Methotrexate resistance is directly associated with glutathione concentration in acute lymphoblastic leukemia cell lines

Author(s)

Rafael Renatino Canevarolo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2017-01-27

Examining board members

José Andrés Yunes; Carlos Alberto Scrideli; Rui Manuel Vieira Reis; Sara Teresinha Olalla Saad; Jörg Kobarg

• Advisor: José Andrés Yunes

Abstract

Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, accounting for 25% of all cancers in this age group. One of the chemotherapeutics used in the therapy of ALL (and autoimmune diseases such as rheumatoid arthritis) is methotrexate (MTX), a folic acid antagonist (antifolate). As a chemotherapeutic agent, MTX's mechanism of action is primarily attributed to the inhibition of the dihydrophate reductase enzyme, which synthesizes tetrahydrofolate from dihydrofolate ¿ a key step in the de novo synthesis of purine nucleotides used in cell division. In rheumatoid arthritis, lower doses of MTX inhibit the 5-aminoimidazole-4-ribonucleotide-carboxamide formyltransferase (ATIC) enzyme, which culminates in the production of high levels of adenosine, a potent anti-inflammatory. However, recent works continue to present previously unknown mechanisms and effects through which MTX acts within the cell, attesting that MTX's mechanisms of action appear to be as multiple as complex. Using several techniques of molecular biology, this work sought to expand the existing knowledge of the action of MTX in ALL. For this purpose, several biological parameters were measured under or without MTX treatment in a panel of 13 ALL cell lines. Proliferation tests, metabolic studies, drug synergism, quantification of cellular respiration and the production of reactive oxygen species (ROS) were performed, as well as the measurement of the activation of the NF-?B signaling pathway. Resistance of the MTX strains within 48 h of treatment (but not 96 h) was related to the proliferation rate of the cells. Treatment with MTX altered the concentration of 28 intracellular metabolites, highlights for a consistent increase in glycine concentration. Intracellular concentrations of asparagine, guanosine and glutathione ¿ including the expression of genes from glutathione pathway ¿ were associated with MTX resistance. Supplementation of the culture medium with N-acetylcysteine, a precursor metabolite of glutathione, promoted proliferation and resistance to MTX; however, cell treatment with piperlongumine or hydrogen peroxide, two glutathione scavengers and ROS promoters, did not potentiate the effect of MTX. MTX produced small amounts of ROS in ALL cell lines. In 1 h treatment, MTX most sensitive cells produced more ROS than the most resistant ones; after 3 h this dynamics inverted and thus remained until 6 h. The oxygen uptake of the cell lines was not associated with MTX resistance and a preliminary test showed that MTX did not alter cellular respiration. MTX activated the transcription factor NF-?B in some ALL cell lines and, interestingly, the activation of this transcription factor by tumor necrosis factor alpha (TNF-?) was positively correlated with the resistance of leukemic lines to MTX. A wide bibliographic review allowed both the integration of the obtained results to the most current knowledge on the subject, and the identification of new paths to be explored in future stages (AU)

• FAPESP's process: 12/11952-0 - Methotrexate resistance is directly associated with glutathione concentration in acute lymphoblastic leukemia cell lines
• Grantee: Rafael Renatino Canevarolo
• Support Opportunities: Scholarships in Brazil - Doctorate