RESUMO
Glioblastoma (GBM) is the most frequent and lethal brain tumor, whose therapeutic outcome - only partially effective with current schemes - places this disease among the unmet medical needs, and effective therapeutic approaches are urgently required. In our attempts to identify repositionable drugs in glioblastoma therapy, we identified the neuroleptic drug chlorpromazine (CPZ) as a very promising compound. Here we aimed to further unveil the mode of action of this drug. We performed a supervised recognition of the signal transduction pathways potentially influenced by CPZ via Reverse-Phase Protein microArrays (RPPA) and carried out an Activity-Based Protein Profiling (ABPP) followed by Mass Spectrometry (MS) analysis to possibly identify cellular factors targeted by the drug. Indeed, the glycolytic enzyme PKM2 was identified as one of the major targets of CPZ. Furthermore, using the Seahorse platform, we analyzed the bioenergetics changes induced by the drug. Consistent with the ability of CPZ to target PKM2, we detected relevant changes in GBM energy metabolism, possibly attributable to the drug's ability to inhibit the oncogenic properties of PKM2. RPE-1 non-cancer neuroepithelial cells appeared less responsive to the drug. PKM2 silencing reduced the effects of CPZ. 3D modeling showed that CPZ interacts with PKM2 tetramer in the same region involved in binding other known activators. The effect of CPZ can be epitomized as an inhibition of the Warburg effect and thus malignancy in GBM cells, while sparing RPE-1 cells. These preclinical data enforce the rationale that allowed us to investigate the role of CPZ in GBM treatment in a recent multicenter Phase II clinical trial.
Assuntos
Glioblastoma , Humanos , Glioblastoma/patologia , Clorpromazina/farmacologia , Clorpromazina/uso terapêutico , Piruvato Quinase/metabolismo , Linhagem Celular Tumoral , Metabolismo EnergéticoRESUMO
With the aim to find novel partners of human Cyclin T2a, we performed a two-hybrid screening in yeast using the full-length cDNA of this cyclin as bait, and a human heart cDNA library as preys source. Upon several interesting genes selected, our attention has been focused on the cDNA coding for PKNalpha, a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. Co-immunoprecipitation and in vitro pull-down assays independently confirmed the interaction between the two proteins. Luciferase assays, performed on NIH3T3 cell extracts after transfection with a MyoD-responsive promoter, pointed out that PKNalpha was able to enhance MyoD-dependent transcription, and that this effect was further increased when cyclin T2a was co-overexpressed. Finally, overexpression of both Cyclin T2a and PKNalpha in C2C12 cells strongly enhanced the expression of myogenic differentiation markers, such as Myogenin and Myosin Heavy Chain, during starvation-induced differentiation. Taken together, our data strengthen the hypothesis that Cyclin T2a plays a role in muscle differentiation, and propose PKNalpha as a novel partner of Cyclin T2a in this process.
Assuntos
Diferenciação Celular , Ciclinas/metabolismo , Células Musculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Sítios de Ligação/genética , Ligação Competitiva , Linhagem Celular , Ciclina T , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Ciclinas/genética , DNA Complementar/genética , Expressão Gênica/genética , Humanos , Camundongos , Células Musculares/citologia , Mutação , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Células NIH 3T3 , Plasmídeos/genética , Ligação Proteica , Proteína Quinase C , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
PURPOSE: Employing an in vitro model system of human melanoma progression, we previously reported ferritin light chain (L-ferritin) gene overexpression in the metastatic phenotype. Here, we attempted to characterize the role of ferritin in the biology of human melanoma and in the progression of this disease. EXPERIMENTAL DESIGN: Starting from the LM human metastatic melanoma cell line, we engineered cell clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. These cells were then assayed for their growth capabilities, chemoinvasive properties, and sensitivity to oxidative stress. Additionally, ferritin protein content in primary and metastatic human melanomas was determined by immunohistochemistry. RESULTS: Artificial L-ferritin down-regulation in the LM cells strongly inhibited proliferation and chemoinvasion in vitro and cell growth in vivo. In addition, L-ferritin down-regulated cells displayed enhanced sensitivity to oxidative stress and to apoptosis. Concurrently, immunohistochemical analysis of a human melanoma tissue array revealed that ferritin expression level in metastatic lesions was significantly higher (P < 0.0001) than in primary melanomas. Furthermore, ferritin expression was constantly up-regulated in autologous lymph node melanoma metastases when compared with the respective primary tumors in a cohort of 11 patients. CONCLUSIONS: These data suggest that high ferritin expression can enhance cell growth and improve resistance to oxidative stress in metastatic melanoma cells by interfering with their cellular antioxidant system. The potential significance of these findings deserves to be validated in a clinical setting.