Montenegro Arce, María Fernanda

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Montenegro Arce, María Fernanda

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Montenegro Arce, María Fernanda

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Bioquímica y Biología Molecular "B" e Inmunología

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Open Access
Targeting protein methylation in pancreatic cancer cells results in KRAS signaling imbalance and inhibition of autophagy
(Springer Nature, 2023-11-23) Martí Díaz, Román; Navarro, Ana; Tolivia, Jorge; Cabezas Herrera, Juan; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
Pancreatic cancer cells with mutant KRAS require strong basal autophagy for viability and growth. Here, we observed that some processes that allow the maintenance of basal autophagy in pancreatic cancer cells are controlled by protein methylation. Thus, by maintaining the methylation status of proteins such as PP2A and MRAS, these cells can sustain their autophagic activity. Protein methylation disruption by a hypomethylating treatment (HMT), which depletes cellular S-adenosylmethionine levels while inducing S-adenosylhomocysteine accumulation, resulted in autophagy inhibition and endoplasmic reticulum stress-induced apoptosis in pancreatic cancer cells. We observed that by reducing the membrane localization of MRAS, hypomethylation conditions produced an imbalance in KRAS signaling, resulting in the partial inactivation of ERK and hyperactivation of the PI3K/AKT–mTORC1 pathway. Interestingly, HMT impeded CRAF activation by disrupting the ternary SHOC2 complex (SHOC2/MRAS/PP1), which functions as a CRAF-S259 holophosphatase. The demethylation events that resulted in PP2A inactivation also favored autophagy inhibition by preventing ULK1 activation while restoring the cytoplasmic retention of the MiT/TFE transcription factors. Since autophagy provides pancreatic cancer cells with metabolic plasticity to cope with various metabolic stress conditions, while at the same time promoting their pathogenesis and resistance to KRAS pathway inhibitors, this hypomethylating treatment could represent a therapeutic opportunity for pancreatic adenocarcinomas.
Open Access
Acetyl-and butyrylcholinesterase activities decrease in human colon adenocarcinoma
(Springer, 2006-02) Montenegro Arce, María Fernanda; Ruiz Espejo, Francisco; Campoy Menéndez, Francisco Javier; Muñoz Delgado, Encarnación; Páez de la Cadena, María; Cabezas Herrera, Juan; Vidal, Cecilio J.; Bioquímica y Biología Molecular A
Apart from the hydrolysis of acetylcholine (ACh), acetyl- (AChE) and butyrylcholinesterase (BChE), through noncatalytic mechanisms, intervene in hematopoiesis, morphogenesis, and neurogenesis (Layer and Willbold, 1995; Soreq and Seidman, 2001). Cholinesterase (ChE) molecules occur as globular (G1, G2, and G4) and asymmetric (A4, A8, and A12) forms (Legay, 2000; Massoulié, 2002). The G species might display amphiphilic (GA) or hydrophilic (GH) properties (Perrier et al., 2002). The involvement of ChEs in tumorigenesis is supported by the measurement of ChE activity in tumors (García-Ayllón et al., 2001; Ruiz-Espejo et al., 2003), the amplification of ChE genes in leukemias and ovarian tumors, and the relationship between the expression of AChE and the aggressiveness of astrocytomas(Perry et al., 2002). This research was undertaken to determine whether ChE activity is altered in gut carcinomas.
Open Access
Human butyrylcholinesterase components differ in aryl acylamidase activity
(De Gruyter Brill, 2008-03-27) Montenegro Arce, María Fernanda; Moral Naranjo, María T.; Páez de la Cadena, María; Campoy Menéndez, Francisco Javier; Muñoz Delgado, Encarnación; Vidal, Cecilio J.; Bioquímica y Biología Molecular A
Apart from its esterase activity, butyrylcholinesterase (BuChE) displays aryl acylamidase (AAA) activity able to hydrolyze o-nitroacetanilide (ONA) and its trifluoro-derivative (F-ONA). We report here that, despite amidase and esterase sites residing in the same protein, in human samples depleted of acetylcholinesterase the ratio of amidase to esterase activity varied depending on the source of BuChE. The much faster degradation of ONA and F-ONA by BuChE monomers (G1) of colon and kidney than by the tetramers (G4) suggests aggregation-driven differences in the AAA site between single and polymerized subunits. The similar ratio of F-ONAto butyrylthiocholine hydrolysis by serum G1 and G4 forms support structural differences in the amidase site according to the source of BuChE. The changing ratios of amidase to esterase activities in the human sources probably arise from post-translational modifications in BuChE subunits, the specific proportion of monomers and oligomers and the variable capacity of the tetramers for degrading ONA and F-ONA. The elevated amidase activity of BuChE monomers and the scant activity of the tetramers justify the occurrence of single BuChE subunits in cells as a means to sustain the AAA activity of BuChE which otherwise could be lost by tetramerization.
Open Access
Melanoma Activation of 3-O-(3,4,5-Trimethoxybenzoyl)-(-)-Epicatechin to a Potent Irreversible Inhibitor of Dihydrofolate Reductase
(2009-04-09) Tárraga Tomás, Alberto; Cabezas Herrera, Juan; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
Human melanoma is a significant clinical problem because it is resistant to treatment by most chemotherapeutic agents, including antifolates. It is therefore a desirable goal to develop a second generation of low-toxicity antifolate drugs to overcome acquired resistance to the prevention and treatment of this skin pathology. In our efforts to improve the stability and bioavailability of green tea polyphenols for cancer therapy, we synthesized a trimethoxy derivative of epicatechin-3-gallate, which showed high anti proliferative and proapoptotic activity against melanoma. This derivative, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), is a prodrug that is selectively activated by the specific melanocyte enzyme tyrosinase. Upon activation, TMECG generates a stable quinone methide product that strongly inhibits dihydrofolate reductase in an irreversible manner. The treatment of melanoma cells with TMECG also affected cellular folate transport and the gene expression of DHFR, which supported the antifolate nature of this compound. In addition, its pharmacological efficacy has been confirmed in a mouse melanoma model, in which tumor growth and metastasis were inhibited, significantly enhancing the mean survival of the treated groups. TMECG, therefore, shows a potential for clinical use in melanoma therapy.
Open Access
Molecular docking studies of ortho-substituted phenols to tyrosinase helps discern if a molecule can be an enzyme substrate
(MDPI, 2024-06-23) Garcia-Molina, Pablo; Garcia-Canovas, Francisco; Garcia-Molina, Francisco; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Tudela Serrano, José; Bioquímica y Biología Molecuar A
Phenolic compounds with a position ortho to the free phenolic hydroxyl group occupied can be tyrosinase substrates. However, ortho-substituted compounds are usually described as inhibitors. The mechanism of action of tyrosinase on monophenols is complex, and if they are ortho-substituted, it is more complicated. It can be shown that many of these molecules can become substrates of the enzyme in the presence of catalytic o-diphenol, MBTH, or in the presence of hydrogen peroxide. Docking studies can help discern whether a molecule can behave as a substrate or inhibitor of the enzyme. Specifically, phenols such as thymol, carvacrol, guaiacol, eugenol, isoeugenol, and ferulic acid are substrates of tyrosinase, and docking simulations to the active center of the enzyme predict this since the distance of the peroxide oxygen from the oxy-tyrosinase form to the ortho position of the phenolic hydroxyl is adequate for the electrophilic attack reaction that gives rise to hydroxylation occurring.
Open Access
The critical role of alpha-folate receptor in the resistance of melanoma to methotrexate
(WILEY, 2009-01-10) Cabezas Herrera, Juan; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
Although methotrexate (MTX) is an effective drug for several types of cancer, it is not active against melanoma. Experiments following methotrexate treatment indicated a reduced accumulation of the drug in the cytosolic compartment in melanoma cells, suggesting that the mechanisms that control the transport and retention of this drug could be altered in melanoma. For this reason, we analyzed the presence and function of folate receptor-alpha (FR alpha) in melanoma cells. In this study, we have identified the presence of FR alpha in normal and pathological melanocytes and demonstrated that MTX is preferentially transported through this receptor in melanoma cells. FR alpha-induced endocytic transport of MTX, together with drug melanosomal sequestration and cellular exportation, ensures reduced accumulation of this cytotoxic compound in intracellular compartments. The critical role of FR alpha in this mechanism of resistance and the therapeutic consequences of these findings are also discussed.
Open Access
Directed Phenotype Switching as an Effective Antimelanoma Strategy
(Cell Press, 2013-06-20) Sáez Ayala, Magalí; Fernández Pérez, María Piedad; Chazarra Parres, Soledad; Freter, Rasmus; Middleton, Mark; Piñero Madrona, Antonio; Cabezas Herrera, Juan; Goding, Colin R.; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A; Cirugía, Pediatría y Obstetricia y Ginecología
Therapeutic resistance in melanoma and other cancers arises via irreversible genetic, and dynamic phenotypic, heterogeneity. Here, we use directed phenotype switching in melanoma to sensitize melanoma cells to lineage-specific therapy. We show that methotrexate (MTX) induces microphthalmia-associated transcription factor (MITF) expression to inhibit invasiveness and promote differentiation-associated expression of the melanocyte-specific Tyrosinase gene. Consequently, MTX sensitizes melanomas to a tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(−)-epicatechin (TMECG), that inhibits the essential enzyme DHFR with high affinity. The combination of MTX and TMECG leads to depletion of thymidine pools, double-strand DNA breaks, and highly efficient E2F1-mediated apoptosis in culture and in vivo. Importantly, this drug combination delivers an effective and tissue-restricted antimelanoma therapy in vitro and in vivo irrespective of BRAF, MEK, or p53 status.
Open Access
Acriflavine, a Potent Inhibitor of HIF-1α, Disturbs Glucose Metabolism and Suppresses ATF4-Protective Pathways in Melanoma under Non-Hypoxic Conditions
(MDPI, 2020-12-31) Martí Díaz, Román; Cabezas Herrera, Juan; Goding, Colin; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
Hypoxia-inducible factor (HIF)-1α is constitutively expressed in melanoma cells under normoxic conditions and its elevated expression correlates with the aggressiveness of melanoma tumors. Here, we used acriflavine, a potent inhibitor of HIF-1α dimerization, as a tool to investigate whether HIF-1α-regulated pathways contribute to the growth of melanoma cells under normoxia. We observed that acriflavine differentially modulated HIF-1α-regulated targets in melanoma under normoxic conditions, although acriflavine treatment resulted in over-expression of vascular endothelial growth factor (VEGF), its action clearly downregulated the expression of pyruvate dehydrogenase kinase 1 (PDK1), a well-known target of HIF-1α. Consequently, downregulation of PDK1 by acrifavine resulted in reduced glucose availability and suppression of the Warburg effect in melanoma cells. In addition, by inhibiting the AKT and RSK2 phosphorylation, acriflavine also avoided protective pathways necessary for survival under conditions of oxidative stress. Interestingly, we show that acriflavine targets activating transcription factor 4 (ATF4) for proteasomal degradation while suppressing the expression of microphthalmia-associated transcription factor (MITF), a master regulator of melanocyte development and a melanoma oncogene. Since acriflavine treatment results in the consistent death of melanoma cells, our results suggest that inhibition of HIF-1α function in melanoma could open new avenues for the treatment of this deadly disease regardless of the hypoxic condition of the tumor.
Open Access
Ex vivo engineering of phagocytic signals in breast cancer cells for a whole tumor cell-based vaccine
(2025-07-01) Martí Díaz, Román; Piñero Madrona, Antonio; Cabezas Herrera, Juan; Montenegro Arce, María Fernanda; Hernández Caselles, Trinidad; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
Background Today, cell therapies are constantly evolving and providing new options for cancer patients. These therapies are mostly based on the inoculation of immune cells extracted from a person’s own tumor; however, some studies using whole tumor cell-based vaccines are approaching the level of maturity required for clinical use. Although these latest therapies will have to be developed further and adapted to overcome many ethical barriers, there is no doubt that therapeutic cancer vaccines are the next frontier of immunotherapy. Methods Ionizing radiation and CD47 knockout via CRISPR-Cas9 genome editing were used to optimize the macrophage-mediated phagocytosis of breast cancer cells. These cells were subsequently used in several mouse models to determine their potential as novel whole-cell-based vaccines to drive antitumor immunity. To improve the recognition of tumor cells by activated immune cells, this cellular therapy was combined with anti-PD-1 antibody treatments. Results Here, we showed that irradiation of 4T1 breast cancer cells increases their immunogenicity and, when injected into the blood of immunocompetent mice, elicits a complete antitumor immune response mediated, in part, by the adaptive immune system. Next, to improve the macrophage-mediated phagocytosis of breast cancer cells, we knocked out CD47 in 4T1 cells. When injected in the bloodstream, irradiated CD47 knockout cells activated both the adaptive and the innate immune systems. Therefore, we used these ex vivo engineered cells as a whole tumor cellbased vaccine to treat breast tumors in immunocompetent mice. A better response was obtained when these cells were combined with an anti-PD-1 antibody. Conclusion These results suggest that tumor cells obtained from surgical samples of a breast cancer patient could be engineered ex vivo and used as a novel cell therapy to drive antitumor immunity.
Metadata only
Acetilcolinesterasa y butirilcolinesterasa en el intestino grueso humano: cambios en sus actividades, propiedades moleculares y cantidad de ARN mensajeros en carcinomas de colón / Mª Fernanda Montenegro Arce; directores Jesús Vidal Moreno y María Páez de la Cadena Tortosa
(Murcia : Universidad de Murcia, Departamento de Bioquímica y Biología Molecular-A,, 2006) Montenegro Arce, María Fernanda