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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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
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
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
Ex vivo engineering of phagocytic signals in breast cancer cells for a whole tumor cell-based vaccine
(2025-07-01) Martí Díaz, Román; Cabezas Herrera, Juan; Montenegro Arce, María Fernanda; Hernández Caselles, Trinidad; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Piñero Madrona, A.; 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
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; Cabezas Herrera, Juan; Goding, Colin R.; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Piñero Madrona, A.; 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
Apuntes Metodología Bioquímica
(2009-10-28T11:53:53Z) Rodríguez López, José Neptuno; Facultad de Biología
En este tema se introduce a los alumnos en los antecedentes sobre la alcachofa, su valor nutritivo y los residuos generados en su transformación. Así mismo, se plantea el uso de los mismos como alternativa a los ya establecidos.
Open Access
Effects of folate cycle disruption by the green tea polyphenol epigallocatechin-3-gallate
(PERGAMON-ELSEVIER SCIENCE LTD, 2007) Navarro Perán, Encarnación María; Cabezas Herrera, Juan; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
We demonstrate that the tea polyphenol, epigallocatechin-3-gallate, is an efficient inhibitor of human dihydrofolate reductase. Like other antifolate compounds, epigallocatechin-3-gallate acts by disturbing folic acid metabolism in cells, causing the inhibition of DNA and RNA synthesis and altering DNA methylation. Epigallocatechin-3-gallate was seen to inhibit the growth of a human colon carcinoma cell line in a concentration and time dependent manner. Rescue experiments using leucovorin and hypoxanthine-thymine medium were the first indication that epigallocatechin-3-gallate could disturb the folate metabolism within cells. Epigallocatechin-3-gallate increased the uptake of [H-3]-thymidine and showed synergy with 5-fluorouracil, while its inhibitory action was strengthened after treatment with hypoxanthine, which indicates that epigallocatechin-3-gallate decreases the cellular production of nucleotides, thus, disturbing DNA and RNA synthesis. In, addition to its effects on nucleotide biosynthesis, antifolate treatment has been linked to a decrease in cellular methylation. Here, we observed that epigallocatechin-3-gallate altered the p16 methylation pattern from methylated to unmethylated as a result of folic acid deprivation. Finally, we demonstrate that epigallocatechin-3-gallate causes adenosine to be released from the cells because it disrupts the purine metabolism. By binding to its specific receptors, adenosine can modulate different signalling pathways. This proposed mechanism should help us to understand most of the molecular and cellular effects described for this tea polyphenol
Open Access
Targeting the methionine cycle for melanoma therapy with 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin
(WILEY, 2008-11-15) Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
The higher expression of methionine cycle genes in melanoma cells than in normal melanocytes may be related with increased protein synthesis and transmethylation reactions and the subsequent need for high levels of methionine. 3-O-(3,4,5-trimethoxybenzoyl)-(-)epicatechin (TMECG), a trimethoxy derivative of epicatechin-3-gallate (ECG), effectively suppressed proliferation of melanoma cells in cultures by inducing apoptosis. TMECG modulates the expression of genes involved in methionine metabolism, cellular methylation and glutathione synthesis in.,melanoma cells. TMECG treatment of melanoma cells resulted in the downregulation of antiapoptotic Bcl-2, the upregulation of proapoptotic Bax and the activation of caspase-3; however, it did not induce the expression of the apoptosis protease-activating factor-1 (Apaf-1). Having elucidated the effects of TMECG on the melanoma methionine cycle, we designed therapeuthical strategies to increase its effectiveness. Combinations of TMECG with S-adenosylmethionine or compounds that modulate the intracellular concentration of adenosine strongly increase the anti proliferative effects of TMECG. The ability of TMECG to target multiple aspects related with melanoma survival, with a high degree of potency, points to its clinical value in melanoma therapy.
Open Access
Binding of Natural and Synthetic Polyphenols to Human Dihydrofolate Reductase
(MDPI, 2009-12-18) Sáez Ayala, Magalí; Chazarra Parres, Soledad; Cabezas Herrera, Juan; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for antifolate drugs. Fluorescence quenching experiments show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent inhibitors of DHFR with dissociation constants (K-D) of 0.9 and 1.8 mu M, respectively, while polyphenols lacking the ester bound gallate moiety [e.g., (-)-epigallocatechin (EGC) and (-)-epicatechin (EC)] did not bind to this enzyme. To avoid stability and bioavailability problems associated with tea catechins we synthesized a methylated derivative of ECG (3-O-(3,4,5-trimethoxybenzoyl)(-)-epicatechin; TMECG), which effectively binds to DHFR (K-D = 2.1 mu M). In alkaline solution, TMECG generates a stable quinone methide product that strongly binds to the enzyme with a K-D of 8.2 nM. Quercetin glucuronides also bind to DHFR but its effective binding was highly dependent of the sugar residue, with quercetin-3-xyloside being the stronger inhibitor of the enzyme with a K-D of 0.6 mu M. The finding that natural polyphenols are good inhibitors of human DHFR could explain the epidemiological data on their prophylactic effects for certain forms of cancer and open a possibility for the use of natural and synthetic polyphenols in cancer chemotherapy.