RESUMO
Aberrant activation of the extracellular signalregulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway is frequently determined in various human tumor types, including liver cancer, and has been considered as a promising target for cancer chemoprevention and therapy. In the present study, using computeraided virtual screening and molecular docking, isobavachalcone (IBC), a natural chalcone compound, was identified to be an ATPcompetitive inhibitor targeting ERK1/2 and RSK2. Cell Counting Kit8, EdU incorporation and colony formation assays were used to detect the effects of IBC on cell viability and proliferation, and the results demonstrated that IBC effectively inhibited the proliferation of liver cancer HepG2 and Hep3B cells, whereas it had no notable cytotoxic effect on immortal liver L02 cells. Flow cytometric analysis and western blotting further revealed that IBC caused significant levels of apoptosis on liver cancer cells via the caspasedependent mitochondria pathway. The computer prediction was confirmed with pulldown and in vitro kinase assays, in which IBC directly bound with ERK1/2 and RSK2, and dosedependently blocked RSK2 kinase activity in liver cancer cells. Treatment of HepG2 or Hep3B cells with IBC significantly attenuated epidermal growth factorinduced phosphorylation of RSK2 and resulted in the reduced activation of its downstream substrates including cAMP response elementbinding protein, activating transcription factor 1, histone H3 and activating protein1. Enforced RSK2 expression in L02 cells could increase the effect of IBC on suppressing cell growth. Conversely, knockdown of RSK2 reduced the inhibitory effect of IBC on HepG2 cell proliferation. Overall, the present data indicated that ERKs/RSK2 signaling serves a pivotal role in IBCinduced suppression of liver cancer cells and that IBC may be a potential therapeutic candidate for human cancer with elevated ERKs/RSK2 activity.
Assuntos
Neoplasias Hepáticas/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/química , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chalconas/química , Chalconas/farmacologia , Células Hep G2 , Humanos , Ligantes , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Medicina Tradicional Chinesa , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Interface Usuário-ComputadorRESUMO
Dimethyl fumarate (DMF) has been applied for decades in the treatment of psoriasis and now also multiple sclerosis. However, the mechanism of action has remained obscure and involves high dose over long time of this small, reactive compound implicating many potential targets. Based on a 1.9 Å resolution crystal structure of the C-terminal kinase domain of the mouse p90 Ribosomal S6 Kinase 2 (RSK2) inhibited by DMF we describe a central binding site in RSKs and the closely related Mitogen and Stress-activated Kinases (MSKs). DMF reacts covalently as a Michael acceptor to a conserved cysteine residue in the αF-helix of RSK/MSKs. Binding of DMF prevents the activation loop of the kinase from engaging substrate, and stabilizes an auto-inhibitory αL-helix, thus pointing to an effective, allosteric mechanism of kinase inhibition. The biochemical and cell biological characteristics of DMF inhibition of RSK/MSKs are consistent with the clinical protocols of DMF treatment.
Assuntos
Fumarato de Dimetilo/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Animais , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Cisteína/química , Fumarato de Dimetilo/química , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Mutação , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologiaRESUMO
The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.