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GSK3ß interacting protein (GSKIP) is a small A-kinase anchor protein previously reported to mediate the N-cadherin/ß-catenin pool for differentiation in SH-SY5Y cells through overexpression of GSKIP to present the neuron outgrowth phenotype. To further investigate how GSKIP functions in neurons, CRISPR/Cas9 technology was utilized to knock out GSKIP (GSKIP-KO) in SH-SY5Y. Several GSKIP-KO clones resulted in an aggregation phenotype and reduced cell growth without retinoic acid (RA) treatment. However, neuron outgrowth was still observed in GSKIP-KO clones treated with RA. The GSKIP-KO clones exhibited an aggregation phenotype through suppression of GSK3ß/ß-catenin pathways and cell cycle progression rather than cell differentiation. Gene set enrichment analysis indicated that GSKIP-KO was related to epithelial mesenchymal transition/mesenchymal epithelial transition (EMT/MET) and Wnt/ß-catenin/cadherin signaling pathways, suppressing cell migration and tumorigenesis through the inhibition of Wnt/ß-catenin mediated EMT/MET. Conversely, reintroduction of GSKIP into GSKIP-KO clones restored cell migration and tumorigenesis. Notably, phosphor-ß-catenin (S675) and ß-catenin (S552) but not phosphor-ß-catenin (S33/S37/T41) translocated into the nucleus for further gene activation. Collectively, these results suggested that GSKIP may function as an oncogene to form an aggregation phenotype for cell survival in harsh environments through EMT/MET rather than differentiation in the GSKIP-KO of SH-SY5Y cells. GSKIP Implication in Signaling Pathways with Potential Impact on SHSY-5Y Cell Aggregation.
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BACKGROUND: Despite advances in treatment, patients with refractory colorectal cancer (CRC) still have poor long-term survival, so there is a need for more effective therapeutic options. METHODS: To evaluate the HDAC8 inhibition efficacy as a CRC treatment, we examined the effects of various HDAC8 inhibitors (HDAC8i), including BMX (NBM-T-L-BMX-OS01) in combination with temozolomide (TMZ) or other standard CRC drugs on p53 mutated HT29 cells, as well as wild-type p53 HCT116 and RKO cells. RESULTS: We showed that HDAC8i with TMZ cotreatment resulted in HT29 arrest in the S and G2/M phase, whereas HCT116 and RKO arrest in the G0/G1 phase was accompanied by high sub-G1. Subsequently, this combination approach upregulated p53-mediated MGMT inhibition, leading to apoptosis. Furthermore, we observed the cotreatment also enabled triggering of cell senescence and decreased expression of stem cell biomarkers. Mechanistically, we found down-expression levels of ß-catenin, cyclin D1 and c-Myc via GSK3ß/ß-catenin signaling. Intriguingly, autophagy also contributes to cell death under the opposite status of ß-catenin/p62 axis, suggesting that there exists a negative feedback regulation between Wnt/ß-catenin and autophagy. Consistently, the Gene Set Enrichment Analysis (GSEA) indicated both apoptotic and autophagy biomarkers in HT29 and RKO were upregulated after treating with BMX. CONCLUSIONS: BMX may act as a HDAC8 eraser and in combination with reframed-TMZ generates a remarkable synergic effect, providing a novel therapeutic target for various CRCs. Video Abstract.
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Apoptose , Neoplasias Colorretais , Inibidores de Histona Desacetilases , Temozolomida , Humanos , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Temozolomida/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização Wnt , Inibidores de Histona Desacetilases/farmacologia , Células HT29RESUMO
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.
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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
Beta2-microglobulin (B2M) a key component of major histocompatibility complex class I molecules, which aid cytotoxic T-lymphocyte (CTL) immune response. However, the majority of studies of B2M have focused only on amyloid fibrils in pathogenesis to the neglect of its role of antimicrobial activity. Indeed, B2M also plays an important role in innate defense and does not only function as an adjuvant for CTL response. A previous study discovered that human aggregated B2M binds the surface protein structure in Streptococci, and a similar study revealed that sB2M-9, derived from native B2M, functions as an antibacterial chemokine that binds Staphylococcus aureus. An investigation of sB2M-9 exhibiting an early lymphocyte recruitment in the human respiratory epithelium with bacterial challenge may uncover previously unrecognized aspects of B2M in the body's innate defense against Mycobactrium tuberculosis. B2M possesses antimicrobial activity that operates primarily under pH-dependent acidic conditions at which B2M and fragmented B2M may become a nucleus seed that triggers self-aggregation into distinct states, such as oligomers and amyloid fibrils. Modified B2M can act as an antimicrobial peptide (AMP) against a wide range of microbes. Specifically, these AMPs disrupt microbe membranes, a feature similar to that of amyloid fibril mediated cytotoxicity toward eukaryotes. This study investigated two similar but nonidentical effects of B2M: the physiological role of B2M, in which it potentially acts against microbes in innate defense and the role of B2M in amyloid fibrils, in which it disrupts the membrane of pathological cells. Moreover, we explored the pH-governing antibacterial activity of B2M and acidic pH mediated B2M amyloid fibrils underlying such cytotoxicity.
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Amiloide/toxicidade , Antibacterianos/farmacologia , Microglobulina beta-2/metabolismo , Sequência de Aminoácidos , Animais , Morte Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Microglobulina beta-2/químicaRESUMO
Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of ß-catenin was reversed by proteasome inhibitor via the ß-catenin/ GSK3ß signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the ß-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.
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Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioblastoma/tratamento farmacológico , Histona Desacetilases/genética , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , beta Catenina/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Proteínas Repressoras/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Temozolomida/efeitos adversos , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Therapeutic resistance in recurrent glioblastoma multiforme (GBM) after concurrent chemoradiotherapy (CCRT) is a challenging issue. Although standard fractionated radiation is essential to treat GBM, it has led to local recurrence along with therapy-resistant cells in the ionizing radiation (IR) field. Lines of evidence showed cancer stem cells (CSCs) play a vital role in therapy resistance in many cancer types, including GBM. However, the molecular mechanism is poorly understood. Here, we proposed that autophagy could be involved in GSC induction for radioresistance. In a clinical setting, patients who received radiation/chemotherapy had higher LC3II expression and showed poor overall survival compared with those with low LC3 II. In a cell model, U87MG and GBM8401 expressed high level of stemness markers CD133, CD44, Nestin, and autophagy marker P62/LC3II after receiving standard fractionated IR. Furthermore, Wnt/ß-catenin proved to be a potential pathway and related to P62 by using proteasome inhibitor (MG132). Moreover, pharmacological inhibition of autophagy with BAF and CQ inhibit GSC cell growth by impairing autophagy flux as demonstrated by decrease Nestin, CD133, and SOX-2 levels. In conclusion, we demonstrated that fractionated IR could induce GSCs with the stemness phenotype by P62-mediated autophagy through the Wnt/ß-catenin for radioresistance. This study offers a new therapeutic strategy for targeting GBM in the future.
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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.
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Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinaminas/metabolismo , Dinâmica Mitocondrial , Mitofagia , Mitose , Proteínas Quinases/metabolismo , Fuso Acromático/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Antimicina A/farmacologia , Aurora Quinase A/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Células HeLa , Humanos , Hidrazonas/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Estresse Oxidativo , Fosforilação , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Quinazolinonas/metabolismo , Rotenona/farmacologia , Quinase 1 Polo-LikeRESUMO
Based on the protein kinase A (PKA)/GSK3ß interaction protein (GSKIP)/glycogen synthase kinase 3ß (GSK3ß) axis, we hypothesized that these might play a role in Tau phosphorylation. Here, we report that the phosphorylation of Tau Ser409 in SHSY5Y cells was increased by overexpression of GSKIP WT more than by PKA- and GSK3ß-binding defective mutants (V41/L45 and L130, respectively). We conducted in vitro assays of various kinase combinations to show that a combination of GSK3ß with PKA but not Ca2+/calmodulin-dependent protein kinase II (CaMK II) might provide a conformational shelter to harbor Tau Ser409. Cerebrospinal fluid (CSF) was evaluated to extend the clinical significance of Tau phosphorylation status in Alzheimer's disease (AD), neurological disorders (NAD), and mild cognitive impairment (MCI). We found higher levels of different PKA-Tau phosphorylation sites (Ser214, Ser262, and Ser409) in AD than in NAD, MCI, and normal groups. Moreover, we used the CRISPR/Cas9 system to produce amyloid precursor protein (APPWT/D678H) isogenic mutants. These results demonstrated an enhanced level of phosphorylation by PKA but not by the control. This study is the first to demonstrate a transient increase in phosphor-Tau caused by PKA, but not GSK3ß, in the CSF and induced pluripotent stem cells (iPSCs) of AD, implying that both GSKIP and GSK3ß function as anchoring proteins to strengthen the cAMP/PKA/Tau axis signaling during AD pathogenesis.
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Non-small cell lung cancer (NSCLC) is a type of malignant cancer, and 85% of metastatic NSCLC patients have a poor prognosis. C2-ceramide induces G2/M phase arrest and cytotoxicity in NSCLC cells. In this study, the autophagy-inducing effect of C2-ceramide was demonstrated, and cotreatment with the autophagy inhibitor chloroquine (CQ) was investigated in NSCLC H460 and H1299 cells. The results suggested that C2-ceramide exhibited dose-dependent anticancer effects in H460 and H1299 cells and autophagy induction. Zebrafish-based acridine orange staining confirmed the combined effects in vivo. Importantly, the combination of a sublethal dose of C2-ceramide and CQ resulted in additive cytotoxicity and autophagy in both cell lines. Alterations of related signaling factors, including Src and SIRT1 inhibition and activation of the autophagic regulators LAMP2 and LC3-I/II, contributed to the autophagy-dependent apoptosis. We found that C2-ceramide continuously initiated autophagy; however, CQ inhibited autophagosome maturation and degradation during autophagy progression. Accumulated and non-degraded autophagosomes increased NSCLC cell stress, eventually leading to cell death. This study sheds light on improvements to NSCLC chemotherapy to reduce the chemotherapy dose and NSCLC patient burden.
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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.
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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
BH3 domains, classified initially as BCL2 homology domains, participate in both apoptosis and autophagy. Beclin1 contains a BH3 domain, which is required for binding to antiapoptotic BCL2 homologs and BCL2mediated inhibition of autophagy. BCL2like 12 (BCL2L12) also harbors a BH3like domain, which is 12 residues long and contains a LXXXAE/D motif. In a yeast twohybrid system performed in the present study, BCL2L12 shared similar binding partnerships to antiapoptotic BCL2 homologs, such as Beclin1. In addition, this BH3like domain was involved in antiapoptosis and druginduced autophagy in glioma cell lines. Mutations in S156 and hydrophobic L213 to alanine counteracted the antiapoptotic properties of BCL2L12 and downregulated the activation of microtubule associated protein 1 light chain 3B (LC3B), autophagyrelated (ATG)12ATG5 conjugates and Beclin1, compared with a BCL2L12 wildtype group. Molecular dynamics simulations revealed that phosphorylation at Ser156 of BCL2L12 (within α6 and α7 helices) influenced the BH3like domain conformation (α9 helix), indicating that glycogen synthase kinase (GSK) 3ßmediated Ser156 phosphorylation modulated a BH3like domain in BCL2L12. Altogether, the present findings indicated that BCL2L12 may participate in antiapoptosis and autophagy via a BH3like domain and GSK3ßmediated phosphorylation at Ser156. Furthermore, blockade of temozolomide (TMZ)induced autophagy by 3methyladenine (3MA) resulted in enhanced activation of apoptotic markers, as well as tumor suppresor protein p53 (p53) expression in U87MG cells. The present results suggested that p53 and O6methylguanine DNA methyltransferase activation, and BCL2, BCLextra large, Beclin1 and BCL2L12 expression may be used as a detection panel to determine which patients can benefit from TMZ and ABT737 combination treatment.
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Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Dacarbazina/análogos & derivados , Glioma/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Musculares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linhagem Celular Tumoral , Dacarbazina/farmacologia , Glioma/metabolismo , Glioma/patologia , Humanos , Modelos Moleculares , Proteínas Musculares/química , Fosforilação/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/química , TemozolomidaRESUMO
GSK3ß interacting protein (GSKIP) is a naturally occurring negative regulator of GSK3ß and retains both the Protein Kinase A Regulatory subunit binding (PKA-RII) domain and GSK3ß interacting domain. Of these two domains, we found that PKA-RII is required for forming a working complex comprising PKA/GSKIP/GSK3ß/Drp1 to influence phosphorylation of Drp1 Ser637. In this study, bioinformatics and experimental explorations re-analyzing GSKIP's biofunctions suggest that the evolutionarily conserved Domain of Unknown Function (DUF727) is an ancestral prototype of GSKIP in prokaryotes, and acquired the C-terminal GSK3ß binding site (tail) in invertebrates except for Saccharomyces spp., after which the N-terminal PKA-RII binding region (head) evolved in vertebrates. These two regions mutually influence each other and modulate GSKIP binding to GSK3ß in yeast two-hybrid assays and co-immunoprecipitation. Molecular modeling showed that mammalian GSKIP could form a dimer through the L130 residue (GSK3ß binding site) rather than V41/L45 residues. In contrast, V41/L45P mutant facilitated a gain-of-function effect on GSKIP dimerization, further influencing binding behavior to GSK3ß compared to GSKIP wild-type (wt). The V41/L45 residues are not only responsible for PKA RII binding that controls GSK3ß activity, but also affect dimerization of GSKIP monomer, with net results of gain-of-function in GSKIP-GSK3ß interaction. In addition to its reported role in modulating Drp1, Ser637 phosphorylation caused mitochondrial elongation; we postulated that GSKIP might be involved in the Wnt signaling pathway as a scavenger to recruit GSK3ß away from the ß-catenin destruction complex and as a competitor to compete for GSK3ß binding, resulting in accumulation of S675 phosphorylated ß-catenin.
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Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt , Sítios de Ligação , Biologia Computacional , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinaminas , Evolução Molecular , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Fosforilação , Filogenia , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Repressoras/genética , Serina/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Since 2015, widespread Zika virus outbreaks in Central and South America have caused increases in microcephaly cases, and this acute problem requires urgent attention. We employed molecular dynamics and Gaussian accelerated molecular dynamics techniques to investigate the structure of Zika NS5 protein with S-adenosyl-L-homocysteine (SAH) and an RNA analogue, namely 7-methylguanosine 5'-triphosphate (m7GTP). For the binding motif of Zika virus NS5 protein and SAH, we suggest that the four Zika NS5 substructures (residue orders: 101-112, 54-86, 127-136 and 146-161) and the residues (Ser56, Gly81, Arg84, Trp87, Thr104, Gly106, Gly107, His110, Asp146, Ile147, and Gly148) might be responsible for the selectivity of the new Zika virus drugs. For the binding motif of Zika NS5 protein and m7GTP, we suggest that the three Zika NS5 substructures (residue orders: 11-31, 146-161 and 207-218) and the residues (Asn17, Phe24, Lys28, Lys29, Ser150, Arg213, and Ser215) might be responsible for the selectivity of the new Zika virus drugs.
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Metiltransferases/genética , Proteínas não Estruturais Virais/metabolismo , Zika virus/genética , Antivirais/química , Sítios de Ligação , Cristalografia por Raios X , Metiltransferases/metabolismo , Simulação de Dinâmica Molecular , Distribuição Normal , Ligação Proteica , RNA/metabolismo , Análogos de Capuz de RNA/genética , Análogos de Capuz de RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , S-Adenosil-Homocisteína/metabolismo , Proteínas não Estruturais Virais/ultraestrutura , Zika virus/metabolismo , Infecção por Zika virus/genéticaRESUMO
Pluchea indica (L.) Less. is a perennial plant known for its versatile uses in traditional medicine. Previous findings have shown that the extracts of Pluchea indica possess significant anti-inflammatory, anti-ulcer and anti-tuberculosis activity. The aim of this study was to demonstrate the anticancer activity of the hexane fraction of P. indica root extract (H-PIRE) in human glioblastoma cells using flow cytometric and western blot analysis. The results shoewd that, H-PIRE suppressed the growth of glioblastoma cells in a dose-dependent manner. H-PIRE treatment markedly decreased the population of cells in S and G2/M phases. The significant upregulation of acidic vesicular organelle (AVO) was detected during H-PIRE treatment. The expression levels of microtubule-associated light chain 3-II (LC3-II) protein, phosphorylated JNK and phosphorylated p38 were significantly increased, confirming the occurrence of autophagy during the process. Finally, the combination treatment of H-PIRE and LY294002, a pan PI3K inhibitor, further decreased cell viability, suggesting an additive anticancer effect. Taken together, our results suggest that H-PIRE suppresses the proliferation of glioblastoma cells by inducing cell cycle arrest and autophagy.
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Hepatocellular carcinoma (HCC) is a leading cancer worldwide. Advanced HCCs are usually resistant to anticancer drugs, causing unsatisfactory chemotherapy outcomes. In this study, we showed that a 4-phenoxyphenol derivative, 4-[4-(4-hydroxyphenoxy)phenoxy]phenol (4-HPPP), exerts an inhibitory activity against two HCC cell lines, Huh7 and Ha22T. We further investigated the anti-HCC activities of 4-HPPP, including anti-proliferation and induction of apoptosis. Our results showed that higher dosage of 4-HPPP downregulates the expression of α-tubulin and causes nuclear enlargement in both the Huh-7 and Ha22T cell lines. Interestingly, the colony formation results showed a discrepancy in the inhibitory effect of 4-HPPP on HCC and rat liver epithelial Clone 9 cells, suggesting the selective cytotoxicity of 4-HPPP toward HCC cells. Furthermore, the cell proliferation and apoptosis assay results illustrated the differences between the two HCC cell lines. The results of cellular proliferation assays, including trypan blue exclusion and colony formation, revealed that 4-HPPP inhibits the growth of Huh7 cells, but exerts less cytotoxicity in Ha22T cells. Furthermore, the annexin V assay performed for detecting the apoptosis showed similar results. Western blotting results showed 4-HPPP caused the increase of pro-apoptotic factors including cleaved caspase-3, Bid and Bax in HCC cells, especially in Huh-7. Furthermore, an increase of autophagy-associated protein microtubule-associated protein-1 light chain-3B (LC3B)-II and the decrease of Beclin-1 and p62/SQSTM1 were observed following 4-HPPP treatment. Additionally, the level of γH2A histone family, member X (γH2AX), an endogenous DNA damage biomarker, was dramatically increased in Huh7 cells after 4-HPPP treatment, suggesting the involvement of DNA damage pathway in 4-HPPP-induced apoptosis. On the contrary, the western blotting results showed that treatment up-regulates pro-survival proteins, including the phosphorylation of protein kinase B (Akt) and the level of survivin on Ha22T cells, which may confer a resistance toward 4-HPPP. Notably, the blockade of extracellular signal-regulated kinases (ERK), but not Akt, enhanced the cytotoxicity of 4-HPPP against Ha22T cells, indicating the pro-survival role of ERK in 4-HPPP-induced anti-HCC effect. Our present work suggests that selective anti-HCC activity of 4-HPPP acts through induction of DNA damage. Accordingly, the combination of ERK inhibitor may significantly enhance the anti-cancer effect of 4-HPPP for those HCC cells which overexpress ERK in the future.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Éteres Fenílicos/farmacologia , Tubulina (Proteína)/genética , Animais , Antineoplásicos/síntese química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/patologia , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Especificidade de Órgãos , Éteres Fenílicos/síntese química , Ratos , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Tubulina (Proteína)/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
We analyzed a panel of cationic molecules secreted in the culture medium of human respiratory epithelial cells (REC) upon activation by IL-1ß and different pathogen-associated molecular patterns. A 9 kDa fragment derived from ß2-microglobulin (B2M) was identified and named shed 9 kDa B2M (sB2M-9). The primary structure of sB2M-9 was revealed to increase its pI value that potentially could play an important role in innate defense. sB2M-9 exhibits antibacterial activity against Gram positive Staphylococcus aureus (SA) but not against Gram negative Klebsiella pneumonia (KP). Upon its binding to SA, sB2M-9 induces clumps, a phenomenon not observed with B2M. Migration of THP-1 monocytes exposed to SA clumps was significantly greater than that to SA without clumps. sB2M-9 binds to SA, more likely as a chemokine, to facilitate THP-1 migration. As a whole, we demonstrated that REC release a novel chemokine with antibacterial activity that is shed from B2M to facilitate THP-1 migration.
Assuntos
Antibacterianos/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Quimiocinas/imunologia , Mucosa Respiratória/imunologia , Microglobulina beta-2/imunologia , Sequência de Aminoácidos , Linhagem Celular , Quimiocina CXCL5/imunologia , Humanos , Imunidade Inata , Interleucina-1beta/imunologia , Monócitos/imunologia , Proteínas Nucleares/imunologia , Moléculas com Motivos Associados a Patógenos/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Mucosa Respiratória/microbiologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade , Proteínas Supressoras da Sinalização de Citocina/imunologia , Células THP-1 , Microglobulina beta-2/química , Microglobulina beta-2/genéticaRESUMO
Areca nut (AN) chewing is associated with chronic kidney disease (CKD). However, the molecular mechanisms of AN-induced CKD are not known. Thus, we studied the effects of arecoline, a major alkaloid of AN, on proximal tubule (LLC-PK1) cells in terms of cytotoxicity, fibrosis, transforming growth factor-ß (TGF-ß) and c-Jun N-terminal kinase (JNK). We found that arecoline dose (0.1-0.5mM) and time (24-72h)-dependently induced cytotoxicity without causing cell death. Arecoline (0.25 mM) also time-dependently (24-72h) increased fibronectin and plasminogen activator inhibitor-1 (PAI1) protein expressions. Arecoline (0.25 mM) time-dependently (24-72h) increased TGF-ß gene transcriptional activity and supernatant levels of active TGF-ß1. Moreover, arecoline (0.25 mM) activated JNK while SP600125 (a JNK inhibitor) attenuated arecoline-induced TGF-ß gene transcriptional activity. SP600125, but not SB431542 (a TGF-ß receptor type I kinase inhibitor), attenuated arecoline-induced fibronectin and PAI1 protein expressions. Finally, tubulointerstitial fibrosis occurred and renal cortical expressions of fibronectin and PAI1 proteins increased in arecoline-fed mice at 24 weeks. We concluded that arecoline induced tubulointerstitial fibrosis in mice while arecoline-induced TGF-ß and pro-fibrotic proteins (fibronectin, PAI1) are dependent on JNK in LLC-PK1 cells.
Assuntos
Arecolina/toxicidade , Fibrose/induzido quimicamente , Fibrose/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/enzimologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Relação Dose-Resposta a Droga , Fibronectinas/biossíntese , Humanos , Células LLC-PK1 , Masculino , Camundongos , Camundongos Endogâmicos ICR , Inibidor 1 de Ativador de Plasminogênio/biossíntese , SuínosRESUMO
ß2-microglobulin (ß2M) functions as a chaperon to maintain structural stability of MHC class I complex that is associated with antigen presentation to cytotoxic (CD8+) T lymphocytes. Cancerous cells in ß2M loss-of-function are thought to avoid immune surveillance. As increased level of ß2M present in tissue/serum is significantly associated with tumor status in various cancers, ß2M may become an important prognostic and survival factor in a range of malignancies. It is believed that ß2M acts as hormone-like molecule to trigger a pleiotropic signaling via a ligand-to-receptor binding mechanism. Anti- ß2M monoclonal antibodies successfully induce apoptosis in malignant cells, suggesting a surprising therapeutic approach. Of note, ß2M is largely localized in the cytoplasm of advanced oral cavity squamous cell carcinoma (OCSCC), in contrast to that in the plasma membrane of normal oral mucosa. This suggests that ß2M-derived intracellular signaling might be preceded by its accumulation in the cytoplasm of epithelial cells of tumors. Hence, translocation of ß2M from cell surface to cytoplasm in advanced tumors may shed light on the mechanism of ß2M-mediated tumorigenesis.
Assuntos
Mutação/genética , Neoplasias/genética , Microglobulina beta-2/genética , Animais , Humanos , Neoplasias/metabolismoRESUMO
Transforming growth factor-ß (TGF-ß) is pivotal in the pathogenesis of diabetic nephropathy. Type 1 TGF-ß receptor (TGF-ßR1) is degraded by Smad7-dependent ubiquitination-proteasomal pathway, which is deubiquitinated by ubiquitin C-terminal hydrolase-L5 (UCHL5). Therefore, we studied the role of UCHL5 in high glucose (27.8mM)-induced TGF-ßR1 protein expression in mouse mesangial (MES13) cells. UCHL5 short hairpin RNA (shRNA) was used to knock down UCHL5 while LY294002 and the dominant-negative p85 were used to inhibit phosphatidylinositol-3-kinase (PI3K). We found that high glucose increased phospho-Akt, TGF-ßR1 mRNA and protein expression. High glucose also increased UCHL5 protein expression, which was attenuated by LY294002, the dominant-negative p85 and the dominant-negative CREB. High glucose-induced TGF-ßR1 protein expression and TGF-ßR1 protein deubiquitination were attenuated by UCHL5 shRNA. Additionally, high glucose-induced p21(WAF1), fibronectin protein expression and cell hypertrophy were attenuated by UCHL5 shRNA. However, high glucose-induced TGF-ßR1 mRNA, p27(kip1) protein expression and growth inhibition were not affected by UCHL5 shRNA. Finally, glomerular UCHL5 and TGF-ßR1 protein expression were increased in streptozotocin-diabetic rats at 8weeks. We conclude that PI3K-dependent UCHL5 is required for high glucose-induced TGF-ßR1 protein expression in mesangial cells. UCHL5 is also required for high glucose-induced TGF-ßR1 protein deubiquitination, p21(WAF1) and fibronectin protein expression and cell hypertrophy.
Assuntos
Carboxipeptidases/metabolismo , Glucose/metabolismo , Células Mesangiais/citologia , Células Mesangiais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Animais , Linhagem Celular , Tamanho Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Diabetes Mellitus Experimental/metabolismo , Fibronectinas/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais/metabolismo , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo I , Ubiquitina TiolesteraseRESUMO
Transforming growth factor-ß (TGF-ß), TGF-ß receptor (TGF-ßR), and epidermal growth factor receptor (EGFR) are important in the pathogenesis of kidney fibrosis, a result of renal fibroblast activation. The EGFR kinase inhibitor gefitinib attenuates glomerular fibrosis in hypertensive rats whereas dominant-negative EGFR attenuates interstitial fibrosis in mouse with acute renal ischemia. Thus, we studied the effects and molecular mechanisms of gefitinib in TGF-ß1-induced mitogenesis and collagen production in normal rat kidney interstitial fibroblast (NRK-49F) cells. We found that TGF-ß1 increased cell mitogenesis. TGF-ß1 also time-dependently increased cyclin D1 protein expression. TGF-ß1 rapidly transactivated EGFR. SB431542 (a type I TGF-ßR kinase inhibitor) and SB203580 (a p38 kinase inhibitor) attenuated TGF-ß1-induced phosphorylation of Smad2/3 protein. SB431542 and gefitinib attenuated TGF-ß1-induced phosphorylation of ERK1/2 and p38 kinase. SB431542 and gefitinib also attenuated TGF-ß1-induced cyclin D1 protein expression. Moreover, SB431542, gefitinib, PD98059 (an ERK1/2 inhibitor), and SB203580 attenuated TGF-ß1-induced cell mitogenesis. Finally, SB431542 and gefitinib attenuated TGF-ß1-induced collagen production. We concluded that gefitinib attenuates TGF-ß1-induced cell mitogenesis via the EGFR-ERK1/2/p38 kinase pathway in NRK-49F cells. Moreover, gefitinib attenuates TGF-ß1-induced cyclin D1 protein expression and collagen production. Thus, gefitinib attenuates TGF-ß1-induced mitogenesis and collagen production in vitro.