RESUMO
BACKGROUND AND PURPOSE: Follicular lymphoma is the second most common non-Hodgkin's lymphoma and, despite the introduction of rituximab for its treatment, this disease is still considered incurable. Besides genetic alterations involving Bcl-2, Bcl-6 or c-Myc, follicular lymphoma cells often display altered B-cell receptor signalling pathways including overactive PKC and PI3K/Akt systems. EXPERIMENTAL APPROACH: The effect of enzastaurin, an inhibitor of PKC, was evaluated both in vitro on follicular lymphoma cell lines and in vivo on a xenograft murine model. Using pharmacological inhibitors and siRNA transfection, we determined the different signalling pathways after enzastaurin treatment. KEY RESULTS: Enzastaurin inhibited the serine-threonine kinase p90RSK which has downstream effects on GSK3ß. Bad and p70S6K. These signalling proteins control follicular lymphoma cell survival and apoptosis; which accounted for the inhibition by enzastaurin of cell survival and its induction of apoptosis of follicular lymphoma cell lines in vitro. Importantly, these results were replicated in vivo where enzastaurin inhibited the growth of follicular lymphoma xenografts in mice. CONCLUSIONS AND IMPLICATIONS: The targeting of p90RSK by enzastaurin represents a new therapeutic option for the treatment of follicular lymphoma.
Assuntos
Antineoplásicos/farmacologia , Linfoma Folicular/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Linfoma Folicular/enzimologia , Linfoma Folicular/genética , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Fosforilação , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Follicular lymphomas (FLs) account for 35-40% of all adult lymphomas. Treatment typically involves chemotherapy combined with the anti-CD20 monoclonal antibody (MAb) rituximab (RTX). The development of the type II anti-CD20 MAb obinutuzumab (GA101) aims to further improve treatment. Here, using FL cells we show that RTX and GA101 display a similar activity on RL cells cultured in 2D. However, 2D culture cannot mimic tumor spatial organization and conventional 2D models may not reflect the effects of antibodies as they occur in vivo. Thus, we created a non-Hodgkin's lymphoma (NHL) 3D culture system, termed multicellular aggregates of lymphoma cells (MALC), and used it to compare RTX and GA101 activity. Our results show that both antibodies display greater activity towards FL cells in 3D culture compared with 2D culture. Moreover, we observed that in the 3D model GA101 was more effective than RTX both in inhibiting MALC growth through induction of (lysosomal) cell death and senescence and in inhibiting intracellular signaling pathways, such as mammalian target of rapamycin, Akt, PLCgamma (Phospholipase C gamma) and Syk. Altogether, our study demonstrates that spatial organization strongly influences the response to antibody treatment, supporting the use of 3D models for the testing of therapeutic agents in NHL.
RESUMO
Opiate withdrawal leads to the emergence of an aversive state that can be conditioned to a specific environment. Reactivation of these withdrawal memories has been suggested to be involved in relapse to drug-seeking of abstinent opiate addicts. Among the limbic areas that are likely to mediate these features of opiate dependence, amygdala nuclei represent critical neural substrates. Using a conditioned place aversion paradigm (CPA), we have previously shown specific opposite patterns of reactivity in the basolateral (BLA) and the central (CeA) amygdala, when comparing the experience of acute opiate withdrawal with the re-exposure to a withdrawal-paired environment. These data gave clues about the potential mechanisms by which amygdala nuclei may be involved in withdrawal memories. To extend these results, the present study aimed to assess the cellular reactivity and plasticity of amygdala nuclei during the opiate withdrawal conditioning process. For this, we have quantified c-fos and arc expression using in situ hybridization in rats, following each of the three conditioning sessions during CPA, and after re-exposure to the withdrawal-paired environment. BLA output neurons showed an increase in the expression of the plasticity-related arc gene during conditioning that was also increased by re-exposure to the withdrawal-paired environment. Interestingly, the CeA showed an opposite pattern of responding, and the intercalated cell masses (ITC), a possible inhibitory interface between the BLA and CeA, showed a persistent activation of c-fos and arc mRNA. We report here specific c-fos and arc patterns of reactivity in amygdala nuclei during withdrawal conditioning. These findings improve our understanding of the involvement of the amygdala network in the formation and retrieval of withdrawal memories. Plasticity processes within BLA output neurons during conditioning, may participate in increasing the BLA reactivity to conditioned stimuli, which could in turn (by the control of downstream nuclei) reinforce and drive the motivational properties of withdrawal over drug consumption.
Assuntos
Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiopatologia , Proteínas do Citoesqueleto/genética , Genes fos/genética , Entorpecentes/efeitos adversos , Proteínas do Tecido Nervoso/genética , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/fisiopatologia , Animais , Genes fos/efeitos dos fármacos , Hibridização In Situ , Masculino , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Fenótipo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
In an attempt to investigate the molecular basis of pyrazinamide hydrolysis by the PncA protein from Mycobacterium tuberculosis, we determined the pyrazinamidase activity of nine PncA mutants bearing a single amino acid substitution. Among them, three mutants (D8G, K96T and S104R) had virtually no activity (< or =0.004 unit/mg), five (F13S, T61P, P69L, Y103S and A146V) retained a low level of activity (0.06-0.25 unit/mg) and one (T167L) exhibited a wild-type activity (1.51 units/mg). The possible structural effects of these substitutions were assessed by analysing a three-dimensional model of the PncA protein constructed on the basis of the crystal structure of the N-carbamoylsarcosine amidohydrolase (CSHase) from Arthrobacter sp., an amidohydrolase which was found by hydrophobic cluster analysis to be closely related to PncA. In the PncA model, five of the mutated residues, Asp-8, Phe-13, Lys-96, Tyr-103 and Ser-104, were located within a 6 A sphere around the cysteine residue Cys-138, which could be the counterpart of the active cysteine residue Cys-177 found in the CSHase. Among the remaining mutated residues, Thr-61, Pro-69 and Ala-146 were found to be more distant from Cys-138 but were associated with structural elements contributing to the catalytic centre, whereas Thr-167 was situated in an alpha-helix located far from the putative active site. These data suggest that the decrease in pyrazinamidase activity observed in the PncA mutant proteins is well correlated with the structural modifications the mutations can cause in the environment of the putative active cysteine Cys-138.