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Fibroblast growth factor receptor (FGFR) overamplification/activation in cancer leads to increased cell proliferation. AZD4547, a FGFR selective inhibitor, hinders breast cancer cells growth. Although luminal B breast tumors may respond to chemotherapy and endocrine therapy, this subtype is associated with poor prognosis, inadequate response and/or acquired drug resistance. Calcitriol, the vitamin D most active metabolite, exerts anti-neoplastic effects and enhances chemotherapeutic drugs activity. In this study, we sought to decrease the concentration of AZD4547 needed to inhibit the luminal-B breast cancer cell line BT-474 proliferation by its combination with calcitriol. Anti-proliferative inhibitory concentrations, combination index and dose-reduction index were analyzed from Sulforhodamine B assays. Western blot and qPCR were used to study FGFR molecular targets. The compound's ability to inhibit BT-474 cells tumorigenic capacity was assessed by tumorspheres formation. Results: BT-474 cells were dose-dependently growth-inhibited by calcitriol and AZD4547 (IC 50 = 2.9 nM and 3.08 μM, respectively). Calcitriol at 1 nM synergistically improved AZD4547 antiproliferative effects, allowing a 2-fold AZD4547 dose-reduction. Mechanistically, AZD4547 downregulated p-FGFR1, p-Akt and tumorsphere formation. Calcitriol also decreased tumorspheres, while induced cell differentiation. Both compounds inhibited MYC and CCND1 expression, as well as ALDH, a stemness marker that positively correlated with FGFR1 and negatively with VDR expression in breast cancer transcriptomic data. In conclusion, the drugs impaired self-aggregation capacity, reduced stemness features, induced cell-differentiation and when combined, synergistically inhibited cell proliferation. Overall, our results suggest that calcitriol, at low pharmacological doses, may be a suitable candidate to synergize AZD4547 effects in luminal B breast tumors, allowing to reduce dose and adverse effects., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Chemotherapy is a standard therapeutic option for triple-negative breast cancer (TNBC); however, its effectiveness is often compromised by drug-related toxicity and resistance development. Herein, we aimed to evaluate whether an improved antineoplastic effect could be achieved in vitro and in vivo in TNBC by combining dovitinib, a multi-kinase inhibitor, with calcitriol, a natural anticancer hormone. In vitro, cell proliferation and cell-cycle distribution were studied by sulforhodamine B-assays and flow cytometry. In vivo, dovitinib/calcitriol effects on tumor growth, angiogenesis, and endothelium activation were evaluated in xenografted mice by caliper measures, Itgb3/VEGFR2-immunohistochemistry and 99m Tc-Ethylenediamine-N,N-diacetic acid/hydrazinonicotinamyl-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)] 2 ( 99m Tc-RGD 2 )-tumor uptake. The drug combination elicited a synergistically improved antiproliferative effect in TNBC-derived cells, which allowed a 7-fold and a 3.3-fold dovitinib dose-reduction in MBCDF-Tum and HCC-1806 cells, respectively. Mechanistically, the co-treatment induced a cell cycle profile suggestive of cell death and DNA damage (accumulation of cells in SubG1, S, and G2/M phases), increased the number of multinucleated cells and inhibited tumor growth to a greater extent than each compound alone. Tumor uptake of 99m Tc-RGD 2 was reduced by dovitinib, suggesting angiogenesis inhibition, which was corroborated by decreased endothelial cell growth, tumor-vessel density and VEGFR2 expression. In summary, calcitriol synergized dovitinib anticancer effects in vitro and in vivo, allowing for a significant dose-reduction of dovitinib while maintaining its antiproliferative potency. Our results suggest the beneficial convergence of independent antitumor mechanisms of dovitinib and calcitriol to inhibit TNBC-tumor growth., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Calcitriol and transforming growth factors beta (TGF-β) are involved in several biological pathways such as cell proliferation, differentiation, migration and invasion. Their cellular effects could be similar or opposite depending on the genetic target, cell type and context. Despite the reported association of calcitriol deficiency and disruption of the TGF-β pathway in prostate cancer and the well-known independent effects of calcitriol and TGF-βs on cancer cells, there is limited information regarding the cellular effects of calcitriol and TGF-β in combination. In this study, we in vitro analyze the combinatory effects of calcitriol and TGF-β on cell growth and apoptosis using PC-3 and DU145 human prostate cancer cell lines. Using high-throughput microarray profiling of PC-3 cells upon independent and combinatory treatments, we identified distinct transcriptional landscapes of each intervention, with a higher effect established by the combinatorial treatment, following by TGF-β1 and later by calcitriol. A set of genes and enriched pathways converge among the treatments, mainly between the combinatory scheme and TGF-β1, but the majority were treatment-specific. Of note, CYP24A1, IGFBP3, CDKN1A, NOX4 and UBE2D3 were significantly up-regulated upon the combinatorial treatment whereas CCNA1, members of the CT45A and APOBEC3 family were down-regulated. By public RNA signatures, we were able to confirm the regulation by the co-treatment over cell proliferation and cell cycle. We finally investigated the possible clinical impact of genes modulated by the combinatorial treatment using benchmark prostate cancer data. This comprehensive analysis reveals that the combinatory treatment impairs cell growth without affecting apoptosis and their combinatory actions might synergize and improved their individual effects to reprogram prostate cancer signaling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright: © 2019 Permanyer.)

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Calcitriol is a multitarget anticancer hormone; however, its effects on angiogenesis remain contradictory. Herein, we tested whether the antiangiogenic phytochemicals curcumin or resveratrol improved calcitriol antitumorigenic effects in vivo. Triple-negative breast cancer tumoral cells (MBCDF-T) were xenografted in nude mice, maintaining treatments for 3 weeks. Tumor onset, volume and microvessel density were significantly reduced in mice coadministered with calcitriol and curcumin (Cal+Cur). Vessel count was also reduced in mice simultaneously treated with calcitriol and resveratrol (Cal+Rsv). Cal+Cur and Cal+Rsv treatments resulted in less tumor activated endothelium, as demonstrated by decreased tumor uptake of integrin-targeted biosensors in vivo. The renal gene expression of Cyp24a1 and Cyp27b1 suggested increased calcitriol bioactivity in the combined regimens. In vitro, the phytochemicals inhibited both MBCDF-T and endothelial cells proliferation, while potentiated calcitriol's ability to reduce MBCDF-T cell-growth and endothelial cells migration. Resveratrol induced endothelial cell death, as deduced by increased sub-G1 cells accumulation, explaining the reduced tumor vessel number in resveratrol-treated mice, which further diminished when combined with calcitriol. In conclusion, the concomitant administration of calcitriol with curcumin or resveratrol synergistically promoted anticancer effects in vitro and in vivo in human mammary tumor cells. Whereas the results suggest different mechanisms of action of the phytochemicals when coadministered with calcitriol, the converging biological effect was inhibition of tumor neoangiogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Tamoxifen is a selective estrogen receptor modulator widely used in oncology and reproductive endocrinology. In order to decrease its non-desirable effects and elucidate mechanisms of action, permanently charged tamoxifen derivatives (PCTDs) have been reported. Whether PCTDs have genomic effects remains controversial. Since the clinical relevance of tamoxifen, the necessity to have new anticancer drugs, and in order to gain insights into the mechanisms of action of PCTDs, we obtained six quaternary ammonium salts derived from tamoxifen including three new compounds. We characterized them by nuclear magnetic resonance, X-ray diffraction, electron microscopy, and/or high performance liquid chromatography, and detected them in cell lysates by liquid chromatography coupled to mass spectrometry. We evaluated their binding to estrogen receptor-α (ERα, their effect on the transcriptional activity mediated by ERα (gene reporter assays), and the proliferation of cancer cells (MCF-7 and cells from a cervical cancer primary culture). Structural studies demonstrated the expected identity of the molecules. All PCTDs did bind to ERα, one of them induced ERα-mediated transcription while two others inhibited such genomic action. Accordingly, PCTDs were detected in cell lysates. PCTDs inhibited cell proliferation, noteworthy, two of them displayed higher inhibition than tamoxifen. Structure–activity analysis suggests that PCTDs permanent positive charge and the length of the aliphatic chain might be associated to the biological responses studied. We suggest genomic effects as a mechanism of action of PCTDs. The experimental approaches here used could lead to a better design of new therapeutic molecules and help to elucidate molecular mechanisms of new anticancer drugs. [ABSTRACT FROM AUTHOR]