In this study, we provide evidence that apigenin significantly delays the appearance of progestin-accelerated breast tumors in a DMBA-induced tumor model, reduces tumor multiplicity, and reduces tumor incidence. While several mechanisms have been proposed for the anticancer effects of apigenin, to our knowledge this is the first report indicating that this naturally occurring flavonoid might inhibit in vivo
tumor development by opposing the effects of progestins. Using cell culture, we previously showed that apigenin inhibits progestin-dependent induction of VEGF in human breast cancer cells (22
). With this in mind, we conducted a series of in vivo
studies aimed at determining whether apigenin might be used to prevent the emergence of mammary tumors, especially in women exposed to synthetic progestins as a component of E+P HRT. Our studies were focused on examining the effects of apigenin on MPA-accelerated mammary tumors in an animal model, because MPA is the most widely prescribed progestin and its use is associated with increased risk of breast cancer and mortality (1
Following the administration of apigenin to DMBA-treated animals that were also given MPA, tumor latency was significantly increased. Furthermore, tumor incidence decreased at least 50% in these animals, compared with animals that were treated with vehicle alone. Importantly, apigenin also reduced tumor multiplicity to levels below those normally observed following progestin treatment. It was interesting to note that, even though apigenin suppressed tumor development, it did not prevent the MPA-induced morphological changes and hyperplasia in the mammary gland of DMBA-treated animals, a finding in direct contrast to the effects of curcumin (16
), a natural plant derived curcuminoid with anti-cancer properties (30
). Surprisingly though, with respect to its ability to increase tumor latency and reduce the incidence of MPA-accelerated tumors in this model, apigenin was a more effective preventive compound than curcumin.
Apigenin has been reported to exert its anticancer effects via a variety of mechanisms, including induction of cell cycle arrest and apoptosis, through both tumor necrosis factor- α induced NFκB-mediated and intrinsic apoptosis pathways (31
) and attenuation of the phosphorylation of epidermal growth factor receptor and MAP kinase (18
). Compelling evidence from a variety of model systems suggests that VEGF is essential for tumor development and progression (11
), and it has been postulated that, unless tumors develop blood vessels, they will not grow beyond 2 mm3
). Our earlier in vitro
studies using human breast cancer cell lines (22
) have demonstrated that apigenin significantly reduces the expression of both VEGF and its receptor (VEGFR-2), an essential mediator of VEGF-dependent tumor cell proliferation (33
). It is therefore likely that, by suppressing VEGF expression, apigenin renders the mammary tissue microenvironment less conducive to tumor development.
Accumulating evidence has shown that many types of cancer, including breast cancer, are initiated by a small population of cancer stem cells (35
). Due to the rapid rate at which breast tumors develop in postmenopausal women taking E+P HRT, we and others have suggested that progestins most likely increase the proliferation of existing tumor cells in the breast (9
). Recently, Horwitz and Sartorius (14
) suggested that progestins may promote tumor development through activation and transformation of dormant breast cancer stem cells into intermediate subpopulations, which then differentiate into breast cancer cells. Because we found that apigenin did not prevent MPA-induced hyperplasia, but did inhibit the emergence of tumors and reduce the incidence and multiplicity of tumors normally associated with MPA treatment, apigenin may target a subset of cells essential for tumor development (cancer stem cells) in this model system. Recent studies showing that sulphorane, a component of broccoli, prevents tumor development by targeting and killing critical cancer stem cells (35
) support this idea. Furthermore, a number of dietary compounds such as curcumin (30
), quercetin, and epigallocatechin-gallate (37
) have long been recognized as agents capable of suppressing cancer stem cell proliferation. Further studies are necessary to confirm whether apigenin might prevent the development of progestin-accelerated DMBA-induced mammary tumors via a mechanism that targets breast cancer stem cells.
Breinholt et al. (38
) reported that apigenin reduced levels of endogenous ER in mouse uterus. With this in mind, we sought to determine whether apigenin might have a similar effect in mammary gland. ER plays a vital role in controlling mammary levels of PR (27
), a fact which could explain the lack of MPA effects, because the presence of adequate levels of PR is essential if MPA is to exert effects such as induction of VEGF (9
). However, we did not detect any differences in the expression levels of either ER or PR in the mammary gland of MPA-, MPA +apigenin-, or vehicle-treated animals. Apigenin has been shown to function in both an ERα-dependent and independent manner (39
) and thus it is possible that in our study apigenin mediates its effect in an ERα independent manner, although this remains to be tested. Interestingly, we observed that while the percentage of cells expressing ERα was similar in all treatment groups, the percentage of PR-positive cells was significantly lower in MPA-treated animals (both those treated with and without apigenin) compared with the placebo group. However, insufficient PR expression is unlikely to explain the antiprogestin effects of apigenin, and we cannot rule out the possibility that it blocks PR-mediated functions by modifying its activity, either through suppressing phosphorylation of the PR protein or through an unidentified mechanism. These scenarios remain to be examined.
Although data on the bioavailability of dietary apigenin in humans is extremely limited, Meyer et al. (40
) reported that, following ingestion of parsley, which is rich in apigenin, plasma levels of the flavonoid increased to a concentration of 0.34 μmol/l. Consequently, it appears that concentrations of apigenin sufficient to be biologically effective can be obtained orally through the diet. Furthermore, it has been reported that apigenin’s slow pharmacokinetics allow it to remain in the circulation for a prolonged period (24
), suggesting that it may accumulate within tissues at sufficient levels to exert chemopreventive effects. Consumption of apigenin-rich foods may be chemopreventive, particularly with respect to progestin-accelerated tumors. Furthermore, regular intake of foods rich in the flavonoid may decrease the cancer risk in postmenopausal women undergoing HRT with a progestin component, as well as in those who have already been exposed to combined HRT. Fortunately, apigenin appears to be nontoxic, since it appears to have different effects on normal versus cancerous cells (20
), suggesting that the intake of high doses will likely prove benign. Indeed, we administered apigenin repeatedly to animals at levels up to 50 mg/kg for 10–13 days and observed no signs of toxicity.
In this study, our main focus was to determine the effects of short-term apigenin administration (approx 10 days) on preventing the development of progestin-accelerated tumors in a model of DMBA-induced mammary carcinogenesis. In the future, we will perform long-term studies (30–60 days of apigenin administration) with a view to determining whether continuous treatment with apigenin can further prevent the appearance of tumors while remaining nontoxic to animals. Such studies will be valuable in helping to determine whether the reduction in tumor incidence (approximately 50%) seen following short-term treatment with apigenin (10 days) can be further improved upon in a long-term preventive strategy. The present study is also limited in that we have yet to determine the maximum tolerated dose that might be used to prevent the emergence of MPA-accelerated tumors in DMBA-treated animals. In the future, we will also look into whether administration of apigenin before the carcinogenic insult protects against the initial formation of DMBA-induced mammary tumors, as well as opposing the effects of MPA on tumor development. Additional preclinical and clinical studies are required to further investigate the efficacy, pharmacokinetics, and suitability of apigenin as a chemopreventive candidate for progestin-accelerated breast cancer and other hormone-dependent cancers in humans.