RESUMO
Glioblastoma patients have a poor prognosis mainly due to temozolomide (TMZ) resistance. NRF2 is an important transcript factor involved in chemotherapy resistance due to its protective role in the transcription of genes involved in cellular detoxification and prevention of cell death processes, such as ferroptosis. However, the relation between NRF2 and iron-dependent cell death in glioma is still poorly understood. Therefore, in this study, we analyzed the role of NRF2 in ferroptosis modulation in glioblastoma cells. Two human glioblastoma cell lines (U251MG and T98G) were examined after treatment with TMZ, ferroptosis inducers (Erastin, RSL3), and ferroptosis inhibitor (Ferrostatin-1). Our results demonstrated that T98G was more resistant to chemotherapy compared to U251MG and showed elevated levels of NRF2 expression. Interestingly, T98G revealed higher sensitivity to ferroptosis, and significant GSH depletion upon system xc- blockage. NRF2 silencing in T98G cells (T98G-shNRF2) significantly reduced the viability upon TMZ treatment. On the other hand, T98G-shNRF2 was resistant to ferroptosis and reverted intracellular GSH levels, indicating that NRF2 plays a key role in ferroptosis induction through GSH modulation. Moreover, silencing of ABCC1, a well-known NRF2 target that diminishes GSH levels, has demonstrated a similar collateral sensitivity. T98G-siABCC1 cells were more sensitive to TMZ and resistant to Erastin. Furthermore, we found that NRF2 positively correlates with ABCC1 expression in tumor tissues of glioma patients, which can be associated with tumor aggressiveness, drug resistance, and poor overall survival. Altogether, our data indicate that high levels of NRF2 result in collateral sensitivity on glioblastoma via the expression of its pro-ferroptotic target ABCC1, which contributes to GSH depletion when the system xc- is blocked by Erastin. Thus, ferroptosis induction could be an important therapeutic strategy to reverse drug resistance in gliomas with high NRF2 and ABCC1 expression.
Assuntos
Ferroptose , Glioblastoma , Glioma , Fator 2 Relacionado a NF-E2/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/metabolismo , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Fator 2 Relacionado a NF-E2/genética , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Regulação para CimaRESUMO
Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3-7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating tumors.
Assuntos
Neoplasias Encefálicas/patologia , Cisplatino/farmacologia , Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioma/patologia , Glutationa/deficiência , Animais , Apoptose/efeitos dos fármacos , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Dacarbazina/farmacologia , Progressão da Doença , Feminino , Humanos , Camundongos Nus , Temozolomida , Proteína Supressora de Tumor p53/metabolismoRESUMO
Primary familial brain calcification (PFBC) is identified by mineralization of the basal ganglia and other brain regions in the absence of known causes. The condition is often inherited in an autosomal dominant pattern and can manifest itself clinically with neuropsychiatric symptoms such as Parkinsonism, headaches, psychosis, and mood swings. Mutations in the SLC20A2 gene account for ~40% of inherited cases, and this gene encodes an inorganic phosphate transporter (PiT-2), a transmembrane protein associated with Pi homeostasis. The p.Y386X mutation in SLC20A2 was identified in a patient who presented migraines, brain calcification, and mild but chronic hypovitaminosis D. SLC20A2 c.1158C > G single-nucleotide heterozygous mutation results in a premature stop codon and a putative truncated protein of 385 amino acids. Proband parents do not present the mutation, which is also not present in major public SNP databases, suggesting a de novo sporadic trait. This study describes for the first time a de novo SLC20A2 mutation in a PFBC patient with migraine and mild hypovitaminosis D. This data further reinforces the pathogenic role of SLC20A2 mutations as causal factors in PFBC physiopathology.
Assuntos
Encéfalo/patologia , Calcinose/genética , Mutação , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Adulto , Calcinose/diagnóstico , Códon de Terminação , Feminino , Humanos , MasculinoRESUMO
This study investigated how 5'-UTR of DEFB1 gene (encoding for the human beta-defensin-1) affects mRNA secondary structure and its correlation with translation efficiency in the susceptibility of diseases. It was possible to determine DEFB1 mRNA folding under the influence of 5'-UTR SNPs haplotypes and putative alternative transcript lengths. Different DEFB1 mRNAs that fold in a pattern that is haplotype and length-dependent are potentially able to drive changes in peptide expression dynamics.
Assuntos
Regiões 5' não Traduzidas/genética , RNA Mensageiro/genética , beta-Defensinas/metabolismo , Processamento Alternativo/imunologia , Simulação por Computador , Regulação da Expressão Gênica/imunologia , Haplótipos , Humanos , Imunidade Inata , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Ligação Proteica/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/imunologia , Software , Fatores de Transcrição/metabolismo , beta-Defensinas/genéticaRESUMO
We analyzed three functional 5' un-translated region beta-defensin 1 (DEFB1) single nucleotide polymorphism (SNPs) in a group of 170 type 1 diabetes (T1D) patients. In order to evaluate the SNPs influence on the disease onset and the development of other autoimmune disorder, such as celiac disease (CD) and autoimmune thyroid disease (AITD), patients were stratified according to the presence of AITD, CD, and both AITD and CD. As control group, we studied 191 healthy children and adolescent not presenting a familiar historic of T1D, CD or AITD. DEFB1 SNPs were in Hardy-Weinberg equilibrium both in healthy controls and T1D patients, as well in the T1D patients stratified according to the presence of other autoimmune disorder(s). Allele, genotype, and haplotype frequencies of T1D patients globally considered were comparable to healthy controls ones. No evidence of any association of DEFB1 SNPs with the onset of AIDT, CD, and both AITD and CD on T1D patients was evidenced. Only a minor trend was found for an increased frequency of the - 20 G allele in T1D patients only presenting AITD vs. T1D patients not presenting AITD or CD, as well as an increase of those haplotypes comprising the - 20 G allele when compared with the GCA haplotype. We also evaluated the influence of functional DEFB1 SNPs on the age of T1D onset: no significant statistical conclusion was achieved. Further studies are envisaged, in order to elucidate the possible role of functional DEFB1 polymorphisms in the onset of TD1 and other autoimmune-related disorders.
Assuntos
Doença Celíaca/genética , Diabetes Mellitus Tipo 1/genética , Polimorfismo Genético , Tireoidite Autoimune/genética , beta-Defensinas/genética , Adolescente , Idade de Início , Doenças Autoimunes/genética , Doenças Autoimunes/fisiopatologia , Brasil , Doença Celíaca/fisiopatologia , Criança , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Tireoidite Autoimune/fisiopatologiaAssuntos
Hanseníase Virchowiana/genética , Polimorfismo de Nucleotídeo Único , beta-Defensinas/química , beta-Defensinas/genética , Sítios de Ligação , Bioestatística , Causalidade , Regulação da Expressão Gênica , Marcadores Genéticos , Predisposição Genética para Doença , Humanos , Hanseníase Virchowiana/epidemiologia , beta-Defensinas/metabolismoRESUMO
The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. We have cloned the CaCDC35 gene encoding C. albicans adenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p. It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast. The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes. C. albicans cells deleted for both alleles of CaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans. The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages. However, morphogenetic switching was restored by exogenous cAMP. On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p. These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching. Homozygous cacdc35 Delta cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections. These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.
Assuntos
Adenilil Ciclases/metabolismo , Candida albicans/enzimologia , Transdução de Sinais , Adenilil Ciclases/genética , Adenilil Ciclases/imunologia , Adenilil Ciclases/isolamento & purificação , Animais , Sequência de Bases , Candida albicans/crescimento & desenvolvimento , Candida albicans/imunologia , Candida albicans/patogenicidade , Candidíase/microbiologia , Linhagem Celular , Deleção Cromossômica , Cromossomos Fúngicos , DNA Fúngico , Feminino , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , VirulênciaRESUMO
In an effort to investigate the molecular mechanisms responsible for the drastic morphological changes the mitochondria go through during the life cycle of the aquatic fungus Blastocladiella emersonii, the gene encoding the alpha subunit of the mitochondrial processing peptidase (alpha-MPP) was isolated. Nucleotide sequence analysis revealed that the predicted alpha-MPP polypeptide comprises 474 amino acids with a calculated molecular mass of 51,900 Da, presenting a characteristic mitochondrial signal sequence. Northern blot analysis indicated a single 1.4-kb transcript encoding the B. emersonii alpha-MPP, whose levels decrease during sporulation, becoming very low in the zoospore, and increase again during germination. Despite these variations in mRNA concentration, B. emersonii alpha-MPP protein levels do not change significantly during the life cycle of the fungus, as observed in Western blots. Experiments to investigate the submitochondrial localization of B. emersonii alpha-MPP and beta-MPP were also carried out, and the results indicated that both subunits are associated with the mitochondrial inner membrane, possibly as part of the bc1 complex, as described for plants.
Assuntos
Blastocladiella/enzimologia , Blastocladiella/genética , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Blastocladiella/crescimento & desenvolvimento , Western Blotting , Clonagem Molecular , DNA Fúngico/análise , DNA Fúngico/genética , Membranas Intracelulares/enzimologia , Metaloendopeptidases/química , Dados de Sequência Molecular , Análise de Sequência de DNA , Partículas Submitocôndricas/enzimologia , Transcrição Gênica , Peptidase de Processamento MitocondrialRESUMO
A 2.3-kb BamHI-KpnI fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the beta subunit of the Blastocladiella mitochondrial processing peptidase (beta-MPP). The predicted beta-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that beta-MPP mRNA levels decrease significantly during B. emersonii sporulation, reaching basal levels in the zoospore stage. The amount of beta-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle.