Effects of indole-3-carbinol on steroid hormone profile and tumor progression in a mice model of canine inflammatory mammarycancer

The relationship between cancer and nutrition has been largely studied; epidemiologic studies indicate that consumption of vegetables containing dietary phytochemicals reduces the risk of developing cancer [1]. Dietary phytochemicals are a wide variety of biologically active compounds found in plants that contain anti-tumor and anti-inflammatory properties [2‐5]. Many of these substances block tumor growth and inhibit metastasis in animal models [6, 7]. Studies on synergistic effects of different phytochemicals might contribute to establish potential chemopreventive strategies [8, 9]. Indole-3-carbinol (I3C), a natural phytochemical found in cruciferous vegetables (i.e. broccoli, cabbage or cauliflower), is considered a potential anticancer agent that prevents the development of certain types of tumors by activating tumor suppressor genes, genes involved in apoptosis and detoxification [2, 10‐14]. Some in vitro and in vivo studies in breast cancer [15‐20] and other neoplasia types such as colorectal cancer, prostate cancer, ovarian cancer, cervix carcinoma and hepatocarcinoma indicated that I3C suppresses cell proliferation and induces apoptosis [17, 21‐25].

Several in vitro studies with breast cancer cells showed that I3C acts by blocking estrogen receptors among other mechanisms [10, 17, 26]. Thus, some authors showed that I3C might be useful as supplement of tamoxifen preventing or treating estrogen-dependent tumors [27, 28]. There are no data on the effect of I3C in human inflammatory breast cancer (IBC) or in spontaneous and experimental canine inflammatory mammary cancer (IMC). Inflammatory breast cancer and IMC are considered as a special clinic-pathological entity and the most malignant type of breast cancer both in humans and dogs, with a fulminant clinical course and an extremely poor survival rate [29‐34]. Inflammatory breast cancer has been proposed as a natural model to study the human disease [29, 35, 36]. Our group has published the establishment and validation of a xenograft model of canine IMC in mice [37]. Spontaneous and experimental canine IMC have been associated with high levels of steroid hormones in tumor homogenates, suggesting a potential autocrine/paracrine secretion [37‐41].

The aim of this study was to analyze the hypothetical antitumor effect of I3C administration on a SCID mouse xenograft model of canine inflammatory mammary cancer using an equivalent dose to that used as a preventive anti-cancer agent in humans.

Several experimental studies have shown that I3C possesses preventive anti-cancer [15, 17, 22] and disrupting estrogen signalling properties [10, 46‐48]. Indole-3-carbinol is considered a potential agent in the prevention and treatment of hormone-dependent breast tumors [2, 21]. Thus, it is freely available in the stores and supermarkets as a dietary phytochemical (non as an approved pharmaceutical drug) for preventing cancer, diminishing premenstrual syndrome and perimenopause-related disturbances. Nevertheless, the effect of I3C on patients with neoplasms, especially breast cancer is poorly documented [20, 49]. To the best of our knowledge, this is the first study regarding the effects of I3C treatment on experimental or spontaneous IMC. Both IBC and canine IMC are considered a special and very aggressive type of breast cancer due to its particular/unique biological, molecular, pathological, genetic and clinical signature features [30, 32‐34]. In the present study, I3C treatment on a xenograft model of canine IMC reduced tumor growth and increased apoptosis, although metastasis and alterations in the peripheral levels of steroid hormones were also observed in some animals.

Tumor proliferation index has been associated with poor prognosis in human [49] and canine mammary cancer [50]. Our xenografts from the I3C group had lower proliferation index compared with control group, indicating a loss of proliferative capacity after I3C administration. In accordance with our results, several reports using breast cancer cell lines [3, 18, 19, 51, 52] and human breast cancer cell-derived tumor xenografts, have reported an antiproliferative effect of I3C [20, 49]. The antiproliferative property of I3C has been associated to changes in cell signaling, specially disrupting estrogen responsiveness [19, 22, 47, 48]. Indole-3-carbinol could have also a role in the induction of specific carcinogen detoxifying enzymes, such as CYP1A [3, 17, 53], and in cell adhesion, dissemination and invasion of human breast cancer cells [2].

In the present study, the lack of ulceration and increased apoptosis in treated I3C tumors is in accordance with a reduction of tumor growth and with other previous in vitro and in vivo studies in human breast cancer [22, 51, 53, 54], probably through pro-apoptotic and anti-proliferative mechanisms [12, 19, 55, 56]. In addition, it has been reported that I3C promotes apoptosis of breast cancer cells by activating caspase-3 and -9, among other mechanisms [4, 22, 54]. Accordingly, our study showed a high expression of caspase-3 in I3C treated xenografts. Both IBC and IMC are typically angiogenic, lymphangiogenic and lymphangiocentric, being the presence of massive neoplastic emboli in dermal vessels, their main histological feature [29, 35, 57, 58]. The presence of dermal neoplastic emboli in experimental mice IBC/IMC xenotransplant models has been documented but it is not seen in all the samples [37, 57‐61]. It is known that the presence of emboli within the dermal lymphatic vessels in IMC, contributes to the rapid development of metastasis [62‐64], and is responsible for the morbidity of this disease. In spite of the antiproliferative and apoptosic effect, emboli in dermal lymphatic vessels and liver metastasis were observed in some animals. Therefore, the effect of I3C could not be enough to avoid the appearance of tumor embolization and metastasis.

The appearance of metastases indicates that this biomodel is appropriate for the study of metastatic tumors in general and inflammatory mammary cancer in particular, since both IBC and IMC are very aggressive tumors with an extremely high metastatic potential. The capacity of I3C to inhibit cell adhesion, migration and invasion of non-inflammatory ER- breast cancer cell has been previously reported [2]. However, in this study, metastases were observed in some animals. A potential limitation of the current study is the scarce number of animals used. More studies should be performed to evaluate the capacity of I3C to inhibit the appearance of metastasis in this cancer.

The presence of thrombosis and some necrotic/degenerated neoplastic cells inside the emboli has been previously documented in IMC xenografts and suggests a partial destruction of metastatic cells, reducing the possibility of establishing distant metastasis [37]. In our study, only the control group showed thrombosis, which could suggest a reduced defense mechanism in I3C treated IMC xenografts. The possible host defense mechanisms in these xenografts remain to be elucidated.

The effect of I3C treatment on estrogens metabolism has been previously indicated [10, 27, 28, 53, 65]; nevertheless, to the best of our knowledge, this is the first time that the possible effects of this indole on other steroid hormones and general steroidogenesis have been studied. According to previous studies, the alteration of estrogen metabolism by I3C can be mediated by cytochrome P450 [66, 67] or the activation of aryl hydrocarbon receptor (AhR)-mediated pathways [68‐70].

Histopathological evidence of lipid droplets in neoplastic cells has been described in spontaneous and experimental canine IMC, and it has been shown that could be due to steroid secretion [29, 37, 40]. The frequent presence of scattered lipid-rich cells in our murine model has been previously described in another IMC xenograft model [37]. In the present study I3C treatment increased significantly the number of intracellular lipid droplets that appeared forming large areas of lipid-rich cells, which were not present in the control group tumors. According to previous studies, these lipid-rich cells could contain large amounts of steroids that could be locally secreted [37, 38, 40]. The steroid secretion by IBC and IMC cell lines has been recently indicated [71]. The presence of large areas containing a lipid-rich cell subpopulation is in accordance with previous observations and explains the high amounts of steroids found in tumor homogenates of I3C treated xenografts of the present study. According to our results, several steroid hormones (estradiol, estrone sulphate and androstenedione) were significantly increased in I3C treated tumor homogenates while testosterone was diminished. Interestingly, testosterone was significantly increased in the sera of I3C treated mice. Further studies should elucidate if these changes in steroid hormone content are related to a major production of estrogens in situ (via aromatase or sulphatase enzymes).

The mechanisms causing hormonal variations after I3C administration are uncertain. The documented anti-estrogen effect of I3C through ER should be questioned in this particular case, since the model is ERα negative [3, 28, 46, 65]. As pointed out before, I3C can alter the estrogen metabolism through other ER-independent pathways [15, 27, 47, 53]. The role of other receptors such as ERβ should be considered and elucidated in future studies. The relevance of ERβ in the pathogenesis of IMC has been suggested previously [39]. Our study suggests that both E1SO₄ and E2 can be locally synthesized, especially after I3C administration. Following the aromatase or sulphatase pathways, androgens (especially testosterone that is diminished in tissue) could be transformed in high tissue E1SO₄ and E2 concentrations. Estrone sulphate could be a reservoir for the synthesis of active estrogens in the mammary tumors, including the canine IMC [39, 40, 72]. Previous studies indicated that I3C effect on breast cancer cells favors estradiol 2-hydroxylation [46, 73]. Although there are no previous studies on intratumoral E2 levels in I3C treated cells, such the activation of estradiol 2-hydroxylation should induce a low level of E2 [19, 74] but an increase of E2 and E1SO₄ concentrations in tumor homogenates was observed. This particular finding has to be compared in non-inflammatory breast cancer xenografts to elucidate if the high amounts of E2 found in the present study are due to the particular characteristics of IMC. In addition to our results refer to intratumoral levels, as stated above.

The higher concentration of steroids and longer half-life suggests the formation of biologically active estrogens in tumor tissue [40]. In order to proliferate, it is possible that tumor increased estradiol levels from the E1SO₄ reservoir. In our results, serum P4 concentrations increased in I3C group, probably due to a lower expression of progesterone receptors by I3C, and consequently, higher serum P4 levels [10, 65, 75]. As occurs with estrogens and progesterone a local synthesis of androgens by IBC and IMC tumor cell lines, has also been confirmed [71]. The role of androgens in breast cancer is not clear. Some studies suggest an antiproliferative effect [76], but others note an increased proliferation in breast cancer cell lines [77, 78]. The decrease of testosterone levels in I3C-treated group could be due to the inhibitory effect of I3C on the androgen receptors transcription [66]. Similar to E2, a higher content of intratumoral A4 in I3C group could be due to an increased local synthesis and to the binding of I3C to androgen receptors, increasing A4 free levels in tissue homogenates. However, intratumoral T concentrations decreased in I3C group when compared with control group, probably by the I3C-associated stimulatory effect on the aromatase enzyme [79] which stimulates T to E2 conversion.

Our data reveal for the first time that the ingestion of indole-3-carbinol, as administered, diminishes proliferation and increases apoptosis of tumor cells in an experimental model of inflammatory breast cancer, although this effect could not be enough to avoid the appearance of tumor embolization and metastasis. Additionally, stimulated steroid hormones levels and a characteristic lipid-rich cell population were presented. These results could be attributable to the special characteristics of this particular cancer. Future clinical trials and studies will be needed to clarify the usefulness of indole-3-carbinol in this cancer and to understand the molecular mechanisms involved.