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.

Enhancing Tamoxifen Therapy with α-Mangostin: Synergistic Antiproliferative Effects on Breast Cancer Cells and Potential Reduced Endometrial Impact.

Breast cancer is the most prevalent neoplasia among women worldwide. For the estrogen receptor-positive (ER+) phenotype, tamoxifen is the standard hormonal therapy; however, it carries the risk of promoting endometrial carcinoma. Hence, we aimed to evaluate the antiproliferative effect of the phytochemical α-mangostin (AM) as a co-adjuvant alongside tamoxifen on breast cancer cells to improve its efficacy while reducing its adverse effects on endometrium. For this, ER+ breast cancer cells (MCF-7 and T-47D) and endometrial cells (N30) were treated with AM, 4-hydroxytamoxifen (4-OH-TMX), and their combination. Cell proliferation was evaluated using sulforhodamine B assay, and the pharmacological interaction was determined through the combination index and the dose reduction index calculation. The genes KCNH1, CCDN1, MKI67, and BIRC5 were amplified by real-time PCR as indicators of oncogenesis, cell cycle progression, cell proliferation, and apoptosis, respectively. Additionally, genes involved in ER signaling were analyzed. In breast cancer cells, the combination of AM with 4-OH-TMX showed a synergistic antiproliferative effect and favorable dose reduction. AM and 4-OH-TMX decreased KCNH1, CCND1, and BIRC5 gene expression. In endometrial cells, AM decreased MKI-67 gene expression, while it reverted the 4-OH-TMX-dependent CCND1 upregulation. This study establishes the benefits of incorporating AM as a co-adjuvant for first-line ER+ breast cancer therapy.

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.

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 Interaction of Human Papillomavirus Infection and Prostaglandin E2 Signaling in Carcinogenesis: A Focus on Cervical Cancer Therapeutics.

Chronic infection by high-risk human papillomaviruses (HPV) and chronic inflammation are factors associated with the onset and progression of several neoplasias, including cervical cancer. Oncogenic proteins E5, E6, and E7 from HPV are the main drivers of cervical carcinogenesis. In the present article, we review the general mechanisms of HPV-driven cervical carcinogenesis, as well as the involvement of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and downstream effectors in this pathology. We also review the evidence on the crosstalk between chronic HPV infection and PGE2 signaling, leading to immune response weakening and cervical cancer development. Finally, the last section updates the current therapeutic and preventive options targeting PGE2-derived inflammation and HPV infection in cervical cancer. These treatments include nonsteroidal anti-inflammatory drugs, prophylactic and therapeutical vaccines, immunomodulators, antivirals, and nanotechnology. Inflammatory signaling pathways are closely related to the carcinogenic nature of the virus, highlighting inflammation as a co-factor for HPV-dependent carcinogenesis. Therefore, blocking inflammatory signaling pathways, modulating immune response against HPV, and targeting the virus represent excellent options for anti-tumoral therapies in cervical cancer.

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.

The interplay between estrogen receptor beta and protein kinase C, a crucial collaboration for medulloblastoma cell proliferation and invasion.

Medulloblastoma (MB) is the most common and aggressive pediatric intracranial tumor. Estrogen receptor ß (ERß) expression correlates with MB development and its phosphorylation modifies its transcriptional activity in a ligand-dependent or independent manner. Using in silico tools, we have identified several residues in ERß protein as potential targets of protein kinases C (PKCs) α and δ. Using Daoy cells, we observed that PKCα and PKCδ associate with ERß and induce its phosphorylation. The activation of ERß promotes MB cells proliferation and invasion, and PKCs downregulation dysregulates these steroid receptor mediated processes. Our data suggest that these kinases may play a crucial role in the regulation of the ERß transcriptional activity. Overexpression of both PKCα and PKCδ in MB biopsies samples supports their relevance in MB progression.

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.

Vasculogenic Mimicry in Breast Cancer: Clinical Relevance and Drivers.

In solid tumors, vasculogenic mimicry (VM) is the formation of vascular structures by cancer cells, allowing to generate a channel-network able to transport blood and tumor cells. While angiogenesis is undertaken by endothelial cells, VM is assumed by cancer cells. Besides the participation of VM in tumor neovascularization, the clinical relevance of this process resides in its ability to favor metastasis and to drive resistance to antiangiogenic therapy. VM occurs in many tumor types, including breast cancer, where it has been associated with a more malignant phenotype, such as triple-negative and HER2-positive tumors. The latter may be explained by known drivers of VM, like hypoxia, TGFB, TWIST1, EPHA2, VEGF, matrix metalloproteinases, and other tumor microenvironment-derived factors, which altogether induce the transformation of tumor cells to a mesenchymal phenotype with a high expression rate of stemness markers. This review analyzes the current literature in the field, including the participation of some microRNAs and long noncoding RNAs in VM-regulation and tumorigenesis of breast cancer. Considering the clinical relevance of VM and its association with the tumor phenotype and clinicopathological parameters, further studies are granted to target VM in the clinic.

Antitumoral effects of dovitinib in triple-negative breast cancer are synergized by calcitriol in vivo and in vitro.

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 99mTc-Ethylenediamine-N,N-diacetic acid/hydrazinonicotinamyl-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]2 (99mTc-RGD2)-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 99mTc-RGD2 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.

Exploring novel capping framework: high substituent pyridine-hydroxamic acid derivatives as potential antiproliferative agents.

PURPOSE: Histone deacetylases (HDACs) play a vital role in the epigenetic regulation of gene expression due to their overexpression in several cancer forms. Therefore, these enzymes are considered as a potential anticancer drug target. Different synthetic and natural structures have been studied as HDACs inhibitors; based on available structural design information, the capping group is important for the biological activity due to the different interactions in the active site entrance. The present study aimed to analyze high substituted pyridine as a capping group, which included carrying out the synthesis, antiproliferative activity analysis, and docking studies of these novel compounds. METHODS: To achieve the synthesis of these derivatives, four reaction steps were performed, generating desired products 15a-k. Their effects on cell proliferation and gene expression of p21, cyclin D1, and p53 were determined using the sulphorhodamine B (SRB) method and quantitative real-time polymerase chain reaction. The HDAC1, HDAC6, and HDAC8 isoforms were used for performing docking experiments with our 15a-k products. RESULT: The products 15a-k were obtained in overall yields of 40-71%. Compounds 15j and 15k showed the highest antiproliferative activity in the breast (BT-474 and MDA-MB-231) and prostate (PC3) cancer cell lines at a concentration of 10 µM. These compounds increased p21 mRNA levels and decreased cyclin D1 and p53 gene expression. The docking study showed an increment in the strength, and in the number of interactions performed by the capping moiety of the tested molecules compared with SAHA; interactions displayed are mainly van der Waals, π-stacking, and hydrogen bond. CONCLUSION: The synthesized compounds 2-thiophene (15j) and 2-furan (15k) pyridine displayed cell growth inhibition, regulation of genes related to cell cycle progression in highly metastatic cancer cell lines. The molecular coupling analysis performed with HDAC1, HDAC6 and HDAC8 showed an increment in the number of interactions performed by the capping moiety and consequently in the strength of the capping group interaction.

Regulation of anti-tumorigenic pathways by the combinatory treatment of calcitriol and TGF-ß in PC-3 and DU145 cells.

Calcitriol and transforming growth factors beta (TGF-ß) 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-ß pathway in prostate cancer and the well-known independent effects of calcitriol and TGF-ßs on cancer cells, there is limited information regarding the cellular effects of calcitriol and TGF-ß in combination. In this study, we in vitro analyze the combinatory effects of calcitriol and TGF-ß 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-ß1 and later by calcitriol. A set of genes and enriched pathways converge among the treatments, mainly between the combinatory scheme and TGF-ß1, 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.

Calcitriol induces estrogen receptor α expression through direct transcriptional regulation and epigenetic modifications in estrogen receptor-negative breast cancer cells.

Patients with estrogen receptor (ER) α-negative breast tumors have a poor prognosis and are not suitable for hormone therapy. Previously, we demonstrated that calcitriol, the active metabolite of vitamin D, induces ERα expression and re-establishes the response to antiestrogens in ER-negative breast cancer cells. However, the mechanisms involved in this process have not been elucidated. Therefore, the present study was undertaken to investigate the mechanisms implicated in the calcitriol-induced ERα expression in ER-negative breast cancer cells. Using EMSA and ChIP assays, we found that the calcitriol/vitamin D receptor (VDR)/retinoic X receptor (RXR) complex binds to putative vitamin D response elements (VDREs) in the ERα gene promoter region. In addition, we established by a fluorometric assay that calcitriol decreased DNA-methyltransferase and histone deacetylase activities. Flow cytometry and qPCR analyses showed that co-treatment of calcitriol with inhibitors of the histone deacetylase and DNA methyltransferase, and genistein significantly increased ERα expression, compared to that observed with the compounds alone. In conclusion, the calcitriol-dependent ERα induction in ER-negative breast cancer cells results from binding of the VDR-RXR complex to VDREs in the ERα gene promoter region, including the downregulation of enzymes with chromatin-remodeling activities. These results may bring forth novel mechanistic knowledge into the actions of calcitriol in ERα-negative breast cancer.

An organotin indomethacin derivative inhibits cancer cell proliferation and synergizes the antiproliferative effects of lapatinib in breast cancer cells.

It is known that an inflammatory condition in different types of cancer provides a sustained microenvironment that favors tumor growth, invasion, and metastasis. Non-steroidal anti-inflammatory drugs such as indomethacin have demonstrated chemo-preventive, anti-proliferative and cytotoxic effects in a variety of tumors. The aim of this study was to investigate the effects of an organotin indomethacin derivative (OID) on the proliferation of breast and prostate cancer cell lines and the possible mechanisms of action of this compound. Different cancer cell lines were treated in the presence of OID and cell proliferation was measured by quantification of the DNA content, changes in the cell cycle profile and the activation of caspase 3 were evaluated by flow cytometry, interleukin 6 (IL-6) gene expression was evaluated by qPCR and protein expression was analyzed by ELISA and Western blot assays. OID inhibited the cell proliferation of a panel of cancer cell lines in a concentration-dependent manner. Moreover, the addition of OID to lapatinib treatment, targeted therapy for breast cancer, significantly enhanced its antiproliferative response. The effects on cell proliferation of these compounds involved, among others, the induction of apoptosis, the downregulation of IL-6 and a decrease of the MAPK activation pathway. Our results suggest that the use of OID alone or in combination with tyrosine kinase inhibitors could be considered as adjuvants in the treatment of cancer.

The chemical environmental pollutants BPA and BPS induce alterations of the proteomic profile of different phenotypes of human breast cancer cells: A proposed interactome.

Breast cancer is one of the most common malignancies and the second leading cause of death in women. Despite efforts for its early detection, its worldwide incidence continues to increase. Thus, identification of risk factors for its development and new targets for its therapy are of vital importance. Environmental pollutants derived from human activity have been associated with predisposition to the development of cancer. Bisphenol A (BPA) is an endocrine disruptor compound (EDC) widely used in the manufacture of polycarbonates, and it has affinity for the estrogen receptor (ER). Scientific evidence has proposed an association between increased incidence of breast cancer and BPA exposure at lower doses. Among worldwide concerns with BPA exposure, different industries proceeded to replace BPA with analogs such as bisphenol S (BPS), which is now employed in products labelled as BPA-free. Nevertheless, recent studies exhibit that its exposure results in altered mammary gland development and morphogenesis; and promotes breast cancer cell proliferation. Of note, most of the effects of both BPA and BPS have been performed in estrogen-dependent breast cancer models. However, gaps in knowledge still exist on the roles and mechanisms that both compounds, specifically BPS, may play in cancer initiation and development in hormone-dependent and other types of breast cancer. Thus, the aim of the present study was to deepen the understanding of biological targets modulated by these ubiquitous pollutants in different breast cancer cell lines, representing two scenarios of this pathology: hormone-dependent and hormone-independent breast cancer. Results point out that both compounds induced proliferation in ER positive cells, not showing this effect in the ER-negative breast cancer cells. Different targets modified at the proteomic level in both breast cancer scenarios were also identified. Stem cell markers (eg. CD44) and invasion proteins (eg. MMP-14) were importantly increased by BPA and BPS in ER-positive breast cancer cells. In contrast, growth factors and associated receptors such as EGFR and TGF-ß were induced by BPS in the ER-negative breast cancer cells; both pollutants induced an increase of vascular endothelial growth factor (VEGF) protein secretion. This finding suggests that the use of BPS must be considered with more caution than BPA, since it can act independently of the presence of the hormonal receptor. These findings show new evidence that BPA and BPS exposure can contribute to breast cancer development and progression. Our results suggest that both BPA and BPS must be considered equally as outstanding risk factors for this pathology.

Design of Fluorescent Coumarin-Hydroxamic Acid Derivatives as Inhibitors of HDACs: Synthesis, Anti-Proliferative Evaluation and Docking Studies.

Coumarin-hydroxamic acid derivatives 7a-k were herein designed with a dual purpose: as antiproliferative agents and fluorescent probes. The compounds were synthesized in moderate yields (30-87%) through a simple methodology, biological evaluation was carried out on prostate (PC3) and breast cancer (BT-474 and MDA-MB-231) cell lines to determine the effects on cell proliferation and gene expression. For compounds 7c, 7e, 7f, 7i and 7j the inhibition of cancer cell proliferation was similar to that found with the reference compound at a comparable concentration (10 µM), in addition, their molecular docking studies performed on histone deacetylases 1, 6 and 8 showed strong binding to the respective active sites. In most cases, antiproliferative activity was accompanied by greater levels of cyclin-dependent kinase inhibitor p21, downregulation of the p53 tumor suppressor gene, and regulation of cyclin D1 gene expression. We conclude that compounds 7c, 7e, 7f, 7i and 7j may be considered as potential anticancer agents, considering their antiproliferative properties, their effect on the regulation of the genes, as well as their capacity to dock to the active sites. The fluorescent properties of compound 7j and 7k suggest that they can provide further insight into the mechanism of action.

Synergistic Antitumorigenic Activity of Calcitriol with Curcumin or Resveratrol is Mediated by Angiogenesis Inhibition in Triple Negative Breast Cancer Xenografts.

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.

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.

Astemizole, an inhibitor of ether-à-go-go-1 potassium channel, increases the activity of the tyrosine kinase inhibitor gefitinib in breast cancer cells

Abstract 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 cytometry. 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.