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Estrogenic and Antiproliferative Properties of Glabridin from Licorice in Human Breast Cancer Cells
Tamir S, Eizenberg M, Somjen D, et al. Cancer Res October 15, 2000 60; 5704
There is an increasing demand for natural compounds that improve women’s health by mimicking the critical benefits of estrogen to the bones and the cardiovascular system but avoiding its deleterious effects on the breast and uterus. The estrogenic properties of glabridin, the major isoflavan in licorice root, were tested in view of the resemblance of its structure and lipophilicity to those of estradiol. The results indicate that glabridin is a phytoestrogen, binding to the human estrogen receptor and stimulating creatine kinase activity in rat uterus, epiphyseal cartilage, diaphyseal bone, aorta, and left ventricle of the heart. The stimulatory effects of 2.5–25μ g/animal glabridin were similar to those of 5 μg/animal estradiol. Chemical modification of glabridin showed that the position of the hydroxyl groups has a significant role in binding to the human estrogen receptor and in proliferation-inducing activity. Glabridin was found to be three to four times more active than 2′-O-methylglabridin and 4′-O-methylglabridin, and both derivatives were more active than 2′,4′-O-methylglabridin. The effect of increasing concentrations of glabridin on the growth of breast tumor cells was biphasic. Glabridin showed an estrogen receptor-dependent, growth-promoting effect at low concentrations (10 nm–10μ m) and estrogen receptor-independent antiproliferative activity at concentrations of >15 μm. This is the first study to indicate that isoflavans have estrogen-like activities. Glabridin and its derivatives exhibited varying degrees of estrogen receptor agonism in different tests and demonstrated growth-inhibitory actions on breast cancer cells.
In the present study we characterized glabridin, a novel phytoestrogen isolated from licorice extract. Glabridin and its derivatives bind to the human ER and were found to act as an estrogen agonist in the induction of an estrogen response marker, such as CK activity, in vivo, to induce uterus wet weight, and to stimulate human breast cancer cell growth.

Glabridin bound to the human ER with about the same affinity as genistein, the best known phytoestrogen, 104 times lower than estradiol (16 , 31) . It not only competed with 3H-labeled estradiol in binding the human ER but also enhanced the proliferation of estrogen-dependent human breast cancer cells in vitro. Growth stimulation of ER+ cells by glabridin closely correlated to its binding affinity to the ER. Stimulation of cell proliferation was optimal at a concentration at which about half of the ER sites were saturated. The concentrations in which we observed the proliferative effects of glabridin (100 nm–10 μm) are well within the reported in vitro range of other phytoestrogens, such as genistein, diadezein, and resveratrol from grapes (32, 33, 34, 35) .

To provide some more insight into what effect glabridin has on breast tumor cells stimulated by estradiol and cell proliferation arrested by antiestrogen, we treated cells with glabridin in the presence of estradiol or tamoxifen. We found that glabridin, like genistein, had little effect on the growth-promoting effect of estradiol in the range of 0.1 nm–1.0 μm. Tamoxifen at 1μ m inhibited the optimal growth of cells treated with glabridin by 50%, and at 5 μm the effect of glabridin was blocked completely. This suggests that the growth-promoting effect of glabridin, like that of other phytoestrogens, is ER mediated (15 , 36) .

To further confirm that glabridin is a phytoestrogen acting via an ER mechanism, we evaluated in vivo the stimulation of CK activity in estrogen-responsive tissues. This specific activity, as a sensitive and rapid postreceptor response marker, was used in other ER-containing cells, such as skeletal cells, containing a low concentration of steroid hormone receptors (30) . The brain type isoenzyme of CK, the major component of the estrogen-induced protein of rat uterus, is part of the energy buffer system that regenerates ATP from ADP and has been a useful marker for the action of steroids and their analogues (30) . Our results demonstrated that the administration of 25 μg/rat glabridin doubled CK activity in skeletal and cardiovascular tissues. These results not only confirm that glabridin acts through the ER but also suggest that it has the potential to mimic the beneficial activities of estrogen in bone and cardiovascular tissues.

It was also shown in vivo that glabridin acts as estrogen agonist in the uterus. Two hundred μg/rat glabridin increased the uterine wet weight to the same extent as 5 μg of estradiol. The determination of uterine wet and/or dry weights has also been used to demonstrate estrogenic activity by other phytoestrogens (37 , 38) . Markaverich et al. (39) reported that an increase in uterine wet and dry weight in ovariectomized animals induced by coumesterol is not indicative of uterine hyperplasia, as determined by a doubling in DNA content, but reflects an increase in water and protein content. Therefore, the potential estrogenicity of glabridin requires reassessment before defining the relationships between glabridin exposure and neoplasia in uterine endometrium.

In contrast to the ER-regulated, growth-promoting effects of glabridin at concentrations ranging from 100 nm to 10μ m, higher concentrations (>10 μm) abruptly inhibited the proliferation of ER+ and ER− breast cancer cells. The same biphasic effect was demonstrated in the anchorage-independent growth of human breast cancer cells in soft agar. Interestingly, neither estradiol nor tamoxifen reversed the antiproliferative effect of glabridin. These results are consistent with those previously reported (32 , 33) , observing growth stimulation by genistein in a concentration-dependent manner between 10 nm and 1 μm and growth inhibition of MCF7 cells at concentrations of >10 μm. Fioravanti et al. (40) and Shao et al. (41) reported that genistein-treated cells accumulated in S and G2-M and underwent apoptosis. On the other hand, in preliminary results, glabridin treatment using two different methods suggested that apoptosis may not be involved (data not shown). The most plausible explanation for this biphasic effect of glabridin on human breast cancer cells is not only that it mediates its actions not only via the ER as an estrogen agonist but also that at higher concentrations it interacts with other ER-independent cellular mechanisms to inhibit cell proliferation induced by glabridin via ER pathways. Recent studies have observed antiproliferative effects of genistein in other, non-breast carcinoma cell lines (42) . In the present study, glabridin inhibited the growth of ER− cells (MDA-MB-468), supporting the hypothesis that the actions of phytoestrogens on ER and on cell growth inhibition occur via different molecular mechanisms (36 , 41 , 43) . Some studies suggest that high concentrations of phytoestrogens may function as estrogen antagonists and inhibit cell growth by competing with estradiol on binding to the ER site (44) . In the present study, glabridin overrode the growth-inhibitory effects of tamoxifen, demonstrating that the inhibitory action of glabridin on tumor growth is different from that of other known antagonists, such as tamoxifen, because the mechanism of its action is not ER dependent. Further studies for understanding the mechanism are required.

To shed some light on the role of the two hydroxyl groups attached to the glabridin molecule in its ability to bind to the human ER and in its growth-promoting effect, natural and semisynthetic glabridin derivatives were tested. Our results showed that, among the isoflavans examined, glabridin has higher affinity to ER and also showed optimal cell growth stimulation. Weaker estrogen agonists than glabridin, 2′-O-MG and 4′-O-MG, were nearly as potent as glabridin as growth inhibitors. However, 2′,4′-O-MG did not bind to the human ER and demonstrated no proliferative activity. This suggests that the two hydroxyl groups in the glabridin are essential to binding and to promoting cell growth. The agonist effects were higher when both hydroxyl groups were present than with a single group. The data also demonstrate that the growth-inhibitory effects of these compounds are not related to their binding or proliferative capability. Previous reports on the involvement of the two hydroxyl groups of estradiol in binding to the human ER demonstrated that both hydroxyl groups 3 and 17β are required for binding (45 , 46) . Our data suggest that hydroxyl 4′ of glabridin may have the same role as hydroxyl 3 of estradiol. Glabridin lacks the additional hydroxyl group of estradiol at position 17β, but it has an ether oxygen on a parallel position, which could contribute a weaker hydrogen bond to histidine 524 at the ligand-binding domain.

The present study demonstrates for the first time that the isoflavan glabridin is a new phytoestrogen. It bound to human ER and activated CK in estrogen-responsive tissues in vivo. The results also reveal that glabridin inhibits the growth of breast cancer cells independently of ERs. This suggests that isoflavans may serve as natural estrogen agonists in preventing the symptoms and diseases associated with estrogen deficiency.

Glabridin, an isoflavan from licorice root, inhibits migration, invasion and angiogenesis of MDA-MB-231 human breast adenocarcinoma cells by inhibiting focal adhesion kinase/Rho signaling pathway.
Hsu YL, Wu LY, Hou MF, et al. Mol Nutr Food Res. 2011 Feb;55(2):318-27. doi: 10.1002/mnfr.201000148.
In this study we first report the antimigration, antiinvasive effect of glabridin, a flavonoid obtained from licorice, in MDA-MB-231 human breast adenocarcinoma cells.
Glabridin exhibited effective inhibition of cell metastasis by decreasing cancer cell migration and invasion of MDA-MB-231 cells. In addition, glabridin also blocked human umbilical vein endothelial cells (HUVEC) migration and decreased MDA-MB-231-mediated angiogenesis. Further investigation revealed that the inhibition of cancer angiogenesis by glabridin was also evident in a nude mice model. Blockade of MDA-MB-231 cells and HUVEC migration was associated with an increase of αγβ3 integrin proteosome degradation. Glabridin also decreased the active forms of FAK and Src, and enhanced levels of inactivated phosphorylated Src (Tyr 416), decreasing the interaction of FAK and Src. Inhibition of the FAK/Src complex by glabridin also blocked AKT and ERK1/2 activation, resulting in reduced activation of RhoA as well as myosin light chain phosphorylation.
This study demonstrates that glabridin may be a novel anticancer agent for the treatment of breast cancer in three different ways: inhibition of migration, invasion and angiogenesis.

 
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