RESUMO
Thioridazine (THD) is a common phenothiazine antipsychotic drug reported to suppress growth in several types of cancer cells. We previously showed that THD acts as an antiglioblastoma and anticancer stem-like cell agent. However, the signaling pathway underlying autophagy and apoptosis induction remains unclear. THD treatment significantly induced autophagy with upregulated AMPK activity and engendered cell death with increased sub-G1 in glioblastoma multiform (GBM) cell lines. Notably, through whole gene expression screening with THD treatment, frizzled (Fzd) proteins, a family of G-protein-coupled receptors, were found, suggesting the participation of Wnt/ß-catenin signaling. After THD treatment, Fzd-1 and GSK3ß-S9 phosphorylation (inactivated form) was reduced to promote ß-catenin degradation, which attenuated P62 inhibition. The autophagy marker LC3-II markedly increased when P62 was released from ß-catenin inhibition. Additionally, the P62-dependent caspase-8 activation that induced P53-independent apoptosis was confirmed by inhibiting T-cell factor/ß-catenin and autophagy flux. Moreover, treatment with THD combined with temozolomide (TMZ) engendered increased LC3-II expression and caspase-3 activity, indicating promising drug synergism. In conclusion, THD induces autophagy in GBM cells by not only upregulating AMPK activity, but also enhancing P62-mediated autophagy and apoptosis through Wnt/ß-catenin signaling. Therefore, THD is a potential alternative therapeutic agent for drug repositioning in GBM.
Assuntos
Autofagia/efeitos dos fármacos , Cateninas/metabolismo , Glioma/metabolismo , Tioridazina/farmacologia , Apoptose/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
We previously revealed the origin of mammalian simple-type glycogen synthase kinase interaction protein (GSKIP), which served as a scavenger and a competitor in the Wnt signaling pathway during evolution. In this study, we investigated the conserved and nonconserved regions of the composite-type GSKIP by utilizing bioinformatics tools, site-directed mutagenesis, and yeast two-hybrid methods. The regions were denoted as the pre-GSK3ß binding site, which is located at the front of GSK3ß-binding sites. Our data demonstrated that clustered mitochondria protein 1 (CLU1), a type of composite-type GSKIP that exists in the mitochondria of all eukaryotic organisms, possesses the protein known as domain of unknown function 727 (DUF727), with a pre-GSK3ß-binding site and a mutant GSK3ß-binding flanking region. Another type of composite-type GSKIP, armadillo repeat containing 4 (ARMC4), which is known for cilium movement in vertebrates, contains an unintegrated DUF727 flanking region with a pre-GSK3ß-binding site (115SPxF118) only. In addition, the sequence of the GSK3ß-binding site in CLU1 revealed that Q126L and V130L were not conserved, differing from the ideal GSK3ß-binding sequence of simple-type GSKIP. We further illustrated two exceptions, namely 70 kilodalton heat shock proteins (Hsp70/DnaK) and Mitofilin in nematodes, that presented an unexpected ideal GSK3ß-binding region with a pre-GSK3ß sequence; this composite-type GSKIP could only occur in vertebrate species. Furthermore, we revealed the importance of the pre-GSK3ß-binding site (118F or 118Y) and various mutant GSK3ß-binding sites of composite-type GSKIP. Collectively, our data suggest that the new composite-type GSKIP starts with a DUF727 domain followed by a pre-GSK3ß-binding site, with the subsequent addition of the GSK3ß-binding site, which plays vital roles for CLU1, Mitofilin, and ARMC4 in mitochondria and Wnt signaling pathways during evolution.
Assuntos
Proteínas do Domínio Armadillo/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Mitocôndrias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/genética , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Evolução Molecular , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Filogenia , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/química , Análise de Sequência de DNA , Técnicas do Sistema de Duplo-Híbrido , Via de Sinalização WntRESUMO
Mitochondrial fission and fusion cycles are integrated with cell cycle progression. Here we first re-visited how mitochondrial ETC inhibition disturbed mitosis progression, resulting in multipolar spindles formation in HeLa cells. Inhibitors of ETC complex I (rotenone, ROT) and complex III (antimycin A, AA) decreased the phosphorylation of Plk1 T210 and Aurora A T288 in the mitotic phase (M-phase), especially ROT, affecting the dynamic phosphorylation status of fission protein dynamin-related protein 1 (Drp1) and the Ser637/Ser616 ratio. We then tested whether specific Drp1 inhibitors, Mdivi-1 or Dynasore, affected the dynamic phosphorylation status of Drp1. Similar to the effects of ROT and AA, our results showed that Mdivi-1 but not Dynasore influenced the dynamic phosphorylation status of Ser637 and Ser616 in Drp1, which converged with mitotic kinases (Cdk1, Plk1, Aurora A) and centrosome-associated proteins to significantly accelerate mitotic defects. Moreover, our data also indicated that evoking mito-Drp1-Ser637 by protein kinase A (PKA) rather than Drp1-Ser616 by Cdk1/Cyclin B resulted in mitochondrial fission via the PINK1/Parkin pathway to promote more efficient mitophagy and simultaneously caused multipolar spindles. Collectively, this study is the first to uncover that mito-Drp1-Ser637 by PKA, but not Drp1-Ser616, drives mitophagy to exert multipolar spindles formation during M-phase.