen (18
). In the previous study, we used a 0.25 mg estrogen implant with a 2.5 mg (E:T = 1:10) or a 5.0 mg (E:T = 1:20) tamoxifen implant. Here, to better represent plasma estrogen level in postmenopausal women, we reduced the amount of estrogen in the implant to 0.08 mg and used a 3 mg tamoxifen silastic tubing (E:T = 1:37.5) to block the action of exogenous estrogen. Thus, the 1000 p.p.m. genistein diet acted differently in the presence of high or low ratio of E to T implants, suggesting that the interaction of dietary genistein with tamoxifen was possibly affected by the ratio of E to T implants, thus, by the plasma estrogen and tamoxifen levels.
Previous analyses of plasma estrogens in postmenopausal breast cancer patients treated with tamoxifen have provided conflicting results (44
). In animal models, a trend of decrease in plasma estrogen levels was observed in the tamoxifen-treated mice, although it did not reach a level of statistical significance (18
). Results from the present study indicated that with the lower ratio of E to T implants, tamoxifen significantly reduced plasma estrogen level increased by E implants. Dietary genistein further reduced plasma estrogen levels, which was consistent with what was shown previously (18
). These results suggested that genistein can, in an undefined manner, reduce circulating estrogen levels, which may be an important factor in determining the overall effect of genistein on tumor growth.
Acquired resistance of tumor cells to tamoxifen treatment has been reported both in vitro
and in vivo
). Tumor cells may not react to tamoxifen if they progress from the estrogen-dependent stage to the estrogen-independent stage as observed in tamoxifen-resistant breast cancer (49
). Recently, tamoxifen downregulation of miR-451 and consequent elevation of the key survival factor 14-3-3zeta have been identified as a mechanistic basis of tamoxifen-associated development of endocrine resistance (51
), whereas genistein (25–50 μM) has been shown to inhibit the expression of 14-3-3 protein in head and neck cancer cells (52
). Although there is no report on the association of low doses of dietary genistein and the expression of 14-3-3 protein, based on what we have seen, it is possible that low doses of dietary genistein increases 14-3-3 expression in MCF-7 tumors and then elicits an additive effect with tamoxifen on endocrine resistance. Thus, to confirm that the abrogating effect of dietary genistein on tamoxifen-treated tumor growth was not due to tamoxifen resistance, we evaluated estrogen responsiveness of the tumors excised from mice treated with tamoxifen and genistein. The result indicated that these tumors retained estrogen responsiveness of the progenitor MCF-7 cells as the estrogen antagonists, tamoxifen and ICI 182
780, significantly inhibited cell proliferation induced by estrogen. This suggested that the growth of tumors in genistein-treated animals was most probably due to the effect from dietary genistein rather than a diminished response to tamoxifen.
Genistein has been reported to regulate MCF-7 cell growth at the transcriptional level. Genistein (1–10 μM) increased progesterone receptor levels in MCF-7 cells in a dose-dependent manner (39
) and increased cyclin D1
synthesis and then stimulated MCF-7 cells to enter the cell cycle in a manner similar to estrogen (53
). Thus, we further investigated the effect of genistein on the expression of PR
and cyclin D1
genes in the tumor cells in order to determine the possible underlying mechanisms by which genistein negated the inhibitory effect of tamoxifen on MCF-7 tumor growth. Our observation revealed that the interaction between dietary genistein and tamoxifen on gene expression, in addition to tumor growth, was also complicated. Tamoxifen treatment decreased the expression of PR
and cyclin D1
genes, while the effect of genistein on the inhibitory effect of tamoxifen on the gene expression was not consistent. Genistein at 1000 p.p.m. reversed the inhibitory effect of tamoxifen on PR
expression. Low doses of genistein negated the effect of tamoxifen on cyclin D1
expression in a dose-dependent manner, whereas 1000 p.p.m. genistein did not have the same effect. The observation on the gene expression of cyclin D1
was consistent with that observed on the PI:AI ratio, tumor prevalence and tumor growth, suggesting that genistein modulated the effect of tamoxifen on estrogen-induced tumor growth, potentially through the regulation of the cell cycle, in addition to acting through estrogen signaling.
In summary, the current study presents evidence that the low doses of dietary genistein negate the inhibitory effect of tamoxifen, in the presence of low blood estrogen levels, on ER+ MCF-7 tumor growth in athymic nude mice by regulating cell proliferation and apoptosis. This effect is also probably, at least in part, due to the regulation of the ER-dependent signaling pathway. Although the high dose of dietary genistein does not negate the inhibitory effect of tamoxifen on gross tumor size, it still results in high tumor prevalence. Therefore, the results from this study raise safety concerns of consuming dietary isoflavone supplements across a broad range of internal exposures in breast cancer patients, especially postmenopausal women, while on tamoxifen treatment.