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

Effects of indole-3-carbinol on steroid hormone profile and tumor progression in a mice model of... Background: Indole-3-carbinol, derived from Cruciferous vegetables is an estrogen receptor antagonist considered a preventive agent that is naturally present in diet. There are no previous studies on its effects in human inflammatory breast cancer or canine inflammatory mammary cancer that is the most aggressive type of breast cancer. Methods: The aim of this study was to analyze the effect of indole-3-carbinol on a SCID mice xenograft model of canine inflammatory mammary cancer, using equivalent human oral dose as a preventive therapy in humans for 3 weeks. Results: Indole-3-carbinol treatment decreased tumor proliferation and increased apoptosis, although tumor embolization and liver metastasis were observed in some animals. There was a characteristic subpopulation of lipid-rich cells and increased contents of select steroid hormones in tumor homogenates and serum. Conclusions: 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. Future clinical trials will be needed to clarify the usefulness of indole-3-carbinol in this cancer and to understand the molecular mechanisms involved. Keywords: Indole-3-carbinol (I3C), Canine inflammatory mammary cancer (IMC), Inflammatory breast cancer (IBC), Steroid hormones, Mice model, Xenograft Background substances block tumor growth and inhibit metastasis in The relationship between cancer and nutrition has been animal models [6, 7]. Studies on synergistic effects of largely studied; epidemiologic studies indicate that con- different phytochemicals might contribute to establish po- sumption of vegetables containing dietary phytochemi- tential chemopreventive strategies [8, 9]. Indole-3-carbinol cals reduces the risk of developing cancer [1]. Dietary (I3C), a natural phytochemical found in cruciferous vege- phytochemicals are a wide variety of biologically active tables (i.e. broccoli, cabbage or cauliflower), is considered compounds found in plants that contain anti-tumor and a potential anticancer agent that prevents the develop- anti-inflammatory properties [2–5]. Many of these ment of certain types of tumors by activating tumor suppressor genes, genes involved in apoptosis and detoxi- fication [2, 10–14]. Some in vitro and in vivo studies in breast cancer [15–20]and otherneoplasia types such * Correspondence: jcillera@ucm.es Department of Animal Physiology, Veterinary Medicine School, Complutense University of Madrid, Madrid, Spain Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 2 of 9 as colorectal cancer, prostate cancer, ovarian cancer, carcinoma, following a protocol previously established cervix carcinoma and hepatocarcinoma indicated that [37] as follows: Fragments (3 mm × 2 mm of diameter) I3C suppresses cell proliferation and induces apop- from canine primary mammary tumor obtained at tosis [17, 21–25]. necropsy were immediately placed in MEM (Minimum Several in vitro studies with breast cancer cells showed Essential Medium) with Earle’s Salts, L-Glutamine and that I3C acts by blocking estrogen receptors among other Penicillin/Streptomycin [100×] (PAA Cell Culture Com- mechanisms [10, 17, 26]. Thus, some authors showed that pany, BioPath Stores, Cambridge) before they were sub- I3C might be useful as supplement of tamoxifen prevent- cutaneously implanted into the ventral side of 3 female ing or treating estrogen-dependent tumors [27, 28]. There SCID mice. The mice were previously anesthetized with are no data on the effect of I3C in human inflammatory isoflurane (IsoVet 1000 mg/g, B Braun VetCare SA, Barce- breast cancer (IBC) or in spontaneous and experimen- lona, Spain) at 4% for induction and at 1.5% for maintain- tal canine inflammatory mammary cancer (IMC). In- ing anesthesia. Isoflurane was supplied in a fresh gas flow flammatory breast cancer and IMC are considered as a rate of 0.5 l of oxygen/minute. The mice were monitored special clinic-pathological entity and the most malig- for tumor growth and, whenever palpable, the xenograft nant type of breast cancer both in humans and dogs, tumor volumes were measured twice weekly with a with a fulminant clinical course and an extremely poor caliper-like instrument throughout the experiment, and survival rate [29–34]. Inflammatory breast cancer has the tumor size was estimated using the following formula: been proposed as a natural model to study the human (L × W )/2,where W = width and L = length. When the disease [29, 35, 36]. Our group has published the estab- implanted tumors reached approximately 1.0 cm ,they lishment and validation of a xenograft model of canine were successively transplanted into three SCID mice IMC in mice [37]. Spontaneous and experimental ca- (second passage) and from each of these animals to three nine IMC have been associated with high levels of new animals (third passage, n = 30), in order to verify that steroid hormones in tumor homogenates, suggesting a the xenograft model was stable and that these tumors did potential autocrine/paracrine secretion [37–41]. not present histopathological modifications between the The aim of this study was to analyze the hypothetical consecutive passages. The engraftment efficiency of the antitumor effect of I3C administration on a SCID mouse second and third passages was 90% (n =24). xenograft model of canine inflammatory mammary can- cer using an equivalent dose to that used as a preventive Treatment groups anti-cancer agent in humans. Animals of third passage were randomly assigned to an I3C treatment/control group (n = 12/12). All mice from the Methods two groups were treated by oral gavage: the control Animals and xenograft establishment group, administered with 200 μl of distilled water/poly- The protocol was approved by the Committee of the Uni- ethylene glycol (Panreac Quim. S.A, Barcelona, Spain) versidad Complutense of Madrid, and Animal Protection (6:4 ratio, n = 12/24) and the I3C group (administered Area of the Community of Madrid, Spain (Ref. Protocol with 200 μl of distilled water/polyethylene glycol contain- Number: PROEX 31/15). Twenty-four non-ovariectomized ing 150 mg I3C/kg/day) (Indole-3-carbinol, Sigma-Aldrich scid female SCID mice (BALB/cJHan®Hsd-Prkdc , Harlan Co., Madrid, Spain), (n = 12/24). The dose was chosen Laboratories Models,S.L.),6–8 weeks of age and from previous studies [42, 43]. Mice were given the doses weighing between 20 and 22 g were used. The animals through an oral feeding tube (18G x ½”) for 3 weeks (the were housed in a flexible-film isolator (Racks IVC de first dose was given 7 days after the xenografts, when Allentown Inc. Panlab Harvard Apparatus) in cages (2–3 tumors were palpable), following a dosage of 5 consecutive animals per cage), each measuring 330 cm ×12 cm, days of administration and 2 days of rest. The operator in a room with controlled environmental conditions was blinded to the treatment group assignment of each (20 °C to 22 °C; 50 to 55% relative humidity; 10 to 15 animal at all times. air changes per hour; and a 12:12 h light: dark cycle). Pre-sterilized food and water were provided ad libitum. All experimental procedures were performed between Histopathology and immunohistochemistry 11:30 a.m. and 12:30 a.m. The method of euthanasia Fragments from the treated tumors were fixed in neu- used to sacrifice the mice was isoflurane flow rate tral formalin and then embedded in paraffin for tumor adjusted to 5% until one minute after breathing stops. histopathology. The samples were histologically diag- Then, cervical dislocation was applied as method of nosed on HE-stained sections following the histological confirmation of euthanasia. classification of canine mammary tumors [44]. Histology The xenograft was directly established from a 9-year-old and immunohistochemistry of samples were evaluated by female dog with a spontaneous inflammatory mammary an experienced veterinary pathologist. Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 3 of 9 Immunohistochemistry for Ki-67, caspase-3 and estro- stored at − 20 °C until assayed for the presence of gen receptor (ER) was performed on deparaffinised hormones. Frozen tissue specimens of approximately sections and using the streptavidin–biotin–complex per- 0.1–0.5 g from individual mice were homogenized in oxidase method or detection kits. High-temperature 5 ml of PBS (pH = 7.2) and the homogenate centrifuged antigen retrieval with 10 mM citrate buffer pH = 6.0 was at 1200 x g, for 20 min at 4 °C. The supernatants were har- performed. Table 1 shows the primary antibodies and vested and stored at − 80 °C until hormone assays [45]. developing systems used. In the case of caspase-3, the Levels of estrone sulphate (E1SO ), estradiol (E2), slides were incubated with streptavidin-HRP conjugated androstenedione (A4), testosterone (T), dehydroepian- anti-caspase-3 antibody (TermoFisher Scientific ref. 43– drosterone (DHEA) and progesterone (P4) in tumor 4323, dilution 1/4000) for 30 min at room temperature homogenates and serum samples were assayed by ampli- (RT) and developed with a chromogen solution containing fied Enzyme-Immunoassay (EIA) previously validated in 3, 3′-diaminobenzidine tetrachloride (DAB). Thereafter, our laboratory [45]. the slides were counterstained with hematoxylin. Washes and dilutions were made in Tris-buffered saline (pH = 7.4) Statistical analysis for each marker; corresponding positive and negative con- Statistical analyses were performed by the IBM SPSS trol slides were performed. Statistics, 19.0 for Windows (Chicago, IL, USA). The Tumor proliferation index (PI) was determined by relationship between continuous variables (plasma and tu- counting Ki-67 positive and negative nuclei in 8–10 moral steroid hormone levels, Ki-67 and caspase index) selected High Power Fields (HPF) with the highest per- and categorical variables (pathological parameters such as centage of labeling (minimum 1000 cells). Every immu- ulceration, sebaceous hyperplasia, dermal emboli, tumor nostained nucleus was considered positive regardless of emboli, degenerated emboli, thrombosis, lipid-rich sub- the intensity of stain. The PI or proportion of positive population, distant metastases, liver metastasis and estro- neoplastic cells in each sample was calculated as previ- gen receptor alpha) was established using the analysis of ously described [44]. variance (ANOVA) followed by appropriate post hoc tests Caspase-3 immunostaining was performed to analyze for similar variances (Duncan Test) or different ones the presence of apoptotic bodies in the xenograft tissue (Games-Howell test). The relationship between continu- and it was semi-quantitatively assessed by the intensity ous variables was assessed using the Pearson correlation. of immunoexpression, which was evaluated as low (+), The association between categorical variables was ana- moderate (++), or intense (+++), by the percentage of lyzed using Pearson’s chi-squared test (χ ). Differences positive cells (apoptotic index). were considered significant at p-value <0.05. Finally, the expression of ER was considered positive when more than 10% of the cells within the tumors were Results positive independently of the intensity of the staining [29]. Macroscopic growth and metastases A stable serial transplantable xenograft was successfully Steroid hormone concentrations in serum samples and established with a constant and rapid growth in all mice. tumor homogenates At the end of the experiment, in 4 weeks, the tumors At the time of necropsy of the control and experimental had an approximate size of 0.9–1.2 cm . The size and SCID mice, 1 ml of blood was obtained by cardiac punc- weight of the xenograft tumors in I3C treated and un- ture using a 25G needle from each mouse. Blood samples treated mice (controls) did not show significant differ- were collected in special tubes (microtube Serum-Gel ences (p > 0.05). Clotting Activator, Sarstedt, Nümbrecht, Germany)and Immediately after sacrifice, complete necropsy was per- spun down in a refrigerated centrifuge (Hettich Zentrifu- formed in each case. Common target organs for IMC such gen Universal 320 R, Germany) at 4 °C and a speed of as bone, lung, liver, brain or distant lymph nodes were 1200 x g (RCF) for 20 min. Serum was harvested and extensively examined. At necropsy, one xenotrasplanted Table 1 Primary and secondary antibodies/kits used for immunohistochemistry Primary Antibody Type Source Incubation Secondary Antibody/kit Source Incubation Ki-67 clon MIB-1 Mab Dako 60 min, RT EnVision+SystemHRP (DAB) Dako 40 min, RT (Ref. M7240) 1:75 (Ref. K4007) kit ERα clon 1D5 Mab Dako Overnight EnVision + SystemHRP (DAB) Dako 40 min, RT (Ref. M7047) 1:15 4° C (Ref. K4007) kit Caspase-3 Pab R&D Systems Overnight Swine anti-rabbit biotinylated Dako 1:200 30 min, RT (Ref. AF835) 1:1200 4° C (Ref. E0353) Mab mouse monoclonal antibody, Pab rabbit polyclonal antibody Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 4 of 9 control mouse presented pulmonary and mesenteric me- areas of isolated cells; the stroma was scant. Scattered tastases (1/12, 8.3% of the control group) and one out of tumor cells showed a lipid-rich cytoplasm. The neo- 12 (8.3%) I3C treated xenografts had pulmonary and mes- plastic cells exhibited a high pleomorphism with enteric metastases. Four animals in the I3C group showed marked cell and nuclear atypia, and an elevated mitotic metastasis in the liver (4/12, 33.3%, p = 0.028). All metas- index (anaplastic cells). Binucleated and multinucleated tases were histologically confirmed. cells, as well as atypical mitoses were frequently found. Large intratumor areas of necrosis, dermal lymphan- Histopathological findings giectasias, and severe edema in the dermis were also Primary canine IMC and control xenografts present. According to the clinical characteristics and the histo- logical invasion of dermal lymphatic vessels by tumor/ neoplastic emboli (Fig. 1a), the primary canine mam- Histological and immunohistochemical findings in the I3C mary tumor was diagnosed as an inflammatory mam- treated xenografts mary cancer originated by a highly undifferentiated Histological and immunohistochemical findings of anaplastic/solid mammary carcinoma grade III with both experimental groups are depicted in Table 2. scattered lipid-rich cells. Control xenografts reproduced Indole-3-carbinol treatment diminished proliferation the histological features of the primary canine mam- and increased apoptosis of tumor cells although mary tumor. Hence, emboli at peripheral lymphatic and embolization of neoplastic cells (7/12), liver metastasis blood vessels, as well as inside the tumor were observed and presence of tumor cells showing a lipid-rich cyto- (2/12, 16.7%) (Fig. 1b). The control xenografts were plasm (8/12) (Figs. 1c-i) together with hyperplasia of highly infiltrative in the dermis and striated muscle and dermal sebaceous glands (10/12) were also observed in were partially surrounded by a fibro-myxoid tissue. some animals. This lipid-rich cell subpopulation fre- Neoplastic cells displayed a solid pattern together with quently presented degeneration (8/12) and necrosis. Fig. 1 Representative histological features. a IMC with neoplastic emboli in superficial dermal lymphatic vessels and infiltration of carcinomatous scid cells in the female dog origin of the xenografts. b IMC with tumor emboli in SCID mouse (BALB/cJHan®Hsd-Prkdc ) xenograft control group. c IMC with tumor emboli in dermis in SCID mouse xenograft I3C group (p = 0.012, compared with control group), and non-dermal tumor emboli in I3C mice (p = 0.035). d IMC with abundant lipid-rich cells in SCID mouse xenograft I3C group (× 20), (× 40) (p = 0.001). e IMC with liver metastasis in SCID mouse xenograft I3C group (× 2), (× 20) (p = 0.028). f and g IMC showing caspase-3 positive immunolabeling in a low number of cells in SCID mouse xenograft control group versus I3C group where positive immunolabeling in numerous cells (× 4), (× 20) (p < 0.001). h IMC showing positive Ki-67 immunolabeling in numerous cells in xenograft control group, and i lower number of Ki-67 positive cells in the I3C group SCID mouse xenograft (× 20) (p < 0.001). Analysis of variance followed by appropriate post hoc tests for similar variances (Duncan Test) or different ones (Games Howell test) was used. IMC inflammatory mammary carcinoma, I3C indole-3-carbinol Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 5 of 9 Table 2 Histopathological and immunohistochemical characteristics in control and indole-3-carbinol (I3C) xenograft groups Control xenograft (%) I3C xenograft (%) p-value Ulceration 33.3 0.0 p = 0.028 Sebaceous hyperplasia 8.3 83.3 p < 0.001 Dermal emboli 0.0 41.7 p = 0.012 Tumor emboli (non-dermal) 16.7 58.3 p = 0.035 Degenerated emboli 0.0 8.3 p > 0.05 Thrombosis 33.3 0.0 p = 0.036 Lipid-rich subpopulation 0.0 66.7 p = 0.001 Distant metastases (any location) 8.3 33.3 p > 0.05 Liver metastases 0.0 33.3 p = 0.028 Control xenograft I3C xenograft p-value (%, mean ± standard deviation) (%, mean ± standard deviation) Ki-67 index 62.2 ± 4.55 54.5 ± 3.79 p < 0.001 Caspase index 6.9 ± 1.35 27.5 ± 2.33 p < 0.001 P values in boldface are significant. These values are p < 0.05 Primary canine tumor and all xenografts samples were Discussion negative for ER immunostaining. The positive control Several experimental studies have shown that I3C was canine uterus. possesses preventive anti-cancer [15, 17, 22]and dis- rupting estrogen signalling properties [10, 46–48]. Determination of steroid hormones Indole-3-carbinol is considered a potential agent in the The content of steroid hormones in serum and tumor prevention and treatment of hormone-dependent homogenates of both control and I3C xenograft groups are breast tumors [2, 21]. Thus, it is freely available in the showninTable 3. Significantly higher levels of estrone stores and supermarkets as a dietary phytochemical sulphate (p = 0.009), estradiol (p < 0.001) and androstene- (non as an approved pharmaceutical drug) for prevent- dione (p = 0.049) and lower level of testosterone (p =0.048) ing cancer, diminishing premenstrual syndrome and in I3C xenograft homogenates compared with the control perimenopause-related disturbances. Nevertheless, the group were found. Serum progesterone (p = 0.003) and tes- effect of I3C on patients with neoplasms, especially tosterone levels (p = 0.022) were also higher in I3C group. breast cancer is poorly documented [20, 49]. To the best of our knowledge, this is the first study regarding Table 3 Serum (S) (ng/ml) and tumor homogenate (T) (ng/g) the effects of I3C treatment on experimental or spontan- steroid hormone concentrations in control and indole-3- eous IMC. Both IBC and canine IMC are considered a carbinol (I3C) groups after 3 weeks of treatment special and very aggressive type of breast cancer due to its Control xenograft I3C xenograft particular/unique biological, molecular, pathological, gen- a,1 a,1 Estrone sulphate (E1SO4) S 0.12 ± 0.03 0.24 ± 0.05 etic and clinical signature features [30, 32–34]. In the a,2 b,2 present study, I3C treatment on a xenograft model of ca- T 13.71 ± 2.42 27.09 ± 3.09 a,1 a,1 nine IMC reduced tumor growth and increased apoptosis, Estradiol (E2) S 11.98 ± 0.82 12.62 ± 1.01 although metastasis and alterations in the peripheral levels a,2 b,1 T 7.44 ± 0.60 13.89 ± 0.99 of steroid hormones were also observed in some animals. a,1 a,1 Androstenedione (A4) S 5.96 ± 0.77 6.10 ± 1.28 Tumor proliferation index has been associated with a,1 b,2 T 5.16 ± 0.99 20.46 ± 5.17 poor prognosis in human [49] and canine mammary a,1 b,1 Testosterone (T) S 1.20 ± 0.09 1.76 ± 0.25 cancer [50]. Our xenografts from the I3C group had a,2 b,1 lower proliferation index compared with control group, T 12.16 ± 3.07 4.02 ± 1.21 a,1 a,2 indicating a loss of proliferative capacity after I3C Dehydroepiandrosterone (DHEA) S 2.96 ± 0.16 3.06 ± 0.37 administration. In accordance with our results, several a,1 a,2 T 2.32 ± 0.11 2.86 ± 0.63 reports using breast cancer cell lines [3, 18, 19, 51, 52] a,1 b,1 Progesterone (P4) S 0.69 ± 0.07 1.15 ± 0.10 and human breast cancer cell-derived tumor xenografts, a,2 a,2 T 5.37 ± 1.04 7.75 ± 1.98 have reported an antiproliferative effect of I3C [20, 49]. All values expressed as mean ± standard error. Different letters denote The antiproliferative property of I3C has been associated significant differences (p < 0.05) among groups. Different numbers denote to changes in cell signaling, specially disrupting estrogen significant differences (p < 0.05) between serum samples and tumor tissue homogenates, in the same treatment group responsiveness [19, 22, 47, 48]. Indole-3-carbinol could Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 6 of 9 have also a role in the induction of specific carcinogen hormones and general steroidogenesis have been studied. detoxifying enzymes, such as CYP1A [3, 17, 53], and in According to previous studies, the alteration of estrogen cell adhesion, dissemination and invasion of human metabolism by I3C can be mediated by cytochrome P450 breast cancer cells [2]. [66, 67] or the activation of aryl hydrocarbon receptor In the present study, the lack of ulceration and in- (AhR)-mediated pathways [68–70]. creased apoptosis in treated I3C tumors is in accordance Histopathological evidence of lipid droplets in neo- with a reduction of tumor growth and with other previ- plastic cells has been described in spontaneous and ex- ous in vitro and in vivo studies in human breast cancer perimental canine IMC, and it has been shown that [22, 51, 53, 54], probably through pro-apoptotic and could be due to steroid secretion [29, 37, 40]. The fre- anti-proliferative mechanisms [12, 19, 55, 56]. In quent presence of scattered lipid-rich cells in our mur- addition, it has been reported that I3C promotes apop- ine model has been previously described in another tosis of breast cancer cells by activating caspase-3 and IMC xenograft model [37]. In the present study I3C -9, among other mechanisms [4, 22, 54]. Accordingly, treatment increased significantly the number of intra- our study showed a high expression of caspase-3 in I3C cellular lipid droplets that appeared forming large areas treated xenografts. Both IBC and IMC are typically an- of lipid-rich cells, which were not present in the control giogenic, lymphangiogenic and lymphangiocentric, being group tumors. According to previous studies, these the presence of massive neoplastic emboli in dermal ves- lipid-rich cells could contain large amounts of steroids sels, their main histological feature [29, 35, 57, 58]. The that could be locally secreted [37, 38, 40]. The steroid presence of dermal neoplastic emboli in experimental secretion by IBC and IMC cell lines has been recently mice IBC/IMC xenotransplant models has been docu- indicated [71]. The presence of large areas containing a mented but it is not seen in all the samples [37, 57–61]. It lipid-rich cell subpopulation is in accordance with previ- is known that the presence of emboli within the dermal ous observations and explains the high amounts of lymphatic vessels in IMC, contributes to the rapid devel- steroids found in tumor homogenates of I3C treated xeno- opment of metastasis [62–64], and is responsible for the grafts of the present study. According to our results, sev- morbidity of this disease. In spite of the antiproliferative eral steroid hormones (estradiol, estrone sulphate and and apoptosic effect, emboli in dermal lymphatic vessels androstenedione) were significantly increased in I3C and liver metastasis were observed in some animals. treated tumor homogenates while testosterone was dimin- Therefore, the effect of I3C could not be enough to avoid ished. Interestingly, testosterone was significantly in- the appearance of tumor embolization and metastasis. creased in the sera of I3C treated mice. Further studies The appearance of metastases indicates that this biomo- should elucidate if these changes in steroid hormone con- del is appropriate for the study of metastatic tumors in tent are related to a major production of estrogens in situ general and inflammatory mammary cancer in particular, (via aromatase or sulphatase enzymes). since both IBC and IMC are very aggressive tumors with The mechanisms causing hormonal variations after an extremely high metastatic potential. The capacity of I3C administration are uncertain. The documented an- I3C to inhibit cell adhesion, migration and invasion of ti-estrogen effect of I3C through ER should be questioned non-inflammatory ER- breast cancer cell has been previ- in this particular case, since the model is ERα negative ously reported [2]. However, in this study, metastases were [3, 28, 46, 65]. As pointed out before, I3C can alter the observed in some animals. A potential limitation of the estrogen metabolism through other ER-independent path- current study is the scarce number of animals used. More ways [15, 27, 47, 53]. The role of other receptors such as studies should be performed to evaluate the capacity of ERβ should be considered and elucidated in future studies. I3C to inhibit the appearance of metastasis in this cancer. The relevance of ERβ in the pathogenesis of IMC has been The presence of thrombosis and some necrotic/degener- suggested previously [39]. Our study suggests that both ated neoplastic cells inside the emboli has been previously E1SO and E2 can be locally synthesized, especially after documented in IMC xenografts and suggests a partial I3C administration. Following the aromatase or sulpha- destruction of metastatic cells, reducing the possibility of tase pathways, androgens (especially testosterone that establishing distant metastasis [37]. In our study, only the is diminished in tissue) could be transformed in high control group showed thrombosis, which could suggest a tissue E1SO and E2 concentrations. Estrone sulphate reduced defense mechanism in I3C treated IMC xeno- could be a reservoir for the synthesis of active estrogens grafts. The possible host defense mechanisms in these in the mammary tumors, including the canine IMC xenografts remain to be elucidated. [39, 40, 72]. Previous studies indicated that I3C effect on The effect of I3C treatment on estrogens metabolism breast cancer cells favors estradiol 2-hydroxylation [46, 73]. has been previously indicated [10, 27, 28, 53, 65]; never- Although there are no previous studies on intratumoral theless, to the best of our knowledge, this is the first time E2 levels in I3C treated cells, such the activation of es- that the possible effects of this indole on other steroid tradiol 2-hydroxylation should induce a low level of E2 Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 7 of 9 [19, 74] but an increase of E2 and E1SO concentrations Acknowledgements We are grateful to Pedro Aranda for the histological technical assistance and in tumor homogenates was observed. This particular to Carmen Pilar Garcia for animal’s care. finding has to be compared in non-inflammatory breast cancer xenografts to elucidate if the high amounts of E2 Funding This research was supported by the Spanish Ministry of Science and found in the present study are due to the particular Education (research project SAF2009–10572). The funding body did not characteristics of IMC. In addition to our results refer to contribute in any of the following: design of the study, data collection, data intratumoral levels, as stated above. analysis, interpretation of data, or in writing the manuscript. The higher concentration of steroids and longer Availability of data and materials half-life suggests the formation of biologically active es- All data analyzed for this study are included in this article. Supplementary trogens in tumor tissue [40]. In order to proliferate, it is information files are available upon request to the corresponding author. possible that tumor increased estradiol levels from the Authors’ contributions E1SO reservoir. In our results, serum P4 concentrations LP and JCI conceived of the study. LP, AG and JCI participated in its design increased in I3C group, probably due to a lower expres- and coordination, interpretation of results and helped to draft the sion of progesterone receptors by I3C, and consequently, manuscript. AM generated the animal model and AM, LDC and SC carried out the experimental study and collected all data. LP, JCI, AG and AM wrote higher serum P4 levels [10, 65, 75]. As occurs with the manuscript. SC and AM carried out the hormonal assays and LDC the estrogens and progesterone a local synthesis of andro- immunohistochemical techniques. All authors read and approved the final gens by IBC and IMC tumor cell lines, has also been con- manuscript, and all of them agree that they are accountable for all aspects of the work. firmed [71]. The role of androgens in breast cancer is not clear. Some studies suggest an antiproliferative effect [76], Ethics approval but others note an increased proliferation in breast cancer All procedures were carried out according to European and Spanish legislative and regulatory guidelines (European convention ETS 1 2 3, on cell lines [77, 78]. The decrease of testosterone levels in the use and protection of vertebrate mammals in experimentation and for I3C-treated group could be due to the inhibitory effect of other scientific purposes and Spanish Law 32/2007, and R.D. 1201/2005 on I3C on the androgen receptors transcription [66]. Similar the protection and use of animals in scientific research). The protocol was approved by the Committee of the Universidad Complutense of Madrid, to E2, a higher content of intratumoral A4 in I3C group and Animal Protection Area of the Community of Madrid, Spain (Ref. could be due to an increased local synthesis and to the Protocol Number: PROEX 31/15). binding of I3C to androgen receptors, increasing A4 free levels in tissue homogenates. However, intratumoral T Competing interests None of the authors of this paper has a financial or personal relationship concentrations decreased in I3C group when compared with other people or organizations that could inappropriately influence or with control group, probably by the I3C-associated stimu- bias the content of the paper. latory effect on the aromatase enzyme [79] which stimu- lates T to E2 conversion. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Conclusions Our data reveal for the first time that the ingestion of Author details Department of Animal Physiology, Veterinary Medicine School, indole-3-carbinol, as administered, diminishes prolifera- Complutense University of Madrid, Madrid, Spain. Department of Animal tion and increases apoptosis of tumor cells in an experi- Medicine, Surgery and Pathology, Veterinary Medicine School, Complutense mental model of inflammatory breast cancer, although University of Madrid, Madrid, Spain. this effect could not be enough to avoid the appearance Received: 6 February 2018 Accepted: 18 May 2018 of tumor embolization and metastasis. Additionally, stimulated steroid hormones levels and a characteristic lipid-rich cell population were presented. These results References 1. McCullough ML, Giovannucci EL. Diet and cancer prevention. 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Effects of indole-3-carbinol on steroid hormone profile and tumor progression in a mice model of canine inflammatory mammarycancer

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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine, general; Medicine/Public Health, general
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Abstract

Background: Indole-3-carbinol, derived from Cruciferous vegetables is an estrogen receptor antagonist considered a preventive agent that is naturally present in diet. There are no previous studies on its effects in human inflammatory breast cancer or canine inflammatory mammary cancer that is the most aggressive type of breast cancer. Methods: The aim of this study was to analyze the effect of indole-3-carbinol on a SCID mice xenograft model of canine inflammatory mammary cancer, using equivalent human oral dose as a preventive therapy in humans for 3 weeks. Results: Indole-3-carbinol treatment decreased tumor proliferation and increased apoptosis, although tumor embolization and liver metastasis were observed in some animals. There was a characteristic subpopulation of lipid-rich cells and increased contents of select steroid hormones in tumor homogenates and serum. Conclusions: 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. Future clinical trials will be needed to clarify the usefulness of indole-3-carbinol in this cancer and to understand the molecular mechanisms involved. Keywords: Indole-3-carbinol (I3C), Canine inflammatory mammary cancer (IMC), Inflammatory breast cancer (IBC), Steroid hormones, Mice model, Xenograft Background substances block tumor growth and inhibit metastasis in The relationship between cancer and nutrition has been animal models [6, 7]. Studies on synergistic effects of largely studied; epidemiologic studies indicate that con- different phytochemicals might contribute to establish po- sumption of vegetables containing dietary phytochemi- tential chemopreventive strategies [8, 9]. Indole-3-carbinol cals reduces the risk of developing cancer [1]. Dietary (I3C), a natural phytochemical found in cruciferous vege- phytochemicals are a wide variety of biologically active tables (i.e. broccoli, cabbage or cauliflower), is considered compounds found in plants that contain anti-tumor and a potential anticancer agent that prevents the develop- anti-inflammatory properties [2–5]. Many of these ment of certain types of tumors by activating tumor suppressor genes, genes involved in apoptosis and detoxi- fication [2, 10–14]. Some in vitro and in vivo studies in breast cancer [15–20]and otherneoplasia types such * Correspondence: jcillera@ucm.es Department of Animal Physiology, Veterinary Medicine School, Complutense University of Madrid, Madrid, Spain Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 2 of 9 as colorectal cancer, prostate cancer, ovarian cancer, carcinoma, following a protocol previously established cervix carcinoma and hepatocarcinoma indicated that [37] as follows: Fragments (3 mm × 2 mm of diameter) I3C suppresses cell proliferation and induces apop- from canine primary mammary tumor obtained at tosis [17, 21–25]. necropsy were immediately placed in MEM (Minimum Several in vitro studies with breast cancer cells showed Essential Medium) with Earle’s Salts, L-Glutamine and that I3C acts by blocking estrogen receptors among other Penicillin/Streptomycin [100×] (PAA Cell Culture Com- mechanisms [10, 17, 26]. Thus, some authors showed that pany, BioPath Stores, Cambridge) before they were sub- I3C might be useful as supplement of tamoxifen prevent- cutaneously implanted into the ventral side of 3 female ing or treating estrogen-dependent tumors [27, 28]. There SCID mice. The mice were previously anesthetized with are no data on the effect of I3C in human inflammatory isoflurane (IsoVet 1000 mg/g, B Braun VetCare SA, Barce- breast cancer (IBC) or in spontaneous and experimen- lona, Spain) at 4% for induction and at 1.5% for maintain- tal canine inflammatory mammary cancer (IMC). In- ing anesthesia. Isoflurane was supplied in a fresh gas flow flammatory breast cancer and IMC are considered as a rate of 0.5 l of oxygen/minute. The mice were monitored special clinic-pathological entity and the most malig- for tumor growth and, whenever palpable, the xenograft nant type of breast cancer both in humans and dogs, tumor volumes were measured twice weekly with a with a fulminant clinical course and an extremely poor caliper-like instrument throughout the experiment, and survival rate [29–34]. Inflammatory breast cancer has the tumor size was estimated using the following formula: been proposed as a natural model to study the human (L × W )/2,where W = width and L = length. When the disease [29, 35, 36]. Our group has published the estab- implanted tumors reached approximately 1.0 cm ,they lishment and validation of a xenograft model of canine were successively transplanted into three SCID mice IMC in mice [37]. Spontaneous and experimental ca- (second passage) and from each of these animals to three nine IMC have been associated with high levels of new animals (third passage, n = 30), in order to verify that steroid hormones in tumor homogenates, suggesting a the xenograft model was stable and that these tumors did potential autocrine/paracrine secretion [37–41]. not present histopathological modifications between the The aim of this study was to analyze the hypothetical consecutive passages. The engraftment efficiency of the antitumor effect of I3C administration on a SCID mouse second and third passages was 90% (n =24). xenograft model of canine inflammatory mammary can- cer using an equivalent dose to that used as a preventive Treatment groups anti-cancer agent in humans. Animals of third passage were randomly assigned to an I3C treatment/control group (n = 12/12). All mice from the Methods two groups were treated by oral gavage: the control Animals and xenograft establishment group, administered with 200 μl of distilled water/poly- The protocol was approved by the Committee of the Uni- ethylene glycol (Panreac Quim. S.A, Barcelona, Spain) versidad Complutense of Madrid, and Animal Protection (6:4 ratio, n = 12/24) and the I3C group (administered Area of the Community of Madrid, Spain (Ref. Protocol with 200 μl of distilled water/polyethylene glycol contain- Number: PROEX 31/15). Twenty-four non-ovariectomized ing 150 mg I3C/kg/day) (Indole-3-carbinol, Sigma-Aldrich scid female SCID mice (BALB/cJHan®Hsd-Prkdc , Harlan Co., Madrid, Spain), (n = 12/24). The dose was chosen Laboratories Models,S.L.),6–8 weeks of age and from previous studies [42, 43]. Mice were given the doses weighing between 20 and 22 g were used. The animals through an oral feeding tube (18G x ½”) for 3 weeks (the were housed in a flexible-film isolator (Racks IVC de first dose was given 7 days after the xenografts, when Allentown Inc. Panlab Harvard Apparatus) in cages (2–3 tumors were palpable), following a dosage of 5 consecutive animals per cage), each measuring 330 cm ×12 cm, days of administration and 2 days of rest. The operator in a room with controlled environmental conditions was blinded to the treatment group assignment of each (20 °C to 22 °C; 50 to 55% relative humidity; 10 to 15 animal at all times. air changes per hour; and a 12:12 h light: dark cycle). Pre-sterilized food and water were provided ad libitum. All experimental procedures were performed between Histopathology and immunohistochemistry 11:30 a.m. and 12:30 a.m. The method of euthanasia Fragments from the treated tumors were fixed in neu- used to sacrifice the mice was isoflurane flow rate tral formalin and then embedded in paraffin for tumor adjusted to 5% until one minute after breathing stops. histopathology. The samples were histologically diag- Then, cervical dislocation was applied as method of nosed on HE-stained sections following the histological confirmation of euthanasia. classification of canine mammary tumors [44]. Histology The xenograft was directly established from a 9-year-old and immunohistochemistry of samples were evaluated by female dog with a spontaneous inflammatory mammary an experienced veterinary pathologist. Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 3 of 9 Immunohistochemistry for Ki-67, caspase-3 and estro- stored at − 20 °C until assayed for the presence of gen receptor (ER) was performed on deparaffinised hormones. Frozen tissue specimens of approximately sections and using the streptavidin–biotin–complex per- 0.1–0.5 g from individual mice were homogenized in oxidase method or detection kits. High-temperature 5 ml of PBS (pH = 7.2) and the homogenate centrifuged antigen retrieval with 10 mM citrate buffer pH = 6.0 was at 1200 x g, for 20 min at 4 °C. The supernatants were har- performed. Table 1 shows the primary antibodies and vested and stored at − 80 °C until hormone assays [45]. developing systems used. In the case of caspase-3, the Levels of estrone sulphate (E1SO ), estradiol (E2), slides were incubated with streptavidin-HRP conjugated androstenedione (A4), testosterone (T), dehydroepian- anti-caspase-3 antibody (TermoFisher Scientific ref. 43– drosterone (DHEA) and progesterone (P4) in tumor 4323, dilution 1/4000) for 30 min at room temperature homogenates and serum samples were assayed by ampli- (RT) and developed with a chromogen solution containing fied Enzyme-Immunoassay (EIA) previously validated in 3, 3′-diaminobenzidine tetrachloride (DAB). Thereafter, our laboratory [45]. the slides were counterstained with hematoxylin. Washes and dilutions were made in Tris-buffered saline (pH = 7.4) Statistical analysis for each marker; corresponding positive and negative con- Statistical analyses were performed by the IBM SPSS trol slides were performed. Statistics, 19.0 for Windows (Chicago, IL, USA). The Tumor proliferation index (PI) was determined by relationship between continuous variables (plasma and tu- counting Ki-67 positive and negative nuclei in 8–10 moral steroid hormone levels, Ki-67 and caspase index) selected High Power Fields (HPF) with the highest per- and categorical variables (pathological parameters such as centage of labeling (minimum 1000 cells). Every immu- ulceration, sebaceous hyperplasia, dermal emboli, tumor nostained nucleus was considered positive regardless of emboli, degenerated emboli, thrombosis, lipid-rich sub- the intensity of stain. The PI or proportion of positive population, distant metastases, liver metastasis and estro- neoplastic cells in each sample was calculated as previ- gen receptor alpha) was established using the analysis of ously described [44]. variance (ANOVA) followed by appropriate post hoc tests Caspase-3 immunostaining was performed to analyze for similar variances (Duncan Test) or different ones the presence of apoptotic bodies in the xenograft tissue (Games-Howell test). The relationship between continu- and it was semi-quantitatively assessed by the intensity ous variables was assessed using the Pearson correlation. of immunoexpression, which was evaluated as low (+), The association between categorical variables was ana- moderate (++), or intense (+++), by the percentage of lyzed using Pearson’s chi-squared test (χ ). Differences positive cells (apoptotic index). were considered significant at p-value <0.05. Finally, the expression of ER was considered positive when more than 10% of the cells within the tumors were Results positive independently of the intensity of the staining [29]. Macroscopic growth and metastases A stable serial transplantable xenograft was successfully Steroid hormone concentrations in serum samples and established with a constant and rapid growth in all mice. tumor homogenates At the end of the experiment, in 4 weeks, the tumors At the time of necropsy of the control and experimental had an approximate size of 0.9–1.2 cm . The size and SCID mice, 1 ml of blood was obtained by cardiac punc- weight of the xenograft tumors in I3C treated and un- ture using a 25G needle from each mouse. Blood samples treated mice (controls) did not show significant differ- were collected in special tubes (microtube Serum-Gel ences (p > 0.05). Clotting Activator, Sarstedt, Nümbrecht, Germany)and Immediately after sacrifice, complete necropsy was per- spun down in a refrigerated centrifuge (Hettich Zentrifu- formed in each case. Common target organs for IMC such gen Universal 320 R, Germany) at 4 °C and a speed of as bone, lung, liver, brain or distant lymph nodes were 1200 x g (RCF) for 20 min. Serum was harvested and extensively examined. At necropsy, one xenotrasplanted Table 1 Primary and secondary antibodies/kits used for immunohistochemistry Primary Antibody Type Source Incubation Secondary Antibody/kit Source Incubation Ki-67 clon MIB-1 Mab Dako 60 min, RT EnVision+SystemHRP (DAB) Dako 40 min, RT (Ref. M7240) 1:75 (Ref. K4007) kit ERα clon 1D5 Mab Dako Overnight EnVision + SystemHRP (DAB) Dako 40 min, RT (Ref. M7047) 1:15 4° C (Ref. K4007) kit Caspase-3 Pab R&D Systems Overnight Swine anti-rabbit biotinylated Dako 1:200 30 min, RT (Ref. AF835) 1:1200 4° C (Ref. E0353) Mab mouse monoclonal antibody, Pab rabbit polyclonal antibody Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 4 of 9 control mouse presented pulmonary and mesenteric me- areas of isolated cells; the stroma was scant. Scattered tastases (1/12, 8.3% of the control group) and one out of tumor cells showed a lipid-rich cytoplasm. The neo- 12 (8.3%) I3C treated xenografts had pulmonary and mes- plastic cells exhibited a high pleomorphism with enteric metastases. Four animals in the I3C group showed marked cell and nuclear atypia, and an elevated mitotic metastasis in the liver (4/12, 33.3%, p = 0.028). All metas- index (anaplastic cells). Binucleated and multinucleated tases were histologically confirmed. cells, as well as atypical mitoses were frequently found. Large intratumor areas of necrosis, dermal lymphan- Histopathological findings giectasias, and severe edema in the dermis were also Primary canine IMC and control xenografts present. According to the clinical characteristics and the histo- logical invasion of dermal lymphatic vessels by tumor/ neoplastic emboli (Fig. 1a), the primary canine mam- Histological and immunohistochemical findings in the I3C mary tumor was diagnosed as an inflammatory mam- treated xenografts mary cancer originated by a highly undifferentiated Histological and immunohistochemical findings of anaplastic/solid mammary carcinoma grade III with both experimental groups are depicted in Table 2. scattered lipid-rich cells. Control xenografts reproduced Indole-3-carbinol treatment diminished proliferation the histological features of the primary canine mam- and increased apoptosis of tumor cells although mary tumor. Hence, emboli at peripheral lymphatic and embolization of neoplastic cells (7/12), liver metastasis blood vessels, as well as inside the tumor were observed and presence of tumor cells showing a lipid-rich cyto- (2/12, 16.7%) (Fig. 1b). The control xenografts were plasm (8/12) (Figs. 1c-i) together with hyperplasia of highly infiltrative in the dermis and striated muscle and dermal sebaceous glands (10/12) were also observed in were partially surrounded by a fibro-myxoid tissue. some animals. This lipid-rich cell subpopulation fre- Neoplastic cells displayed a solid pattern together with quently presented degeneration (8/12) and necrosis. Fig. 1 Representative histological features. a IMC with neoplastic emboli in superficial dermal lymphatic vessels and infiltration of carcinomatous scid cells in the female dog origin of the xenografts. b IMC with tumor emboli in SCID mouse (BALB/cJHan®Hsd-Prkdc ) xenograft control group. c IMC with tumor emboli in dermis in SCID mouse xenograft I3C group (p = 0.012, compared with control group), and non-dermal tumor emboli in I3C mice (p = 0.035). d IMC with abundant lipid-rich cells in SCID mouse xenograft I3C group (× 20), (× 40) (p = 0.001). e IMC with liver metastasis in SCID mouse xenograft I3C group (× 2), (× 20) (p = 0.028). f and g IMC showing caspase-3 positive immunolabeling in a low number of cells in SCID mouse xenograft control group versus I3C group where positive immunolabeling in numerous cells (× 4), (× 20) (p < 0.001). h IMC showing positive Ki-67 immunolabeling in numerous cells in xenograft control group, and i lower number of Ki-67 positive cells in the I3C group SCID mouse xenograft (× 20) (p < 0.001). Analysis of variance followed by appropriate post hoc tests for similar variances (Duncan Test) or different ones (Games Howell test) was used. IMC inflammatory mammary carcinoma, I3C indole-3-carbinol Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 5 of 9 Table 2 Histopathological and immunohistochemical characteristics in control and indole-3-carbinol (I3C) xenograft groups Control xenograft (%) I3C xenograft (%) p-value Ulceration 33.3 0.0 p = 0.028 Sebaceous hyperplasia 8.3 83.3 p < 0.001 Dermal emboli 0.0 41.7 p = 0.012 Tumor emboli (non-dermal) 16.7 58.3 p = 0.035 Degenerated emboli 0.0 8.3 p > 0.05 Thrombosis 33.3 0.0 p = 0.036 Lipid-rich subpopulation 0.0 66.7 p = 0.001 Distant metastases (any location) 8.3 33.3 p > 0.05 Liver metastases 0.0 33.3 p = 0.028 Control xenograft I3C xenograft p-value (%, mean ± standard deviation) (%, mean ± standard deviation) Ki-67 index 62.2 ± 4.55 54.5 ± 3.79 p < 0.001 Caspase index 6.9 ± 1.35 27.5 ± 2.33 p < 0.001 P values in boldface are significant. These values are p < 0.05 Primary canine tumor and all xenografts samples were Discussion negative for ER immunostaining. The positive control Several experimental studies have shown that I3C was canine uterus. possesses preventive anti-cancer [15, 17, 22]and dis- rupting estrogen signalling properties [10, 46–48]. Determination of steroid hormones Indole-3-carbinol is considered a potential agent in the The content of steroid hormones in serum and tumor prevention and treatment of hormone-dependent homogenates of both control and I3C xenograft groups are breast tumors [2, 21]. Thus, it is freely available in the showninTable 3. Significantly higher levels of estrone stores and supermarkets as a dietary phytochemical sulphate (p = 0.009), estradiol (p < 0.001) and androstene- (non as an approved pharmaceutical drug) for prevent- dione (p = 0.049) and lower level of testosterone (p =0.048) ing cancer, diminishing premenstrual syndrome and in I3C xenograft homogenates compared with the control perimenopause-related disturbances. Nevertheless, the group were found. Serum progesterone (p = 0.003) and tes- effect of I3C on patients with neoplasms, especially tosterone levels (p = 0.022) were also higher in I3C group. breast cancer is poorly documented [20, 49]. To the best of our knowledge, this is the first study regarding Table 3 Serum (S) (ng/ml) and tumor homogenate (T) (ng/g) the effects of I3C treatment on experimental or spontan- steroid hormone concentrations in control and indole-3- eous IMC. Both IBC and canine IMC are considered a carbinol (I3C) groups after 3 weeks of treatment special and very aggressive type of breast cancer due to its Control xenograft I3C xenograft particular/unique biological, molecular, pathological, gen- a,1 a,1 Estrone sulphate (E1SO4) S 0.12 ± 0.03 0.24 ± 0.05 etic and clinical signature features [30, 32–34]. In the a,2 b,2 present study, I3C treatment on a xenograft model of ca- T 13.71 ± 2.42 27.09 ± 3.09 a,1 a,1 nine IMC reduced tumor growth and increased apoptosis, Estradiol (E2) S 11.98 ± 0.82 12.62 ± 1.01 although metastasis and alterations in the peripheral levels a,2 b,1 T 7.44 ± 0.60 13.89 ± 0.99 of steroid hormones were also observed in some animals. a,1 a,1 Androstenedione (A4) S 5.96 ± 0.77 6.10 ± 1.28 Tumor proliferation index has been associated with a,1 b,2 T 5.16 ± 0.99 20.46 ± 5.17 poor prognosis in human [49] and canine mammary a,1 b,1 Testosterone (T) S 1.20 ± 0.09 1.76 ± 0.25 cancer [50]. Our xenografts from the I3C group had a,2 b,1 lower proliferation index compared with control group, T 12.16 ± 3.07 4.02 ± 1.21 a,1 a,2 indicating a loss of proliferative capacity after I3C Dehydroepiandrosterone (DHEA) S 2.96 ± 0.16 3.06 ± 0.37 administration. In accordance with our results, several a,1 a,2 T 2.32 ± 0.11 2.86 ± 0.63 reports using breast cancer cell lines [3, 18, 19, 51, 52] a,1 b,1 Progesterone (P4) S 0.69 ± 0.07 1.15 ± 0.10 and human breast cancer cell-derived tumor xenografts, a,2 a,2 T 5.37 ± 1.04 7.75 ± 1.98 have reported an antiproliferative effect of I3C [20, 49]. All values expressed as mean ± standard error. Different letters denote The antiproliferative property of I3C has been associated significant differences (p < 0.05) among groups. Different numbers denote to changes in cell signaling, specially disrupting estrogen significant differences (p < 0.05) between serum samples and tumor tissue homogenates, in the same treatment group responsiveness [19, 22, 47, 48]. Indole-3-carbinol could Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 6 of 9 have also a role in the induction of specific carcinogen hormones and general steroidogenesis have been studied. detoxifying enzymes, such as CYP1A [3, 17, 53], and in According to previous studies, the alteration of estrogen cell adhesion, dissemination and invasion of human metabolism by I3C can be mediated by cytochrome P450 breast cancer cells [2]. [66, 67] or the activation of aryl hydrocarbon receptor In the present study, the lack of ulceration and in- (AhR)-mediated pathways [68–70]. creased apoptosis in treated I3C tumors is in accordance Histopathological evidence of lipid droplets in neo- with a reduction of tumor growth and with other previ- plastic cells has been described in spontaneous and ex- ous in vitro and in vivo studies in human breast cancer perimental canine IMC, and it has been shown that [22, 51, 53, 54], probably through pro-apoptotic and could be due to steroid secretion [29, 37, 40]. The fre- anti-proliferative mechanisms [12, 19, 55, 56]. In quent presence of scattered lipid-rich cells in our mur- addition, it has been reported that I3C promotes apop- ine model has been previously described in another tosis of breast cancer cells by activating caspase-3 and IMC xenograft model [37]. In the present study I3C -9, among other mechanisms [4, 22, 54]. Accordingly, treatment increased significantly the number of intra- our study showed a high expression of caspase-3 in I3C cellular lipid droplets that appeared forming large areas treated xenografts. Both IBC and IMC are typically an- of lipid-rich cells, which were not present in the control giogenic, lymphangiogenic and lymphangiocentric, being group tumors. According to previous studies, these the presence of massive neoplastic emboli in dermal ves- lipid-rich cells could contain large amounts of steroids sels, their main histological feature [29, 35, 57, 58]. The that could be locally secreted [37, 38, 40]. The steroid presence of dermal neoplastic emboli in experimental secretion by IBC and IMC cell lines has been recently mice IBC/IMC xenotransplant models has been docu- indicated [71]. The presence of large areas containing a mented but it is not seen in all the samples [37, 57–61]. It lipid-rich cell subpopulation is in accordance with previ- is known that the presence of emboli within the dermal ous observations and explains the high amounts of lymphatic vessels in IMC, contributes to the rapid devel- steroids found in tumor homogenates of I3C treated xeno- opment of metastasis [62–64], and is responsible for the grafts of the present study. According to our results, sev- morbidity of this disease. In spite of the antiproliferative eral steroid hormones (estradiol, estrone sulphate and and apoptosic effect, emboli in dermal lymphatic vessels androstenedione) were significantly increased in I3C and liver metastasis were observed in some animals. treated tumor homogenates while testosterone was dimin- Therefore, the effect of I3C could not be enough to avoid ished. Interestingly, testosterone was significantly in- the appearance of tumor embolization and metastasis. creased in the sera of I3C treated mice. Further studies The appearance of metastases indicates that this biomo- should elucidate if these changes in steroid hormone con- del is appropriate for the study of metastatic tumors in tent are related to a major production of estrogens in situ general and inflammatory mammary cancer in particular, (via aromatase or sulphatase enzymes). since both IBC and IMC are very aggressive tumors with The mechanisms causing hormonal variations after an extremely high metastatic potential. The capacity of I3C administration are uncertain. The documented an- I3C to inhibit cell adhesion, migration and invasion of ti-estrogen effect of I3C through ER should be questioned non-inflammatory ER- breast cancer cell has been previ- in this particular case, since the model is ERα negative ously reported [2]. However, in this study, metastases were [3, 28, 46, 65]. As pointed out before, I3C can alter the observed in some animals. A potential limitation of the estrogen metabolism through other ER-independent path- current study is the scarce number of animals used. More ways [15, 27, 47, 53]. The role of other receptors such as studies should be performed to evaluate the capacity of ERβ should be considered and elucidated in future studies. I3C to inhibit the appearance of metastasis in this cancer. The relevance of ERβ in the pathogenesis of IMC has been The presence of thrombosis and some necrotic/degener- suggested previously [39]. Our study suggests that both ated neoplastic cells inside the emboli has been previously E1SO and E2 can be locally synthesized, especially after documented in IMC xenografts and suggests a partial I3C administration. Following the aromatase or sulpha- destruction of metastatic cells, reducing the possibility of tase pathways, androgens (especially testosterone that establishing distant metastasis [37]. In our study, only the is diminished in tissue) could be transformed in high control group showed thrombosis, which could suggest a tissue E1SO and E2 concentrations. Estrone sulphate reduced defense mechanism in I3C treated IMC xeno- could be a reservoir for the synthesis of active estrogens grafts. The possible host defense mechanisms in these in the mammary tumors, including the canine IMC xenografts remain to be elucidated. [39, 40, 72]. Previous studies indicated that I3C effect on The effect of I3C treatment on estrogens metabolism breast cancer cells favors estradiol 2-hydroxylation [46, 73]. has been previously indicated [10, 27, 28, 53, 65]; never- Although there are no previous studies on intratumoral theless, to the best of our knowledge, this is the first time E2 levels in I3C treated cells, such the activation of es- that the possible effects of this indole on other steroid tradiol 2-hydroxylation should induce a low level of E2 Martín-Ruiz et al. BMC Cancer (2018) 18:626 Page 7 of 9 [19, 74] but an increase of E2 and E1SO concentrations Acknowledgements We are grateful to Pedro Aranda for the histological technical assistance and in tumor homogenates was observed. This particular to Carmen Pilar Garcia for animal’s care. finding has to be compared in non-inflammatory breast cancer xenografts to elucidate if the high amounts of E2 Funding This research was supported by the Spanish Ministry of Science and found in the present study are due to the particular Education (research project SAF2009–10572). The funding body did not characteristics of IMC. In addition to our results refer to contribute in any of the following: design of the study, data collection, data intratumoral levels, as stated above. analysis, interpretation of data, or in writing the manuscript. The higher concentration of steroids and longer Availability of data and materials half-life suggests the formation of biologically active es- All data analyzed for this study are included in this article. Supplementary trogens in tumor tissue [40]. In order to proliferate, it is information files are available upon request to the corresponding author. possible that tumor increased estradiol levels from the Authors’ contributions E1SO reservoir. In our results, serum P4 concentrations LP and JCI conceived of the study. LP, AG and JCI participated in its design increased in I3C group, probably due to a lower expres- and coordination, interpretation of results and helped to draft the sion of progesterone receptors by I3C, and consequently, manuscript. AM generated the animal model and AM, LDC and SC carried out the experimental study and collected all data. LP, JCI, AG and AM wrote higher serum P4 levels [10, 65, 75]. As occurs with the manuscript. SC and AM carried out the hormonal assays and LDC the estrogens and progesterone a local synthesis of andro- immunohistochemical techniques. All authors read and approved the final gens by IBC and IMC tumor cell lines, has also been con- manuscript, and all of them agree that they are accountable for all aspects of the work. firmed [71]. The role of androgens in breast cancer is not clear. Some studies suggest an antiproliferative effect [76], Ethics approval but others note an increased proliferation in breast cancer All procedures were carried out according to European and Spanish legislative and regulatory guidelines (European convention ETS 1 2 3, on cell lines [77, 78]. The decrease of testosterone levels in the use and protection of vertebrate mammals in experimentation and for I3C-treated group could be due to the inhibitory effect of other scientific purposes and Spanish Law 32/2007, and R.D. 1201/2005 on I3C on the androgen receptors transcription [66]. Similar the protection and use of animals in scientific research). The protocol was approved by the Committee of the Universidad Complutense of Madrid, to E2, a higher content of intratumoral A4 in I3C group and Animal Protection Area of the Community of Madrid, Spain (Ref. could be due to an increased local synthesis and to the Protocol Number: PROEX 31/15). binding of I3C to androgen receptors, increasing A4 free levels in tissue homogenates. However, intratumoral T Competing interests None of the authors of this paper has a financial or personal relationship concentrations decreased in I3C group when compared with other people or organizations that could inappropriately influence or with control group, probably by the I3C-associated stimu- bias the content of the paper. latory effect on the aromatase enzyme [79] which stimu- lates T to E2 conversion. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Conclusions Our data reveal for the first time that the ingestion of Author details Department of Animal Physiology, Veterinary Medicine School, indole-3-carbinol, as administered, diminishes prolifera- Complutense University of Madrid, Madrid, Spain. Department of Animal tion and increases apoptosis of tumor cells in an experi- Medicine, Surgery and Pathology, Veterinary Medicine School, Complutense mental model of inflammatory breast cancer, although University of Madrid, Madrid, Spain. this effect could not be enough to avoid the appearance Received: 6 February 2018 Accepted: 18 May 2018 of tumor embolization and metastasis. Additionally, stimulated steroid hormones levels and a characteristic lipid-rich cell population were presented. These results References 1. McCullough ML, Giovannucci EL. Diet and cancer prevention. 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BMC CancerSpringer Journals

Published: Jun 4, 2018

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