MCF-7 Drug Resistant Cell Lines Switch Their Lipid Metabolism to Triple Negative Breast Cancer Signature.

Obesity and adipose tissue have been closely related to a poor cancer prognosis, especially in prostate and breast cancer patients. The ability of transferring lipids from the adipose tissue to the tumor cells is actively linked to tumor progression. However, different types of breast tumor seem to use these lipids in different ways and metabolize them in different pathways. In this study we have tracked by mass spectrometry how palmitic acid from the adipocytes is released to media being later incorporated in different breast cancer cell lines (MDA-MB-231, SKBR3, BT474, MCF-7 and its resistant MCF-7 EPIR and MCF-7 TAXR). We have observed that different lines metabolize the palmitic acid in a different way and use their carbons in the synthesis of different new lipid families. Furthermore, we have observed that the lipid synthesis pattern varied according to the cell line. Surprisingly, the metabolic pattern of the resistant cells was more related to the TNBC cell line compared to their sensitive cell line MCF-7. These results allow us to determine a specific lipid pattern in different cell lines that later might be used in breast cancer diagnosis and to find a better treatment according to the cancer molecular type.

Altered Serum Metabolic Profile Assessed by Advanced 1H-NMR in Breast Cancer Patients.

BACKGROUND: Altered lipid metabolism has been described in some types of cancer. To analyse in depth the metabolic modifications in breast cancer patients, advanced 1H-nuclear magnetic resonance was performed in these patients. The main objective of this paper was to define a specific lipidomic signature for these cancer patients. MATERIALS AND METHODS: Serum from 240 women (171 breast cancer patients and 69 control women) were studied and analysed by nuclear magnetic resonance. RESULTS: Triglyceride-enriched particles, specifically very low-density lipoprotein triglycerides, intermediate-density lipoprotein triglycerides, low-density lipoprotein triglycerides, and high-density lipoprotein triglycerides, were positively associated with breast cancer. Moreover, alanine, tyrosine, and branched amino acids were also associated with increased risk of breast cancer. CONCLUSIONS: Breast cancer patients showed a modified metabolome, giving a very interesting tool to draw different radar charts between control women and breast cancer patients. To our knowledge, this is the first time that advanced nuclear magnetic resonance profiling has been used to identify relevant and specifically altered lipid or amino acid metabolites in BC serum samples. The altered metabolic signature could be analysed for early and reliable BC patient diagnosis and prognosis.

Hepatic Lipidomics and Molecular Imaging in a Murine Non-Alcoholic Fatty Liver Disease Model: Insights into Molecular Mechanisms.

An imbalance between hepatic fatty acid uptake and removal results in ectopic fat accumulation, which leads to non-alcoholic fatty liver disease (NAFLD). The amount and type of accumulated triglycerides seem to play roles in NAFLD progression; however, a complete understanding of how triglycerides contribute to NAFLD evolution is lacking. Our aim was to evaluate triglyceride accumulation in NAFLD in a murine model and its associations with molecular mechanisms involved in liver damage and adipose tissue-liver cross talk by employing lipidomic and molecular imaging techniques. C57BL/6J mice fed a high-fat diet (HFD) for 12 weeks were used as a NAFLD model. Standard-diet (STD)-fed animals were used as controls. Standard liver pathology was assessed using conventional techniques. The liver lipidome was analyzed by liquid chromatography-mass spectrometry (LC-MS) and laser desorption/ionization-mass spectrometry (LDI-MS) tissue imaging. Liver triglycerides were identified by MS/MS. The transcriptome of genes involved in intracellular lipid metabolism and inflammation was assessed by RT-PCR. Plasma leptin, resistin, adiponectin, and FABP4 levels were determined using commercial kits. HFD-fed mice displayed increased liver lipid content. LC-MS analyses identified 14 triglyceride types that were upregulated in livers from HFD-fed animals. Among these 14 types, 10 were identified in liver cross sections by LDI-MS tissue imaging. The accumulation of these triglycerides was associated with the upregulation of lipogenesis and inflammatory genes and the downregulation of ß-oxidation genes. Interestingly, the levels of plasma FABP4, but not of other adipokines, were positively associated with 8 of these triglycerides in HFD-fed mice but not in STD-fed mice. Our findings suggest a putative role of FABP4 in the liver-adipose tissue cross talk in NAFLD.

OZF is a Claspin-interacting protein essential to maintain the replication fork progression rate under replication stress.

DNA replication is essential for cell proliferation and is one of the cell cycle stages where DNA is more vulnerable. Replication stress is a prominent property of tumor cells and an emerging target for cancer therapy. Although it is not directly involved in nucleotide incorporation, Claspin is a protein with relevant functions in DNA replication. It harbors a DNA-binding domain that interacts preferentially with branched or forked DNA molecules. It also acts as a platform for the interaction of proteins related to DNA damage checkpoint activation, DNA repair, DNA replication origin firing, and fork progression. In order to find new proteins potentially involved in the regulation of DNA replication, we performed a two-hybrid screen to discover new Claspin-binding proteins. This system allowed us to identify the zinc-finger protein OZF (ZNF146) as a new Claspin-interacting protein. OZF is also present at replication forks and co-immunoprecipitates not only with Claspin but also with other replisome components. Interestingly, OZF depletion does not affect DNA replication in a normal cell cycle, but its depletion induces a reduction in the fork progression rate under replication stress conditions. Our results suggest that OZF is a Claspin-binding protein with a specific function in fork progression under replication stress.

Niveles plasmáticos de glucosa, triglicéridos, VLDL, leptina y resistina como potenciales biomarcadores de la grasa miocárdica en ratones / Plasma glucose, triglycerides, VLDL, leptin and resistin levels as potential biomarkers for myocardial fat in mice

Introducción: El incremento de grasa miocárdica ha sido propuesto como uno de los principales precursores de la disfunción miocárdica de etiología diabética independiente de la enfermedad arterial coronaria. Sin embargo, actualmente se carece de biomarcadores que reflejen el contenido de grasa miocárdica para la detección clínica de esta patología. Métodos: Las correlaciones entre el contenido de triglicéridos cardíacos y los niveles plasmáticos de las principales moléculas alteradas durante la diabetes y los niveles cardíacos de ARNm de genes implicados en el metabolismo cardíaco (Cd36 y Pdk4) han sido exploradas en un modelo murino de resistencia a la insulina inducida por una dieta con alto contenido en grasas. Resultados: En ratones resistentes a la insulina, la dieta grasa aumentó los niveles de triglicéridos del miocardio, en comparación con animales controles alimentados con una dieta estándar. El contenido de triglicéridos cardíacos se encontró directamente asociado con los niveles plasmáticos de glucosa, triglicéridos, VLDL, resistina y leptina. Además, se observó una correlación inversa entre el contenido de triglicéridos y los niveles cardíacos de ARNm de Cd36 y Pdk4. Conclusiones: Nuestros datos revelan que el contenido cardíaco de triglicéridos se encuentra asociado con un perfil bioquímico plasmático alterado y con una reprogramación de la expresión de genes dirigida a atenuar el impacto de la acumulación ectópica de lípidos en miocardio

Introduction: The increase in myocardial fat has been proposed as one of the main precursors of myocardial dysfunction due to diabetic aetiology, independently of coronary artery disease. However, biomarkers reflecting the myocardial fat content for the clinical detection of this pathology are currently lacking. Methods: Correlations between 4cardiac triglyceride content and plasma levels of major altered molecules during diabetes and cardiac mRNA levels of genes involved in cardiac metabolism (Cd36 and Pdk4) have been explored in a murine model of insulin resistance induced by a high-fat diet. Results: In insulin-resistant mice, the fatty diet increased myocardial triglyceride levels, compared to control animals fed with a standard diet. The content of cardiac triglycerides was directly associated with plasma levels of glucose, triglycerides, VLDL, resistin and leptin. In addition, an inverse correlation was observed between the content of cardiac triglycerides and the cardiac mRNA levels of Cd36 and Pdk4. Conclusions: Our data reveal that the cardiac triglyceride content is associated with altered plasma biochemical profile and reprogramming of gene expression aimed to mitigate the impact of ectopic lipid accumulation in the myocardium

Plasma glucose, triglycerides, VLDL, leptin and resistin levels as potential biomarkers for myocardial fat in mice. / Niveles plasmáticos de glucosa, triglicéridos, VLDL, leptina y resistina como potenciales biomarcadores de la grasa miocárdica en ratones.

INTRODUCTION: The increase in myocardial fat has been proposed as one of the main precursors of myocardial dysfunction due to diabetic aetiology, independently of coronary artery disease. However, biomarkers reflecting the myocardial fat content for the clinical detection of this pathology are currently lacking. METHODS: Correlations between cardiac triglyceride content and plasma levels of major altered molecules during diabetes and cardiac mRNA levels of genes involved in cardiac metabolism (Cd36 and Pdk4) have been explored in a murine model of insulin resistance induced by a high-fat diet. RESULTS: In insulin-resistant mice, the fatty diet increased myocardial triglyceride levels, compared to control animals fed with a standard diet. The content of cardiac triglycerides was directly associated with plasma levels of glucose, triglycerides, VLDL, resistin and leptin. In addition, an inverse correlation was observed between the content of cardiac triglycerides and the cardiac mRNA levels of Cd36 and Pdk4. CONCLUSIONS: Our data reveal that the cardiac triglyceride content is associated with altered plasma biochemical profile and reprogramming of gene expression aimed to mitigate the impact of ectopic lipid accumulation in the myocardium.

The Circulating GRP78/BiP Is a Marker of Metabolic Diseases and Atherosclerosis: Bringing Endoplasmic Reticulum Stress into the Clinical Scenario.

BACKGROUND: Glucose-regulated protein 78/Binding immunoglobulin protein (GRP78/BiP) is a protein associated with endoplasmic reticulum stress and is upregulated by metabolic alterations at the tissue-level, such as hypoxia or glucose deprivation, and it is hyper-expressed in fat tissue of obese individuals. OBJECTIVE: To investigate the role of the GRP78/BiP level as a metabolic and vascular disease biomarker in patients with type 2 diabetes (DM), obesity and metabolic syndrome (MS). METHODS: Four hundred and five patients were recruited, of whom 52.5% were obese, 72.8% had DM, and 78.6% had MS. The intimae media thickness (cIMT) was assessed by ultrasonography. The plasma GRP78/BiP concentration was determined, and its association with metabolic and vascular parameters was assessed. Circulating GRP78/BiP was also prospectively measured in 30 DM patients before and after fenofibrate/niacin treatment and 30 healthy controls. RESULTS: In the cross-sectional study, the GRP78/BiP level was significantly higher in the patients with obesity, DM, and MS. Age-, gender- and BMI-adjusted GRP78/BiP was directly associated with LDL-cholesterol, non-HDL-cholesterol, triglycerides, apoB, and cIMT. GRP78/BiP was positively associated to carotid plaque presence in the adjusted model, irrespective of obesity, DM and MS. In the prospective study, nicotinic acid treatment produced a significant reduction in the GRP78/BiP levels that was not observed with fenofibrate. CONCLUSIONS: GRP78/BiP plasma concentrations are increased in patients with both metabolic derangements and subclinical atherosclerosis. GRP78/BiP could be a useful marker of metabolic and cardiovascular risk.

Fatty acid binding protein 4 (FABP4) as a potential biomarker reflecting myocardial lipid storage in type 2 diabetes.

OBJECTIVE: Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone involved in the crosstalk between adipose and peripheral tissues, and it contributes to widespread insulin resistance in cells, including cardiac cells. However, the role of this adipokine in regulating cardiac metabolism and myocardial neutral lipid content in patients with type 2 diabetes has not been elucidated. METHODS: The impact of circulating FABP4 on the cardiac neutral lipid content was measured by proton magnetic resonance spectroscopy (1H-MRS) in patients with type 2 diabetes. Additionally, circulating FABP4 and the cardiac triglyceride content were analysed in high-fat diet (HFD)-fed mice, and the impact of the exogenous FABP4 was explored in HL-1 cardiac cells. RESULTS: Serum FABP4 levels were higher in type 2 diabetic patients compared to healthy individuals. Circulating FABP4 levels were associated with myocardial neutral lipid content in type 2 diabetic patients. In HFD-fed mice, both serum FABP4 and myocardial triglyceride content were increased. In FABP4-challenged HL-1 cells, extracellular FABP4 increased intracellular lipid accumulation, which led to impairment of the insulin-signalling pathway and reduced insulin-stimulated glucose uptake. However, these effects were partially reversed by FABP4 inhibition with BMS309403, which attenuated the intracellular lipid content and improved insulin signalling and insulin-stimulated glucose uptake. CONCLUSIONS: Taken together, our results identify FABP4 as a molecule involved in diabetic/lipid-induced cardiomyopathy and indicate that this molecule may be an emerging biomarker for diabetic cardiomyopathy-related disturbances, such as myocardial neutral lipid accumulation. Additionally, FABP4 inhibition may be a potential therapeutic target for metabolic-related cardiac dysfunctions.

FABP4 inhibitor BMS309403 decreases saturated-fatty-acid-induced endoplasmic reticulum stress-associated inflammation in skeletal muscle by reducing p38 MAPK activation.

AIMS: Fatty acid binding protein 4 (FABP4) inhibitors have been proposed as potential therapeutic approaches against insulin resistance-related inflammation and type 2 diabetes mellitus. However, the underlying molecular mechanisms by which these molecules drive these effects in skeletal muscle remain unknown. Here, we assessed whether the FABP4 inhibitor BMS309403 prevented lipid-induced endoplasmic reticulum (ER) stress-associated inflammation in skeletal muscle. MATERIALS AND METHODS: The BMS309403 treatment was assessed both in the skeletal muscle of high-fat diet (HFD)-fed mice and in palmitate-stimulated C2C12 myotubes. RESULTS: HFD feeding promoted insulin resistance, which is characterized by increased plasma levels of glucose, insulin, non-esterified fatty acids, triglycerides, resistin, and leptin and reduced plasma levels of adiponectin compared with control mice fed a standard diet. Additionally, insulin-resistant animals showed increased FABP4 plasma levels. In line with this evidence, recombinant FABP4 attenuated the insulin-induced AKT phosphorylation in C2C12 myotubes. Treatment with BMS309403 reduced lipid-induced ER stress and inflammation in both mouse skeletal muscle and C2C12 myotubes. The effects of the FABP4 inhibitor reducing lipid-induced ER stress-associated inflammation were related to the reduction of fatty acid-induced intramyocellular lipid deposits, ROS and nuclear factor-kappaB (NF-κB) nuclear translocation. Accordingly, BMS309403 reduced lipid-induced p38 MAPK phosphorylation, which is upstream of NF-κB activation. CONCLUSION: Overall, these findings indicate that BMS309403 reduces fatty acid-induced ER stress-associated inflammation in skeletal muscle by reducing p38 MAPK activation.

Clinical and pathophysiological evidence supporting the safety of extremely low LDL levels-The zero-LDL hypothesis.

While the impact of very low concentrations of low-density lipoprotein cholesterol (LDL-C) on cardiovascular prevention is very reassuring, it is intriguing to know what effect these extremely low LDL-C concentrations have on lipid homoeostasis. The evidence supporting the safety of extremely low LDL levels comes from genetic studies and clinical drug trials. Individuals with lifelong low LDL levels due to mutations in genes associated with increased LDL-LDL receptor (LDLR) activity reveal no safety issues. Patients achieving extremely low LDL levels in the IMPROVE-IT and FOURIER, and the PROFICIO and ODYSSEY programs seem not to have an increased prevalence of adverse effects. The main concern regarding extremely low LDL-C plasma concentrations is the adequacy of the supply of cholesterol, and other molecules, to peripheral tissues. However, LDL proteomic and kinetic studies reaffirm that LDL is the final product of endogenous lipoprotein metabolism. Four of 5 LDL particles are cleared through the LDL-LDLR pathway in the liver. Given that mammalian cells have no enzymatic systems to degrade cholesterol, the LDL-LDLR pathway is the main mechanism for removal of cholesterol from the body. Our focus, therefore, is to review, from a physiological perspective, why such extremely low LDL-C concentrations do not appear to be detrimental. We suggest that extremely low LDL-C levels due to increased LDLR activity may be a surrogate of adequate LDL-LDLR pathway function.

Metabolomics reveals novel blood plasma biomarkers associated to the BRCA1-mutated phenotype of human breast cancer.

Hereditary breast and ovarian cancer syndrome (HBOC) is partly due to the presence of mutations in the BRCA genes. Triple-negative (TN) breast cancer (BC) shares histological characteristics with germline BRCA1 mutation-associated tumours. We have investigated the metabolic profiles of human breast cancer (BC) cell lines carrying BRCA1 pathogenic mutations by non-targeted liquid chromatography coupled to mass spectrometry technology. Based on our in vitro results, we performed a targeted metabolomic analysis of plasma samples from TN HBOC patients taking into account their BRCA1 genotype. BRCA1 promoter hypermethylation and the BRCAness phenotype of BC cell lines were also studied. The purpose of this study was to determine the metabolic signature of HBOC syndrome and TNBC patients and to evaluate the potential contribution of the metabolites identified to the genetic diagnosis of breast cancer. The present results show the existence of a differential metabolic signature for BC cells based on the BRCA1 functionality. None of the studied BC cell lines presented hypermethylation of the BRCA1 promoter region. We provide evidence of the existence of free methylated nucleotides capable of distinguishing plasma samples from HBOC patients as BRCA1-mutated and BRCA1 non-mutated, suggesting that they might be considered as BRCA1-like biomarkers for TNBC and HBOC syndrome.

Adipose-Derived Fatty Acid-Binding Proteins Plasma Concentrations Are Increased in Breast Cancer Patients.

BACKGROUND: Adipose tissue is an endocrine organ that could play a role in tumor progression via its secreted adipokines. The role of adipose-derived fatty acid-binding protein (FABP) 4 and FABP5 in breast cancer is presently under study, but their circulating levels in this pathology are poorly known. We analyzed the blood concentrations of FABP4 and FABP5 in breast cancer patients to determine whether there is an association between them and breast cancer. MATERIALS AND METHODS: We studied 294 women in the oncology department with a family history of breast cancer; 198 of the women had breast cancer, and 96 were healthy controls. The levels of FABP4, FABP5, lipid profile, standard biochemical parameter, and high-sensitivity C-reactive protein (hsCRP) were determined. We analyzed the association of FABP4 and FABP5 with breast cancer, while adjusting for demographic, anthropometric, and biochemical parameters. RESULTS: Breast cancer patients had a 24.8% (p < .0001) and 11.4% (p < .05) higher blood concentration of FABP4 and FABP5, respectively. Fatty acid-binding protein 4 was positively associated with age, body mass index (BMI), FABP5, very-low-density lipoprotein cholesterol (VLDLc), non-high-density lipoprote in cholesterol (non-HDLc), Apolipoprotein B 100 (ApoB100), triglycerides, glycerol, glucose, and hsCRP (p < .05), and was negatively associated with HDLc (p < .005) in breast cancer patients. Fatty acid-binding protein 5 was positively associated with BMI, FABP4, VLDLc, triglycerides, glycerol, and hsCRP (p < .05), and was negatively associated with HDLc and Apolipoprotein AI (ApoAI) (p < .05) in breast cancer patients. Using a logistic regression analysis and adjusting for age, BMI, hsCRP, non-HDLc, and triglycerides, FABP4 was independently associated with breast cancer (odds ratio [OR]: 1.091 [95% CI: 1.037-1.149]). Moreover, total cholesterol, VLDLc, non-HDLc, ApoB100, triglycerides, and hsCRP were significantly increased in breast cancer patients (p < .005). In contrast, the non-esterified fatty acids concentrations were significantly decreased in breast cancer patients (p < .05). CONCLUSION: Circulating FABP4 and FABP5 levels were increased in breast cancer patients compared with controls. The positive association of FABP4 with breast cancer was maintained after adjusting for important covariates, while the association with FABP5 was lost. Our data reinforce the role of adipose tissue and their adipokines in breast cancer. Despite these data, further studies must be performed to better explain the prognosis or diagnostic value of these blood parameters and their possible role in breast cancer. IMPLICATIONS FOR PRACTICE: We focus on the effect of adipose tissue on cancer, which is increasingly recognized. The association between adipocyte-derived adipokines and breast cancer opens new diagnosis and therapy perspectives. In this study, we provide original data concerning FABP4 and FABP5 plasma concentrations in breast cancer patients. Compared to control group, breast cancer patients show higher FABP4 and FABP5 blood levels. Our data suggest that, particularly, circulating FABP4 levels could be considered a new independent breast cancer biomarker. Our work translates basic science data to clinic linking the relationship between adipose tissue and lipid metabolism to breast cancer.

Exogenous FABP4 increases breast cancer cell proliferation and activates the expression of fatty acid transport proteins.

Adipose tissue plays an important role in tumor progression, because it provides nutrients and adipokines to proliferating cells. Fatty acid binding protein 4 (FABP4) is a key adipokine for fatty acid transport. In metabolic pathologies, plasma levels of FABP4 are increased. However, the role of this circulating protein is unknown. Recent studies have demonstrated that FABP4 might have a role in tumor progression, but the molecular mechanisms involved are still unclear. In this study, we analysed the role of eFABP4 (exogenous FABP4) in breast cancer progression. MCF-7 and MDA-MB-231 breast cancer cells did not express substantial levels of FABP4 protein, but intracellular FABP4 levels increased after eFABP4 incubation. Moreover, eFABP4 enhanced the proliferation of these breast cancer cells but did not have any effect on MCF-7 and MDA-MB-231 cell migration. Additionally, eFABP4 induced the AKT and MAPK signaling cascades in breast cancer cells, and the inhibition of these pathways reduced the eFBAP4-mediated cell proliferation. Interestingly, eFABP4 treatment in MCF-7 cells increased levels of the transcription factor FoxM1 and the fatty acid transport proteins CD36 and FABP5. In summary, we showed that eFABP4 plays a key role in tumor proliferation and activates the expression of fatty acid transport proteins in MCF-7 breast cancer cells. © 2016 Wiley Periodicals, Inc.

Circulating PCSK9 levels and CETP plasma activity are independently associated in patients with metabolic diseases.

BACKGROUND: PCSK9 inhibition is a new powerful cholesterol-lowering strategy. Recently, it was reported that CETP inhibitors influence PCSK9 levels as an off-target effect. We explored the relationship between circulating PCSK9 levels and CETP activity in patients with metabolic disease who were not on lipid-lowering therapy. METHODS: Plasma CETP activity and PCSK9 levels were measured in 450 participants (median age, 58 years; 49 % women) who attended the metabolism unit because of metabolic syndrome (MetS) (78 %), atherogenic dyslipidemia (32 %), obesity (50 %), type 2 diabetes mellitus (72 %), and other risk factors (13 %). A 6 week lipid-lowering drug wash-out period was established in treated patients. RESULTS: Both PCSK9 levels and CETP activity were higher in patients with an increasing number of MetS components. PCSK9 levels were positively correlated with CETP activity in the entire cohort (r = 0.256, P < 0.0001) independent of age, gender, body mass index (BMI), systolic blood pressure (SBP), LDL cholesterol (LDL-C), triglycerides and glucose. Individuals with the loss-of-function PCSK9 genetic variant rs11591147 (R46L) had lower levels of PCSK9 (36.5 %, P < 0.0001) and LDL-C (17.8 %, P = 0.010) as well as lower CETP activity (10.31 %, P = 0.009). This association remained significant in the multiple regression analysis even after adjusting for gender, age, BMI, LDL-C, triglycerides, glucose, lecithin-cholesterol acyltransferase, SBP and MetS (P = 0.003). CONCLUSIONS: Our data suggest a metabolic association between PCSK9 and CETP independent of lipid-lowering treatment. The clinical implications of this metabolic relationship could be relevant for explaining the effect of PCSK9 and CETP inhibition on overall lipid profiles.

Exogenous FABP4 induces endoplasmic reticulum stress in HepG2 liver cells.

BACKGROUND AND AIMS: Fatty acid binding protein 4 (FABP4) is an intracellular fatty acid (FA) carrier protein that is, in part, secreted into circulation. Circulating FABP4 levels are increased in obesity, diabetes and other insulin resistance (IR) diseases. FAs contribute to IR by promoting endoplasmic reticulum stress (ER stress) and altering the insulin signaling pathway. The effect of FABP4 on ER stress in the liver is not known. The aim of this study was to investigate whether exogenous FABP4 (eFABP4) is involved in the lipid-induced ER stress in the liver. METHODS: HepG2 cells were cultured with eFABP4 (40 ng/ml) with or without linoleic acid (LA, 200 µM) for 18 h. The expression of ER stress-related markers was determined by Western blotting (ATF6, EIF2α, IRE1 and ubiquitin) and real-time PCR (ATF6, CHOP, EIF2α and IRE1). Apoptosis was studied by flow cytometry using Annexin V-FITC and propidium iodide staining. RESULTS: eFABP4 increased the ER stress markers ATF6 and IRE1 in HepG2 cells. This effect led to insulin resistance mediated by changes in AKT and JNK phosphorylation. Furthermore, eFABP4 significantly induced both apoptosis, as assessed by flow cytometry, and CHOP expression, without affecting necrosis and ubiquitination. The presence of LA increased the ER stress response induced by eFABP4. CONCLUSIONS: eFABP4, per se, induces ER stress and potentiates the effect of LA in HepG2 cells, suggesting that FABP4 could be a link between obesity-associated metabolic abnormalities and hepatic IR mechanisms.

In Vitro Biocompatibility of Surface-Modified Porous Alumina Particles for HepG2 Tumor Cells: Toward Early Diagnosis and Targeted Treatment.

Porous alumina photoluminescence-inherent particles are produced and proposed for the development of biomarkers detectors and localized treatment of HepG2 cells. Nanoporous alumina particles (NPAPs) are amorphous, consist of hexagonally ordered nanometric pores in an alumina matrix, have high chemical stability in physiological pH, and exhibit a high inherent photoluminescence in the visible spectrum independently of their size, selectable from nanometers to tens of micrometers. The surface of NPAPs is chemically modified using two different functionalization methods, a multistep method with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GLTA) and a novel simplified-step method with silane-PEG-NHS. Fourier Transform infrared spectroscopy analysis confirmed the proper surface modification of the particles for both functionalization methods. HepG2 cells were cultured during different times with growing concentrations of particles. The analysis of cytotoxicity and cell viability of HepG2 cells confirmed the good biocompatibility of NPAPs in all culture conditions. The results prove the suitability of NPAPs for developing new label-free biomarker detectors and advantageous carriers for localized drug delivery.

The stress-activated protein kinases p38α/ß and JNK1/2 cooperate with Chk1 to inhibit mitotic entry upon DNA replication arrest.

Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G 2/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38ß isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.

Functional cooperation between Snail1 and twist in the regulation of ZEB1 expression during epithelial to mesenchymal transition.

Snail1 and Zeb1 are E-cadherin-transcriptional repressors induced during epithelial mesenchymal transition (EMT). In this article we have analyzed the factors controlling Zeb1 expression during EMT. In NMuMG cells treated with TGF-ß, Snail1 RNA and protein are induced 1 h after addition of the cytokine preceding Zeb1 up-regulation that requires 6-8 h. Zeb1 gene expression is caused by increased RNA levels but also by enhanced protein stability and is markedly dependent on Snail1 because depletion of this protein prevents Zeb1 protein and RNA up-regulation. In addition to Snail1, depletion of the Twist transcriptional factor retards Zeb1 stimulation by TGF-ß or decreases Zeb1 expression in other cellular models indicating that this factor is also required for Zeb1 expression. Accordingly, Snail1 and Twist cooperate in the induction of Zeb1: co-transfection of both cDNAs is required for the maximal expression of ZEB1 mRNA. Unexpectedly, the expression of Snail1 and Twist shows a mutual dependence although to a different extent; whereas Twist depletion retards Snail1 up-regulation by TGF-ß, Snail1 is necessary for the rapid increase in Twist protein and later up-regulation of Twist1 mRNA induced by the cytokine. Besides this effect on Twist, Snail1 also induces the nuclear translocation of Ets1, another factor required for Zeb1 expression. Both Twist and Ets1 bind to the ZEB1 promoter although to different elements: whereas Ets1 interacts with the proximal promoter, Twist does it with a 700-bp sequence upstream of the transcription start site. These results indicate that Snail1 controls Zeb1 expression at multiple levels and acts cooperatively with Twist in the ZEB1 gene transcription induction.

Different S/M checkpoint responses of tumor and non tumor cell lines to DNA replication inhibition.

Cell cycle checkpoint abrogation, especially the inhibition of Chk1 in combination with DNA-damaging treatments, has been proposed as a promising way of sensitizing cancer cells. However, less is known about the possibility to selectively affect tumor cells when they are treated with agents that block DNA synthesis in combination with replication checkpoint inhibitors. Here, we present clear insights in the different responses of tumor and non-transformed cells to the inhibition of DNA replication with hydroxyurea in combination with checkpoint abrogation via inhibition of Ataxia telangiectasia-mutated- (ATM) and Rad3-related/ATM (ATR/ATM) and Chk1 kinases. Interestingly, we find that non-transformed cell lines activate ATR/ATM- and Chk1-independent pathways in response to replication inhibition to prevent mitotic entry with unreplicated DNA. In contrast, tumor cell lines such as HCT116 and HeLa cells rely entirely on Chk1 activity for a proper response to replication inhibitors. Our results show that p38 is activated in response to hydroxyurea treatment and collaborates with Chk1 to prevent mitotic entry in non-transformed cell lines by maintaining cyclin B1/Cdk1 complexes inactive. Furthermore, DNA replication arrest down-regulates cyclin B1 promoter activity in non-transformed cells, but not in tumor cells in a Chk1- and p38-independent way. Thus, our data show that non-transformed cells present a more robust DNA replication checkpoint response compared with tumor cells that involves activation of the p38 pathway. We show that some of these responses to replication block can be lost in tumor cells, causing a defective checkpoint and providing a rationale for tumor-selective effects of combined therapies.

Chk1- and claspin-dependent but ATR/ATM- and Rad17-independent DNA replication checkpoint response in HeLa cells.

When DNA synthesis is inhibited, DNA replication checkpoint is activated to prevent mitosis entry without fully replicated DNA. In Xenopus, caffeine-sensitive kinases [ataxia telangiectasia mutated (ATM) and ATM-related protein (ATR)] are essential in this checkpoint response, but in mammal cells an ATR/ATM-independent checkpoint response to DNA synthesis inhibition exists. Using HeLa cells, which have a caffeine-insensitive checkpoint response, we have analyzed here which molecules known to be involved in the DNA replication checkpoint participate in the caffeine-insensitive response. When DNA synthesis was inhibited in the presence of UCN01 or after knocking down Chk1 expression [Chk1 small interfering RNA (siRNA)], HeLa cells entered into aberrant mitosis. Consequently, Chk1 is essential for both the ATR/ATM-dependent and ATR/ATM-independent checkpoint response in HeLa cells. Neither wortmannin, Ly294002, nor SB202190 abrogated the caffeine-insensitive checkpoint response, indicating that DNA-PK and p38 alpha,beta are not involved in the ATR/ATM-independent Chk1 activation upon DNA synthesis inhibition. Using siRNA to knock down Rad17 and claspin, two molecules involved in sensing stalled replication forks, we also showed that claspin but not Rad17 is essential for the ATR/ATM-independent checkpoint response. Inhibition of DNA synthesis in HeLa cells led to a decrease in cyclin B1 protein accumulation that was abrogated when UCN01 was added or when claspin was knocked down. We conclude that upon DNA synthesis inhibition, Chk1 can be activated in a claspin-dependent manner independently of ATR and ATM, leading to cyclin B1 down-regulation and providing the cells of an additional mechanism to inhibit mitosis entry.