Abstract
BackgroundTumours 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.MethodsFour 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-β1 were used. For steroidogenic pathway hormones, an ELISA assay was performed.ResultsThe 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.ConclusionsThis 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.
Highlights
Tumours in mammary glands represent the most common neoplasia in bitches, as in humans
We conclude that the association of DOXO with Canine amniotic membrane stem cells (AMC) (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC366 triple-negative cells
This treatment promoted estrogen receptor (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
Summary
Tumours in mammary glands represent the most common neoplasia in bitches, as in humans. Tumours in canine mammary glands represent the most common neoplasm in female dogs [2] These types of tumours in female dogs are very similar to tumours presented in humans regarding their biological behaviour, their response to cytotoxic agents and their histological characteristics, but their incidence rate is three times higher than in women [3]. This high incidence results in part from the stimulation by sex hormones of the mammary glands of female dogs and women exposed to them during their reproductive life [4]. When acting together in combination with other factors, for example, environmental and epigenetic factors at different stages of tumour formation, it is possible that they play a determining role in tumour formation [5]
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