RESUMO
BACKGROUND: Glioblastomas are invasive therapy resistant brain tumors with extremely poor prognosis. The Glioma initiating cell (GIC) population contributes to therapeutic resistance and tumor recurrence. Targeting GIC-associated gene candidates could significantly impact GBM tumorigenicity. Here, we investigate a protein kinase, PBK/TOPK as a candidate for regulating growth, survival and in vivo tumorigenicity of GICs. METHODS: PBK is highly upregulated in GICs and GBM tissues as shown by RNA and protein analyses. We knocked down PBK using shRNA vectors and inhibited the function of PBK protein with a pharmacological PBK inhibitor, HITOPK-032. We assessed viability, tumorsphere formation and apoptosis in three patient derived GIC cultures. RESULTS: Gene knockdown of PBK led to decreased viability and sphere formation and in one culture an increase in apoptosis. Treatment of cells with inhibitor HITOPK-032 (5 µM and 10 µM) almost completely abolished growth and elicited a large increase in apoptosis in all three cultures. HI-TOPK-032 treatment (5 mg/kg and 10 mg/kg bodyweight) in vivo resulted in diminished growth of experimentally induced subcutaneous GBM tumors in mice. We also carried out multi-culture assays of cell survival to investigate the relative effects on GICs compared with the normal neural stem cells (NSCs) and their differentiated counterparts. Normal NSCs seemed to withstand treatment slightly better than the GICs. CONCLUSION: Our study of identification and functional validation of PBK suggests that this candidate can be a promising molecular target for GBM treatment.
Assuntos
Glioblastoma/metabolismo , Glioblastoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Indolizinas/farmacologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Inibidores de Proteínas Quinases/farmacologia , Quinoxalinas/farmacologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mouse bone marrow cells were exposed to specific monoclonal antibodies, so that lineage positive (Lin+) cells could be removed with magnetic beads. The Lin- cells were cultured with Sca-1 or CD90 (Thy-1) monoclonal antibodies (MoAbs) in semi-solid medium for 7 d. We found that Sca-1 MoAb suppressed colony formation (20-30%), and the effect was largely abolished by N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase. Similar results were obtained with antibodies to CD90. The findings suggest that the unknown physiological ligands to Sca-1 and Thy-1 markers on haematopoietic progenitor cells can inhibit colony formation, with NO as a pivotal mediator. Primitive progenitors may be a primary target of this Sca-1 ligand, as the Sca-1+ cell population contains the major part of the multipotent haematopoietic stem cells.
