In the present study, we found that a low concentration of genistein (1 μg/ml) did not alter cell morphology in UtLM cells and UtSMCs; however, it did alter cell proliferation dynamics in UtLM cells, but not UtSMCs. Although in vitro
and in vivo
reports on the morphologic and proliferative effects of genistein in human uterine leiomyoma and myometrial cells are lacking, a few studies have assessed the effects of structurally similar compounds on these cell types. Kawaguchi et al. (1985)
investigated the effects of estradiol (E2
M) on smooth muscle cells from the explants of myometrium and leiomyomas, and found that a low concentration of E2
caused both cell types to proliferate, as indicated by a marked increase in cell numbers from day 7 to 14. Furthermore, both cell types treated with E2
showed similar ultrastructural features to controls, and E2
did not significantly affect the “differentiation” or change the shape or size of either cell type (Kawaguchi et al., 1985
). Our findings are similar to the above study. However, the absence of a proliferative effect in the UtSMCs is in contrast with the E2
-treated cells in the Kawaguchi et al. (1985)
study and could be related to the fact that genistein is weakly estrogenic and a less potent inducer of cell growth in UtSMC. Our findings are also in agreement with an in vitro
study of Eker rat leiomyoma cells in which low concentrations of genistein were proliferative and high concentrations were inhibitory (Hunter et al., 1999
). Taken together, these data suggest that low concentrations of an estrogenic compound have no effect on the morphology of uterine leiomyoma or myometrial cells.
Newbold et al. (2002)
found that CD-1 mice prenatally exposed to low (2.5 μg/kg and 10 μg/kg) concentrations of the synthetic, environmental estrogen, diethylstilbestrol (DES), had an increased incidence later in life of uterine leiomyomas that had histologic characteristics typical of uterine leiomyomas in women (Newbold et al., 2002
). Interestingly, previous findings by McLachlan et al. (1980)
revealed that low (2.5 μg/kg and 10 μg/kg) concentrations of DES in CD-1 mice treated at days 9 and 16 of gestation lead to the development of uterine leiomyomas in 12 to 18-month-old offspring; whereas a high (100 μg/kg) concentration of DES was often teratogenic and caused smooth muscle hypoplasia and underdevelopment, and thus no leiomyomas. The effects of prenatal DES exposure have also been evaluated in women. One study has shown that premenopausal women who reported prenatal exposure to DES show increased risk for fibroids compared with non-exposed women, and that the tumors tend to be larger in the exposed women (Baird and Newbold, 2005
). In contrast, Wise et al. (2005)
found no association between prenatal exposure and risk of fibroids in women, and instead determined that prenatal DES exposure was associated with an increased risk of paraovarian cysts. Collectively, however, the human data support the concept that estrogenic exposures during development can promote reproductive tract abnormalities later in life.
Genistein is believed to elicit a concentration dependent dual threshold effect with regard to growth stimulation or inhibition. Studies using MCF-7 cells have shown that concentrations ≤1 μM genistein stimulate cell growth (Wang et al., 1996
; Chen and Wong, 2004
), whereas concentrations ≥10 μM inhibit growth (Le Bail et al., 1998
; Maggiolini et al., 2001
). The lower concentrations of 0.001 (0.0037 μM)-1 μg/ml (3.7 μM) of genistein, selected for our study, are within the physiological range (reported to be ≤1 μM) (Bouker and Hilakivi-Clarke, 2000
), and the higher concentrations of 10 (37 μM)-50 μg/ml (185 μM) are within pharmacological (>10 μM) concentrations (Bouker and Hilakivi-Clarke, 2000
). It appears to be unclear whether high in vitro
concentrations can be achieved in vivo
(de Lemos, 2001
). Most of the in vitro
experiments have used concentrations that have exceeded 10 μM; however, based on pharmacokinetic calculations involving daily intake of isoflavones, following absorption from the gut, distribution to peripheral tissues and excretion, it is unlikely that blood isoflavone concentrations even in high soy consumers would be greater than in the 1-5 μM range (Barnes et al., 1996
). Although a study has shown that healthy Japanese men and women consuming a single, high dose of genistein have plasma concentrations of >20 μM at 4 h after initial consumption, but by 24 h the plasma levels decrease (< 5 μM) (Izumi et al., 2000
) and are within the physiological range.
In order to ascertain a mechanism by which genistein produced stimulatory and inhibitory effects in the UtLM cells and UtSMC, we assessed cell proliferation using a MTS-based assay and PCNA labeling. Flow cytometry was used to evaluate percentage of cells in the S-phase and apoptosis. We found that UtLM cells, but not UtSMC, treated with a low concentration of genistein showed significant increases in cell proliferation and PCNA labeling; whereas a high concentration of genistein resulted in significant decreases in cell proliferation and PCNA labeling and significant increases in apoptosis for both cell types, with the percentage of apoptosis in UtSMC being triple that of UtLM cells. Our findings are in agreement with two studies using MCF-7 cells in that treatment of MCF-7 cells with a low concentration (1 μM) of genistein stimulated cell growth (Le Bail et al., 1998
) and increased the percentage of S-phase cells, while decreasing the percentage of quiescent to gap 1 (G0/G1; resting) phase cells (Chen and Wong, 2004
). Chen and Wong (2004)
attributed the increased proliferation to the mitogenic ability of genistein to stimulate gap 1 to synthesis (G1/S) transition in the cells. However, another study has shown that treatment of MCF-7 cells with 1 μM of genistein caused increased synthesis of cyclin D1
, activation of Cdk2 and induced hyperphosphorylation of pRb105, indicating genistein stimulated the entrance of MCF-7 cells into the cell cycle (Dees et al., 1997
). Our results suggest that genistein induces the progression of UtLM cells from the G0/G1 phase to the S-phase, ultimately leading to cell proliferation. Contrary to the effects of a low dose of genistein, in our study, higher concentrations (10-50 μg/ml) of genistein reduced cell growth in both the UtLM cells and UtSMCs, which is similar to what has been reported for MCF-7 cells (Le Bail et al., 1998
). Po et al. (2002)
also showed that treatment of MCF-7 cells with 10150 μM genistein resulted in a significant increase in apoptosis compared with control cells.
Indicators of apoptosis such as caspase-3 activity and nuclear morphology were assessed in both cell types in our study. Caspase-3 is an important biological mediator in the apoptotic pathway (Thornberry et al., 1997
). This executioner protein is involved in the extrinsic and intrinsic apoptotic pathways (Boatright and Salvesen, 2003
). We found that a high concentration of genistein significantly increased caspase-3 activity and apoptosis in both cell types. Furthermore, at the high concentration, UtSMCs caspase-3 activity was nearly double that observed in UtLM cells and supports the flow cytometry data in which we found nearly eight times the percentage of apoptotic UtSMCs compared with UtLM cells. Confocal microscopy and the Hoechst stain confirmed the presence of apoptotic bodies, primarily in the UtSMCs, whereas the UtLM cells did show some characteristics of apoptotic cells, although this was not a predominant feature of dying cells.
In summary, genistein’s concentration determines its effects on the morphology and cell proliferation of uterine leiomyoma and normal myometrial cells. Low (0.1 μg/ml and 1.0 μg/ml) concentrations of genistein significantly stimulated UtLM cell growth, but not UtSMC growth; high concentrations (10-50 μg/ml) of genistein inhibited the growth of both UtLM cells and UtSMCs. Our data show that UtLM cells are more responsive than UtSMCs to the proliferative effects of a low concentration of genistein, as indicated by an increase absorbance of cells in the MTS-based assay, enhanced PCNA labeling and the increased percentage of cells in the S-phase of the cell cycle. The increased responsiveness observed in UtLM cells could be due, in part, to enhanced transactivation of the ER, and/or activation or up-regulation of transcription factors, growth factor peptides, receptor tyrosine kinases and their downstream effector kinases (MAPK/ERK 1/2), which have all been shown to be up-regulated or activated in response to treatment with 17β E2
in UtLM cells, but not in UtSMCs (Barbarisi et al., 2001
; Swartz et al., 2005
). Conversely, a high dose of genistein induced more severe apoptotic effects in the UtSMCs possibly due to early (data not shown) and sustained up-regulation of caspase-3 activity leading to activation of apoptotic pathways which was not observed in the UtLM cells. Although cell death occurred in the UtLM cells at the higher concentration, it appears that the mechanism of death was mainly by a non-apoptotic route. UtLM cells are neoplastic cells and may be programmed towards cell survival and proliferation; therefore, they might be less susceptible to the apoptotic inducing effects of genistein. Our in vitro
model provides some insight on the differential effects of low and high concentrations of a phytoestrogen on uterine leiomyoma and uterine smooth muscle cell proliferation and death.