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1.
Chemistry ; 25(59): 13531-13536, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31393030

RESUMO

Unusual lipid modification of K-Ras makes Ras-directed cancer therapy a challenging task. Aiming to disrupt electrostatic-driven protein-protein interactions (PPIs) of K-Ras with FTase and GGTase I, a series of bivalent dual inhibitors that recognize the active pocket and the common acidic surface of FTase and GGTase I were designed. The structure-activity-relationship study resulted in 8 b, in which a biphenyl-based peptidomimetic FTI-277 was attached to a guanidyl-containing gallate moiety through an alkyl linker. Cell-based evaluation demonstrated that 8 b exhibited substantial inhibition of K-Ras processing without apparent interference with Rap-1A processing. Fluorescent imaging showed that 8 b disrupts localization of K-Ras to the plasma membrane and impairs interaction with c-Raf, whereas only FTI-277 was found to be inactive. These results suggest that targeting the PPI interface of K-Ras may provide an alternative method of inhibiting K-Ras.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Metionina/análogos & derivados , Proteínas Serina-Treonina Quinases/química , Proteínas ras/química , Metionina/química , Metionina/farmacologia , Peptidomiméticos , Prenilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas ras/metabolismo
2.
Nat Commun ; 10(1): 2532, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182717

RESUMO

Targeted inhibition of the ERK-MAPK pathway, upregulated in a majority of human cancers, has been hindered in the clinic by drug resistance and toxicity. The MRAS-SHOC2-PP1 (SHOC2 phosphatase) complex plays a key role in RAF-ERK pathway activation by dephosphorylating a critical inhibitory site on RAF kinases. Here we show that genetic inhibition of SHOC2 suppresses tumorigenic growth in a subset of KRAS-mutant NSCLC cell lines and prominently inhibits tumour development in autochthonous murine KRAS-driven lung cancer models. On the other hand, systemic SHOC2 ablation in adult mice is relatively well tolerated. Furthermore, we show that SHOC2 deletion selectively sensitizes KRAS- and EGFR-mutant NSCLC cells to MEK inhibitors. Mechanistically, SHOC2 deletion prevents MEKi-induced RAF dimerization, leading to more potent and durable ERK pathway suppression that promotes BIM-dependent apoptosis. These results present a rationale for the generation of SHOC2 phosphatase targeted therapies, both as a monotherapy and to widen the therapeutic index of MEK inhibitors.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Quinases raf/metabolismo , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos Knockout , Camundongos Nus , Mutação , Transplante de Neoplasias , Multimerização Proteica , Quinases raf/antagonistas & inibidores , Quinases raf/genética , Proteínas ras/metabolismo
3.
Artif Cells Nanomed Biotechnol ; 47(1): 2343-2351, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31184227

RESUMO

Recent papers suggest that oncogenic Ras participate in regulating tumour cells proliferation and metastasis. This work linked Ras with H1.4 modification in non-small-cell lung carcinoma (NSCLC), to better understand the oncogenic effects of Ras. A plasmid for expressing Ras mutated at G13D and T35S was transfected into NCI-H2126 and A549 cells. Phosphorylation of H1.4S36 was determined by immunoblotting. Effects of phosphorylation of H1.4 at serine (S) 36 (H1.4S36ph) on NCI-H2126 and A549 cells were tested by MTT assay, soft-agar colony formation assay, flow cytometry and transwell assay. Chromatin-immunoprecipitation (ChIP) and RT-qPCR were conducted to measure the effects of H1.4S36ph on Ras downstream genes. The catalyzing enzymes participate in H1.4S36 phosphorylation were further studied. We found that Ras-ERK signalling repressed the phosphorylation of H1.4 at S36. H1.4S36ph functioned as a tumour suppressor, as its overexpression repressed NCI-H2126 and A549 cells viability, colony formation, S-phase arrest, migration and invasion. H1.4S36ph was able to mediate the transcription of Ras downstream genes. Ras-ERK signalling repressed H1.4S36ph through degradation of PKA, and the degradation was mediated by MDM2. In conclusion, Ras-ERK signalling repressed H1.4 phosphorylation at S36 to participate in NSCLC cells growth, migration and invasion. Ras-ERK signalling repressed H1.4S36ph through MDM2-dependent degradation of PKA. This study provides a novel explanation for Ras-ERK's tumour-promoting function. Highlights: H1.4S36 phosphorylation is repressed by Ras-ERK activation; H1.4S36ph inhibits the phenotype of NSCLC cells; H1.4S36ph regulates the transcription of Ras downstream genes; Ras-ERK represses H1.4S36ph by MDM2-dependent degradation of PKA.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Histonas/metabolismo , Neoplasias Pulmonares/patologia , Transdução de Sinais , Proteínas ras/metabolismo , Células A549 , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Neoplásica da Expressão Gênica , Histonas/química , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fenótipo , Fosforilação , Proteólise , Serina/metabolismo
4.
Nat Commun ; 10(1): 2197, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097693

RESUMO

In colorectal cancer (CRC), aberrant Wnt signalling is essential for tumorigenesis and maintenance of cancer stem cells. However, how other oncogenic pathways converge on Wnt signalling to modulate stem cell homeostasis in CRC currently remains poorly understood. Using large-scale compound screens in CRC, we identify MEK1/2 inhibitors as potent activators of Wnt/ß-catenin signalling. Targeting MEK increases Wnt activity in different CRC cell lines and murine intestine in vivo. Truncating mutations of APC generated by CRISPR/Cas9 strongly synergize with MEK inhibitors in enhancing Wnt responses in isogenic CRC models. Mechanistically, we demonstrate that MEK inhibition induces a rapid downregulation of AXIN1. Using patient-derived CRC organoids, we show that MEK inhibition leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stemness and cancer relapse. Our study demonstrates that clinically used MEK inhibitors inadvertently induce stem cell plasticity, revealing an unknown side effect of RAS pathway inhibition.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Antineoplásicos/uso terapêutico , Biópsia , Sistemas CRISPR-Cas/genética , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Plasticidade Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Intestinos/citologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismo
5.
BMC Cancer ; 19(1): 441, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088402

RESUMO

BACKGROUND: Glioblastoma is a disease with high heterogeneity that has long been difficult for doctors to identify and treat. ARHI is a remarkable tumor suppressor gene in human ovarian cancer and many other cancers. We found over-expression of ARHI can also inhibit cancer cell proliferation, decrease tumorigenicity, and induce autophagic cell death in human glioma and inhibition of the late stage of autophagy can further enhance the antitumor effect of ARHI through inducing apoptosis in vitro or vivo. METHODS: Using MTT assay to detect cell viability. The colony formation assay was used to measure single cell clonogenicity. Autophagy associated morphological changes were tested by transmission electron microscopy. Flow cytometry and TUNEL staining were used to measure the apoptosis rate. Autophagy inhibitor chloroquine (CQ) was used to study the effects of inhibition at late stage of autophagy on ARHI-induced autophagy and apoptosis. Protein expression were detected by Western blot, immunofluorescence and immunohistochemical analyses. LN229-derived xenografts were established to observe the effect of ARHI in vivo. RESULTS: ARHI induced autophagic death in glioma cells, and blocking late-stage autophagy markedly enhanced the antiproliferative activites of ARHI. In our research, we observed the inhibition of RAS-AKT-mTOR signaling in ARHI-glioma cells and blockade of autophagy flux at late stage by CQ enhanced the cytotoxicity of ARHI, caused accumulation of autophagic vacuoles and robust apoptosis. As a result, the inhibition of RAS augmented autophagy of glioma cells. CONCLUSION: ARHI may also be a functional tumor suppressor in glioma. And chloroquine (CQ) used as an auxiliary medicine in glioma chemotherapy can enhance the antitumor effect of ARHI, and this study provides a novel mechanistic basis and strategy for glioma therapy.


Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Proteínas ras/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Autofagia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Cloroquina/farmacologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas ras/genética , Proteínas rho de Ligação ao GTP/genética
6.
PLoS Genet ; 15(5): e1008056, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31086367

RESUMO

The six C. elegans vulval precursor cells (VPCs) are induced to form the 3°-3°-2°-1°-2°-3° pattern of cell fates with high fidelity. In response to EGF signal, the LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK canonical MAP kinase cascade is necessary to induce 1° fate and synthesis of DSL ligands for the lateral Notch signal. In turn, LIN-12/Notch receptor is necessary to induce neighboring cells to become 2°. We previously showed that, in response to graded EGF signal, the modulatory LET-60/Ras-RGL-1/RalGEF-RAL-1/Ral signal promotes 2° fate in support of LIN-12. In this study, we identify two key differences between RGL-1 and RAL-1. First, deletion of RGL-1 confers no overt developmental defects, while previous studies showed RAL-1 to be essential for viability and fertility. From this observation, we hypothesize that the essential functions of RAL-1 are independent of upstream activation. Second, RGL-1 plays opposing and genetically separable roles in VPC fate patterning. RGL-1 promotes 2° fate via canonical GEF-dependent activation of RAL-1. Conversely, RGL-1 promotes 1° fate via a non-canonical GEF-independent activity. Our genetic epistasis experiments are consistent with RGL-1 functioning in the modulatory 1°-promoting AGE-1/PI3-Kinase-PDK-1-AKT-1 cascade. Additionally, animals lacking RGL-1 experience 15-fold higher rates of VPC patterning errors compared to the wild type. Yet VPC patterning in RGL-1 deletion mutants is not more sensitive to environmental perturbations. We propose that RGL-1 functions to orchestrate opposing 1°- and 2°-promoting modulatory cascades to decrease developmental stochasticity. We speculate that such switches are broadly conserved but mostly masked by paralog redundancy or essential functions.


Assuntos
Caenorhabditis elegans/genética , Fator de Crescimento Epidérmico/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/genética , Vulva/metabolismo , Animais , Padronização Corporal/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Epistasia Genética , Feminino , Fertilidade/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Vulva/citologia , Vulva/crescimento & desenvolvimento , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
7.
Int J Oncol ; 54(6): 2117-2126, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31081052

RESUMO

Transforming growth factor-ß1 (TGF-ß1) is a multifunctional cytokine that functions as a growth suppressor in normal epithelial cells and early stage tumors, but acts as a tumor promoter during malignant progression. However, the molecular basis underlying the conversion of TGF­ß1 function remains largely undefined. X­linked inhibitor of apoptosis­associated factor 1 (XAF1) is a pro­apoptotic tumor suppressor that frequently displays epigenetic inactivation in various types of human malignancies, including colorectal cancer. The present study explored whether the anti­apoptotic effect of TGF­ß1 is linked to its regulatory effect on XAF1 induction in human colon cancer cells under stressful conditions. The results revealed that TGF­ß1 treatment protected tumor cells from various apoptotic stresses, including 5­fluorouracil, etoposide and γ­irradiation. XAF1 expression was activated at the transcriptional level by these apoptotic stresses and TGF­ß1 blocked the stress­mediated activation of the XAF1 promoter. The study also demonstrated that mitogen­activated protein kinase kinase inhibition or extracellular signal­activated kinase (Erk)1/2 depletion induced XAF1 induction, while the activation of K­Ras (G12C) led to its reduction. In addition, TGF­ß1 blocked the stress­mediated XAF1 promoter activation and induction of apoptosis. This effect was abrogated if Erk1/2 was depleted, indicating that TGF­ß1 represses XAF1 transcription through Erk activation, thereby protecting tumor cells from apoptotic stresses. These findings point to a novel molecular mechanism underlying the tumor­promoting function of TGF­ß1, which may be utilized in the development of a novel therapeutic strategy for the treatment of colorectal cancer.


Assuntos
Neoplasias do Colo/metabolismo , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias do Colo/genética , Progressão da Doença , Etoposídeo/farmacologia , Fluoruracila/farmacologia , Raios gama/efeitos adversos , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Proteínas ras/metabolismo
8.
Nat Commun ; 10(1): 1710, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979884

RESUMO

Whether fragile X mental retardation protein (FMRP) target mRNAs and neuronal activity contributing to elevated basal neuronal protein synthesis in fragile X syndrome (FXS) is unclear. Our proteomic experiments reveal that the de novo translational profile in FXS model mice is altered at steady state and in response to metabotropic glutamate receptor (mGluR) stimulation, but the proteins expressed differ under these conditions. Several altered proteins, including Hexokinase 1 and Ras, also are expressed in the blood of FXS model mice and pharmacological treatments previously reported to ameliorate phenotypes modify their abundance in blood. In addition, plasma levels of Hexokinase 1 and Ras differ between FXS patients and healthy volunteers. Our data suggest that brain-based de novo proteomics in FXS model mice can be used to find altered expression of proteins in blood that could serve as disease-state biomarkers in individuals with FXS.


Assuntos
Síndrome do Cromossomo X Frágil/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Adolescente , Adulto , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Feminino , Síndrome do Cromossomo X Frágil/genética , Hexoquinase/sangue , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Adulto Jovem , Proteínas ras/metabolismo
9.
Artif Cells Nanomed Biotechnol ; 47(1): 1207-1215, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30942624

RESUMO

Histone H4 acetylation at lysine 12 (H4K12ac) has been reported to be associated with the poor prognosis of pancreatic cancer. The study intends to study whether H4K12ac participates in regulating the carcinogenic effect of Ras-ERK1/2 on osteosarcoma (OS). The plasmids of pEGFP-N1, pEGFP-RasWT and pEGFP-K-RasG12V/T35S were transfected into MG-63 cells, the protein levels of H4K12ac and ERK1/2 were analyzed by Western blot. Effects of H4K12ac on cell proliferation and migration of MG-63 cells were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide solution (MTT), transwell, soft-agar colony formation and flow cytometry assays. Effect of H4K12ac on the transcription of ERK1/2 downstream genes was analyzed by RT-qPCR and ChIP assays. The involvements of HDAT6, HAT1 and MDM2 in cell proliferation and migration of MG-63 cells were finally studied. We found that H4K12ac was specifically down-regulated by Ras-ERK1/2 activation in MG-63 cells. H4K12ac suppressed Ras-ERK1/2-induced cell viability, colony formation and migration in MG-63 cells. Additionally, HDAC6 silence recovered Ras-ERK1/2-repressed H4K12ac expression, as well as inhibited the carcinogenic effect of Ras-ERK1/2 on MG-63 cells. Besides, down-regulated H4K12ac induced by Ras-ERK1/2 was found to be associated with MDM2-mediated HAT1 degradation. In conclusion, these results testified that Ras-ERK1/2 signalling promoted the development of OS by mediating H4K12ac through MDM2-mediated HAT1 degradation.


Assuntos
Histona Acetiltransferases/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Sistema de Sinalização das MAP Quinases , Osteossarcoma/patologia , Acetilação , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Ativação Enzimática , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transcrição Genética , Proteínas ras/metabolismo
10.
Nat Commun ; 10(1): 1897, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015486

RESUMO

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Cromatina/química , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Animais , Butadienos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nitrilos/farmacologia , Piperazinas/farmacologia , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
11.
Artif Cells Nanomed Biotechnol ; 47(1): 1524-1532, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30982359

RESUMO

Proteinuria is one of the most important clinical features of nephrotic syndrome (NS). Injury of podocyte has been proved to contribute to the occurrence of proteinuria. This study explored the effects of geniposide (GEN) on lipopolysaccharide (LPS)-caused murine kidney podocyte MPC5 apoptosis and autophagy. Viability and apoptosis of MPC5 cells were respectively detected with the help of CCK-8 assay and Guava Nexin assay. 3-Methyladenine (3-MA) was used as an autophagy inhibitor, while rapamycin as autophagy activator. Si-Beclin-1 was transfected in MPC5 cells to down-regulate the expression of Beclin-1. We found that LPS stimulation significantly caused MPC5 cell viability reduction, apoptosis and autophagy (P < .05 or P < .01). GEN treatment remarkably alleviated the LPS-caused MPC5 cell viability reduction and apoptosis, but promoted cell autophagy (P < .05). Moreover, 3-MA incubation or si-Beclin-1 transfection notably weakened the effects of GEN on LPS-caused MPC5 cell apoptosis and autophagy (P < .05), while rapamycin had opposite effects (P < .05). Furthermore, GEN activated Ras/Raf/MEK/ERK pathway in LPS-treated MPC5 cells (P < .05). In conclusion, this research verified the protective effects of GEN on podocytes damage. GEN alleviates LPS-caused apoptosis of murine kidney podocytes by activating Ras/Raf/MEK/ERK-mediated cell autophagy. Highlights: LPS causes podocyte MPC5 apoptosis and autophagy. GEN alleviates LPS-caused MPC5 cell apoptosis, but promotes cell autophagy. 3-MA or si-Beclin-1 weakens the effects of GEN on LPS-treated MPC5 cells. Rapamycin strengthens the effects of GEN on LPS-treated MPC5 cells. GEN activates Ras/Raf/MEK/ERK pathway in LPS-treated MPC5 cells.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Iridoides/farmacologia , Rim/citologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Inativação Gênica , Camundongos , Podócitos/citologia , Podócitos/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismo
12.
Cancer Sci ; 110(5): 1573-1586, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30815935

RESUMO

The testis-specific protein, Y-linked 1 (TSPY1), a newly recognized cancer/testis antigen, has been suggested to accelerate tumor progression. However, the mechanisms underlying TSPY1 cancer-related function remain limited. By mining the RNA sequencing data of lung and liver tumors from The Cancer Genome Atlas, we found frequent ectopic expression of TSPY1 in lung adenocarcinoma (LUAD) and liver hepatocellular carcinoma (LIHC), and the male-specific protein was associated with higher mortality rate and worse overall survival in patients with LUAD and LIHC. Overexpression of TSPY1 promotes cell proliferation, invasiveness, and cycle transition and inhibits apoptosis, whereas TSPY1 knockdown has the opposite effects on these cancer cell phenotypes. Transcriptomic analysis revealed the involvement of TSPY1 in PI3K/AKT and RAS signaling pathways in both LUAD and LIHC cells, which was further confirmed by the increase in the levels of phosphorylated proteins in the PI3K-AKT and RAS signaling pathways in TSPY1-overexpressing cancer cells, and by the suppression on the activity of these two pathways in TSPY1-knockdown cells. Further investigation identified that TSPY1 could directly bind to the promoter of insulin growth factor binding protein 3 (IGFBP3) to inhibit IGFBP3 expression and that downregulation of IGFBP3 increased the activity of PI3K/AKT/mTOR/BCL2 and RAS/RAF/MEK/ERK/JUN signaling in LUAD and LIHC cells. Taken together, the observations reveal a novel mechanism by which TSPY1 could contribute to the progression of LUAD and LIHC. Our finding is of importance for evaluating the potential of TSPY1 in immunotherapy of male tumor patients with TSPY1 expression.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/genética , Transdução de Sinais , Células A549 , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , Masculino , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Análise de Sequência de RNA , Análise de Sobrevida , Proteínas ras/metabolismo
13.
Cancer Sci ; 110(5): 1525-1535, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30874331

RESUMO

The Ras/Raf/ERK pathway is one of the most frequently dysregulated signaling pathways in various cancers. In some such cancers, Ras and Raf are hotspots for mutations, which cause continuous activation of this pathway. However, in some other cancers, it is known that negative regulators of the Ras/Raf/ERK pathway are responsible for uncontrolled activation. The Sprouty/Spred family is broadly recognized as important negative regulators of the Ras/Raf/ERK pathway, and its expression is downregulated in many malignancies, leading to hyperactivation of the Ras/Raf/ERK pathway. After the discovery of this family, intensive research investigated the mechanism by which it suppresses the Ras/Raf/ERK pathway and its roles in developmental and pathophysiological processes. In this review, we discuss the complicated roles of the Sprouty/Spred family in tumor initiation, promotion, and progression and its future therapeutic potential.


Assuntos
Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Família Multigênica , Proteínas ras/metabolismo
14.
Expert Rev Mol Med ; 21: e2, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30862318

RESUMO

Deviations from the precisely coordinated programme of human head development can lead to craniofacial and orofacial malformations often including a variety of dental abnormalities too. Although the aetiology is still unknown in many cases, during the last decades different intracellular signalling pathways have been genetically linked to specific disorders. Among these pathways, the RAS/extracellular signal-regulated kinase (ERK) signalling cascade is the focus of this review since it encompasses a large group of genes that when mutated cause some of the most common and severe developmental anomalies in humans. We present the components of the RAS/ERK pathway implicated in craniofacial and orodental disorders through a series of human and animal studies. We attempt to unravel the specific molecular targets downstream of ERK that act on particular cell types and regulate key steps in the associated developmental processes. Finally we point to ambiguities in our current knowledge that need to be clarified before RAS/ERK-targeting therapeutic approaches can be implemented.


Assuntos
Anormalidades Craniofaciais/metabolismo , Sistema de Sinalização das MAP Quinases , Anormalidades Dentárias/metabolismo , Proteínas ras/metabolismo , Animais , Humanos
15.
J Photochem Photobiol B ; 194: 46-55, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30925276

RESUMO

Human papillomavirus (HPV) infection is linked to several diseases, the most prominent of which are cervical cancer and genital condyloma acuminatum. Previous studies have suggested an effective role for 5-aminolevulinic acid photodynamic therapy (ALA-PDT) against various cancers by the induction of autophagy and apoptosis. However, few reports have focused on the effectiveness of ALA-PDT on HPV related disorders. To identify the role of ALA-PDT in the context of HPV infection, we initially investigated 111 patients suffering from genital condyloma acuminatum. HPV viral load detected before and after ALA-PDT treatment was compared during this procedure; a significant difference was noted. HeLa (HPV18) cells were exposed to ALA-PDT in vitro to further explore the underlying mechanisms. Western blot analysis showed that ALA-PDT induces LC3II and p62 expression, along with the up regulation of caspase-3 and cleaved caspase-3. Our study also demonstrated that ALA-PDT treatment inhibits the proliferation of HeLa cells in a dose dependent manner and effectively reduces HPV viral load via autophagy and apoptosis by regulating the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways. Hydroxychloroquine (HCQ), although it inhibited autophagy degradation, functioned to activate reactive oxygen species (ROS) levels of ALA-PDT to enhance the observed effect. These findings suggest strategies for the improvement of PDT efficacy in patients.


Assuntos
Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Ácidos Levulínicos/farmacologia , Papillomaviridae/fisiologia , Fotoquimioterapia , Carga Viral/efeitos dos fármacos , Carga Viral/efeitos da radiação , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Autofagia/efeitos dos fármacos , Autofagia/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Papillomaviridae/efeitos dos fármacos , Papillomaviridae/efeitos da radiação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismo
16.
Science ; 363(6432): 1226-1230, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30872527

RESUMO

RIT1 oncoproteins have emerged as an etiologic factor in Noonan syndrome and cancer. Despite the resemblance of RIT1 to other members of the Ras small guanosine triphosphatases (GTPases), mutations affecting RIT1 are not found in the classic hotspots but rather in a region near the switch II domain of the protein. We used an isogenic germline knock-in mouse model to study the effects of RIT1 mutation at the organismal level, which resulted in a phenotype resembling Noonan syndrome. By mass spectrometry, we detected a RIT1 interactor, leucine zipper-like transcription regulator 1 (LZTR1), that acts as an adaptor for protein degradation. Pathogenic mutations affecting either RIT1 or LZTR1 resulted in incomplete degradation of RIT1. This led to RIT1 accumulation and dysregulated growth factor signaling responses. Our results highlight a mechanism of pathogenesis that relies on impaired protein degradation of the Ras GTPase RIT1.


Assuntos
Síndrome de Noonan/genética , Proteínas Oncogênicas/metabolismo , Proteólise , Fatores de Transcrição/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Técnicas de Introdução de Genes , Mutação em Linhagem Germinativa , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Camundongos Mutantes , Proteínas Oncogênicas/genética , Fatores de Transcrição/genética
17.
Mol Med Rep ; 19(4): 3105-3113, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816458

RESUMO

Autophagy regulates the metabolism, survival and function of numerous types of cell, including cells that comprise the cardiovascular system. The dysfunction of autophagy has been demonstrated in atherosclerosis, restenotic lesions and hypertensive vessels. As a member of the Ras GTPase superfamily, Rab7 serves a significant role in the regulation of autophagy. The present study evaluated how Rab7 affects the proliferation and invasion, and phenotypic transformations of aortic dissection (AD) smooth muscle cells (SMCs) via autophagy. Rab7 was overexpressed in AD tissues and the percentage of synthetic human aortic SMCs (HASMCs) was higher in AD tissues compared with NAD tissues. Downregulation of Rab7 decreased cell growth, reduced the number of invasive cells and decreased the percentage cells in the G1 phase. Autophagy of HASMCs was inhibited following Rab7 knockdown. Inhibition of autophagy with 3­methyladenine or Rab7 knockdown suppressed the phenotypic conversion of contractile to synthetic HASMCs. The action of Rab7 may be mediated by inhibiting the Ras/Raf/mitogen­activated protein kinase (MAPK) kinase (MEK)/extracellular signal related kinase (ERK) signaling pathway. In conclusion, the results revealed that Rab7­mediated autophagy regulated the behavior of SMCs and the phenotypic transformations in AD via activation of the Ras/Raf/MEK/ERK signaling pathway. The findings of the present study may improve understanding of the role Rab7 in the molecular etiology of AD and suggests the application of Rab7 as a novel therapeutic target in the treatment of human AD.


Assuntos
Aneurisma Dissecante/etiologia , Aneurisma Dissecante/metabolismo , Autofagia , Miócitos de Músculo Liso/metabolismo , Fenótipo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismo , Ciclo Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismo
18.
Mol Med Rep ; 19(4): 3255-3262, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816469

RESUMO

Repulsive guidance molecule a (RGMa) is a membrane­associated glycoprotein that regulates axonal guidance and inhibits axon outgrowth. In our previous study, we hypothesized that RGMa may be involved in temporal lobe epilepsy (TLE) via the repulsive guidance molecule a (RGMa)­focal adhesion kinase (FAK)­Ras signaling pathway. To investigate the role of RGMa in epilepsy, recombinant RGMa protein and FAK inhibitor 14 was intracerebroventricularly injected into a pentylenetetrazol (PTZ) kindling model and Timm staining, co­immunoprecipitation and western blotting analyses were subsequently performed. The results of the present study revealed that intracerebroventricular injection of recombinant RGMa protein reduced the phosphorylation of FAK (Tyr397) and intracerebroventricular injection of FAK inhibitor 14 reduced the interaction between FAK and p120GAP, as wells as Ras expression. Recombinant RGMa protein and FAK inhibitor 14 exerted seizure­suppressant effects; however, recombinant RGMa protein but not FAK inhibitor 14 suppressed mossy fiber sprouting in the PTZ kindling model. Collectively, these results demonstrated that RGMa may be considered as a potential therapeutic agent for epilepsy, and that RGMa may exert the aforementioned biological effects partly via the FAK­p120GAP­Ras signaling pathway.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Glicoproteínas de Membrana/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Transdução de Sinais , Proteína p120 Ativadora de GTPase/metabolismo , Proteínas ras/metabolismo , Animais , Modelos Animais de Doenças , Expressão Gênica , Masculino , Glicoproteínas de Membrana/administração & dosagem , Fibras Musgosas Hipocampais/fisiopatologia , Proteínas do Tecido Nervoso/administração & dosagem , Pentilenotetrazol/efeitos adversos , Fosforilação , Ligação Proteica , Ratos , Proteínas Recombinantes , Convulsões/tratamento farmacológico , Convulsões/etiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/genética
19.
Nutrients ; 11(3)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871060

RESUMO

Glycyrrhetinic acid (GA), a bioactive component in the human diet, has been reported to improve hyperglycemia, dyslipidemia, insulin resistance and obesity in rats with metabolic syndrome. However, GA-specific target proteins and the mechanisms involved in the downstream signaling and cross-talk to improve insulin sensitivity have not been fully elucidated. In this study, the potential targets of GA were identified by chemical proteomics strategies using serial GA probes for target fishing and cell molecular imaging. Intracellular enzyme activity evaluation and insulin resistance models were used for validating the function of the target proteins on the downstream insulin signaling pathways. Collectively, our data demonstrate that GA improved the insulin-responsive pathway and glucose consumption levels via multiple diabetogenic factors that activated the insulin signaling pathway in HepG2 cells. GA improved Glucose transporter 4(GLUT4) expression by targeting the Ras protein to regulate the mitogen-activated protein kinase (MAPK) pathway. GA exhibited a strong inhibitory effect on IRS1ser307 phosphorylation in cells treated with the Protein kinase C (PKC) activator Phorbol 12-myristate 13-acetate (PMA.) Consistently, IRS1ser307 phosphorylation was also inhibited by GA in Free fatty acid (FFA)-treated HepG2 cells. GA also inhibited the PMA-induced phosphorylation of IκB kinase α/ß (IKKα/ß), c-Jun N-terminal kinase (JNK) and p38 proteins (P38), suggesting that IKKα/ß, JNK and P38 activation is dependent on PKC activity.


Assuntos
Ácido Glicirretínico/farmacologia , Insulina/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas ras/metabolismo , Animais , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose/administração & dosagem , Glucose/metabolismo , Células Hep G2 , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas ras/genética
20.
Cells ; 8(3)2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875834

RESUMO

Human papillomaviruses (HPV) replicate their DNA in the suprabasal layer of the infected mucosa or skin. In order to create a suitable environment for vegetative viral DNA replication HPV delay differentiation and sustain keratinocyte proliferation that can lead to hyperplasia. The mechanism underlying cell growth stimulation is not well characterized. Here, we show that the E6 oncoprotein of the ßHPV type 8 (HPV8), which infects the cutaneous skin and is associated with skin cancer in Epidermodysplasia verruciformis patients and immunosuppressed organ transplant recipients, binds to the protein tyrosine phosphatase H1 (PTPH1), which resulted in increased protein expression and phosphatase activity of PTPH1. Suppression of PTPH1 in immortalized keratinocytes reduced cell proliferation as well as the level of epidermal growth factor receptor (EGFR). Furthermore, we report that HPV8E6 expressing keratinocytes have increased level of active, GTP-bound Ras. This effect was independent of PTPH1. Therefore, HPV8E6-mediated targeting of PTPH1 might result in higher level of EGFR and enhanced keratinocyte proliferation. The HPV8E6-mediated stimulation of Ras may be an additional step to induce cell growth. Our results provide novel insights into the mechanism how ßHPVE6 proteins support proliferation of infected keratinocytes, thus creating an environment with increased risk of development of skin cancer particularly upon UV-induced DNA mutations.


Assuntos
Queratinócitos/citologia , Queratinócitos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Oncogenes , Proteína Tirosina Fosfatase não Receptora Tipo 3/metabolismo , Proliferação de Células , Receptores ErbB/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/metabolismo
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