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1.
Sci Rep ; 11(1): 6618, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758209

RESUMO

Cancer metastasis is a major cause of the high mortality rate in lung cancer patients. The cytoskeletal rearrangement and degradation of extracellular matrix are required to facilitate cell migration and invasion and the suppression of these behaviors is an intriguing approach to minimize cancer metastasis. Even though Erianthridin (ETD), a phenolic compound isolated from the Thai orchid Dendrobium formosum exhibits various biological activities, the molecular mechanism of ETD for anti-cancer activity is unclear. In this study, we found that noncytotoxic concentrations of ETD (≤ 50 µM) were able to significantly inhibit cell migration and invasion via disruption of actin stress fibers and lamellipodia formation. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 was markedly downregulated in a dose-dependent manner after ETD treatment. Mechanistic studies revealed that protein kinase B (Akt) and its downstream effectors mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70S6K) were strongly attenuated. An in silico study further demonstrated that ETD binds to the protein kinase domain of Akt with both hydrogen bonding and van der Waals interactions. In addition, an in vivo tail vein injection metastasis study demonstrated a significant effect of ETD on the suppression of lung cancer cell metastasis. This study provides preclinical information regarding ETD, which exhibits promising antimetastatic activity against non-small-cell lung cancer through Akt/mTOR/p70S6K-induced actin reorganization and MMPs expression.


Assuntos
Antineoplásicos/farmacologia , Fenantrenos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Actinas/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Humanos , Metaloproteinases da Matriz/metabolismo , Modelos Moleculares , Fenantrenos/química , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/química
2.
Arch Biochem Biophys ; 674: 108105, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31518555

RESUMO

Currently, there is a lack of investigation into the initial signaling events underlying the development of disuse muscle atrophy. The study was aimed to (i) identify an assumed relationship between AMPK dephosphorylation and p70S6K hyperphosphorylation in the initial period of hindlimb unloading (HS), and (ii) assess the signaling consequences of p70S6K hyperphosphorylation following 24-h HS. For experiment 1, rats were treated with AMPK activator (AICAR) for 6 d before HS as well as during 24-h HS. For experiment 2, rats were treated with mTORC1 inhibitor rapamycin during 24-h HS. The key signaling markers implicated in protein turnover were assessed using WB and RT-PCR. One-day HS resulted in a significant upregulation of MuRF-1 and MAFbx expression, increase in p70S6K (Thr389) and IRS-1 (Ser639) phosphorylation and a significant decrease in phosphorylated AMPK, AKT, FOXO3, total IRS-1 content, and HDAC5 nuclear content. AMPK and p70S6K phosphorylation did not differ from control in AICAR-treated unloaded rats. Rapamycin treatment during unloading abolished p70S6K and E3 ligases upregulation and increased HDAC5 nuclear accumulation. The results of the study suggest that mTORC-1/p70S6K signaling pathway in rat soleus muscle is activated following 24-h mechanical unloading. This activation is facilitated by a decrease in AMPK phosphorylation. Increased p70S6K activity at the initial stage of hindlimb unloading could lead to the upregulation of E3 ligases MAFbx/atrogin-1 and MuRF-1 via nuclear export of HDAC5.


Assuntos
Músculo Esquelético/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Elevação dos Membros Posteriores , Histona Desacetilases/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Ratos Wistar , Ribonucleotídeos/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Sirolimo/farmacologia , Treonina/química , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima
3.
BMC Cancer ; 19(1): 773, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387554

RESUMO

BACKGROUND: The mTOR/S6K1 signaling pathway is often activated in cervical cancer, and thus considered a molecular target for cervical cancer therapies. Inhibiting mTOR is cytotoxic to cervical cancer cells and creates a synergistic anti-tumor effect with conventional chemotherapy agents. In this study, we identified a novel S6K1 inhibitor, rosmarinic acid methyl ester (RAME) for the use of therapeutic agent against cervical cancer. METHODS: Combined structure- and ligand-based virtual screening was employed to identify novel S6K1 inhibitors among the in house natural product library. In vitro kinase assay and immunoblot assay was used to examine the effects of RAME on S6K1 signaling pathway. Lipidation of LC3 and mRNA levels of ATG genes were observed to investigate RAME-mediated autophagy. PARP cleavage, mRNA levels of apoptotic genes, and cell survival was measured to examine RAME-mediated apoptosis. RESULTS: RAME was identified as a novel S6K1 inhibitor through the virtual screening. RAME, not rosmarinic acid, effectively reduced mTOR-mediated S6K1 activation and the kinase activity of S6K1 by blocking the interaction between S6K1 and mTOR. Treatment of cervical cancer cells with RAME promoted autophagy and apoptosis, decreasing cell survival rate. Furthermore, we observed that combination treatment with RAME and cisplatin greatly enhanced the anti-tumor effect in cisplatin-resistant cervical cancer cells, which was likely due to mTOR/S6K1 inhibition-mediated autophagy and apoptosis. CONCLUSIONS: Our findings suggest that inhibition of S6K1 by RAME can induce autophagy and apoptosis in cervical cancer cells, and provide a potential option for cervical cancer treatment, particularly when combined with cisplatin.


Assuntos
Antineoplásicos/farmacologia , Cinamatos/farmacologia , Depsídeos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cinamatos/química , Cisplatino/farmacologia , Depsídeos/química , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Técnicas de Silenciamento de Genes , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Neoplasias do Colo do Útero
4.
Future Oncol ; 15(1): 95-102, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30730779

RESUMO

S6K2, the newer member of S6 Kinase family, is a crucial modulator of Akt/mTOR signaling pathway and is a member of AGC kinase family that regulates cellular growth and survival. S6K1 and S6K2 share high sequence similarity; therefore, S6K2 had been underestimated. However, recent studies displayed distinct functions of S6K2. Activated by both Akt/mTOR and Ras/Raf/Mek/Erk signaling pathways, S6K2 regulates cancer cell survival via different routes. Complexation with antiapoptotic proteins BRAF and PKCε avoids non-small-cell lung cancer cells  from apoptosis upon FGF-2 stimulation. Indirect upregulation of the translation of antiapoptotic proteins Bcl-XL and XIAP in HEK293T cells and interference with TNF-induced apoptosis in MCF-7 cells are other routes of cancer cell survival. The aforementioned studies on S6K2 necessitate the development of therapies targeting only on S6K2. Studies targeting S6K2 may help to build important roads for cancer therapy.


Assuntos
Neoplasias/enzimologia , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proliferação de Células , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/patologia
5.
Mol Carcinog ; 57(11): 1540-1552, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30035335

RESUMO

Esophageal squamous cell carcinoma (ESCC) is highly prevalent in Asia, especially in China. Research findings indicate that nitrosamines, malnutrition, unhealthy living habits, and genetics contribute to esophageal carcinogenesis. Currently, the 5-year survival rate for ESCC patients remains low, owing in part to a lack of a clear understanding of mechanisms involved. Chemoprevention using natural or synthesized compounds might be a promising strategy to reduce esophageal cancer incidence. The epidermal growth factor receptor (EGFR) can activate downstream pathways including the phosphatidylinositol 3-kinase (PI3K) pathway and the Ras/mitogen-activated protein kinase (MAPK) pathways. Among the important players, AKT and ERKs have an important relationship with cancer initiation and progression. Here, we found that phosphorylated (p)-AKT and p-ERKs were highly expressed in esophageal cancer cell lines and in esophageal cancer patients. Human phospho-kinase array and pull-down assay results showed that quercetin-3-methyl ether (Q3ME) is a natural flavonoid compound that interacted with AKT and ERKs and inhibited their kinase activities. At the cellular level, Q3ME attenuated esophageal cancer cell proliferation and anchorage-independent growth. Western blot analysis showed that this compound suppressed the activation of AKT and ERKs downstream signaling pathways, subsequently inhibiting activating protein-1 (AP-1) activity. Importantly, Q3ME inhibited the formation of esophageal preneoplastic lesions induced by N-nitrosomethylbenzylamine (NMBA). The inhibition by Q3ME was associated with decreased inflammation and esophageal cancer cell proliferation in vivo. Collectively, our data suggest that Q3ME is a promising chemopreventive agent against esophageal carcinogenesis by targeting AKT and ERKs.


Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Esofágicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quercetina/análogos & derivados , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Mucosa Esofágica/metabolismo , Neoplasias Esofágicas/patologia , Humanos , Inflamação/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Estrutura Molecular , Proteínas Proto-Oncogênicas c-akt/química , Quercetina/química , Quercetina/farmacologia , Ratos , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/química
6.
Cell Death Dis ; 9(3): 407, 2018 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-29540819

RESUMO

Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70 S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1S757 phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPKT172 and ULK1S555 phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-ß-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1G93A protein aggregates with autophagosomes and accelerated SOD1G93A protein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.


Assuntos
Compostos de Anilina/farmacologia , Autofagia/efeitos dos fármacos , Hidroxibutiratos/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Superóxido Dismutase-1/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Motivos de Aminoácidos , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular , Crotonatos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Nitrilas , Agregados Proteicos/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Superóxido Dismutase-1/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Toluidinas
7.
Int J Biol Macromol ; 102: 625-629, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28431943

RESUMO

The p70ΔCT104 S6K is a 421 amino acid residue long truncated form of p70S6 kinase, with 104 amino acids residues cleaved from the carboxyl terminal end of the original protein. The p70ΔCT104 S6K was cloned in E. coli DH5α and successfully expressed in E. coli BL21 (DE3) strain. Western blot with rabbit polyclonal anti-GST antibody was used to follow the protein during expression and purification. The protein purification was achieved by affinity chromatography using Glutathione resin-agarose beads, followed by chromatography on a spin concentration column. The purified protein was confirmed by rabbit polyclonal anti-p70S6 kinase antibody. MALDI/MS Peptide mass fingerprinting confirmed identity of the expressed product.


Assuntos
Escherichia coli/genética , Glutationa Transferase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/isolamento & purificação , Deleção de Sequência , Clonagem Molecular , Expressão Gênica , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química
8.
J Biol Chem ; 291(35): 18410-8, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27365393

RESUMO

Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser(36) as the major p70S6k phosphorylation site, along with a low frequency site at Thr(40), using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 µmol/min/mg) of the activity of WT enzyme (95 µmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser(36) is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser(36) upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg(104) in the adjacent subunit. Because Arg(104) is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser(36) inhibits the catalytic function of LTC4S by interference with the catalytic machinery.


Assuntos
Glutationa Transferase/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Catálise , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Leucotrieno A4/biossíntese , Leucotrieno A4/química , Leucotrieno A4/genética , Camundongos , Mutação de Sentido Incorreto , Fosforilação , Estrutura Secundária de Proteína , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina/química , Serina/genética , Serina/metabolismo
9.
Proc Natl Acad Sci U S A ; 113(28): 7876-81, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27342859

RESUMO

Viruses depend upon the host cell for manufacturing components of progeny virions. To mitigate the inextricable dependence on host cell protein synthesis, viruses can modulate protein synthesis through a variety of mechanisms. We report that the viral protein kinase (vPK) encoded by open reading frame 36 (ORF36) of Kaposi's sarcoma-associated herpesvirus (KSHV) enhances protein synthesis by mimicking the function of the cellular protein S6 kinase (S6KB1). Similar to S6KB1, vPK phosphorylates the ribosomal S6 protein and up-regulates global protein synthesis. vPK also augments cellular proliferation and anchorage-independent growth. Furthermore, we report that both vPK and S6KB1 phosphorylate the enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) and that both kinases promote endothelial capillary tubule formation.


Assuntos
Herpesvirus Humano 8/enzimologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Virais/metabolismo , Simulação por Computador , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Moleculares , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Especificidade por Substrato , Proteínas Virais/química
10.
PLoS One ; 10(12): e0145013, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26698305

RESUMO

BACKGROUND: The AKT/mTORC1/S6K pathway is frequently overstimulated in breast cancer, constituting a promising therapeutic target. The benefit from mTOR inhibitors varies, likely as a consequence of tumour heterogeneity, and upregulation of several compensatory feed-back mechanisms. The mTORC1 downstream effectors S6K1, S6K2, and 4EBP1 are amplified and overexpressed in breast cancer, associated with a poor outcome and divergent endocrine treatment benefit. S6K1 and S6K2 share high sequence homology, but evidence of partly distinct biological functions is emerging. The aim of this work was to explore possible different roles and treatment target potentials of S6K1 and S6K2 in breast cancer. MATERIALS AND METHODS: Whole-genome expression profiles were compared for breast tumours expressing high levels of S6K1, S6K2 or 4EBP1, using public datasets, as well as after in vitro siRNA downregulation of S6K1 and/or S6K2 in ZR751 breast cancer cells. In silico homology modelling of the S6K2 kinase domain was used to evaluate its possible structural divergences to S6K1. RESULTS: Genome expression profiles were highly different in S6K1 and S6K2 high tumours, whereas S6K2 and 4EBP1 profiles showed significant overlaps, both correlated to genes involved in cell cycle progression, among these the master regulator E2F1. S6K2 and 4EBP1 were inversely associated with IGF1 levels, and their prognostic value was shown to be restricted to tumours positive for IGFR and/or HER2. In vitro, S6K1 and S6K2 silencing resulted in upregulation of genes in the mTORC1 and mTORC2 complexes. Isoform-specific silencing also showed distinct patterns, e.g. S6K2 downregulation lead to upregulation of several cell cycle associated genes. Structural analyses of the S6K2 kinase domain showed unique structure patterns, deviating from those of S6K1, facilitating the development of isoform-specific inhibitors. Our data support emerging proposals of distinct biological features of S6K1 and S6K2, suggesting their importance as separate oncogenes and clinical markers, where specific targeting in different breast cancer subtypes could facilitate further individualised therapies.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Serina-Treonina Quinases TOR/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Conformação Proteica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Taxa de Sobrevida , Células Tumorais Cultivadas
11.
Clin Cancer Res ; 21(21): 4922-34, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26080838

RESUMO

PURPOSE: To investigate the involvement of hsa-miRNA-195-5p (miR-195) in progression and prognosis of human prostate cancer. EXPERIMENTAL DESIGN: qRT-PCR was performed to detect miR-195 expression in both prostate cancer cell lines and clinical tissue samples. Its clinical significance was statistically analyzed. The roles of miR-195 and its candidate target gene, ribosomal protein S6 kinase, 70 kDa, polypeptide 1 (RPS6KB1) in prostate cancer progression were confirmed on the basis of both in vitro and in vivo systems. RESULTS: miR-195 downregulation in prostate cancer tissues was significantly associated with high Gleason score (P = 0.001), positive metastasis failure (P < 0.001), and biochemical recurrence (BCR, P < 0.001). Survival analysis identified miR-195 as an independent prognostic factor for BCR-free survival of prostate cancer patients (P = 0.022). Then, we confirmed the tumor suppressive role of miR-195 through prostate cancer cell invasion, migration, and apoptosis assays in vitro, along with tumor xenograft growth, angiogenesis, and invasion in vivo according to both gain-of-function and loss-of-function experiments. In addition, RPS6KB1 was identified as a novel direct target of miR-195 through proteomic expression profiling combined with bioinformatic target prediction and luciferase reporter assay. Moreover, the reexpression and knockdown of RPS6KB1 could respectively rescue and imitate the effects induced by miR-195. Importantly, RPS6KB1 expression was closely correlated with aggressive progression and poor prognosis in prostate cancer patients as opposed to miR-195. Furthermore, we identified MMP-9, VEGF, BAD, and E-cadherin as the downstream effectors of miR-195-RPS6KB1 axis. CONCLUSION: The newly identified miR-195-RPS6KB1 axis partially illustrates the molecular mechanism of prostate cancer progression and represents a novel potential therapeutic target for prostate cancer treatment.


Assuntos
MicroRNAs/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Regiões 3' não Traduzidas , Animais , Apoptose/genética , Sequência de Bases , Sítios de Ligação , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , MicroRNAs/química , Neovascularização Patológica/genética , Prognóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Proteômica/métodos , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Carga Tumoral/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo
12.
J Med Chem ; 58(1): 305-14, 2015 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-25356520

RESUMO

Aberrant activation of S6 kinase 1 (S6K1) is found in many diseases, including diabetes, aging, and cancer. We developed ATP competitive organometallic kinase inhibitors, EM5 and FL772, which are inspired by the structure of the pan-kinase inhibitor staurosporine, to specifically inhibit S6K1 using a strategy previously used to target other kinases. Biochemical data demonstrate that EM5 and FL772 inhibit the kinase with IC50 value in the low nanomolar range at 100 µM ATP and that the more potent FL772 compound has a greater than 100-fold specificity over S6K2. The crystal structures of S6K1 bound to staurosporine, EM5, and FL772 reveal that the EM5 and FL772 inhibitors bind in the ATP binding pocket and make S6K1-specific contacts, resulting in changes to the p-loop, αC helix, and αD helix when compared to the staurosporine-bound structure. Cellular data reveal that FL772 is able to inhibit S6K phosphorylation in yeast cells. Together, these studies demonstrate that potent, selective, and cell permeable S6K1 inhibitors can be prepared and provide a scaffold for future development of S6K inhibitors with possible therapeutic applications.


Assuntos
Compostos Organometálicos/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Western Blotting , Linhagem Celular Tumoral , Descoberta de Drogas , Células HEK293 , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Rutênio/química
13.
PLoS One ; 9(12): e114389, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25486532

RESUMO

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.


Assuntos
Antracenos/farmacologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Antracenos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Humanos , Isoquinolinas/farmacologia , Modelos Moleculares , Conformação Molecular , Mutação , Fosforilação/efeitos dos fármacos , Poliploidia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Sulfonamidas/farmacologia
14.
Cell Signal ; 26(3): 461-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24316235

RESUMO

The mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1) pathway integrates signals generated by hormones and nutrients to control cell growth and metabolism. The activation state of mTORC1 is regulated by a variety of GTPases including Rheb and Rags. Recently, Rho1, the yeast ortholog of RhoA, was shown to interact directly with TORC1 and repress its activation state in yeast. Thus, the purpose of the present study was to test the hypothesis that the RhoA GTPase modulates signaling through mTORC1 in mammalian cells. In support of this hypothesis, exogenous overexpression of either wild type or constitutively active (ca)RhoA repressed mTORC1 signaling as assessed by phosphorylation of p70S6K1 (Thr389), 4E-BP1 (Ser65) and ULK1 (Ser757). Additionally, RhoA·GTP repressed phosphorylation of mTORC1-associated mTOR (Ser2481). The RhoA·GTP mediated repression of mTORC1 signaling occurred independent of insulin or leucine induced stimulation. In contrast to the action of Rho1 in yeast, no evidence was found to support a direct interaction of RhoA·GTP with mTORC1. Instead, expression of caRheb, but not caRags, was able to rescue the RhoA·GTP mediated repression of mTORC1 suggesting RhoA functions upstream of Rheb to repress mTORC1 activity. Consistent with this suggestion, RhoA·GTP repressed phosphorylation of TSC2 (Ser939), PRAS40 (Thr246), Akt (Ser473), and mTORC2-associated mTOR (Ser2481). Overall, the results support a model in which RhoA·GTP represses mTORC1 signaling upstream of Akt and mTORC2.


Assuntos
Complexos Multiproteicos/antagonistas & inibidores , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas de Ciclo Celular , Linhagem Celular , Efrina-A5/biossíntese , Fibroblastos , Células HEK293 , Humanos , Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Leucina/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Proteínas Monoméricas de Ligação ao GTP/biossíntese , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Neuropeptídeos/biossíntese , Neuropeptídeos/metabolismo , Fosfoproteínas/química , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas c-akt/química , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Ratos , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/química , Proteína rhoA de Ligação ao GTP/biossíntese
15.
Mol Divers ; 17(4): 767-72, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23982212

RESUMO

S6K1 has emerged as a potential target for the treatment for obesity, type II diabetes and cancer diseases. Discovery of S6K1 inhibitors has thus attracted much attention in recent years. In this investigation, a hybrid virtual screening method that involves pharmacophore hypothesis, genetic function approximation (GFA) model, and molecular docking technology has been used to discover S6K1 inhibitors especially with novel scaffolds. The common feature pharmacophore hypothesis and GFA regression model of S6K1 inhibitors were first developed and applied in a virtual screen of the Specs database for retrieving S6K1 inhibitors. Then, the molecular docking method was carried out to re-filter these screened compounds. Finally, 60 compounds with promising S6K1 inhibitory activity were carefully selected and have been handed over to the other group to complete the follow-up compound synthesis (or purchase) and activity test.


Assuntos
Modelos Moleculares , Inibidores de Proteínas Quinases/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Bases de Dados Factuais , Desenho de Fármacos , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Relação Estrutura-Atividade
16.
J Biol Regul Homeost Agents ; 27(2): 399-408, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23830390

RESUMO

S6K1 regulation associates a central role with dynamics of sequential phosphorylations at the hydrophobic motif (T412) and activation loop (T252) of the enzyme, such that the hydrophobic motif phosphorylation supposedly brought about by mTOR- kinase, primes the enzyme for PDK1 dependent phosphorylation at the activation loop for its full activation. Accordingly loss of hydrophobic motif phosphorylation attributed to TOR- kinase inhibition, with resultant loss of activation loop phosphorylation is the hypothesis put forward to explain the mechanism of rapamycin inhibition. Our recent observation that rapamycin continues to inhibit S6K1 in the absence of either phosphorylation, together with the evidence that phosphorylation at activation loop may occur prior to that of hydrophobic motif raises serious questions about the proposed mechanism of rapamycin inhibition. Here, we show that rapamycin fails to effect preferential loss of either phosphorylation and the two instead exhibit equal sensitivity to rapamycin both in time and quantum. We further show that of activation loop and hydrophobic motif phosphorylations turnover in an interdependent manner so as to exhibit all or none pattern of loss to rapamycin. Using insect cell expression system, we further substantiate their interdependent turnover and provide evidence that the two phosphorylations are brought about in a coordinate and not sequential manner. These data together with the observation that both kinases that cause hydrophobic motif and activation loop phosphorylations in insect or mammalian cells are completely insensitive to inhibition by rapamycin, suggest that their loss is a consequence and not the mechanism of rapamycin inhibition in accordance with the model proposed herein.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Sirolimo/farmacologia , Motivos de Aminoácidos , Células HEK293 , Humanos , Modelos Moleculares , Fosforilação , Estrutura Terciária de Proteína , Proteínas Quinases S6 Ribossômicas 70-kDa/química
17.
Food Chem Toxicol ; 59: 325-33, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23793038

RESUMO

Hep3B cells were treated with DEHP at various concentrations (62.5, 125.0, 250.0, 500.0 and 1000.0 µM). After 24 h exposure to DEHP only, increased Hep3B cell viability was observed (p<0.05 or p<0.01). However, after 24 h co-exposure to DEHP at indicated concentrations plus 50.0 µM LY294002 (PI3K inhibitor), cell viability was significantly decreased compared to the corresponding DEHP treated groups. DEHP increased mitochondrial membrane potential level and induced oxidative DNA damage in Hep3B cells, DEHP also increased DNA replication rate and accelerated the cell cycle. The PI3K inhibitor LY294002 could recover the mitochondrial membrane potential and attenuate the oxidative stress in Hep3B cells; however, it could not protect the cells from oxidation of DNA damage. The findings showed that LY294002 attenuated DEHP-induced up-regulation of the selected genes (pi3k, akt, mtor and p70s6k) involved in PI3K-AKT-mTOR signaling pathway at both mRNA and protein levels thus inhibited the cell abnormal proliferation.


Assuntos
Carcinógenos Ambientais/toxicidade , Dietilexilftalato/toxicidade , Hepatócitos/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/agonistas , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Carcinógenos Ambientais/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromonas , Dano ao DNA , Dietilexilftalato/antagonistas & inibidores , Inibidores Enzimáticos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Morfolinas , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Concentração Osmolar , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/química , Fosfatidilinositol 3-Quinase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Plastificantes/química , Plastificantes/toxicidade , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
18.
Biochem J ; 454(1): 39-47, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23731517

RESUMO

The activity of S6K1 (p70 ribosomal protein subunit 6 kinase 1) is stimulated by phosphorylation of Thr389 in the hydrophobic motif by mTORC1 (mammalian target of rapamycin complex 1) and phosphorylation of Thr229 in the activation loop by PDK1 (phosphoinositide-dependent kinase 1); however, the order of the two events is still ambiguous. In the present paper we report six crystal structures of the S6K1 kinase domain alone or plus the hydrophobic motif in various forms, in complexes with a highly specific inhibitor. The structural data, together with the biochemical data, reveal in vivo phosphorylation of Thr389 in the absence of Thr229 phosphorylation and demonstrate the importance of two conserved residues, Gln140 and Arg121, in the establishment of a hydrogen-bonding network between the N-lobe (N-terminal lobe) and the hydrophobic motif. Phosphorylation of Thr389 or introduction of a corresponding negatively charged group leads to reinforcement of the network and stabilization of helix αC. Furthermore, comparisons of S6K1 with other AGC (protein kinase A/protein kinase G/protein kinase C) family kinases suggest that the structural and sequence differences in the hydrophobic motif and helix αC account for their divergence in PDK1 dependency. Taken together, the results of the present study indicate that phosphorylation of the hydrophobic motif in S6K1 is independent of, and probably precedes and promotes, phosphorylation of the activation loop.


Assuntos
Química Encefálica/fisiologia , Interações Hidrofóbicas e Hidrofílicas , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Cristalização , Humanos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica/fisiologia
19.
Curr Cancer Drug Targets ; 13(3): 252-66, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23297824

RESUMO

Humans are exposed to heavy metals through a variety of occupational and non-occupational means. Growing evidence has accumulated that prolonged exposure to these heavy metals is associated with cancer occurrence at various body sites including lung, liver, bladder, colon, and skin. Much research effort has been placed on discovering the mechanisms by which heavy metals induce different kinds of cancers. Results from these mechanistic studies have varied for different metals, but increased activation of signaling pathways is often observed. This review will focus on the signaling molecules including epidermal growth factor receptor (EGFR), phosphatidyl inositol 3-kinase (PI3K), AKT, and mammalian target of rapamycin (mTOR) in carcinogenesis and cancer progression; and how these molecules are affected by the exposure to heavy metals: arsenic, chromium, nickel, and cadmium. Furthermore, drug targets for the prevention and therapy of cancers induced by heavy metals will be discussed with a focus on drugs that are currently in clinical trials for these targets.


Assuntos
Receptores ErbB/metabolismo , Metais Pesados/toxicidade , Proteínas de Neoplasias/metabolismo , Neoplasias/induzido quimicamente , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Receptores ErbB/agonistas , Receptores ErbB/antagonistas & inibidores , Humanos , Terapia de Alvo Molecular , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Neoplasias/terapia , Fosfatidilinositol 3-Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/química
20.
Am J Physiol Gastrointest Liver Physiol ; 301(2): G210-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21252047

RESUMO

Leptin modulates the angiogenic properties of hepatic stellate cells (HSC), but the molecular mechanisms involved are poorly understood. We investigated the pathways regulating hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in leptin-stimulated myofibroblastic HSC. Exposure to leptin enhanced the phosphorylation of TSC2 on T1462 residues and of p70 S6 kinase and the translational inhibitor 4E-binding protein-1, indicating the ability of leptin to activate the mammalian target of rapamycin (mTOR) pathway. Similar findings were observed when HSC were exposed to PDGF. Both leptin and PDGF increased the expression of HIF-1α and VEGF in HSC. In the presence of rapamycin, a specific mTOR inhibitor, leptin and PDGF were no longer able to activate mTOR, and expression of VEGF was reduced, whereas HIF-1α abundance was not affected. Moreover, knockdown of Raptor, a component of the mTORC1 complex, reduced the ability of leptin to increase VEGF. mTOR was also necessary for leptin- and PDGF-dependent increase in HSC migration. Leptin increased the generation of reactive oxygen species in HSC, which was reduced by NADP(H) oxidase inhibitors. Both N-acetyl cysteine and diphenylene iodonium, a NADP(H) inhibitor, inhibited the expression of HIF-1α and VEGF stimulated by leptin or PDGF. Finally, conditioned media from HSC treated with leptin or PDGF induced tube formation in cultured human umbilical vein endothelial cells. In conclusion, in HSC exposed to leptin or PDGF, increased expression of VEGF requires both activation of mTOR and generation of reactive oxygen species via NADPH-oxidase. Induction of HIF-1α requires NADP(H) oxidase but not mTOR activation.


Assuntos
Células Estreladas do Fígado/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leptina/fisiologia , Fígado/irrigação sanguínea , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Células Hep G2 , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Leptina/metabolismo , NADPH Oxidases/fisiologia , Neovascularização Patológica , Neovascularização Fisiológica , Fosforilação , Fator de Crescimento Derivado de Plaquetas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/química , Fator A de Crescimento do Endotélio Vascular/fisiologia
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