Chapter Cellular and Molecular Mechanisms of Methotrexate Resistance in Melanoma

Endocrinolog

The Relationship between the IC50 Values and the Apparent Inhibition Constant in the Study of Inhibitors of Tyrosinase Diphenolase Activity Helps Confirm the Mechanism of Inhibition

Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fruits and vegetables. Control of its activity can be carried out using inhibitors, which is interesting in terms of quantitatively understanding the action of these regulators. In the study of the inhibition of the diphenolase activity of tyrosinase, it is intriguing to know the strength and type of inhibition. The strength is indicated by the value of the inhibition constant(s), and the type can be, in a first approximation: competitive, non-competitive, uncompetitive and mixed. In this work, it is proposed to calculate the degree of inhibition (iD), varying the concentration of inhibitor to a fixed concentration of substrate, L-dopa (D). The non-linear regression adjustment of iD with respect to the initial inhibitor concentration I0 allows for the calculation of the inhibitor concentration necessary to inhibit the activity by 50%, at a given substrate concentration (IC50), thus avoiding making interpolations between different values of iD. The analytical expression of the IC50, for the different types of inhibition, are related to the apparent inhibition constant (KIapp). Therefore, this parameter can be used: (a) To classify a series of inhibitors of an enzyme by their power. Determining these values at a fixed substrate concentration, the lower IC50, the more potent the inhibitor. (b) Checking an inhibitor for which the type and the inhibition constant have been determined (using the usual methods), must confirm the IC50 value according to the corresponding analytical expression. (c) The type and strength of an inhibitor can be analysed from the study of the variation in iD and IC50 with substrate concentration. The dependence of IC50 on the substrate concentration allows us to distinguish between non-competitive inhibition (iD does not depend on D0) and the rest. In the case of competitive inhibition, this dependence of iD on D0 leads to an ambiguity between competitive inhibition and type 1 mixed inhibition. This is solved by adjusting the data to the possible equations; in the case of a competitive inhibitor, the calculation of KI1app is carried out from the IC50 expression. The same occurs with uncompetitive inhibition and type 2 mixed inhibition. The representation of iD vs. n, with n=D0/KmD, allows us to distinguish between them. A hyperbolic iD vs. n representation that passes through the origin of coordinates is a characteristic of uncompetitive inhibition; the calculation of KI2app is immediate from the IC50 value. In the case of mixed inhibitors, the values of the apparent inhibition constant of meta-tyrosinase (Em) and oxy-tyrosinase (Eox), KI1app and the apparent inhibition constant of metatyrosinase/Dopa complexes (EmD) and oxytyrosinase/Dopa (EoxD), KI2app are obtained from the dependence of iD vs. n, and the results obtained must comply with the IC50 value

Considerations about the Continuous Assay Methods, Spectro-Photometric and Spectrofluorometric, of the Monophenolase Activity of Tyrosinase

With the purpose to obtain the more useful tyrosinase assay for the monophenolase activity of tyrosinase between the spectrofluorometric and spectrophotometric continuous assays, simulated assays were made by means of numerical integration of the equations that characterize the mechanism of monophenolase activity. These assays showed that the rate of disappearance of monophenol () is equal to the rate of accumulation of dopachrome () or to the rate of accumulation of its oxidized adduct, originated by the nucleophilic attack on o-quinone by a nucleophile such as 3-methyl-2-benzothiazolinone (MBTH), (), despite the existence of coupled reactions. It is shown that the spectrophotometric methods that use MBTH are more useful, as they do not have the restrictions of the L-tyrosine disappearance measurement method, of working at pH = 8 and not having a linear response from 100 μM of L-tyrosine. It is possible to obtain low LODM (limit of detection of the monophenolase activity) values with spectrophotometric methods. The spectrofluorimetric methods had a lower LODM than spectrophotometric methods. In the case of 4-hydroxyphenil-propionic acid, the LODM obtained by us was 0.25 U/mL. Considering the relative sensitivities of 4-hydroxyanisole, compared with 4-hydroxyphenil-propionic acid, LODM values like those obtained by fluorescent methods would be expected

Binding of Natural and Synthetic Polyphenols to Human Dihydrofolate Reductase

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 (KD)of 0.9 and 1.8 μM, 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 (KD = 2.1 μM). In alkaline solution, TMECG generates a stable quinone methide product that strongly binds to the enzyme with a KD 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 KD of 0.6 μM. 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

Apuntes Metodología Bioquímica

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

Proteólisis Intracelular: Recambio Proteico

La concentración celular de cada clase de proteína es consecuencia del equilibrio entre su síntesis y su degradación. Aunque parece derrochador, la degradación y síntesis continua de las proteínas, un proceso que recibe el nombre de recambio proteico, tiene varios fines. El primero de todos es la flexibilidad metabólica, que se consigue mediante cambios relativamente rápidos de la concentración de enzimas reguladoras, hormonas peptídicas y moléculas receptoras. El recambio proteico protege también a las células de la acumulación de proteínas anómalas. Finalmente, numerosos procesos fisiológicos dependen tanto de las reacciones de degradación oportunas como de las de síntesis. Las proteínas se diferencian de forma significativa en sus velocidades de recambio, que se miden como vida media. Las proteínas que desempeñan funciones estructurales suelen tener una vida media más larga. Por ejemplo, algunas proteínas del tejido conjuntivo, como los colágenos, suelen tener una vida media que se mide por años. Por el contrario, la vida media de las enzimas reguladoras suele medirse en minutos. En los últimos años se ha realizado un gran avance en la elucidación de los mecanismos que controlan el recambio proteico. Las proteínas se degradan mediante enzimas proteolíticas que se encuentran por toda la célula. Entre ellas, las calpaínas activadas por Ca2+ y las catepsinas lisosómicas. Además, la ubiquitinación se cree que tiene una función fundamental en el recambio proteico. En la ubiquitinación varias moléculas de una proteína eucariota pequeña de 76 residuos, que se denomina ubiquitina, se unen covaléntemente a algunas proteínas destinadas a la degradación. Una vez que la proteína está ubiquitinada, se degrada por un complejo proteolítico que se denomina proteosoma. A pesar de lo que se ha avanzado en los últimos años, no se conocen bien los mecanismos que dirigen a las proteínas a su destrucción por ubiquitinación o por otros procesos degradativos. Sin embargo, se piensa que la vida media de una proteína está parcialmente determinada por su resto N-terminal, por la existencia de secuencias determinadas o por la presencia de restos de aminoácidos oxidados. Además, el control de estos procesos degradativos es importante para la salud, ya que una disfunción de estos sistemas desembocaría en la aparición de graves enfermedades, entre ellas, el Alzheimer o el cáncer. Estos, y otros aspectos, que recogen los últimos descubrimientos, sobre la degradación intracelular de las proteínas, se discuten en esta revisión

Estudio cinético de la actuación de tirosinasa sobre sustratos mono-Di- y trihidroxilados / José Neptuno Rodríguez López ; directores Francisco García Cánovas, José Tudela Serrano.

Tesis-Universidad de Murcia.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. D 372.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. T.M.-945

Targeting the methionine cycle for melanoma therapy with 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin

This is an Accepted Manuscript version of the following article, accepted for publication in [International Journal of Cancer]. [Sánchez-del-Campo L, Rodríguez-López JN. Targeting the methionine cycle for melanoma therapy with 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin. Int J Cancer. 2008 Nov 15;123(10):2446-55. doi: 10.1002/ijc.23813.]. It is deposited under the terms of the Creative Commons Attribution-Non Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited.©2008. This manuscript version is made available under the CC-BY-NC 4.0 license http://creativecommons.org/licenses/by-nc/4.0/ This document is the Accepted version of a Published Work that appeared in final form in International Journal of Cancer (IJC). To access the final edited and published work see https://doi.org/ 10.1002/ijc.23813The 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