RESUMO
Information on the mechanisms that are associated with tumor resistance has the potential to provide the fundamental basis for novel therapeutic strategies. In glioblastoma (GBM), predictive biomarkers of cellular responses to temozolomide (TMZ) combined with polyADPribose polymerase inhibitor (PARPi) remain largely unidentified. In this context, the influence of MGMT (O6methylguanine DNA methyltransferase) and PTEN (phosphatase and tensin homologue deleted on chromosome ten) has been studied in addition to the occurrence of synthetic lethality involving PTEN and PARPi. The present study investigated whether PARP1 inhibition by NU1025 may increase the cytotoxicity of TMZinduced lesions in GBM cells, and whether these mechanisms can be influenced by MGMT and PTEN status. The impact of PTEN deficiency in repair pathways, and the effects of PARP1 inhibition and PTEN silencing, in terms of synthetic lethality, were also assessed. NU1025 combined with TMZ effectively sensitized TMZresistant cells (T98G PTENmutated and LN18 PTENwildtype) and TMZsensitive cells (U251MG PTENmutated), in contrast to NU1025 alone. However, the sensitizing effects were not observed in U87MG (PTENmutated) cells, suggesting that specific genetic alterations may influence the response to drug treatment. The sensitizing effects occurred independently of MGMT activity, which was evaluated in O6BGtreated cells. PTEN silencing using small interfering (si)RNA did not sensitize PTENproficient cells to TMZ + NU1025, or NU1025 alone, indicating an absence of synthetic lethality. The responses to TMZ + NU1025 involved antiproliferative activity, G2/M arrest, double strand breaks and the induction of apoptosis. Following 20 days of recovery after three consecutive days of TMZ treatment, TMZresistant cells were observed. However, when TMZ was combined with NU1025, the viability of T98G and LN18 cells was extremely decreased, indicating a lethal drug combination. Therefore, independently of MGMT proficiency and PTEN status, TMZ combined with PARPi may be a promising strategy that can be used to overcome TMZ acquired resistance in GBM cells.
Assuntos
Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Glioblastoma/tratamento farmacológico , PTEN Fosfo-Hidrolase/genética , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Quinazolinas/farmacologia , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Antineoplásicos Alquilantes/farmacologia , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Mutação , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
BACKGROUND: Glioblastoma is considered to the most common and malignant brain tumor in adults. Patients have a median survival of approximately one year from diagnosis due to poor response to therapy. OBJECTIVE: We applied bioinformatics approaches to predict transcription factors (TF) that are deregulated in glioblastoma in an attempt to point out molecular targets for therapy. METHODS: Up-regulated genes in glioblastoma selected from public microarray data were submitted to two TF association analyses. Thereafter, the expression levels of TF obtained in the overlap of analyses were assessed by RT-qPCR carried out in seven glioblastoma cell lines (T98, U251, U138, U87, U343, M059J, and M059K). RESULTS: E2F1 and E2F4 were highlighted in both TF analyses. However, only E2F1 was confirmed as significantly up-regulated in all glioblastoma cell lines in vitro. CONCLUSION: E2F1 is a potential common regulator of differentially expressed genes in glioblastoma, despite the genetic heterogeneity of tumor cells.
Assuntos
Neoplasias do Sistema Nervoso Central/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F4/genética , Glioblastoma/genética , Linhagem Celular Tumoral , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para CimaRESUMO
Glioblastoma multiforme (GBM) is a lethal tumor and novel strategies are required to overcome resistance. Transcription factor 12 (HEB) has been associated with neural and stem cell proliferation, is overexpressed in certain tumor types and is induced in irradiated U87MG cells. The present study aimed to determine whether HEB knockdown, with or without irradiation, may sensitize GBM cells. U87MG GBM and ACBRI371 primary human astrocytes were cultured in monolayers or neurospheres. Cell proliferation and death, cell cycle and subG1 detection, and cluster of differentiation (CD) 133 immunofluorescence were analyzed by flow cytometry, whereas HEB protein expression was analyzed by immunocytochemistry and western blotting. Greater HEB protein expression was observed in U87MG neurospheres compared with ACBRI371, and the two cell lines exhibited nuclear HEB expression. HEB silencing in cells grown in monolayers induced a significant reduction in proliferation and decreased the proportion of cells in G0/G1 phase. In addition, HEB silencing reduced (twofold) the number of neurospheres compared with control scrambled (SCR) cells. HEB silencing combined with irradiation reduced U87MG cell proliferation when cultured in monolayers and reduced neurosphere cell number compared with the SCR irradiated group; however, not significantly. Differentiation of U87MG cells from neurospheres was reduced in HEBsilenced cells, whereas in irradiated cells the proportion of CD133+ cells was similar in HEBsilenced cells compared with the SCR control. These results suggest that HEB may contribute to the proliferation and maintenance of GBM cells. However, only limited effects were exerted by irradiation in HEBsilenced cells. HEB may be a potential target to decrease proliferation in U87MG GBM cells, grown as monolayers or neurospheres, and may provide important information for the development of novel strategies for cancer therapy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Inativação Gênica , Apoptose/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Glioblastoma/genética , Humanos , Interferência de RNA , RNA Interferente Pequeno , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Temozolomide (TMZ) is widely used for patients with glioblastoma (GBM); however, tumor cells frequently exhibit drug-resistance. Base excision repair (BER) has been identified as a possible mediator of TMZ resistance, and an attractive approach to sensitizing cells to chemotherapy. Human apurinic/apyrimidinic endonuclease/redox factor-1 (APE1) is an essential enzyme with a role in the BER pathway by repairing abasic sites, and it also acts as a reduction factor, maintaining transcription factors in an active reduced state. Thus, we aimed to investigate whether the down-regulation of APE1 expression by siRNA can interfere with the resistance of GBM to TMZ, being evaluated by several cellular and molecular parameters. We demonstrated that APE1 knockdown associated with TMZ treatment efficiently reduced cell proliferation and clonogenic survival of resistant cells (T98G), which appears to be a consequence of increased DNA damage, S-phase arrest, and H2AX phosphorylation, resulting in apoptosis induction. On the contrary, for those assays, the sensitization effects of APE1 silencing plus TMZ treatment did not occur in the TMZ-sensitive cell line (U87MG). Interestingly, TMZ-treatment and APE1 knockdown significantly reduced cell invasion in both cell lines, but TMZ alone did not reduce the invasion capacity of U87MG cells, as observed for T98G. We also found that VEGF expression was down-regulated by TMZ treatment in T98G cells, regardless of APE1 knockdown, but U87MG showed a different response, since APE1 silencing counteracted VEGF induction promoted by TMZ, suggesting that the APE1-redox function may play an indirect role, depending on the cell line. The present results support the contribution of BER in the GBM resistance to TMZ, with a greater effect in TMZ-resistant, compared with TMZ-sensitive cells, emphasizing that APE1 can be a promising target for modifying TMZ tolerance. Furthermore, genetic characteristics of tumor cells should be considered as critical information to select an appropriate therapeutic strategy.