EB1089 Increases the Antiproliferative Response of Lapatinib in Combination with Antiestrogens in HER2-Positive Breast Cancer Cells.

HER2-positive breast cancer is associated with aggressive behavior and reduced survival rates. Calcitriol restores the antiproliferative activity of antiestrogens in estrogen receptor (ER)-negative breast cancer cells by re-expressing ERα. Furthermore, calcitriol and its analog, EB1089, enhance responses to standard anti-cancer drugs. Therefore, we aimed to investigate EB1089 effects when added to the combined treatment of lapatinib and antiestrogens on the proliferation of HER2-positive breast cancer cells. BT-474 (ER-positive/HER2-positive) and SK-BR-3 (ER-negative/HER2-positive) cells were pre-treated with EB1089 to modulate ER expression. Then, cells were treated with EB1089 in the presence of lapatinib with or without the antiestrogens, and proliferation, phosphorylation array assays, and Western blot analysis were performed. The results showed that EB1089 restored the antiproliferative response to antiestrogens in SK-BR-3 cells and improved the inhibitory effects of the combination of lapatinib with antiestrogens in the two cell lines. Moreover, EB1089, alone or combined, modulated ERα protein expression and reduced Akt phosphorylation in HER2-positive cells. EB1089 significantly enhanced the cell growth inhibitory effect of lapatinib combined with antiestrogens in HER2-positive breast cancer cells by modulating ERα expression and Akt phosphorylation suppression. These results highlight the potential of this therapeutic approach as a promising strategy for managing HER2-positive breast cancer.

The Preventive Role of the Vitamin D Endocrine System in Cervical Cancer.

Vitamin D along with its active metabolite calcitriol and its metabolic and signaling system, known as the vitamin D endocrine system, have been widely recognized as a pivotal regulator of calcium homeostasis in addition to non-calcemic antitumoral effects in a variety of human cancers, including cervical cancer. Several studies have found an inverse relationship between the incidence of cervical neoplasia and vitamin D levels. This narrative review updates the current evidence supporting the notion that the vitamin D endocrine system has a preventive role on cervical cancer, mainly in the early phases of the disease, acting at the level of suppressing cell proliferation, promoting apoptosis, modulating inflammatory responses, and probably favoring the clearance of human papillomavirus-dependent cervical lesions. Although an optimal vitamin D status helps in the prevention and regression of low-grade squamous intraepithelial lesions of the cervix, it appears that vitamin D alone or combined with chemotherapeutic agents has little effectivity once advanced cervical cancer is established. These observations suggest that an optimal vitamin D status might exert beneficial actions in the early phases of cervical cancer by preventing its onset and progression.

The Phytochemical α-Mangostin Inhibits Cervical Cancer Cell Proliferation and Tumor Growth by Downregulating E6/E7-HPV Oncogenes and KCNH1 Gene Expression.

Cervical cancer is the fourth most common cancer among women worldwide. The main factor associated with the onset and progression of this neoplasia is the human papillomavirus (HPV) infection. The HPV-oncogenes E6 and E7 are critical drivers of cellular transformation, promoting the expression of oncogenes such as KCNH1. The phytochemical α-mangostin (AM) is a potent antineoplastic and antiviral compound. However, its effects on HPV oncogenes and KCNH1 gene expression remain unknown. This study evaluated the effects of AM on cell proliferation, cell cycle distribution and gene expression, including its effects on tumor growth in xenografted mice. AM inhibited cell proliferation in a concentration-dependent manner, being the most sensitive cell lines those with the highest number of HPV16 copies. In addition, AM promoted G1-cell cycle arrest in CaSki cells, while led to cell death in SiHa and HeLa cells. Of interest was the finding of an AM-dependent decreased gene expression of E6, E7 and KCNH1 both in vitro and in vivo, as well as the modulation of cytokine expression, Ki-67, and tumor growth inhibition. On these bases, we suggest that AM represents a good option as an adjuvant for the treatment and prevention of cervical cancer.

The Inhibition of the FGFR/PI3K/Akt Axis by AZD4547 Disrupts the Proangiogenic Microenvironment and Vasculogenic Mimicry Arising from the Interplay between Endothelial and Triple-Negative Breast Cancer Cells.

Vasculogenic mimicry (VM), a process in which aggressive cancer cells form tube-like structures, plays a crucial role in providing nutrients and escape routes. Highly plastic tumor cells, such as those with the triple-negative breast cancer (TNBC) phenotype, can develop VM. However, little is known about the interplay between the cellular components of the tumor microenvironment and TNBC cells' VM capacity. In this study, we analyzed the ability of endothelial and stromal cells to induce VM when interacting with TNBC cells and analyzed the involvement of the FGFR/PI3K/Akt pathway in this process. VM was corroborated using fluorescently labeled TNBC cells. Only endothelial cells triggered VM formation, suggesting a predominant role of paracrine/juxtacrine factors from an endothelial origin in VM development. Via immunocytochemistry, qPCR, and secretome analyses, we determined an increased expression of proangiogenic factors as well as stemness markers in VM-forming cancer cells. Similarly, endothelial cells primed by TNBC cells showed an upregulation of proangiogenic molecules, including FGF, VEGFA, and several inflammatory cytokines. Endothelium-dependent TNBC-VM formation was prevented by AZD4547 or LY294002, strongly suggesting the involvement of the FGFR/PI3K/Akt axis in this process. Given that VM is associated with poor clinical prognosis, targeting FGFR/PI3K/Akt pharmacologically may hold promise for treating and preventing VM in TNBC tumors.

High Glucose Promotes Inflammation and Weakens Placental Defenses against E. coli and S. agalactiae Infection: Protective Role of Insulin and Metformin.

Placentas from gestational diabetes mellitus (GDM) patients undergo significant metabolic and immunologic adaptations due to hyperglycemia, which results in an exacerbated synthesis of proinflammatory cytokines and an increased risk for infections. Insulin or metformin are clinically indicated for the treatment of GDM; however, there is limited information about the immunomodulatory activity of these drugs in the human placenta, especially in the context of maternal infections. Our objective was to study the role of insulin and metformin in the placental inflammatory response and innate defense against common etiopathological agents of pregnancy bacterial infections, such as E. coli and S. agalactiae, in a hyperglycemic environment. Term placental explants were cultivated with glucose (10 and 50 mM), insulin (50-500 nM) or metformin (125-500 µM) for 48 h, and then they were challenged with live bacteria (1 × 105 CFU/mL). We evaluated the inflammatory cytokine secretion, beta defensins production, bacterial count and bacterial tissue invasiveness after 4-8 h of infection. Our results showed that a GDM-associated hyperglycemic environment induced an inflammatory response and a decreased beta defensins synthesis unable to restrain bacterial infection. Notably, both insulin and metformin exerted anti-inflammatory effects under hyperglycemic infectious and non-infectious scenarios. Moreover, both drugs fortified placental barrier defenses, resulting in reduced E. coli counts, as well as decreased S. agalactiae and E. coli invasiveness of placental villous trees. Remarkably, the double challenge of high glucose and infection provoked a pathogen-specific attenuated placental inflammatory response in the hyperglycemic condition, mainly denoted by reduced TNF-α and IL-6 secretion after S. agalactiae infection and by IL-1ß after E. coli infection. Altogether, these results suggest that metabolically uncontrolled GDM mothers develop diverse immune placental alterations, which may help to explain their increased vulnerability to bacterial pathogens.

Endothelium-Dependent Induction of Vasculogenic Mimicry in Human Triple-Negative Breast Cancer Cells Is Inhibited by Calcitriol and Curcumin.

In highly aggressive tumors, cancer cells may form channel-like structures through a process known as vasculogenic mimicry (VM). VM is generally associated with metastasis, mesenchymal phenotype, and treatment resistance. VM can be driven by antiangiogenic treatments and/or tumor microenvironment-derived factors, including those from the endothelium. Curcumin, a turmeric product, inhibits VM in some tumors, while calcitriol, the most active vitamin D metabolite, exerts potent antineoplastic effects. However, the effect of these natural products on VM in breast cancer remains unknown. Herein, we studied the effect of both compounds on triple-negative breast cancer (TNBC) VM-capacity in a co-culture model. The process of endothelial cell-induced VM in two human TNBC cell lines was robustly inhibited by calcitriol and partially by curcumin. Calcitriol promoted TNBC cells' morphological change from spindle-like to cobblestone-shape, while curcumin diminished VM 3D-structure. Notably, the treatments dephosphorylated several active kinases, especially those involved in the PI3K/Akt pathway. In summary, calcitriol and curcumin disrupted endothelium-induced VM in TNBC cells partially by PI3K/Akt inactivation and mesenchymal phenotype inhibition. Our results support the possible use of these natural compounds as adjuvants for VM inactivation in patients with malignant tumors inherently capable of forming VM, or those with antiangiogenic therapy, warranting further in vivo studies.

Compartmentalized Innate Immune Response of Human Fetal Membranes against Escherichia coli Choriodecidual Infection.

An infectious process into the uterine cavity represents a major endangered condition that compromises the immune privilege of the maternal-fetal unit and increases the risk for preterm birth (PTB) and premature rupture of membranes (PROM). Fetal membranes are active secretors of antimicrobial peptides (AMP), which limit bacterial growth, such as Escherichia coli. Nevertheless, the antibacterial responses displayed by chorioamniotic membranes against a choriodecidual E. coli infection have been briefly studied. The objective of this research was to characterize the profile of synthesis, activity, and spatial distribution of a broad panel of AMPs produced by fetal membranes in response to E. coli choriodecidual infection. Term human chorioamniotic membranes were mounted in a two independent compartment model in which the choriodecidual region was infected with live E. coli (1 × 105 CFU/mL). Amnion and choriodecidual AMP tissue levels and TNF-α and IL-1ß secretion were measured by the enzyme-linked immunosorbent assay. The passage of bacterium through fetal membranes and their effect on structural continuity was followed for 24 h. Our results showed that E. coli infection caused a progressive mechanical disruption of the chorioamniotic membranes and an activated inflammatory environment. After the challenge, the amnion quickly (2-4 h) induced production of human beta defensins (HBD)-1, HBD-2, and LL-37. Afterwards (8-24 h), the amnion significantly produced HBD-1, HBD-2, HNP-1-3, S100A7, sPLA2, and elafin, whereas the choriodecidua induced LL-37 synthesis. Therefore, we noticed a temporal- and tissue-specific pattern regulation of the synthesis of AMPs by infected fetal membranes. However, fetal membranes were not able to contain the collagen degradation or the bacterial growth and migration despite the battery of produced AMPs, which deeply increases the risk for PTB and PROM. The mixture of recombinant HBDs at low concentrations resulted in increased bactericidal activity compared to each HBD alone in vitro, encouraging further research to study AMP combinations that may offer synergy to control drug-resistant infections in the perinatal period.

Combinations of Calcitriol with Anticancer Treatments for Breast Cancer: An Update.

Preclinical, clinical, and epidemiological studies indicate that vitamin D3 (VD) deficiency is a risk factor for the development of breast cancer. Underlying mechanisms include the ability of calcitriol to induce cell differentiation, inhibit oncogenes expression, and modify different signaling pathways involved in the control of cell proliferation. In addition, calcitriol combined with different kinds of antineoplastic drugs has been demonstrated to enhance their beneficial effects in an additive or synergistic fashion. However, a recognized adjuvant regimen based on calcitriol for treating patients with breast cancer has not yet been fully established. Accordingly, in the present work, we review and discuss the preclinical and clinical studies about the combination of calcitriol with different oncological drugs, aiming to emphasize its main therapeutic benefits and opportunities for the treatment of this pathology.

Immunoendocrine Dysregulation during Gestational Diabetes Mellitus: The Central Role of the Placenta.

Gestational Diabetes Mellitus (GDM) is a transitory metabolic condition caused by dysregulation triggered by intolerance to carbohydrates, dysfunction of beta-pancreatic and endothelial cells, and insulin resistance during pregnancy. However, this disease includes not only changes related to metabolic distress but also placental immunoendocrine adaptations, resulting in harmful effects to the mother and fetus. In this review, we focus on the placenta as an immuno-endocrine organ that can recognize and respond to the hyperglycemic environment. It synthesizes diverse chemicals that play a role in inflammation, innate defense, endocrine response, oxidative stress, and angiogenesis, all associated with different perinatal outcomes.

Ethical Considerations in Animal Research: The Principle of 3R's.

In the last century, progress in the knowledge of human diseases, their diagnosis and treatment have grown exponentially, due in large part to the introduction and use of laboratory animals. Along with this important progress, the need to provide training and guidance to the scientific community in all aspects related to the proper use of experimental animals has been indispensable. Animal research committees play a primary role in evaluating experimental research protocols, from their feasibility to the rational use of animals, but above all in seeking animal welfare. The Institutional Committee for the Care and Use of Animals (IACUC) has endeavored to share several relevant aspects in conducting research with laboratory animals. Here, we present and discuss the topics that we consider of utmost importance to take in the account during the design of any experimental research protocol, so we invite researchers, technicians, and undergraduate and graduate students to dive into the fascinating subject of proper animal care and use for experimentation. The main intention of these contributions is to sensitize users of laboratory animals for the proper and rational use of them in experimental research, as well as to disseminate the permitted and unpermitted procedures in laboratory animals. In the first part, the significance of experimental research, the main functions of IACUC, and the principle of the three R's (replacement, reduction, and refinement) are addressed.

Ethical Considerations in Animal Research: The Principle of 3R's.

In the last century, progress in the knowledge of human diseases, their diagnosis and treatment have grown exponentially, due in large part to the introduction and use of laboratory animals. Along with this important progress, the need to provide training and guidance to the scientific community in all aspects related to the proper use of experimental animals has been indispensable. Animal research committees play a primary role in evaluating experimental research protocols, from their feasibility to the rational use of animals, but above all in seeking animal welfare. The Institutional Committee for the Care and Use of Animals (IACUC) has endeavored to share several relevant aspects in conducting research with laboratory animals. Here, we present and discuss the topics that we consider of utmost importance to take in the account during the design of any experimental research protocol, so we invite researchers, technicians, and undergraduate and graduate students to dive into the fascinating subject of proper animal care and use for experimentation. The main intention of these contributions is to sensitize users of laboratory animals for the proper and rational use of them in experimental research, as well as to disseminate the permitted and unpermitted procedures in laboratory animals. In the first part, the significance of experimental research, the main functions of IACUC, and the principle of the three R's (replacement, reduction, and refinement) are addressed.

Innate Immune Cells and Toll-like Receptor-Dependent Responses at the Maternal-Fetal Interface.

During pregnancy, the placenta, the mother and the fetus exploit several mechanisms in order to avoid fetal rejection and to maintain an immunotolerant environment throughout nine months. During this time, immune cells from the fetal and maternal compartments interact to provide an adequate defense in case of an infection and to promote a tolerogenic milieu for the fetus to develop peacefully. Trophoblasts and decidual cells, together with resident natural killer cells, dendritic cells, Hofbauer cells and other macrophages, among other cell types, contribute to the modulation of the uterine environment to sustain a successful pregnancy. In this review, the authors outlined some of the various roles that the innate immune system plays at the maternal-fetal interface. First, the cell populations that are recruited into gestational tissues and their immune mechanisms were examined. In the second part, the Toll-like receptor (TLR)-dependent immune responses at the maternal-fetal interface was summarized, in terms of their specific cytokine/chemokine/antimicrobial peptide expression profiles throughout pregnancy.

ASTEMIZOLE, AN INHIBITOR OF ETHER-À-GO-GO-1 POTASSIUM CHANNEL, INCREASES THE ACTIVITY OF THE TYROSINE KINASE INHIBITOR GEFITINIB IN BREAST CANCER CELLS.

BACKGROUND: Expression and activity of the potassium channel ether-à-go-go-1 (EAG1) are strongly related to carcinogenesis and tumor progression, which can be exploited for therapeutic purposes. EAG1 activity may be reduced by preventing its phosphorylation with epidermal growth factor receptor (EGFR) kinase inhibitors and by astemizole, which blocks the channel pore and downregulates its gene expression. OBJECTIVE: We aimed to study the potential cooperative antiproliferative effect of the EGFR inhibitor gefitinib and the EAG1-blocker astemizole, in breast cancer cells. MATERIALS AND METHODS: The cells were characterized by immunocytochemistry. Inhibitory concentrations were determined by non-linear regression analysis using dose-response curves. The nature of the pharmacological effect was evaluated by the combination index equation while cell cycle analysis was studied by flow cy-tometry. RESULTS: Astemizole and gefitinib inhibited cell proliferation in a concentration-dependent manner, with inhibitory concentrations (IC 50) values of 1.72 µM and 0.51 µM, respectively. All combinations resulted in a synergistic antiproliferative effect. The combination of astemizole and gefitinib diminished the percentage of cells in G2/M and S phases, while increased accumulation in G0/G1 of the cell cycle. CONCLUSIONS: Astemizole and gefitinib synergistically inhibited proliferation in breast cancer cells expressing both EGFR and EAG1. Our results suggest that the combined treatment increased cell death by targeting the oncogenic activity of EAG1.

The role of the ovarian cycle and the effects of mitogen-induced cytokines on human prolactin gene expression in peripheral blood mononuclear cells.

BACKGROUND: little is known on the influences of normal menstrual cycle on prolactin gene expression in immune cells. AIM OF THE STUDY: to determine the effects of the ovarian cycle on prolactin and its receptor expression. METHODS: peripheral blood mononuclear cells (PBMC) were obtained from twenty-six normal menstruating women at different intervals of their menstrual cycle. The PBMC were incubated during 24 h in the presence or absence of Concanavalin-A (Con-A) and the gene expression of PRL, PRLR and cytokines was evaluated by qPCR. Prolactin, IL-2 and cAMP were determined in each culture by specific immunoassays. RESULTS: neither PRL nor its receptor expression in PBMC changed significantly among groups, including the cytokines (IL-2, IL-10, and IFNG) studied. Similar results, among groups, were obtained, when PRL expression was stimulated by PGE2 or 8-Br-cAMP. Concanavalin A-stimulated PBMC expressed significantly less prolactin and a significant negative correlation between secreted IL-2 and PRL expression was found. The presence of anti-IL-2 antibodies in Con-A stimulated-cultures significantly increased PRL expression when compared to control cells regardless the hormonal status. CONCLUSIONS: these data suggest that the menstrual cycle does not significantly modulate or influence prolactin and cytokines gene expression in PBMC, and indicate that IL-2 may be involved in the Con-A regulation of PRL expression in immune cells.

Preclinical and clinical aspects of TNF-α and its receptors TNFR1 and TNFR2 in breast cancer.

Breast cancer is the most common malignancy in women and a public health problem worldwide. Breast cancer is often accompanied by an inflammatory process characterized by the presence of proinflammatory cytokines such as tumor necrosis factor (TNF-α), which has important implications in the course of the disease. Inflammation has been described primarily as a favorable environment for tumor development. However, under certain conditions TNF-α can promote signals for activation, differentiation, survival or cell death, so the study of the variants of this cytokine, its receptors, the presence of polymorphisms and its implication in different phenotypes of breast cancer is necessary. Although the clinical application of TNF-α has been limited by its toxicity and side effects, preclinical and clinical studies have shown that these effects may partially be avoided via tumor-targeted delivery strategies. In this manner, TNF-α alone or combined with chemotherapy and radiotherapy can function as an adjuvant in the treatment of breast cancer.

Calcitriol stimulates gene expression of cathelicidin antimicrobial peptide in breast cancer cells with different phenotype.

BACKGROUND: In normal and neoplastic cells, growth-promoting, proangiogenic, cytotoxic and pro-apoptotic effects have all been attributed to cathelicidin antimicrobial peptide (CAMP). Nevertheless, little is known about the factors regulating this peptide expression in breast cancer. Herein we asked if the well-known antineoplastic hormone calcitriol could differentially modulate CAMP gene expression in human breast cancer cells depending on the cell phenotype in terms of efficacy and potency. METHODS: The established breast cancer cell lines MCF7, BT-474, HCC1806, HCC1937, SUM-229PE and a primary cell culture generated from invasive ductal breast carcinoma were used in this study. Calcitriol regulation of cathelicidin gene expression in vitro and in human breast cancer xenografts was studied by real time PCR. Tumorigenicity was evaluated for each cell line in athymic mice. RESULTS: Estrogen receptor (ER)α + breast cancer cells showed the highest basal CAMP gene expression. When incubated with calcitriol, CAMP gene expression was stimulated in a dose-dependent and cell phenotype-independent manner. Efficacy of calcitriol was lower in ERα + cells when compared to ERα- cells (<10 vs. >70 folds over control, respectively). Conversely, calcitriol lowest potency upon CAMP gene expression was observed in the ERα-/EGFR+ SUM-229PE cell line (EC50 = 70.8 nM), while the highest was in the basal-type/triple-negative cells HCC1806 (EC50 = 2.13 nM) followed by ERα + cells MCF7 and BT-474 (EC50 = 4.42 nM and 14.6 nM, respectively). In vivo, lower basal CAMP gene expression was related to increased tumorigenicity and lack of ERα expression. Xenografted triple-negative breast tumors of calcitriol-treated mice showed increased CAMP gene expression compared to vehicle-treated animals. CONCLUSIONS: Independently of the cell phenotype, calcitriol provoked a concentration-dependent stimulation on CAMP gene expression, showing greater potency in the triple negative HCC1806 cell line. Efficacy of calcitriol was lower in ERα + cells when compared to ERα- cells in terms of stimulating CAMP gene expression. Lower basal CAMP and lack of ERα gene expression was related to increased tumorigenicity. Our results suggest that calcitriol anti-cancer therapy is more likely to induce higher levels of CAMP in ERα- breast cancer cells, when compared to ERα + breast cancer cells.

Calcitriol increases Dicer expression and modifies the microRNAs signature in SiHa cervical cancer cells.

MicroRNAs play important roles in cancer biology. Calcitriol, the hormonal form of vitamin D3, regulates microRNAs expression in tumor cells. In the present study we asked if calcitriol would modify some of the components of the microRNA processing machinery, namely, Drosha and Dicer, in calcitriol-responsive cervical cancer cells. We found that calcitriol treatment did not affect Drosha mRNA; however, it significantly increased Dicer mRNA and protein expression in VDR-positive SiHa and HeLa cells. In VDR-negative C33-A cells, calcitriol had no effect on Dicer mRNA. We also found a vitamin D response element in Dicer promoter that interacts in vitro to vitamin D and retinoid X receptors. To explore the biological plausibility of these results, we asked if calcitriol alters the microRNA expression profile in SiHa cells. Our results revealed that calcitriol regulates the expression of a subset of microRNAs with potential regulatory functions in cancer pathways, such as miR-22, miR-296-3p, and miR-498, which exert tumor-suppressive effects. In summary, the data indicate that in SiHa cells, calcitriol stimulates the expression of Dicer possibly through the vitamin D response element located in its promoter. This may explain the calcitriol-dependent modulation of microRNAs whose target mRNAs are related to anticancer pathways, further adding to the various anticancer mechanisms of calcitriol.

The expression of RNA helicase DDX5 is transcriptionally upregulated by calcitriol through a vitamin D response element in the proximal promoter in SiHa cervical cells.

The DEAD box RNA helicase DDX5 is a multifunctional protein involved in the regulatory events of gene expression. Herein, we presented evidence indicating that DDX5 is transcriptionally upregulated by calcitriol, the hormonal form of vitamin D3. In silico analysis revealed the presence of two putative vitamin D response elements (VDREs) in the DDX5 promoter region. Using luciferase reporter assays, we demonstrated that the DDX5 promoter containing these putative VDREs significantly increased the luciferase activity in vitamin D receptor (VDR)-positive SiHa cells upon calcitriol treatment. Electrophoretic mobility shift assays showed the ability of VDR and retinoid X receptor to interact only with the most proximal VDRE, while chromatin immunoprecipitation analysis confirmed the occupancy of this VDRE by the VDR. Finally, we demonstrated that calcitriol significantly increased both DDX5 mRNA and protein in SiHa cells. In summary, this study shows that DDX5 gene is transcriptionally upregulated by calcitriol through a VDRE located in its proximal promoter. Given the importance of DDX5 as a master regulator of differentiation programs, our study suggests that the pro-differentiating properties of calcitriol may be related with the induction of DDX5.

In vivo dual targeting of the oncogenic Ether-à-go-go-1 potassium channel by calcitriol and astemizole results in enhanced antineoplastic effects in breast tumors.

BACKGROUND: The oncogenic ether-à-go-go-1 potassium channel (EAG1) activity and expression are necessary for cell cycle progression and tumorigenesis. The active vitamin D metabolite, calcitriol, and astemizole, a promising antineoplastic drug, target EAG1 by inhibiting its expression and blocking ion currents, respectively. We have previously shown a synergistic antiproliferative effect of calcitriol and astemizole in breast cancer cells in vitro, but the effect of this dual therapy in vivo has not been studied. METHODS: In the present study, we explored the combined antineoplastic effect of both drugs in vivo using mice xenografted with the human breast cancer cell line T-47D and a primary breast cancer-derived cell culture (MBCDF). Tumor-bearing athymic female mice were treated with oral astemizole (50 mg/kg/day) and/or intraperitoneal injections of calcitriol (0.03 µg/g body weight twice a week) during 3 weeks. Tumor sizes were measured thrice weekly. For mechanistic insights, we studied EAG1 expression by qPCR and Western blot. The expression of Ki-67 and the relative tumor volume were used as indicators of therapeutic efficacy. RESULTS: Compared to untreated controls, astemizole and calcitriol significantly reduced, while the coadministration of both drugs further suppressed, tumor growth (P < 0.05). In addition, the combined therapy significantly downregulated tumoral EAG1 and Ki-67 expression. CONCLUSIONS: The concomitant administration of calcitriol and astemizole inhibited tumor growth more efficiently than each drug alone, which may be explained by the blocking of EAG1. These results provide the bases for further studies aimed at testing EAG1-dual targeting in breast cancer tumors expressing both EAG1 and the vitamin D receptor.

Calcitriol restores antiestrogen responsiveness in estrogen receptor negative breast cancer cells: a potential new therapeutic approach.

BACKGROUND: Approximately 30% of breast tumors do not express the estrogen receptor (ER) α, which is necessary for endocrine therapy approaches. Studies are ongoing in order to restore ERα expression in ERα-negative breast cancer. The aim of the present study was to determine if calcitriol induces ERα expression in ER-negative breast cancer cells, thus restoring antiestrogen responses. METHODS: Cultured cells derived from ERα-negative breast tumors and an ERα-negative breast cancer cell line (SUM-229PE) were treated with calcitriol and ERα expression was assessed by real time PCR and western blots. The ERα functionality was evaluated by prolactin gene expression analysis. In addition, the effects of antiestrogens were assessed by growth assay using the XTT method. Gene expression of cyclin D1 (CCND1), and Ether-à-go-go 1 (EAG1) was also evaluated in cells treated with calcitriol alone or in combination with estradiol or ICI-182,780. Statistical analyses were determined by one-way ANOVA. RESULTS: Calcitriol was able to induce the expression of a functional ERα in ER-negative breast cancer cells. This effect was mediated through the vitamin D receptor (VDR), since it was abrogated by a VDR antagonist. Interestingly, the calcitriol-induced ERα restored the response to antiestrogens by inhibiting cell proliferation. In addition, calcitriol-treated cells in the presence of ICI-182,780 resulted in a significant reduction of two important cell proliferation regulators CCND1 and EAG1. CONCLUSIONS: Calcitriol induced the expression of ERα and restored the response to antiestrogens in ERα-negative breast cancer cells. The combined treatment with calcitriol and antiestrogens could represent a new therapeutic strategy in ERα-negative breast cancer patients.