Publications by authors named "Raquel Ordoñez"

31 Publications

Design and Synthesis of Novel Epigenetic Inhibitors Targeting Histone Deacetylases, DNA Methyltransferase 1, and Lysine Methyltransferase G9a with Efficacy in Multiple Myeloma.

J Med Chem 2021 Mar 4. Epub 2021 Mar 4.

Small Molecule Discovery Platform, Molecular Therapeutics Program, Center for Applied Medical Research (CIMA), University of Navarra, Avenida Pio XII 55, E-31008 Pamplona, Spain.

Concomitant inhibition of key epigenetic pathways involved in silencing tumor suppressor genes has been recognized as a promising strategy for cancer therapy. Herein, we report a first-in-class series of quinoline-based analogues that simultaneously inhibit histone deacetylases (from a low nanomolar range) and DNA methyltransferase-1 (from a mid-nanomolar range, IC < 200 nM). Additionally, lysine methyltransferase G9a inhibitory activity is achieved (from a low nanomolar range) by introduction of a key lysine mimic group at the 7-position of the quinoline ring. The corresponding epigenetic functional cellular responses are observed: histone-3 acetylation, DNA hypomethylation, and decreased histone-3 methylation at lysine-9. These chemical probes, multitarget epigenetic inhibitors, were validated against the multiple myeloma cell line MM1.S, demonstrating promising activity of (CM-444) with GI of 32 nM, an adequate therapeutic window (>1 log unit), and a suitable pharmacokinetic profile. , achieved significant antitumor efficacy in a xenograft mouse model of human multiple myeloma.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02255DOI Listing
March 2021

A versatile platform for locus-scale genome rewriting and verification.

Proc Natl Acad Sci U S A 2021 Mar;118(10)

Institute for Systems Genetics, NYU Langone Health, New York, NY 10016.

Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.
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http://dx.doi.org/10.1073/pnas.2023952118DOI Listing
March 2021

Characterization of complete lncRNAs transcriptome reveals the functional and clinical impact of lncRNAs in multiple myeloma.

Leukemia 2021 Feb 17. Epub 2021 Feb 17.

Área de Oncología, Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, IDISNA, Pamplona, Spain.

Multiple myeloma (MM) is an incurable disease, whose clinical heterogeneity makes its management challenging, highlighting the need for biological features to guide improved therapies. Deregulation of specific long non-coding RNAs (lncRNAs) has been shown in MM, nevertheless, the complete lncRNA transcriptome has not yet been elucidated. In this work, we identified 40,511 novel lncRNAs in MM samples. lncRNAs accounted for 82% of the MM transcriptome and were more heterogeneously expressed than coding genes. A total of 10,351 overexpressed and 9,535 downregulated lncRNAs were identified in MM patients when compared with normal bone-marrow plasma cells. Transcriptional dynamics study of lncRNAs in the context of normal B-cell maturation revealed 989 lncRNAs with exclusive expression in MM, among which 89 showed de novo epigenomic activation. Knockdown studies on one of these lncRNAs, SMILO (specific myeloma intergenic long non-coding RNA), resulted in reduced proliferation and induction of apoptosis of MM cells, and activation of the interferon pathway. We also showed that the expression of lncRNAs, together with clinical and genetic risk alterations, stratified MM patients into several progression-free survival and overall survival groups. In summary, our global analysis of the lncRNAs transcriptome reveals the presence of specific lncRNAs associated with the biological and clinical behavior of the disease.
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http://dx.doi.org/10.1038/s41375-021-01147-yDOI Listing
February 2021

Sequencing identifies multiple early introductions of SARS-CoV-2 to the New York City region.

Genome Res 2020 12 22;30(12):1781-1788. Epub 2020 Oct 22.

Department of Pathology, NYU Grossman School of Medicine, New York, New York 10016, USA.

Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epidemiological parameters. Here, we report the analysis of 864 SARS-CoV-2 sequences from cases in the New York City metropolitan area during the COVID-19 outbreak in spring 2020. The majority of cases had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that early transmission was most linked to cases from Europe. Our data are consistent with numerous seeds from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of genomic surveillance in addition to traditional epidemiological indicators.
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http://dx.doi.org/10.1101/gr.266676.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706732PMC
December 2020

Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma.

Genome Res 2020 Sep 20;30(9):1217-1227. Epub 2020 Aug 20.

Centro de Investigación Biomédica en Red de Cáncer, CIBERONC, 28029 Madrid, Spain.

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (), a major regulator of cellular redox status and, in addition, identified as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.
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http://dx.doi.org/10.1101/gr.265520.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545147PMC
September 2020

Correction: Ordoñez, et al.; DNA Methylation of Enhancer Elements in Myeloid Neoplasms: Think Outside the Promoters? Cancers 2019, 11, 1424.

Cancers (Basel) 2020 Jul 13;12(7). Epub 2020 Jul 13.

Área de Hemato-Oncología, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Avenida Pío XII-55, 31008 Pamplona, Spain.

The authors would like to make a correction to their published paper [...].
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http://dx.doi.org/10.3390/cancers12071885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409024PMC
July 2020

Sequencing identifies multiple, early introductions of SARS-CoV2 to New York City Region.

medRxiv 2020 Apr 21. Epub 2020 Apr 21.

Department of Pathology, NYU Grossman School of Medicine, New York, USA.

Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epidemiological parameters. Here, we report the analysis of 236 SARS-CoV2 sequences from cases in the New York City metropolitan area during the initial stages of the 2020 COVID-19 outbreak. The majority of cases throughout the region had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that the majority were most related to cases from Europe. Our data are consistent with numerous seed transmissions from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of real-time genomic surveillance in addition to traditional epidemiological indicators.
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http://dx.doi.org/10.1101/2020.04.15.20064931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276014PMC
April 2020

DNA Methylation of Enhancer Elements in Myeloid Neoplasms: Think Outside the Promoters?

Cancers (Basel) 2019 Sep 24;11(10). Epub 2019 Sep 24.

Área de Hemato-Oncología, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Avenida Pío XII-55, 31008 Pamplona, Spain.

Gene regulation through DNA methylation is a well described phenomenon that has a prominent role in physiological and pathological cell-states. This epigenetic modification is usually grouped in regions denominated CpG islands, which frequently co-localize with gene promoters, silencing the transcription of those genes. Recent genome-wide DNA methylation studies have challenged this paradigm, demonstrating that DNA methylation of regulatory regions outside promoters is able to influence cell-type specific gene expression programs under physiologic or pathologic conditions. Coupling genome-wide DNA methylation assays with histone mark annotation has allowed for the identification of specific epigenomic changes that affect enhancer regulatory regions, revealing an additional layer of complexity to the epigenetic regulation of gene expression. In this review, we summarize the novel evidence for the molecular and biological regulation of DNA methylation in enhancer regions and the dynamism of these changes contributing to the fine-tuning of gene expression. We also analyze the contribution of enhancer DNA methylation on the expression of relevant genes in acute myeloid leukemia and chronic myeloproliferative neoplasms. The characterization of the aberrant enhancer DNA methylation provides not only a novel pathogenic mechanism for different tumors but also highlights novel potential therapeutic targets for myeloid derived neoplasms.
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http://dx.doi.org/10.3390/cancers11101424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827153PMC
September 2019

Epigenomic profiling of myelofibrosis reveals widespread DNA methylation changes in enhancer elements and as a potential tumor suppressor gene that is epigenetically regulated.

Haematologica 2019 08 17;104(8):1572-1579. Epub 2019 Jan 17.

Área de Hemato-Oncología, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Pamplona

In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. reporter assay and 5'-azacitidine treatment confirmed the functional relevance of hyper-methylation of enhancer. Furthermore, rescue of expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing as a novel candidate tumor suppressor gene.
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http://dx.doi.org/10.3324/haematol.2018.204917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669145PMC
August 2019

Molecular characterization of autophagic and apoptotic signaling induced by sorafenib in liver cancer cells.

J Cell Physiol 2018 01 21;234(1):692-708. Epub 2018 Aug 21.

Institute of Biomedicine of Seville (IBiS), Hospital University "Virgen del Rocío"/CSIC/University of Seville, Seville, Spain.

Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of endoplasmic reticulum (ER) stress, c-Jun-N-terminal kinase (JNK), Akt, and 5'AMP-activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3-12 hr) ER stress characterized by an increase of P-eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of P-JNK1/2/JNK1/2, P-AMPKα, P-Foxo3a, P-AKt/AKt and P-Foxo3a/Foxo3a ratios, and reduction of P-mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim , Beclin-1, Bcl-xL, Bcl-2, autophagy markers, and reduction of myeloid cell leukemia-1 (Mcl-1) expression. The progressive increase of CHOP expression, and reduction of P-AKt/AKt and P-AKt/AKt ratios were associated with the reduction of autophagic flux and an additional upregulation of Bim expression and caspase-3 activity (24 hr). Small interfering-RNA (si-RNA) assays showed that Bim, but not Bak and Bax, was involved in the induction of caspase-3 in sorafenib-treated HepG2 cells. Sorafenib increased autophagic and apoptotic markers in tumor-derived xenograft model. In conclusion, the early sorafenib-induced ER stress and regulation of JNK and AMPK-dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK-CHOP-dependent rise of Bim , which was associated with the shift from autophagy to apoptosis. The kinetic of Bim expression profile might also be related to the tight balance between AKt- and AMPK-related signaling leading to Foxo3a-dependent BIM upregulation.
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http://dx.doi.org/10.1002/jcp.26855DOI Listing
January 2018

Discovery of Reversible DNA Methyltransferase and Lysine Methyltransferase G9a Inhibitors with Antitumoral in Vivo Efficacy.

J Med Chem 2018 Aug 19;61(15):6518-6545. Epub 2018 Jul 19.

Departmento de Hematología, Clinica Universidad de Navarra , University of Navarra , Avenida Pio XII 36 , E-31008 Pamplona , Spain.

Using knowledge- and structure-based approaches, we designed and synthesized reversible chemical probes that simultaneously inhibit the activity of two epigenetic targets, histone 3 lysine 9 methyltransferase (G9a) and DNA methyltransferases (DNMT), at nanomolar ranges. Enzymatic competition assays confirmed our design strategy: substrate competitive inhibitors. Next, an initial exploration around our hit 11 was pursued to identify an adequate tool compound for in vivo testing. In vitro treatment of different hematological neoplasia cell lines led to the identification of molecules with clear antiproliferative efficacies (GI values in the nanomolar range). On the basis of epigenetic functional cellular responses (levels of lysine 9 methylation and 5-methylcytosine), an acceptable therapeutic window (around 1 log unit) and a suitable pharmacokinetic profile, 12 was selected for in vivo proof-of-concept ( Nat. Commun. 2017 , 8 , 15424 ). Herein, 12 achieved a significant in vivo efficacy: 70% overall tumor growth inhibition of a human acute myeloid leukemia (AML) xenograft in a mouse model.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01926DOI Listing
August 2018

A new background subtraction method for Western blot densitometry band quantification through image analysis software.

J Immunol Methods 2018 06 6;457:1-5. Epub 2018 Mar 6.

Independent Researcher, Madrid, Spain.

Since its first description, Western blot has been widely used in molecular labs. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability.
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http://dx.doi.org/10.1016/j.jim.2018.03.004DOI Listing
June 2018

Melatonin-induced increase in sensitivity of human hepatocellular carcinoma cells to sorafenib is associated with reactive oxygen species production and mitophagy.

J Pineal Res 2016 Oct 19;61(3):396-407. Epub 2016 Aug 19.

Institute of Biomedicine (IBIOMED), University of León, León, Spain.

Effects of sorafenib in hepatocellular carcinoma (HCC) are frequently transient due to tumor-acquired resistance, a phenotype that could be targeted by other molecules to reduce this adaptive response. Because melatonin is known to exert antitumor effects in HCC cells, this study investigated whether and how melatonin reduces resistance to sorafenib. Susceptibility to sorafenib (10 nmol/L to 50 μmol/L) in the presence of melatonin (1 and 2 mmol/L) was assessed in HCC cell lines HepG2, HuH7, and Hep3B. Cell viability was reduced by sorafenib from 1 μmol/L in HepG2 or HuH7 cells, and 2.5 μmol/L in Hep3B cells. Co-administration of melatonin and sorafenib exhibited a synergistic cytotoxic effect on HepG2 and HuH7 cells, while Hep3B cells displayed susceptibility to doses of sorafenib that had no effect when administrated alone. Co-administration of 2.5 μmol/L sorafenib and 1 mmol/L melatonin induced apoptosis in Hep3B cells, increasing PARP hydrolysis and BAX expression. We also observed an early colocalization of mitochondria with lysosomes, correlating with the expression of mitophagy markers PINK1 and Parkin and a reduction of mitofusin-2 and mtDNA compared with sorafenib administration alone. Moreover, increased reactive oxygen species production and mitochondrial membrane depolarization were elicited by drug combination, suggesting their contribution to mitophagy induction. Interestingly, Parkin silencing by siRNA to impair mitophagy significantly reduced cell killing, PARP cleavage, and BAX expression. These results demonstrate that the pro-oxidant capacity of melatonin and its impact on mitochondria stability and turnover via mitophagy increase sensitivity to the cytotoxic effect of sorafenib.
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http://dx.doi.org/10.1111/jpi.12358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018464PMC
October 2016

Modulation of Autophagy by Sorafenib: Effects on Treatment Response.

Front Pharmacol 2016 8;7:151. Epub 2016 Jun 8.

Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd)León, Spain; Institute of Biomedicine (IBIOMED), University of LeónLeón, Spain.

The multikinase inhibitor sorafenib is, at present, the only drug approved for the treatment of hepatocellular carcinoma (HCC), one of the most lethal types of cancer worldwide. However, the increase in the number of sorafenib tumor resistant cells reduces efficiency. A better knowledge of the intracellular mechanism of the drug leading to reduced cell survival could help to improve the benefits of sorafenib therapy. Autophagy is a bulk cellular degradation process activated in a broad range of stress situations, which allows cells to degrade misfolded proteins or dysfunctional organelles. This cellular route can induce survival or death, depending on cell status and media signals. Sorafenib, alone or in combination with other drugs is able to induce autophagy, but cell response to the drug depends on the complex integrative crosstalk of different intracellular signals. In cancerous cells, autophagy can be regulated by different cellular pathways (Akt-related mammalian target of rapamycin (mTOR) inhibition, 5' AMP-activated protein kinase (AMPK) induction, dissociation of B-cell lymphoma 2 (Bcl-2) family proteins from Beclin-1), or effects of some miRNAs. Inhibition of mTOR signaling by sorafenib and diminished interaction between Beclin-1 and myeloid cell leukemia 1 (Mcl-1) have been related to induction of autophagy in HCC. Furthermore, changes in some miRNAs, such as miR-30α, are able to modulate autophagy and modify sensitivity in sorafenib-resistant cells. However, although AMPK phosphorylation by sorafenib seems to play a role in the antiproliferative action of the drug, it does not relate with modulation of autophagy. In this review, we present an updated overview of the effects of sorafenib on autophagy and its related activation pathways, analyzing in detail the involvement of autophagy on sorafenib sensitivity and resistance.
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http://dx.doi.org/10.3389/fphar.2016.00151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896953PMC
July 2016

Melatonin Activates Endoplasmic Reticulum Stress and Apoptosis in Rats with Diethylnitrosamine-Induced Hepatocarcinogenesis.

PLoS One 2015 11;10(12):e0144517. Epub 2015 Dec 11.

Institute of Biomedicine (IBIOMED), University of León, and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), León, Spain.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence, its metastatic potential and the low efficacy of conventional treatment. Inactivation of apoptosis is implicated in tumour progression and chemotherapy resistance, and has been linked to the presence of endoplasmic reticulum stress. Melatonin, the main product of the pineal gland, exerts anti-proliferative, pro-apoptotic and anti-angiogenic effects in HCC cells, but these effects still need to be confirmed in animal models. Male Wistar rats in treatment groups received diethylnitrosamine (DEN) 50 mg/kg intraperitoneally twice/once a week for 18 weeks. Melatonin was given in drinking water at 1 mg/kg/d, beginning 5 or 12 weeks after the start of DEN administration. Melatonin improved survival rates and successfully attenuated liver injury, as shown by histopathology, decreased levels of serum transaminases and reduced expression of placental glutathione S-transferase. Furthermore, melatonin treatment resulted in a significant increase of caspase 3, 8 and 9 activities, polyadenosine diphosphate (ADP) ribose polymerase (PARP) cleavage, and Bcl-associated X protein (Bax)/Bcl-2 ratio. Cytochrome c, p53 and Fas-L protein concentration were also significantly enhanced by melatonin. Melatonin induced an increased expression of activating transcription factor 6 (ATF6), C/EBP-homologous protein (CHOP) and immunoglobulin heavy chain-binding protein (BiP), while cyclooxygenase (COX)-2 expression decreased. Data obtained suggest that induction of apoptosis and ER stress contribute to the beneficial effects of melatonin in rats with DEN-induced HCC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144517PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684373PMC
June 2016

Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis.

J Pineal Res 2015 Oct 9;59(3):292-307. Epub 2015 Aug 9.

Institute of Biomedicine (IBIOMED), University of León, León, Spain.

Endoplasmic reticulum (ER) is a dynamic organelle that participates in a number of cellular functions by controlling lipid metabolism, calcium stores, and proteostasis. Under stressful situations, the ER environment is compromised, and protein maturation is impaired; this causes misfolded proteins to accumulate and a characteristic stress response named unfolded protein response (UPR). UPR protects cells from stress and contributes to cellular homeostasis re-establishment; however, during prolonged ER stress, UPR activation promotes cell death. ER stressors can modulate autophagy which in turn, depending of the situation, induces cell survival or death. Interactions of different autophagy- and apoptosis-related proteins and also common signaling pathways have been found, suggesting an interplay between these cellular processes, although their dynamic features are still unknown. A number of pathologies including metabolic, neurodegenerative and cardiovascular diseases, cancer, inflammation, and viral infections are associated with ER stress, leading to a growing interest in targeting components of the UPR as a therapeutic strategy. Melatonin has a variety of antioxidant, anti-inflammatory, and antitumor effects. As such, it modulates apoptosis and autophagy in cancer cells, neurodegeneration and the development of liver diseases as well as other pathologies. Here, we review the effects of melatonin on the main ER stress mechanisms, focusing on its ability to regulate the autophagic and apoptotic processes. As the number of studies that have analyzed ER stress modulation by this indole remains limited, further research is necessary for a better understanding of the crosstalk between ER stress, autophagy, and apoptosis and to clearly delineate the mechanisms by which melatonin modulates these responses.
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http://dx.doi.org/10.1111/jpi.12264DOI Listing
October 2015

Ceramide metabolism regulates autophagy and apoptotic cell death induced by melatonin in liver cancer cells.

J Pineal Res 2015 Sep 8;59(2):178-89. Epub 2015 Jun 8.

Institute of Biomedicine (IBIOMED), University of León, León, Spain.

Autophagy is a process that maintains homeostasis during stress, although it also contributes to cell death under specific contexts. Ceramides have emerged as important effectors in the regulation of autophagy, mediating the crosstalk with apoptosis. Melatonin induces apoptosis of cancer cells; however, its role in autophagy and ceramide metabolism has yet to be clearly elucidated. This study was aimed to evaluate the effect of melatonin administration on autophagy and ceramide metabolism and its possible link with melatonin-induced apoptotic cell death in hepatocarcinoma (HCC) cells. Melatonin (2 mm) transiently induced autophagy in HepG2 cells through JNK phosphorylation, characterized by increased Beclin-1 expression, p62 degradation, and LC3II and LAMP-2 colocalization, which translated in decreased cell viability. Moreover, ATG5 silencing sensitized HepG2 cells to melatonin-induced apoptosis, suggesting a dual role of autophagy in cell death. Melatonin enhanced ceramide levels through both de novo synthesis and acid sphingomyelinase (ASMase) stimulation. Serine palmitoyltransferase (SPT) inhibition with myriocin prevented melatonin-induced autophagy and ASMase inhibition with imipramine-impaired autophagy flux. However, ASMase inhibition partially protected HepG2 cells against melatonin, while SPT inhibition significantly enhanced cell death. Findings suggest a crosstalk between SPT-mediated ceramide generation and autophagy in protecting against melatonin, while specific ASMase-induced ceramide production participates in melatonin-mediated cell death. Thus, dual blocking of SPT and autophagy emerges as a potential strategy to potentiate the apoptotic effects of melatonin in liver cancer cells.
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http://dx.doi.org/10.1111/jpi.12249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523438PMC
September 2015

Genome-wide microarray expression and genomic alterations by array-CGH analysis in neuroblastoma stem-like cells.

PLoS One 2014 13;9(11):e113105. Epub 2014 Nov 13.

Department of Biochemistry and Genetics, University of Navarra School of Sciences, Pamplona, Spain.

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113105PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231109PMC
December 2015

Inhibition of matrix metalloproteinase-9 and nuclear factor kappa B contribute to melatonin prevention of motility and invasiveness in HepG2 liver cancer cells.

J Pineal Res 2014 Jan 30;56(1):20-30. Epub 2013 Sep 30.

Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), León, Spain; Institute of Biomedicine (IBIOMED), University of León, León, Spain.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence and its metastatic potential. Extracellular matrix degradation by matrix metalloproteinases (MMPs) has been connected with cancer cell invasion, and it has been suggested that inhibition of MMPs by synthetic and natural inhibitors may be of great importance in the HCC therapies. Melatonin, the main product of the pineal gland, exerts antiproliferative, proapoptotic, and antiangiogenic properties in HepG2 human hepatocellular cells, and exhibits anti-invasive and antimetastatic activities by suppressing the enzymatic activity of MMP-9 in different tumor types. However, the underlying mechanism of anti-invasive activity in HCC models has not been fully elucidated. Here, we demonstrate that 1 mm melatonin dosage reduced in IL-1β-induced HepG2 cells MMP-9 gelatinase activity and inhibited cell invasion and motility through downregulation of MMP-9 gene expression and upregulation of the MMP-9-specific inhibitor tissue inhibitor of metalloproteinases (TIMP)-1. No significant changes were observed in the expression and activity of MMP-2, the other proteinase implicated in matrix collagen degradation, and its tissue inhibitor, TIMP-2. Also, melatonin significantly suppressed IL-1β-induced nuclear factor-kappaB (NF-κB) translocation and transcriptional activity. In summary, we demonstrate that melatonin modulates motility and invasiveness of HepG2 cell in vitro through a molecular mechanism that involves TIMP-1 upregulation and attenuation of MMP-9 expression and activity via NF-κB signal pathway inhibition.
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http://dx.doi.org/10.1111/jpi.12092DOI Listing
January 2014

[Considerations on health in the rural Mexican environment].

Gac Med Mex 1981 Nov;117(11):439-41

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November 1981

[Research in public health].

Gac Med Mex 1978 Jun;114(6):292-4

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June 1978

[Environmental pollution and lactation].

Gac Med Mex 1978 Feb;114(2):78-80

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February 1978

[Lead air pollution in industrial areas. II. Epidemiology].

Gac Med Mex 1977 May;113(5):215-21

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May 1977

[Congenital abnormalities and the biological and physical environment].

Gac Med Mex 1975 Jun;109(6):359-68

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June 1975

[Characteristics of life in the country and in the city].

Gac Med Mex 1974 Apr;107(4):288-95

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April 1974

[Urbanization and health].

Gac Med Mex 1972 Mar;103(3):202-12

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March 1972

[The effects of air pollution on the health of man].

Salud Publica Mex 1972 Mar-Apr;14(2):209-16

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July 1974