Uncovering a novel mechanism: Butyrate induces estrogen receptor alpha activation independent of estrogen stimulation in MCF-7 breast cancer cells.

Butyrate is a promising candidate for an antitumoral drug, as it promotes cancer cell apoptosis and reduces hormone receptor activity, while promoting differentiation and proliferation in normal cells. However, the effects of low-dose butyrate on breast cancer cell cultures are unclear. We explored the impact of sub-therapeutic doses of butyrate on estrogen receptor alpha (ERα) transcriptional activity in MCF-7 cells, using RT-qPCR, Western blot, wound-healing assays, and chromatin immunoprecipitation. Our results showed that sub-therapeutic doses of sodium butyrate (0.1 - 0.2 mM) increased the transcription of ESR1, TFF1, and CSTD genes, but did not affect ERα protein levels. Moreover, we observed an increase in cell migration in wound-healing assays. ChIP assays revealed that treatment with 0.1 mM of sodium butyrate resulted in estrogen-independent recruitment of ERα at the pS2 promoter and loss of NCoR. Appropriate therapeutic dosage of butyrate is essential to avoid potential adverse effects on patients' health, especially in the case of estrogen receptor-positive breast tumors. Sub-therapeutic doses of butyrate may induce undesirable cell processes, such as migration due to low-dose butyrate-mediated ERα activation. These findings shed light on the complex effects of butyrate in breast cancer and provide insights for research in the development of antitumoral drugs.

A Quest for New Cancer Diagnosis, Prognosis and Prediction Biomarkers and Their Use in Biosensors Development.

Traditional techniques for cancer diagnosis, such as nuclear magnetic resonance, ultrasound and tissue analysis, require sophisticated devices and highly trained personnel, which are characterized by elevated operation costs. The use of biomarkers has emerged as an alternative for cancer diagnosis, prognosis and prediction because their measurement in tissues or fluids, such as blood, urine or saliva, is characterized by shorter processing times. However, the biomarkers used currently, and the techniques used for their measurement, including ELISA, western-blot, polymerase chain reaction (PCR) or immunohistochemistry, possess low sensitivity and specificity. Therefore, the search for new proteomic, genomic or immunological biomarkers and the development of new noninvasive, easier and cheaper techniques that meet the sensitivity and specificity criteria for the diagnosis, prognosis and prediction of this disease has become a relevant topic. The purpose of this review is to provide an overview about the search for new cancer biomarkers, including the strategies that must be followed to identify them, as well as presenting the latest advances in the development of biosensors that possess a high potential for cancer diagnosis, prognosis and prediction, mainly focusing on their relevance in lung, prostate and breast cancers.

Assistance for Folding of Disease-Causing Plasma Membrane Proteins.

An extensive catalog of plasma membrane (PM) protein mutations related to phenotypic diseases is associated with incorrect protein folding and/or localization. These impairments, in addition to dysfunction, frequently promote protein aggregation, which can be detrimental to cells. Here, we review PM protein processing, from protein synthesis in the endoplasmic reticulum to delivery to the PM, stressing the main repercussions of processing failures and their physiological consequences in pathologies, and we summarize the recent proposed therapeutic strategies to rescue misassembled proteins through different types of chaperones and/or small molecule drugs that safeguard protein quality control and regulate proteostasis.

Resveratrol up-regulates ATP2A3 gene expression in breast cancer cell lines through epigenetic mechanisms.

Resveratrol (RSV) is a phytoestrogen which has been related to chemoprevention of several types of cancer. In this work, we show up to a 6-fold increased expression of ATP2A3 gene induced by RSV that triggers apoptosis and changes of intracellular Ca2+ management in MCF-7 and MDA-MB-231 breast cancer cell lines. We explored epigenetic mechanisms for that RSV-induced ATP2A3 up-regulation. The results indicate that RSV-induced ATP2A3 up-regulation correlates with about 50% of reduced HDAC activity and reduced nuclear HDAC2 expression and occupancy on ATP2A3 promoter, increasing the global acetylation of histone H3 and the enrichment of histone mark H3K27Ac on the proximal promoter of the ATP2A3 gene in MDA-MB-231 cells. We also quantified HAT activity, finding that it can be boosted with RSV treatment; however, pharmacological inhibition of p300, one of the main HATs, did not have significant effects in RSV-mediated ATP2A3 gene expression. Additionally, DNMT activity was also reduced in cells treated with RSV, as well as the expression of Methyl-DNA binding proteins MeCP2 and MBD2. However, analysis of the methylation pattern of ATP2A3 gene promoter showed un-methylated promoter in both cell lines. Taken together, the results of this work help to explain, at the molecular level, how ATP2A3 gene is regulated in breast cancer cells, and the benefits of RSV intake observed in epidemiological data, studies with animals, and in vitro models.

Histone deacetylase inhibitors promote ATP2A3 gene expression in hepatocellular carcinoma cells: p300 as a transcriptional regulator.

Sarco(endo)plasmic reticulum Ca2+-ATPases (SERCA) expression is reduced or absent in several types of cancer and cancer cell lines; however, their expression and regulation in hepatocellular carcinoma (HCC) are unknown. Histone deacetylase inhibitors (HDACi) increase SERCA3 mRNA expression in gastric and breast cancer cell lines by increasing H3K9ac and binding of Sp1 and Sp3 transcription factors to the promoter; however, the molecular mechanism is not fully understood. Our results show that ATP2A3 (SERCA3) gene expression is decreased in human HCC samples and rat HCC AS-30D cells compared to normal liver, and HCC patients with high expression of ATP2A3 had longer overall survival than those with low expression. Sodium butyrate (NaB) and trichostatin A (TSA) increase SERCA3 mRNA expression in AS-30D cells, whereas SERCA2b mRNA expression did not change. NaB and TSA increase H3K9ac and H3K27ac in two ATP2A3 promoter regions. Besides, NaB treated cells increased Sp1 and Sp3 occupancy at ATP2A3 promoter; whereas TSA treated cells showed increased p300 levels at ATP2A3 promoter. Inhibition of p300 by C646, a specific inhibitor, mitigates SERCA3 mRNA induction by TSA, and reduces more than 70% of basal SERCA3 mRNA expression, suggesting that p300 is important for ATP2A3 gene transcription in AS-30D cells. Moreover, inhibition of p300 decreases H3K9ac in TSA treated cells. Our results provide evidence of decreased SERCA3 expression in human HCC samples and rat AS-30D cells and a correlation of SERCA3 expression with overall survival in HCC patients. Also, reveal new insights in SERCA3 transcriptional regulation mediated by HDACi.

Epigenetic regulation of the human ATP2A3 gene promoter in gastric and colon cancer cell lines.

The knowledge about the role of calcium-regulated pathways in cancer cell growth and differentiation could be useful for the development of new therapeutic approaches to diminish its mortality. The ATP2A genes encode for SERCA pumps, which modulate cytosolic Ca2+ concentration, regulating various cellular processes including cell growth. ATP2A3 gene transcriptional down-regulation has been reported in gastric and colon cancer, but there is still a lack of understanding about the epigenetic processes regulating its transcription. In this work, we report that butyrate, trichostatin A, and 5-azacytidine treatments increase SERCA3 expression, increased apoptosis, and decreased cell viability of the KATO-III gastric carcinoma cell line. We analyzed the methylation profile of the ATP2A3 gene promoter CpG island, finding clones with methylated status through -280 to -135 promoter region, harboring Sp1 and AP-2 binding sites, which could have a role in transcriptional repression. Post-translational modifications of histones show a major role in the ATP2A3 transcriptional regulation, and our results show histones marks linked to transcriptional repression associated with the -262 to -135 region, this repressive context changed to transcriptional permissive through SERCA3 re-expressing conditions. These results suggest that the nucleotide sequence from -280 to -135 position is an ATP2A3 epigenetic regulatory CpG region in KATO-III cells. Analyses of online-databases show a decreased SERCA3 expression in gastric and colon tumors, as well as overall survival results, showed that high SERCA3 expression could serve as a favorable prognostic marker for colon and gastric cancer patients.

Correction to: Hyper-response to Novelty Increases c-Fos Expression in the Hippocampus and Prefrontal Cortex in a Rat Model of Schizophrenia.

The original version of this article unfortunately contained a mistake. The spelling of the author Tommaso Ianniti was incorrect and has been corrected as Tommaso Iannitti. The original article has been corrected.

Hyper-response to Novelty Increases c-Fos Expression in the Hippocampus and Prefrontal Cortex in a Rat Model of Schizophrenia.

Schizophrenia is a debilitating disorder that may have a neurodevelopmental origin. For this reason, animal models based on neonatal insults or manipulations have been extensively used to demonstrate schizophrenia-related behaviors. Among those, the neonatal ventral hippocampus lesion (nVHL) is largely used as a model of schizophrenia-related behavior as it mimics behavioral and neurochemical abnormalities often seen in schizophrenic patients including hyperlocomotion in a novel environment. To investigate the neuroanatomical basis of coding novelty in the nVHL rat, we assessed the behavioral locomotor activity paradigm in a novel environment and measured expression of c-Fos, a marker of neural activation, in brain regions involved in the process of coding novelty or locomotion. Upon reaching adulthood, nVHL rats showed hyperlocomotion in the novel environment paradigm. Moreover, in nVHL rats the expression of c-Fos was greater in the prefrontal cortex (PFC) and CA1 region of the dorsal hippocampus compared to sham rats. Whereas similar expression of c-Fos was observed in the basolateral amygdala, nucleus accumbens and dentate gyrus region of  hippocampus of nVHL and sham rats. These results suggest that the nVHL disrupts the neural activity in the PFC and CA1 region of hippocampus in the process of coding novelty in the rat.

ATP2A3 gene as an important player for resveratrol anticancer activity in breast cancer cells.

The Ca2+ -ATPases from the Sarco/endoplasmic reticulum (SERCA) are fundamental for maintaining intracellular [Ca2+ ] homeostasis by pumping Ca2+ into the endoplasmic reticulum (ER) of eukaryotic cells. SERCA enzymes are encoded by three different genes (ATP2A1-3), whose expression occurs in a tissue and development stage-specific manner. It has been reported alterations in the expression of SERCA2 and SERCA3 pumps in different types of cancer: oral, lung, colon, stomach, central nervous system, thyroid, breast, and prostate. Resveratrol (RSV), a phytoalexin produced by a wide variety of plants in response to stress situations can modulate cellular processes involved in all stages of carcinogenesis. In this work, we used breast cancer cell lines (MCF-7 and MDA-MB-231) to evaluate mRNA levels of ATP2A2 and ATP2A3 genes in response to RSV treatment. Our results demonstrate that RSV treatment induced the expression of ATP2A3 gene in both cell lines in a time and concentration-dependent manner, while the expression of ATP2A2 gene remained unaffected. The RSV-induced expression of SERCA3 in these breast cancer cell lines produced decreased cell viability, triggered apoptosis and changes in cytosolic Ca2+ levels, as well as changes in the capacity for Ca2+ release by the ER. These data suggest an important participation of SERCA3 genes in RSV-mediated anti-tumor effect in breast cancer cell lines. Nevertheless, further research is needed to elucidate the molecular mechanisms underlying this effect.

EGF regulates claudin-2 and -4 expression through Src and STAT3 in MDCK cells.

Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2-dependent downregulation of claudin-2 (CLDN-2) and upregulation of claudin-4 (CLDN-4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN-2 downregulation and CLDN-4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN-2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF-induced changes on CLDN-2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus, a process that depends on Src activation. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF-induced downregulation of CLDN-2, but they are also implicated in the EGF-induced CLDN-4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN-2 and -4 proteins and mRNAs through Src and STAT3 activity.

Tristetraprolin represses estrogen receptor α transactivation in breast cancer cells.

Estrogen receptor α (ERα) mediates the effects of 17ß-estradiol (E2) in normal mammary gland, and it is a key participant in breast cancer tumor development. ERα transactivation activity is mediated by the synergistic interaction of two domains designated AF1 and AF2. The function of AF2 is to recruit coactivator and corepressor proteins that allow ERα to oscillate between the roles of transcriptional activator and repressor. In contrast, the mechanism responsible for AF-1 transcriptional activity is not completely understood. In this study, we identified tristetraproline (TTP) as a novel ERα-associated protein. TTP expression in MCF7 cells repressed ERα transactivation and reduced MCF7 cell proliferation and the ability of the cells to form tumors in a mouse model. We show that TTP transcriptional activity is mediated through its recruitment to the promoter region of ERα target genes and its interaction with histone deacetylases, in particular with HDAC1. TTP expression attenuates the coactivating activity of SRC-1, suggesting that exchange between TTP and other coactivators may play an important role in fine-tuning ERα transactivation. These results indicate that TTP acts as a bona fide ERα corepressor and suggest that this protein may be a contributing factor in the development of E2-dependent tumors in breast cancer.

SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells.

The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors.

Holocarboxylase synthetase acts as a biotin-independent transcriptional repressor interacting with HDAC1, HDAC2 and HDAC7.

In human cells, HCS catalyzes the biotinylation of biotin-dependent carboxylases and mediates the transcriptional control of genes involved in biotin metabolism through the activation of a cGMP-dependent signal transduction pathway. HCS also targets to the cell nucleus in association with lamin-B suggesting additional gene regulatory functions. Studies from our laboratory in Drosophila melanogaster showed that nuclear HCS is associated with heterochromatin bands enriched with the transcriptionally repressive mark histone 3 trimethylated at lysine 9. Further, HCS was shown to be recruited to the core promoter of the transcriptionally inactive hsp70 gene suggesting that it may participate in the repression of gene expression, although the mechanism involved remained elusive. In this work, we expressed HCS as a fusion protein with the DNA-binding domain of GAL4 to evaluate its effect on the transcription of a luciferase reporter gene. We show that HCS possesses transcriptional repressor activity in HepG2 cells. The transcriptional function of HCS was shown by in vitro pull down and in vivo co-immunoprecipitation assays to depend on its interaction with the histone deacetylases HDAC1, HDAC2 and HDAC7. We show further that HCS interaction with HDACs and its function in transcriptional repression is not affected by mutations impairing its biotin-ligase activity. We propose that nuclear HCS mediates events of transcriptional repression through a biotin-independent mechanism that involves its interaction with chromatin-modifying protein complexes that include histone deacetylases.

Management of a patient with holocarboxylase synthetase deficiency.

We investigated in a patient with holocarboxylase synthetase deficiency, the relation between the biochemical and genetic factors of the mutant protein with the pharmacokinetic factors of successful biotin treatment. A girl exhibited abnormal skin at birth, and developed in the first days of life neonatal respiratory distress syndrome and metabolic abnormalities diagnostic of multiple carboxylase deficiency. Enzyme assays showed low carboxylase activities. Fibroblast analysis showed poor incorporation of biotin into the carboxylases, and low transfer of biotin by the holocarboxylase synthetase enzyme. Kinetic studies identified an increased Km but a preserved Vmax. Mutation analysis showed the child to be a compound heterozygote for a new nonsense mutation Q379X and for a novel missense mutation Y663H. This mutation affects a conserved amino acid, which is located the most 3' of all recorded missense mutations thus far described, and extends the region of functional biotin interaction. Treatment with biotin 100mg/day gradually improved the biochemical abnormalities in blood and in cerebrospinal fluid (CSF), corrected the carboxylase enzyme activities, and provided clinical stability and a normal neurodevelopmental outcome. Plasma concentrations of biotin were increased to more than 500 nM, thus exceeding the increased Km of the mutant enzyme. At these pharmacological concentrations, the CSF biotin concentration was half the concentration in blood. Measuring these pharmacokinetic variables can aid in optimizing treatment, as individual tailoring of dosing to the needs of the mutation may be required.

Impaired biotinidase activity disrupts holocarboxylase synthetase expression in late onset multiple carboxylase deficiency.

Biotinidase catalyzes the hydrolysis of the vitamin biotin from proteolytically degraded biotin-dependent carboxylases. This key reaction makes the biotin available for reutilization in the biotinylation of newly synthesized apocarboxylases. This latter reaction is catalyzed by holocarboxylase synthetase (HCS) via synthesis of 5'-biotinyl-AMP (B-AMP) from biotin and ATP, followed by transfer of the biotin to a specific lysine residue of the apocarboxylase substrate. In addition to carboxylase activation, B-AMP is also a key regulatory molecule in the transcription of genes encoding apocarboxylases and HCS itself. In humans, genetic deficiency of HCS or biotinidase results in the life-threatening disorder biotin-responsive multiple carboxylase deficiency, characterized by a reduction in the activities of all biotin-dependent carboxylases. Although the clinical manifestations of both disorders are similar, they differ in some unique neurological characteristics whose origin is not fully understood. In this study, we show that biotinidase deficiency not only reduces net carboxylase biotinylation, but it also impairs the expression of carboxylases and HCS by interfering with the B-AMP-dependent mechanism of transcription control. We propose that biotinidase-deficient patients may develop a secondary HCS deficiency disrupting the altruistic tissue-specific biotin allocation mechanism that protects brain metabolism during biotin starvation.

Antitubercular isoniazid and drug resistance of Mycobacterium tuberculosis--a review.

Isoniazid is one of the most potent drugs available for tuberculosis treatment. As a pro-drug it requires activation, which is performed by catalase/peroxidase. The active principle, whose identity has not yet been determined unambiguously, then acts on at least one target molecule, the enoyl-acyl carrier protein, required for the synthesis of the vital mycolic acids present in the cell wall of the bacterium. Some other targets have been proposed in order to explain the unusual potency of isoniazid; however, the supporting data are still controversial. We thoroughly discuss the action of isoniazid, resistance mechanisms, and the possible active product, which includes an isonicotinic acid-NADH adduct as well as a meta-isomer of NADH. Both structures have been probed positively in a 3D modeling analysis.