Tumor Growth Progression in Ectopic and Orthotopic Xenografts from Inflammatory Breast Cancer Cell Lines.

Xenografts can grow in immunosuppressed hosts, such as SCID mice, and tumor material can be injected into hosts either ectopically or orthotopically. Choosing the correct model to use is a crucial step in animal research. The aim of this study was to report the differences between ectopic and orthotopic xenografts in tumor progression, metastasis capacity, histological features, and steroid hormone profiles in xenografts from the cIMC (canine inflammatory mammary cancer) cell line IPC-366 and hIBC (human inflammatory breast cancer) cell line SUM149. To achieve this purpose, 40 female mice 6-8 weeks old were inoculated with IPC-366 and SUM149 cells subcutaneously (ectopic models) or into mammary fat pad (orthotopic models). Mice were monitored for tumor progression and appearance of metastases, and generated tumors were analyzed in terms of histological examination and steroid hormone production. The results revealed differences in tumor appearance and percentage of metastasis between ectopic and orthotopic models, which were higher in the ectopic xenografts from both cell lines. However, both models had similar characteristics of tumor progression, histological features, and steroid hormone secretion profiles. We show that the ectopic model can be validated as a good and useful model of tumor development in addition to, not contrary to, the orthotopic model in breast cancer research.

Effects of doxorubicin associated with amniotic membrane stem cells in the treatment of canine inflammatory breast carcinoma (IPC-366) cells.

BACKGROUND: Tumours in mammary glands represent the most common neoplasia in bitches, as in humans. This high incidence results in part from the stimulation of sex hormones on these glands. Among mammary tumours, inflammatory carcinoma is the most aggressive, presenting a poor prognosis to surgical treatment and chemotherapy. One of the most widely used chemotherapy drugs for breast cancer treatment is doxorubicin (DOXO). Alternative therapies have been introduced in order to assist in these treatments; studies on treatments using stem cells have emerged, since they have anti-inflammatory and immunomodulatory properties. The aim of this study was to evaluate the effects of DOXO and canine amniotic membrane stem cells (AMCs) on the triple-negative canine inflammatory mammary carcinoma cell line IPC-366. METHODS: Four experimental groups were analysed: a control group without treatment; Group I with DOXO, Group II with AMC and Group III with an association of DOXO and AMCs. We performed the MTT assay with DOXO in order to select the best concentration for the experiments. The growth curve was performed with all groups (I-III) in order to verify the potential of treatments to reduce the growth of IPC-366. For the cell cycle, all groups (I-III) were tested using propidium iodide. While in the flow cytometry, antibodies to progesterone receptor (PR), estrogen receptor (ER), PCNA, VEGF, IL-10 and TGF-ß1 were used. For steroidogenic pathway hormones, an ELISA assay was performed. RESULTS: The results showed that cells treated with 10 µg/mL DOXO showed a 71.64% reduction in cellular growth after 72 h of treatment. Reductions in the expression of VEGF and PCNA-3 were observed by flow cytometry in all treatments when compared to the control. The intracellular levels of ERs were also significantly increased in Group III (4.67% vs. 27.1%). Regarding to the levels of steroid hormones, significant increases in the levels of estradiol (E2) and estrone sulphate (S04E1) were observed in Groups I and III. On the other hand, Group II did not show differences in steroid hormone levels in relation to the control. We conclude that the association of DOXO with AMCs (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC-366 triple-negative cells. CONCLUSIONS: This treatment promoted ER positive expression, suggesting that the accumulated oestrogen conducted these cells to a synergistic state, rendering these tumour cells responsive to ERs and susceptible to new hormonal cancer therapies.

Effects of soya milk on reproductive hormones during puberty in male Wistar rats.

Puberty is considered a critical period on development that involved sexual maturation and morphological changes. Isoflavones have been described as endocrine disruptors in male rats. Therefore, the present study attempt to evaluate the effect that daily intake of low and high doses of isoflavones exert into the hormonal regulation that take place during puberty by analyzing the levels of serum and testes steroid and pituitary hormones. 108 male pre-puberal Wistar rats (30 days old) were randomly divided into three groups; control, low and high doses of isoflavones. Experimental animals were daily dosed orally with low and high doses of a mixture of two soy isoflavones (genistein and daidzein) during 6 weeks. An EIA was performed in serum and testes homogenates for analyzing FSH, LH, P5, P4, DHEA, A4, T, DHT, SO4E1 and E2 hormone concentrations. Results revealed a decrease of an oestrogen environment in testes stimulates the secretion of FSH and LH leading to the production of androgens in the testes at the onset of puberty. Low doses of isoflavones resulted in a significant increase of testes oestrogens that consequently produced a delay on the onset of puberty; however at high doses of isoflavones the maintained oestrogenic environment in the testes prevent the stimulation of the secretion of pituitary hormones and the production of T abolishing the onset of puberty. These results clarify the hormonal mechanisms that take place on puberty and determine the effect of high and low doses of isoflavones at the onset of puberty.

Ovary and vaginal epithelium dynamics during the estrous cycle in Dasyprocta prymnolopha Wagler, 1831: ultrasound and cytological examinations.

The objective of the present study was to monitor and characterize morphological alterations in ovaries of agouti (Dasyprocta prymnolopha), reared in captivity, by using abdominal ultrasonography. All animals underwent daily vaginal cytological examination to identify the current cycle phase. For each phase of the estrous cycle, ultrasound examinations were carried out to identify and describe the morphology of both ovaries. Topographic parameters in an ultrasound window were established to locate the ovaries. The agouti estrous cycle lasted an average of 29.94 ± 6.77 days. During vaginal cytology examinations, all cell types were identified, and each phase of the estrous cycle was established by cell counts. No significant alterations were observed in the assessed ovarian morphometry measurements. In 75% of the animals examined, ovarian follicle presence was observed in the proestrus phase.

Steroid Tumor Environment in Male and Female Mice Model of Canine and Human Inflammatory Breast Cancer.

Canine inflammatory mammary cancer (IMC) shares clinical and histopathological characteristics with human inflammatory breast cancer (IBC) and has been proposed as a good model for studying the human disease. The aim of this study was to evaluate the capacity of female and male mice to reproduce IMC and IBC tumors and identify the hormonal tumor environment. To perform the study sixty 6-8-week-old male and female mice were inoculated subcutaneously with a suspension of 10(6)IPC-366 and SUM149 cells. Tumors and serum were collected and used for hormonal analysis. Results revealed that IPC-366 reproduced tumors in 90% of males inoculated after 2 weeks compared with 100% of females that reproduced tumor at the same time. SUM149 reproduced tumors in 40% of males instead of 80% of females that reproduced tumors after 4 weeks. Both cell lines produce distant metastasis in lungs being higher than the metastatic rates in females. EIA analysis revealed that male tumors had higher T and SO4E1 concentrations compared to female tumors. Serum steroid levels were lower than those found in tumors. In conclusion, IBC and IMC male mouse model is useful as a tool for IBC research and those circulating estrogens and intratumoral hormonal levels are crucial in the development and progression of tumors.

Steroid hormone secretion in inflammatory breast cancer cell lines.

Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. Though several IBC cell lines have been established, recently a first IMC cell line was established. The aims of this study were: (1) to validate a highly sensitive, reliable, accurate and direct amplified enzyme immunoassay (EIA) to measure several cell-secreted steroid hormones: progesterone (P4), androstenedione (A4), testosterone (T), 17ß-estradiol (E2) and estrone sulfate (SO4E1) in the culture medium. (2) To assess whether hormone production profile by IPC-366 cells validates the IMC model for human IBC. We validated a non-competitive amplified EIA for inflammatory breast cancer cell lines based on the results of accuracy, precision, sensitivity and parallelism. The low detection limits of the technique were: P4=13.2 pg/well, A4=2.3 pg/well, T=11.4 pg/well, E2=1.9 pg/well and SO4E1=4.5 pg/well. Intra- and inter-assay coefficient of variation percentages were <10%. The mean recovery rate of hormone added to the culture medium was >90%. In all hormones studied SUM149 have higher levels (1.4 times, but not significant) than IPC-366, and the correlation index between SUM149 and IPC-366 concentrations were >97%. We can coclude that cells of both cell lines, IPC-366 and SUM149, are capable to produce steroid hormone in culture media. The presented EIA methodology is very valuable for the detection of steroid production in culture media and could be used in hormone regulation studies and therapeutic agents in cell lines of inflammatory and non-inflammatory mammary carcinoma or other cancer cell lines in preclinical studies.

Establishment and characterization of a new cell line of canine inflammatory mammary cancer: IPC-366.

Canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The cells were grown under adherent conditions. The growth, cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Ten female Balb/SCID mice were inoculated with IPC-366 cells to assess their tumorigenicity and metastatic potential. Chromosome aberration test and Karyotype revealed the presence of structural aberration, numerical and neutral rearrangements, demonstrating a chromosomal instability. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon that has been related to s vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2, E-cadherin, overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). At 2 weeks after inoculating the IPC-366 cells, a tumor mass was found in 100 % of mice. At 4 weeks metastases in lung and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative study of both IMC and IBC.

Characterization of antiestrogenic activity of the Chinese herb, prunella vulgaris, using in vitro and in vivo (Mouse Xenograft) models.

Prunella vulgaris (PV), a commonly used Chinese herb, also known as Self-heal, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182,780 and PV extract. Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner--responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women.

A role for alphavbeta3 integrin during implantation in the rabbit model.

The study of implantation has been facilitated by the identification of specific biomarkers that are associated with uterine receptivity. The alpha(v)beta(3) integrin is a cell surface adhesion receptor, whose expression has been shown to be elevated in the endometrium at the time of implantation in both humans and other mammalian species; however, the distribution of alpha(v)beta(3) in the rabbit model is unknown. The rabbit has been shown to be an excellent model for the study of implantation. As an obligate ovulator, the timing of pregnancy can be precisely established, and embryonic attachment occurs through specialized trophoblast-endometrial structures known as trophoblastic knobs. In the present study, the expression of alpha(v)beta(3) integrin subunit in the rabbit uterus was examined by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization. Expression of the alpha(v)beta(3) integrin was examined in Day 6.5 embryos, flushed from pregnant does. Immunofluorescence demonstrated strong immunostaining on the rabbit blastocyst (Day 6.5). RT-PCR analyses showed higher levels of mRNA for beta(3) subunit at the implantation site, with reduced expression in nonimplantation sites and in nonpregnant adult and immature endometrium. Immunohistochemistry demonstrated little, if any, beta(3) immunoreactivity on the endometrial epithelium. In contrast, in situ hybridization showed expression of the beta(3) integrin subunit mRNA in the uterine myometrium and on the trophoblast. To further determine the functional significance of alpha(v)beta(3) integrin expression during implantation, pregnant female rabbits that underwent ventral laparotomy on the morning of Day 6 received intrauterine injection of the following into the right uterine horn: 1) the monoclonal alpha(v)beta(3) neutralizing antibody (LM609), 2) arg-gly-asp (RGD) hexapeptides (GRGDSP), 3) non-RGD hexapeptides (GRGESP), and 4) IgG isotype matched control antibody. The left horn served as a control and received only saline injections. A significant reduction in the number of implantation sites was observed in the horns receiving anti-alpha(v)beta(3) antibody (P < 0.001) and the RGD peptides (P = 0.03). In the rabbit, the alpha(v)beta(3) integrin is present on the embryo and trophoblast and appears to be involved in early embryo-maternal interaction.

Regulated expression of osteopontin in the peri-implantation rabbit uterus.

Blastocyst attachment to the lining of the mammalian uterus during early implantation involves the initial apposition of the trophoblast to the uterine epithelial surface. Osteopontin (OPN) is a glycoprotein component of the extracellular matrix that is secreted by the glandular epithelium of mammalian uteri at the time of implantation. This protein is recognized by several members of the integrin family and promotes cell-cell attachment and adhesion. In the present study, rabbit uteri were examined using Northern and in situ hybridization to evaluate the temporal and spatial distribution of OPN mRNA during early pregnancy. Northern blot analysis demonstrated a dramatic increase in OPN expression on Days 4-7 of pregnancy, corresponding to the rise in circulating progesterone and the time of initial embryo attachment in this species. In situ hybridization analysis revealed OPN mRNA expression on Day 6.75 of pregnancy, which was most prominent on endometrial epithelium. Using immunofluorescence, OPN protein was present on the glandular epithelium on Day 6.75 of pregnancy, but was absent on blastocysts. Further, no expression of OPN mRNA or protein was found in the nonpregnant endometrium. Induction of endometrial OPN expression was observed in unmated rabbits treated with progesterone alone and was prevented by cotreatment with the antiprogestin ZK137.316. Estradiol-17beta had no effect on OPN expression by itself, and estrogen priming was not necessary to demonstrate the stimulatory effect of progesterone. In The rabbit uterus, as in other mammalian species studied, OPN is expressed in a stage-specific manner by the endometrial glands during the peri-implantation period and is regulated by progesterone.