Rodríguez López, José Neptuno

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Rodríguez López, José Neptuno

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Rodríguez López, José Neptuno

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Universidad de Murcia. Departamento de Bioquímica y Biología MolecularA

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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.
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Estudio cinético de la actuación de tirosinasa sobre sustratos mono-Di- y trihidroxilados / José Neptuno Rodríguez López ; directores Francisco García Cánovas, José Tudela Serrano.
(Murcia : Universidad de Murcia, Departamento de Bioquímica,, 1993) Rodríguez López, José Neptuno
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.
Open Access
Lebein, a snake venom disintegrin, induces apoptosis in human melanoma cells
(MDPI, 2016-07-05) Hammouda, Manel B.; Zakraoui, Ons; Aloui, Zohra; Riahi-Chebbi, Ichrak; Karoui, Habib; Essafi Benkhadir, Khadija; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associated X protein (BAX) and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2). It generated a distinct response in reactive oxygen species (ROS) generation and p53 levels depending on the p53 cell line status (wild type or mutant). Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma.
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
Persister state-directed transitioning and vulnerability in melanoma
(Springer Nature, 2022-06-01) Chauvistré, Heike; Shannan, Batool; Daignault-Mill, Sheena M.; Ju, Robert J.; Picard, Daniel; Egetemaier, Stefanie; Váraljai, Renáta; Gibhardt, Christine; Sechi, Antonio; Kaschani, Farnusch; Keminer, Oliver; Stehbens, Samantha J.; Liu, Qin; Yin, Xiangfan; Jeyakumar, Kirujan; Vogel, Felix C.E.; Krepler, Clemens; Rebecca, Vito W.; Kubat, Linda; Lueong, Smiths S.; Forster, Jan; Horn, Susanne; Remke, Marc; Ehrmann, Michael; Paschen, Annette; Becker, Jürgen C.; Helfrich, Iris; Rauh, Daniel; Kaiser, Markus; Gul, Sheraz; Herlyn, Meenhard; Bogeski, Ivan; Haass, Nikolas; Schadendorf, Dirk; Roesch, Alexander; Rodríguez López, José Neptuno; Bioquímica y Biología Molecular A
Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination
Open Access
Tumor suppressor SET9 guides the epigenetic plasticity of breast cancer cells and serves as an early-stage biomarker for predicting metastasis
(Springer Nature, 2016-05-02) Gonzalez Guerrero, Rebeca; Martínez Barba, Enrique; Piñero Madrona, Antonio; Cabezas Herrera, Juan; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
During the course of cancer progression, neoplastic cells undergo dynamic and reversible transitions between multiple phenotypic states, and this plasticity is enabled by underlying shifts in epigenetic regulation. Our results identified a negative feedback loop in which SET9 controls DNA methyltransferase-1 protein stability, which represses the transcriptional activity of the SET9 promoter in coordination with Snail. The modulation of SET9 expression in breast cancer cells revealed a connection with E2F1 and the silencing of SET9 was sufficient to complete an epigenetic program that favored epithelial–mesenchymal transition and the generation of cancer stem cells, indicating that SET9 plays a role in modulating breast cancer metastasis. SET9 expression levels were significantly higher in samples from patients with pathological complete remission than in samples from patients with disease recurrence, which indicates that SET9 acts as a tumor suppressor in breast cancer and that its expression may serve as a prognostic marker for malignancy
Open Access
Suppression of antifolate resistance by targeting the myosin va trafficking pathway in melanoma
(Elsevier, 2014-02-28) Fernández Pérez, Maria Piedad; Sáez Ayala, Magalí; Piñero Madrona, Antonio; 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. As most melanoma patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is one of the highest priorities to improve melanoma therapy. Melanosomal sequestration and the cellular exportation of cytotoxic drugs have been proposed to be important melanoma-specific mechanisms that contribute to multidrug resistance in melanoma. Concretely, we found that treatment of melanoma with methotrexate (MTX) altered melanogenesis and accelerated the exportation of melanosomes; however, the cellular and molecular processes by which MTX is trapped into melanosomes and exported out of cells have not been elucidated. In this study, we identified myosin Va (MyoVa) as a possible mediator of these cellular processes. The results demonstrated that melanoma treatment with MTX leads to Akt2-dependent MyoVa phosphorylation, which enhances its ability to interact with melanosomes and accelerates their exportation. To understand the mechanism(s) by which MTX activates Akt2, we examined the effects of this drug on the activity of protein phosphatase 2A, an Akt inhibitor activated by the methylation of its catalytic subunit. Taken together, this study identified a novel trafficking pathway in melanoma that promotes tumor resistance through Akt2/MyoVa activation. Because of these findings, we explored several MTX combination therapies to increase the susceptibility of melanoma to this drug. By avoiding MTX exportation, we observed that the E2F1 apoptotic pathway is functional in melanoma, and its induction activates p73 and apoptosis protease-activating factor 1 following a p53-autonomous proapoptotic signaling event.
Open Access
MITF induces escape from innate immunity in melanoma
(BMC, 2021) Martí-Díaz, Román; González-Guerrero, Rebeca; Martínez-Barba, Enrique; Piñero-Madrona, Antonio; Cabezas-Herrera, Juan; Goding, Colin R.; Montenegro Arce, María Fernanda; Hernández Caselles, Trinidad; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular B e Inmunología
Background: The application of immune-based therapies has revolutionized cancer treatment. Yet how the immune system responds to phenotypically heterogeneous populations within tumors is poorly understood. In melanoma, one of the major determinants of phenotypic identity is the lineage survival oncogene MITF that integrates diverse microenvironmental cues to coordinate melanoma survival, senescence bypass, differentiation, proliferation, invasion, metabolism and DNA damage repair. Whether MITF also controls the immune response is unknown. Methods: By using several mouse melanoma models, we examine the potential role of MITF to modulate the anti-melanoma immune response. ChIP-seq data analysis, ChIP-qPCR, CRISPR-Cas9 genome editing, and luciferase reporter assays were utilized to identify ADAM10 as a direct MITF target gene. Western blotting, confocal microscopy, flow cytometry, and natural killer (NK) cytotoxicity assays were used to determine the underlying mechanisms by which MITF-driven phenotypic plasticity modulates melanoma NK cell-mediated killing. Results: Here we show that MITF regulates expression of ADAM10, a key sheddase that cleaves the MICA/B family of ligands for NK cells. By controlling melanoma recognition by NK-cells MITF thereby controls the melanoma response to the innate immune system. Consequently, while melanoma MITFLow cells can be effectively suppressed by NK-mediated killing, MITF-expressing cells escape NK cell surveillance. Conclusion: Our results reveal how modulation of MITF activity can impact the anti-melanoma immune response with implications for the application of anti-melanoma immunotherapies.