RESUMO
Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromonas/farmacologia , Cromonas/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Flavonas/farmacologia , Flavonas/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-pim-1/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient's prognosis. Beside promoter methylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. METHODS: Therefore, we evaluated the proportion and prognostic significance of promoter methylation of MGMT, ABCB1 and ABCG2 in 64 GBM patient samples using pyrosequencing technology. Further, the single nucleotide polymorphisms MGMT C-56 T (rs16906252), ABCB1 C3435T (rs1045642) and ABCG2 C421A (rs2231142) were determined using the restriction fragment length polymorphism method (RFLP). To study a correlation between promoter methylation and gene expression, we analyzed MGMT, ABCB1 and ABCG2 expression in 20 glioblastoma and 7 non-neoplastic brain samples. RESULTS: Despite a significantly increased MGMT and ABCB1 promoter methylation in GBM tissue, multivariate regression analysis revealed no significant association between overall survival of glioblastoma patients and MGMT or ABCB1 promoter methylation. However, a significant negative correlation between promoter methylation and expression could be identified for MGMT but not for ABCB1 and ABCG2. Furthermore, MGMT promoter methylation was significantly associated with the genotypes of the MGMT C-56 T polymorphism showing a higher methylation level in the T allele bearing GBM. CONCLUSIONS: In summary, the data of this study confirm the previous published relation of MGMT promoter methylation and gene expression, but argue for no pivotal role of MGMT, ABCB1 and ABCG2 promoter methylation in GBM patients' survival.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Neoplasias Encefálicas/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética , Glioblastoma/genética , Proteínas de Neoplasias/genética , Proteínas Supressoras de Tumor/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Adulto , Idoso , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Metilação de DNA , Feminino , Expressão Gênica , Glioblastoma/mortalidade , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , RecidivaRESUMO
Patients with glioblastoma multiforme (GBM) are at high risk to develop a relapse despite multimodal therapy. Assumedly, glioma stem cells (GSCs) are responsible for treatment resistance of GBM. Identification of specific GSC markers may help to develop targeted therapies. Here, we performed expression analyses of stem cell (ABCG2, CD44, CD95, CD133, ELF4, Nanog, and Nestin) as well as differentiation and microglia markers (GFAP, Iba1, and Sparc) in GBM compared to nonmalignant brain. Furthermore, the role of these proteins for patient survival and their expression in LN18 stem-like neurospheres was analyzed. At mRNA level, ABCG2 and CD95 were reduced, GFAP was unchanged; all other investigated markers were increased in GBM. At protein level, CD44, ELF4, Nanog, Nestin, and Sparc were elevated in GBM, but only CD133 and Nestin were strongly associated with survival time. In addition, ABCG2 and GFAP expression was decreased in LN18 neurospheres whereas CD44, CD95, CD133, ELF4, Nanog, Nestin, and Sparc were upregulated. Altogether only CD133 and Nestin were associated with survival rates. This raises concerns regarding the suitability of the other target structures as prognostic markers, but makes both CD133 and Nestin candidates for GBM therapy. Nevertheless, a search for more specific marker proteins is urgently needed.