Magnetic layered double hydroxides with carbamazepine for breast cancer treatment.

Current cancer chemotherapy is associated with many side effects and, in some cases, drug resistance, which makes the search for new active molecules and drug delivery strategies imperative. Carbamazepine is an antiepileptic compound that has shown efficacy against breast cancer cell lines. In this study, it was incorporated into layered double hydroxide nanoclays, the percentage of drug loading was increased compared to previous research, and the clays were impregnated with magnetic Fe3O4 nanoparticles. The goal of the magnetic Fe3O4-impregnation was to direct the nanocomposites to the therapeutic target with an external magnetic field. The nanoclay-carbamazepine composites had a carbamazepine loading of 51 %, and the nanoclay-carbamazepine-nanoparticles had a drug loading of 13 % due to the addition of more ingredients. The structure of the composites was analyzed by X-ray diffraction and Scherrer equation, showing a layered double hydroxide organization with crystal sizes of 9-15 nm; from transmission electron microscopy, the final compounds showed a particle size of 97-158 nm, small enough for systemic circulation. In vibrating sample magnetization studies, the composites showed a superparamagnetic behavior with high magnetic saturation (9-17 emu/gr), which should allow a good material attraction by an external magnetic field located near the tumor. In vitro drug release studies were done in Franz cells and measured by UV/Vis spectrophotometry; they showed that carbamazepine release from the nanocomposites responds to the media pH: a good drug release at the lysosome pH and slow release at the blood pH. Finally, the efficacy was tested in vitro in MDA-MB-231 breast cancer cells, and the composites showed an enhanced efficacy in comparison with that produced by the free drug (96 % and 62 % of cell inhibition respectively). Carbamazepine administered with magnetic clays as a carrier is a promising treatment for breast cancer, and further studies should be done to measure the arrival time and the efficacy in vivo.

A Screening Tool for Self-Evaluation of Risk for Age-Related Macular Degeneration: Validation in a Spanish Population.

Purpose: The objectives of this study were the creation and validation of a screening tool for age-related macular degeneration (AMD) for routine assessment by primary care physicians, ophthalmologists, other healthcare professionals, and the general population. Methods: A simple, self-administered questionnaire (Simplified Théa AMD Risk-Assessment Scale [STARS] version 4.0) which included well-established risk factors for AMD, such as family history, smoking, and dietary factors, was administered to patients during ophthalmology visits. A fundus examination was performed to determine presence of large soft drusen, pigmentary abnormalities, or late AMD. Based on data from the questionnaire and the clinical examination, predictive models were developed to estimate probability of the Age-Related Eye Disease Study (AREDS) score (categorized as low risk/high risk). The models were evaluated by area under the receiving operating characteristic curve analysis. Results: A total of 3854 subjects completed the questionnaire and underwent a fundus examination. Early/intermediate and late AMD were detected in 15.9% and 23.8% of the patients, respectively. A predictive model was developed with training, validation, and test datasets. The model in the test set had an area under the curve of 0.745 (95% confidence interval [CI] = 0.705-0.784), a positive predictive value of 0.500 (95% CI = 0.449-0.557), and a negative predictive value of 0.810 (95% CI = 0.770-0.844). Conclusions: The STARS questionnaire version 4.0 and the model identify patients at high risk of developing late AMD. Translational Relevance: The screening instrument described could be useful to evaluate the risk of late AMD in patients >55 years without having an eye examination, which could lead to more timely referrals and encourage lifestyle changes.

A Decade of Research on Coffee as an Anticarcinogenic Beverage.

Coffee consumption has been investigated as a protective factor against cancer. Coffee is a complex beverage that contains more than 1000 described phytochemicals, which are responsible for its pleasant taste, aroma, and health-promoting properties. Many of these compounds have a potential therapeutic effect due to their antioxidant, anti-inflammatory, antifibrotic, and anticancer properties. The roasting process affects the phytochemical content, and undesirable compounds may be formed. In recent years, there have been contradictory publications regarding the effect of coffee drinking and cancer. Therefore, this study is aimed at evaluating the association of coffee consumption with the development of cancer. In PubMed, until July 2021, the terms "Coffee and cancer" resulted in about 2150 publications, and almost 50% of them have been published in the last 10 years. In general, studies published in recent years have shown negative associations between coffee consumption and the risk or development of different types of cancer, including breast, prostate, oral, oral and pharyngeal, melanoma, skin and skin nonmelanoma, kidney, gastric, colorectal, endometrial, liver, leukemic and hepatocellular carcinoma, brain, and thyroid cancer, among others. In contrast, only a few publications demonstrated a double association between coffee consumption and bladder, pancreatic, and lung cancer. In this review, we summarize the in vitro and in vivo studies that accumulate epidemiological evidence showing a consistent inverse association between coffee consumption and cancer.

Antitumor Effects of Freeze-Dried Robusta Coffee (Coffea canephora) Extracts on Breast Cancer Cell Lines.

Coffee consumption is believed to have chemopreventive and chemotherapeutic effects and to contribute to preventing the development and progression of cancer. However, there is still controversy around these claims. As indicated in our previous works, diet can influence the risk of breast cancer. Intake of coffee is hypothesized to reduce this risk, but current scientific evidence is not conclusive. This work is aimed at studying the effects of Robusta coffee bean extract on cell viability, proliferation, and apoptosis of different human cancers, especially breast cancer cell lines. To this end, cell viability was evaluated by Alamar Blue in 2D and 3D models, the cell cycle by PI, apoptosis by annexin V, mitochondrial morphology, and functionality by mitoTracker, and colony formation capacity by the clonogenic assay. Green and dark coffee extract significantly reduced viability in human breast, colorectal, brain, and bone cancer cells. Coffee anticancer activity was clearly evidenced in MDA-MB-231 (ER-) and MCF-7 (ER+) breast cancer cells but not in the normal breast cell line. In addition, coffee extract induces an increase S phase and a decrease G2/M population in breast cancer cells, affected the mitochondrial morphology, and triggered apoptosis. MDA-MB-231 breast cancer cells lost their clonogenic capacity after treatment. The antitumor activity was demonstrated in both 2D and 3D culture cell models.

Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction.

PURPOSE: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds. EXPERIMENTAL DESIGN: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dual-tumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database. RESULTS: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells. CONCLUSIONS: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.

Tumor-Associated Macrophages Induce Endocrine Therapy Resistance in ER+ Breast Cancer Cells.

Antiestrogenic adjuvant treatments are first-line therapies in patients with breast cancer positive for estrogen receptor (ER+). Improvement of their treatment strategies is needed because most patients eventually acquire endocrine resistance and many others are initially refractory to anti-estrogen treatments. The tumor microenvironment plays essential roles in cancer development and progress; however, the molecular mechanisms underlying such effects remain poorly understood. Breast cancer cell lines co-cultured with TNF-α-conditioned macrophages were used as pro-inflammatory tumor microenvironment models. Proliferation, migration, and colony formation assays were performed to evaluate tamoxifen and ICI 182,780 resistance and confirmed in a mouse-xenograft model. Molecular mechanisms were investigated using cytokine antibody arrays, WB, ELISA, ChIP, siRNA, and qPCR-assays. In our simulated pro-inflammatory tumor microenvironment, tumor-associated macrophages promoted proliferation, migration, invasiveness, and breast tumor growth of ER+ cells, rendering these estrogen-dependent breast cancer cells resistant to estrogen withdrawal and tamoxifen or ICI 182,780 treatment. Crosstalk between breast cancer cells and conditioned macrophages induced sustained release of pro-inflammatory cytokines from both cell types, activation of NF-κB/STAT3/ERK in the cancer cells and hyperphosphorylation of ERα, which resulted constitutively active. Our simulated tumor microenvironment strongly altered endocrine and inflammatory signaling pathways in breast cancer cells, leading to endocrine resistance in these cells.

Oxalate induces breast cancer.

BACKGROUND: Microcalcifications can be the early and only presenting sign of breast cancer. One shared characteristic of breast cancer is the appearance of mammographic mammary microcalcifications that can routinely be used to detect breast cancer in its initial stages, which is of key importance due to the possibility that early detection allows the application of more conservative therapies for a better patient outcome. The mechanism by which mammary microcalcifications are formed is still largely unknown but breast cancers presenting microcalcifications are more often associated with a poorer prognosis. METHODS: We combined Capillary Electrochromatography, histology, and gene expression (qRT-PCR) to analyze patient-matched normal breast tissue vs. breast tumor. Potential carcinogenicity of oxalate was tested by its inoculation into mice. All data were subjected to statistical analysis. RESULTS: To study the biological significance of oxalates within the breast tumor microenvironment, we measured oxalate concentration in both human breast tumor tissues and adjoining non-pathological breast tissues. We found that all tested breast tumor tissues contain a higher concentration of oxalates than their counterpart non-pathological breast tissue. Moreover, it was established that oxalate induces proliferation of breast cells and stimulates the expression of a pro-tumorigenic gene c-fos. Furthermore, oxalate generates highly malignant and undifferentiated tumors when it was injected into the mammary fatpad in female mice, but not when injected into their back, indicating that oxalate does not induce cancer formation in all types of tissues. Moreover, neither human kidney-epithelial cells nor mouse fibroblast cells proliferate when are treated with oxalate. CONCLUSIONS: We found that the chronic exposure of breast epithelial cells to oxalate promotes the transformation of breast cells from normal to tumor cells, inducing the expression of a proto-oncogen as c-fos and proliferation in breast cancer cells. Furthermore, oxalate has a carcinogenic effect when injected into the mammary fatpad in mice, generating highly malignant and undifferentiated tumors with the characteristics of fibrosarcomas of the breast. As oxalates seem to promote these differences, it is expected that a significant reduction in the incidence of breast cancer tumors could be reached if it were possible to control oxalate production or its carcinogenic activity.

c-Fos: an AP-1 transcription factor with an additional cytoplasmic, non-genomic lipid synthesis activation capacity.

The mechanisms that co-ordinately activate lipid synthesis when high rates of membrane biogenesis are needed to support cell growth are largely unknown. c-Fos, a well known AP-1 transcription factor, has emerged as a unique protein with the capacity to associate to specific enzymes of the pathway of synthesis of phospholipids at the endoplasmic reticulum and activate their synthesis to accompany genomic decisions of growth. Herein, we discuss this cytoplasmic, non-genomic effect of c-Fos in the context of other mechanisms that have been proposed to regulate lipid synthesis.

c-Fos-activated synthesis of nuclear phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] promotes global transcriptional changes.

c-Fos is a well-recognized member of the AP-1 (activator protein-1) family of transcription factors. In addition to this canonical activity, we previously showed that cytoplasmic c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. c-Fos associates with particular enzymes of the lipid synthesis pathway at the endoplasmic reticulum and increases the Vmax of the reactions without modifying the Km values. This lipid synthesis activation is associated with events of differentiation and proliferation that require high rates of membrane biogenesis. Since lipid synthesis also occurs in the nucleus, and different phospholipids have been assigned transcription regulatory functions, in the present study we examine if c-Fos also acts as a regulator of phospholipid synthesis in the nucleus. Furthermore, we examine if c-Fos modulates transcription through its phospholipid synthesis activator capacity. We show that nuclear-localized c-Fos associates with and activates PI4P5K (phosphatidylinositol-4-monophosphate 5-kinase), but not with PI4KIIIß (type IIIß phosphatidylinositol 4-kinase) thus promoting PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) formation, which, in turn, promotes transcriptional changes. We propose c-Fos as a key regulator of nuclear PtdIns(4,5)P2 synthesis in response to growth signals that results in c-Fos-dependent transcriptional changes promoted by the newly synthesized lipids.

Controlling cytoplasmic c-Fos controls tumor growth in the peripheral and central nervous system.

Some 20 years ago c-Fos was identified as a member of the AP-1 family of inducible transcription factors (Angel and Karin in Biochim Biophys Acta 1072:129-157, 1991). More recently, an additional activity was described for this protein: it associates to the endoplasmic reticulum and activates the biosynthesis of phospholipids (Bussolino et al. in FASEB J 15:556-558, 2001), (Gil et al. in Mol Biol Cell 15:1881-1894, 2004), the quantitatively most important components of cellular membranes. This latter activity of c-Fos determines the rate of membrane genesis and consequently of growth in differentiating PC12 cells (Gil et al. in Mol Biol Cell 15:1881-1894, 2004). In addition, it has been shown that c-Fos is over-expressed both in PNS and CNS tumors (Silvestre et al. in PLoS One 5(3):e9544, 2010). Herein, it is shown that c-Fos-activated phospholipid synthesis is required to support membrane genesis during the exacerbated growth characteristic of brain tumor cells. Specifically blocking c-Fos-activated phospholipid synthesis significantly reduces proliferation of tumor cells in culture. Blocking c-Fos expression also prevents tumor progression in mice intra-cranially xeno-grafted human brain tumor cells. In NPcis mice, an animal model of the human disease Neurofibromatosis Type I (Cichowski and Jacks in Cell 104:593-604, 2001), animals spontaneously develop tumors of the PNS and the CNS, provided they express c-Fos (Silvestre et al. in PLoS One 5(3):e9544, 2010). Treatment of PNS tumors with an antisense oligonucleotide that specifically blocks c-Fos expression also blocks tumor growth in vivo. These results disclose cytoplasmic c-Fos as a new target for effectively controlling brain tumor growth.

Coupling of receptor conformation and ligand orientation determine graded activity.

Small molecules stabilize specific protein conformations from a larger ensemble, enabling molecular switches that control diverse cellular functions. We show here that the converse also holds true: the conformational state of the estrogen receptor can direct distinct orientations of the bound ligand. 'Gain-of-allostery' mutations that mimic the effects of ligand in driving protein conformation allowed crystallization of the partial agonist ligand WAY-169916 with both the canonical active and inactive conformations of the estrogen receptor. The intermediate transcriptional activity induced by WAY-169916 is associated with the ligand binding differently to the active and inactive conformations of the receptor. Analyses of a series of chemical derivatives demonstrated that altering the ensemble of ligand binding orientations changes signaling output. The coupling of different ligand binding orientations to distinct active and inactive protein conformations defines a new mechanism for titrating allosteric signaling activity.

Growth of peripheral and central nervous system tumors is supported by cytoplasmic c-Fos in humans and mice.

BACKGROUND: We have previously shown that the transcription factor c-Fos is also capable of associating to endoplasmic reticulum membranes (ER) and activating phospholipid synthesis. Herein we examined phospholipid synthesis status in brain tumors from human patients and from NPcis mice, an animal model of the human disease Neurofibromatosis Type 1 (NF1). PRINCIPAL FINDINGS: In human samples, c-Fos expression was at the limit of detection in non-pathological specimens, but was abundantly expressed associated to ER membranes in tumor cells. This was also observed in CNS of adult tumor-bearing NPcis mice but not in NPcis fos(-/-) KO mice. A glioblastoma multiforme and a malignant PNS tumor from a NF1 patient (MPNST) showed a 2- and 4- fold c-Fos-dependent phospholipid synthesis activation, respectively. MPNST samples also showed increased cell proliferation rates and abundant c-Fos expression. CONCLUSIONS: Results highlight a role of cytoplasmic c-Fos as an activator of phospholipid synthesis in events demanding high rates of membrane biogenesis as occurs for the exacerbated growth of tumors cells. They also disclose this protein as a potential target for controlling tumor growth in the nervous system.

c-Fos activates glucosylceramide synthase and glycolipid synthesis in PC12 cells.

It has been demonstrated that c-Fos has, in addition to its well recognized AP-1 transcription factor activity, the capacity to associate to the endoplasmic reticulum and activate key enzymes involved in the synthesis of phospholipids required for membrane biogenesis during cell growth and neurite formation. Because membrane genesis requires the coordinated supply of all its integral membrane components, the question emerges as to whether c-Fos also activates the synthesis of glycolipids, another ubiquitous membrane component. We show that c-Fos activates the metabolic labeling of glycolipids in differentiating PC12 cells. Specifically, c-Fos activates the enzyme glucosylceramide synthase (GlcCerS), the product of which, GlcCer, is the first glycosylated intermediate in the pathway of synthesis of glycolipids. By contrast, the activities of GlcCer galactosyltransferase 1 and lactosylceramide sialyltransferase 1 are essentially unaffected by c-Fos. Co-immunoprecipitation experiments in cells co-transfected with c-Fos and a V5-tagged version of GlcCerS evidenced that both proteins participate in a physical association. c-Fos expression is tightly regulated by specific environmental cues. This strict regulation assures that lipid metabolism activation will occur as a response to cell requirements thus pointing to c-Fos as an important regulator of key membrane metabolisms in membrane biogenesis-demanding processes.

NFkappaB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses.

Our understanding of how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems that are common to steroid hormone receptors are circumvented by mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the structure of an apo steroid receptor that reveals a ligand-accessible channel allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFkappaB-selective signaling through the estrogen receptor, thus revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define structural features of signaling specificity that would not be apparent in a single structure.

CBP Is a dosage-dependent regulator of nuclear factor-kappaB suppression by the estrogen receptor.

The estrogen receptor (ER) protects against debilitating effects of the inflammatory response by inhibiting the proinflammatory transcription factor nuclear factor-kappaB (NFkappaB). Heretofore cAMP response element-binding protein (CREB)-binding protein (CBP) has been suggested to mediate inhibitory cross talk by functioning either as a scaffold that links ER and NFkappaB or as a required cofactor that competitively binds to one or the other transcriptional factor. However, here we demonstrate that ER is recruited to the NFkappaB response element of the MCP-1 (monocyte chemoattractant protein-1) and IL-8 promoters and displaces CBP, but not p65, in the MCF-7 breast cancer cell line. In contrast, ER displaced p65 and associated coregulators from the IL-6 promoter, demonstrating a gene-specific role for CBP in integrating inflammatory and steroid signaling. Further, RNA interference and overexpression studies demonstrated that CBP dosage regulates estrogen-mediated suppression of MCP-1 and IL-8, but not IL-6, gene expression. This work further demonstrates that CBP dosage is a critical regulator of gene-specific signal integration between the ER- and NFkappaB-signaling pathways.

Reconstructing signal transduction pathways: challenges and opportunities.

In this chapter, we will review how signal transduction pathways have been assembled in the past, bringing us to our present understanding of this area of research. The methods employed have relied heavily upon the genetics of yeast, worms, flies, mice, and humans. The use of second site suppressors and epistasis has permitted the detection of interacting elements and the sequence of genetic activities. Biochemistry has been employed to elucidate metabolic pathways, demonstrate protein complexes, and identify functions of gene products. The tools of molecular biology-knocking concentration of protein products down or up-have been helpful to trace the function of pathways in vivo. The study of disease states has led to the identification of a set of altered genes and helped define a network that is altered and gives rise to the disease. We will also discuss some serious limitations in these approaches. After reviewing how signal transduction pathways are constructed and investigated, we will turn our attention to an example that demonstrates the inter-relationships between pathways and the regulation of a specific set of pathways. We will examine how the p53 pathway in responding to stress shuts down the AKT-1 and mTOR pathways so as to limit the error frequency of cell growth and division during a stressful time where homeostatic mechanisms are required to respond and increase the fidelity of these processes.

Structural plasticity in the oestrogen receptor ligand-binding domain.

The steroid hormone receptors are characterized by binding to relatively rigid, inflexible endogenous steroid ligands. Other members of the nuclear receptor superfamily bind to conformationally flexible lipids and show a corresponding degree of elasticity in the ligand-binding pocket. Here, we report the X-ray crystal structure of the oestrogen receptor alpha (ERalpha) bound to an oestradiol derivative with a prosthetic group, ortho- trifluoromethlyphenylvinyl, which binds in a novel extended pocket in the ligand-binding domain. Unlike ER antagonists with bulky side groups, this derivative is enclosed in the ligand-binding pocket, and acts as a potent agonist. This work shows that steroid hormone receptors can interact with a wider array of pharmacophores than previously thought through structural plasticity in the ligand-binding pocket.

Detection of functional single-nucleotide polymorphisms that affect apoptosis.

Human EBV-transformed B lymphocyte cell lines (LCLs) were used to measure the apoptotic response of individuals to gamma radiation. The responses form a normal distribution around a median of 35.5% apoptosis with a range of 12-58%. This heterogeneous response has a genetic basis. LCLs from Caucasian donors and African American donors form distinct distributions of apoptotic response; all of the 11 LCLs comprising the lowest responding group (exhibiting between 12-20% apoptosis) are from Caucasian donors. The assay is capable of detecting significant effects of SNPs in two genes, MDM2 and AKT1, whose products are involved in controlling the p53 pathway and cellular response to DNA damage, suggesting that these data and this assay can be used to identify novel SNPs in other genes whose products impact the cellular response to radiation. Finally, the LCLs in the lowest apoptotic response group have the highest concentration of AKT1 protein and all harbor a haplotype in AKT1 that is present in Caucasians but absent in African Americans.

c-Fos activated phospholipid synthesis is required for neurite elongation in differentiating PC12 cells.

We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation.