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1.
Proc Natl Acad Sci U S A ; 119(36): e2202730119, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36044553

RESUMO

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.


Assuntos
Neoplasias Hepáticas , Proteínas R-SNARE , Sinaptotagmina I , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Transporte Proteico , Proteínas R-SNARE/metabolismo , Sinaptotagmina I/metabolismo , Microambiente Tumoral
2.
Cell Struct Funct ; 48(2): 123-133, 2023 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-37380437

RESUMO

When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or ß-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions. Expression levels of enzymes for the uptake and ß-oxidation of fatty acids as well as synthesis of triacylglycerol were elevated, whereas those for the synthesis of cholesterol as well as export of cholesterol and triacylglycerol were decreased under starvation conditions, which explains the accumulation of triacylglycerol in the liver. Our results provide the basis for future research to understand how gene malfunction(s) affects the development of fatty liver, which can lead to nonalcoholic steatohepatitis and then to liver cirrhosis.Key words: amino acid catabolism, ß-oxidation, triacylglycerol, cholesterol, export.


Assuntos
Fígado Gorduroso , Oryzias , Animais , Oryzias/metabolismo , Larva/metabolismo , Proteômica , Fígado Gorduroso/veterinária , Ácidos Graxos/metabolismo , Triglicerídeos/metabolismo , Colesterol , Aminoácidos
3.
J Cell Sci ; 133(6)2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32051283

RESUMO

Membrane lipid biosynthesis is a complex process that occurs in various intracellular compartments. In Drosophila, phosphatidylinositol glycan-B (PIG-B), which catalyzes addition of the third mannose in glycosylphosphatidylinositol (GPI), localizes to the nuclear envelope (NE). Although this NE localization is essential for Drosophila development, the underlying molecular mechanism remains unknown. To elucidate this mechanism, we identified PIG-B-interacting proteins by performing immunoprecipitation followed by proteomic analysis. We then examined which of these proteins are required for the NE localization of PIG-B. Knockdown of Lamin Dm0, a B-type lamin, led to mislocalization of PIG-B from the NE to the endoplasmic reticulum. Lamin Dm0 associated with PIG-B at the inner nuclear membrane, a process that required the tail domain of Lamin Dm0. Furthermore, GPI moieties were distributed abnormally in the Lamin Dm0 mutant. These data indicate that Lamin Dm0 is involved in the NE localization of PIG-B and is required for proper GPI-anchor modification of proteins.


Assuntos
Proteínas de Drosophila , Drosophila , Laminas , Animais , Proteínas de Drosophila/genética , Proteínas Ligadas por GPI , Glicosilfosfatidilinositóis , Proteômica
4.
Blood ; 136(1): 106-118, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32219445

RESUMO

Mutations in JAK2, myeloproliferative leukemia virus (MPL), or calreticulin (CALR) occur in hematopoietic stem cells (HSCs) and are detected in more than 80% of patients with myeloproliferative neoplasms (MPNs). They are thought to play a driver role in MPN pathogenesis via autosomal activation of the JAK-STAT signaling cascade. Mutant CALR binds to MPL, activates downstream MPL signaling cascades, and induces essential thrombocythemia in mice. However, embryonic lethality of Calr-deficient mice precludes determination of a role for CALR in hematopoiesis. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr-deficient mice. CALR deficiency had little effect on the leukocyte count, hemoglobin levels, or platelet count in peripheral blood. However, Calr-deficient mice showed some hematopoietic properties of MPN, including decreased erythropoiesis and increased myeloid progenitor cells in the bone marrow and extramedullary hematopoiesis in the spleen. Transplantation experiments revealed that Calr haploinsufficiency promoted the self-renewal capacity of HSCs. We generated CALRdel52 mutant transgenic mice with Calr haploinsufficiency as a model that mimics human MPN patients and found that Calr haploinsufficiency restored the self-renewal capacity of HSCs damaged by CALR mutations. Only recipient mice transplanted with Lineage-Sca1+c-kit+ cells harboring both CALR mutation and Calr haploinsufficiency developed MPN in competitive conditions, showing that CALR haploinsufficiency was necessary for the onset of CALR-mutated MPNs.


Assuntos
Calreticulina/fisiologia , Transtornos Mieloproliferativos/etiologia , Células-Tronco/patologia , Animais , Medula Óssea/patologia , Calreticulina/deficiência , Calreticulina/genética , Autorrenovação Celular , Eritropoese , Genótipo , Hematopoese Extramedular , Células-Tronco Hematopoéticas/patologia , Camundongos , Camundongos Transgênicos , Transtornos Mieloproliferativos/patologia , Células-Tronco Neoplásicas/patologia , Deleção de Sequência , Transcriptoma
5.
Cancer Sci ; 112(10): 4208-4219, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34363714

RESUMO

Previous studies reported the critical role of the brefeldin A-inhibited guanine nucleotide exchange protein 3-prohibitin 2 (BIG3-PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3-PHB2 in OS malignancy. BIG3-PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen-dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3-PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose-dependent suppression of OS cell growth, migration, and invasion resulting from G2/M-phase arrest and in PARP cleavage, ultimately leading to PARP-1/apoptosis-inducing factor (AIF) pathway activation-dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3-PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3-PHB2 complex might regulate PARP-1/AIF pathway-dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS.


Assuntos
Neoplasias Ósseas/patologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Mitocôndrias/metabolismo , Osteossarcoma/patologia , Proteínas Repressoras/fisiologia , Animais , Apoptose/fisiologia , Fator de Indução de Apoptose/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Bases de Dados Factuais , Regulação para Baixo , Pontos de Checagem da Fase G2 do Ciclo Celular , Inativação Gênica , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Membranas Mitocondriais/metabolismo , Invasividade Neoplásica , Transplante de Neoplasias , Osteossarcoma/metabolismo , Osteossarcoma/terapia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proibitinas , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/metabolismo
6.
Microb Pathog ; 153: 104742, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33460746

RESUMO

Insect parasitic nematodes have developed a mechanism to escape from the cellular immunity of their insect hosts for successful parasitism. However, the detailed mechanism whereby they achieve this remains unclear. In our previous study, we demonstrated that non-parasitic nematodes such as Caenorhabditis elegans potentially have the ability to escape from the cellular immunity of the greater wax moth Galleria mellonella. Here we aimed to clarify the effect of non-parasitic and parasitic nematodes on the spreading of hemocytes-an essential cellular reaction for adhering to a foreign substance -from G. mellonella larvae. The hexane/methanol extract of C. elegans inhibited the spreading of hemocytes. Using 2D-TLC and reversed-phase HPLC, we detected a single peak that inhibited the spreading of hemocytes. In addition, the spreading of hemocytes recovered from C. elegans-injected insects was significantly delayed. Western blotting analysis showed that phosphorylated extracellular signal-regulated protein kinase (ERK) -an essential signaling component for spreading in hemocytes-was decreased by the injection of C. elegans, and that plasma from nematode-injected insects contained the factor that causes the decrease of phosphorylated ERK. We also observed this phenomenon using other non-parasitic and parasitic bacterial-feeding nematodes. These results suggest that the factors inhibiting hemocyte adhesion and delaying the spreading of hemocytes are conserved in bacterial-feeding nematodes and could be a pre-adaptation for parasitism.


Assuntos
Mariposas , Nematoides , Animais , Caenorhabditis elegans , Hemócitos , Larva
7.
Arch Insect Biochem Physiol ; 106(2): e21758, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33145828

RESUMO

Stress enhances the concentration of reactive oxygen species (ROS) in animal plasma. Increased ROS alter various physiological functions, such as development and the immune response, but excessive increases could be harmful. In this study, we tested the hypothesis that abnormally increased plasma ROS levels are associated with animal death. Injection of the nematode Caenorhabditis elegans into insect larvae caused high mortality in Galleria mellonella, and the plasma ROS concentration was four times higher than M9 buffer-injected larvae. There was no difference in plasma antioxidant activity after nematode injection. However, coinjecting nematodes with an antioxidant (ascorbic acid or N-acetylcysteine) suppressed increases in ROS concentrations by the nematodes and increases in the number of nematodes in the larvae, which increased G. mellonella survival. These results suggest that the abnormal elevation of ROS associated with the stress caused by nematode propagation is lethal for G. mellonella.


Assuntos
Interações Hospedeiro-Parasita , Mariposas/parasitologia , Nematoides/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Larva/crescimento & desenvolvimento , Larva/parasitologia , Mariposas/crescimento & desenvolvimento , Plasma/parasitologia
8.
J Emerg Nurs ; 47(4): 669-674, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33931236

RESUMO

OBJECTIVE: The Emergency Severity Index (ESI) is a highly reliable and valid triage scale that is widely used in emergency departments in not only English language regions but also other countries. The Japan Triage and Acuity Scale (JTAS) is frequently used for emergency patients, and the ESI has not been evaluated against the JTAS in Japan. This study aimed to examine the decision accuracy of the ESI for simulated clinical scenarios among nursing specialists in Japan compared with the JTAS. METHOD: A parallel group randomized trial was conducted. In total, 23 JTAS-trained triage nurses from 10 Japanese emergency departments were randomly assigned to the ESI or the JTAS group. Nurses independently assigned triage categories to 80 emergency cases for the assessment of interrater agreement. RESULTS: Interrater agreement between the expert and triage nurses was κ = 0.82 (excellent) in the ESI group and κ = 0.74 (substantial) in the JTAS group. In addition, interrater agreement by acuity was level 2 = 0.42 (moderate) in the ESI group and level 2 = 0.31 (fair) in the JTAS group. Interrater agreement for triage decisions was classified in a higher category in the ESI group than in the JTAS Scale group at level 2. Triage decisions based on the ESI in Japan maintained the same level of interrater agreement and sensitivity as those in other countries. CONCLUSION: These findings suggest that the ESI can be introduced in Japan, despite its different emergency medical background compared with other countries.


Assuntos
Enfermagem em Emergência , Enfermeiras e Enfermeiros , Serviço Hospitalar de Emergência , Humanos , Japão , Organizações , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Triagem
9.
Parasitology ; 147(3): 279-286, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31735173

RESUMO

Insect parasitic nematodes have acquired mechanisms to evade their host immune response for successful parasitism. Despite the importance of understanding of the evolution of evasion mechanisms from host immunity, insect immune response against non-parasitic nematodes has not been well studied. In our previous study, we demonstrated that a non-insect parasitic nematode Caenorhabditis elegans was not encapsulated by haemocytes in the larvae of the greater wax moth Galleria mellonella. To understand how nematodes influence insect haemocytes to escape encapsulation, we examined the effect of C. elegans on haemocytes in the haemocoel of G. mellonella larvae. Injection of nematodes resulted in the decrease of haemocyte density while mortality and spreading ability of haemocytes, the haematopoietic organs were not affected. In vitro co-incubation of haemocytes with nematodes resulted in a decrease of haemocyte density and we observed feeding on haemocytes by nematodes. Injection of C. elegans feeding-delay mutants into insects did not cause the decrease of haemocyte density. The decrease of haemocyte density was due to the nematode's ingestion of haemocytes. Furthermore, an entomopathogenic nematode and other bacterial feeding nematodes also showed similar feeding behaviour. The nematode's ability to feed on haemocytes may have played an important role in the evolution of nematode parasitism in bacterial-feeding nematodes.


Assuntos
Caenorhabditis elegans/fisiologia , Interações Hospedeiro-Parasita , Imunidade Celular , Mariposas/parasitologia , Animais , Hemócitos , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/parasitologia , Mariposas/crescimento & desenvolvimento , Mariposas/imunologia
10.
Parasitology ; 146(6): 708-715, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30567609

RESUMO

Immunity to microbial infections is well understood; however, information regarding the immunity to parasitic multicellular organisms remains lacking. To understand innate host cellular immunity to nematodes, we compared the cellular response of the greater wax moth (Galleria mellonella) larvae against the non-parasitic, bacterial-feeding nematode Caenorhabditis elegans and pathogenic nematode Heterorhabditis bacteriophora. When intact first-instar or dauer larvae of C. elegans were injected into a G. mellonella larva, most of the nematodes were alive and not confined by the surrounding reaction by insect haemocytes (encapsulation), similarly as the pathogenic nematode, whereas most of the heat-killed nematodes of both species were severely encapsulated by 24 h after inoculation. Other non-parasitic nematodes were also not encapsulated. Surprisingly, C. elegans injected into the insect haemocoel grew and propagated in the live insect, resulting in death of the host insect. Our results suggest that C. elegans has some basic mechanisms to evade immunity of G. mellonenlla and grow in the haemocoel.

12.
Proc Natl Acad Sci U S A ; 111(2): 717-22, 2014 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-24379358

RESUMO

Upon DNA damage, tumor suppressor p53 determines cell fate by repairing DNA lesions to survive or by inducing apoptosis to eliminate damaged cells. The decision is based on its posttranslational modifications. Especially, p53 phosphorylation at Ser46 exerts apoptotic cell death. However, little is known about the precise mechanism of p53 phosphorylation on the induction of apoptosis. Here, we show that amphiregulin (AREG) is identified for a direct target of Ser46 phosphorylation via the comprehensive expression analyses. Ser46-phosphorylated p53 selectively binds to the promoter region of AREG gene, indicating that the p53 modification changes target genes by altering its binding affinity to the promoter. Although AREG belongs to a family of the epidermal growth factor, it also emerges in the nucleus under DNA damage. To clarify nuclear function of AREG, we analyze AREG-binding proteins by mass spectrometry. AREG interacts with DEAD-box RNA helicase p68 (DDX5). Intriguingly, AREG regulates precursor microRNA processing (i.e., miR-15a) with DDX5 to reduce the expression of antiapoptotic protein Bcl-2. These findings collectively support a mechanism in which the induction of AREG by Ser46-phosphorylated p53 is required for the microRNA biogenesis in the apoptotic response to DNA damage.


Assuntos
Apoptose/fisiologia , Dano ao DNA/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Anfirregulina , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , RNA Helicases DEAD-box/metabolismo , Família de Proteínas EGF , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Immunoblotting , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Espectrometria de Massas , Análise em Microsséries , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real
13.
Mol Cell Proteomics ; 13(6): 1429-38, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24643969

RESUMO

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors.


Assuntos
Carcinoma Hepatocelular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hepáticas/genética , Serina-Treonina Quinases TOR/biossíntese , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/biossíntese , Niacinamida/análogos & derivados , Niacinamida/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Fosforilação , Proteômica , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sorafenibe , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
14.
Proteomics ; 15(9): 1622-30, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25641790

RESUMO

Lumbar spinal stenosis (LSS) is a syndromic degenerative spinal disease and is characterized by spinal canal narrowing with subsequent neural compression causing gait disturbances. Although LSS is a major age-related musculoskeletal disease that causes large decreases in the daily living activities of the elderly, its molecular pathology has not been investigated using proteomics. Thus, we used several proteomic technologies to analyze the ligamentum flavum (LF) of individuals with LSS. Using comprehensive proteomics with strong cation exchange fractionation, we detected 1288 proteins in these LF samples. A GO analysis of the comprehensive proteome revealed that more than 30% of the identified proteins were extracellular. Next, we used 2D image converted analysis of LC/MS to compare LF obtained from individuals with LSS to that obtained from individuals with disc herniation (nondegenerative control). We detected 64 781 MS peaks and identified 1675 differentially expressed peptides derived from 286 proteins. We verified four differentially expressed proteins (fibronectin, serine protease HTRA1, tenascin, and asporin) by quantitative proteomics using SRM/MRM. The present proteomic study is the first to identify proteins from degenerated and hypertrophied LF in LSS, which will help in studying LSS.


Assuntos
Ligamento Amarelo/química , Ligamento Amarelo/patologia , Proteoma/análise , Estenose Espinal/patologia , Adulto , Idoso , Feminino , Humanos , Deslocamento do Disco Intervertebral/patologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica
15.
Int J Cancer ; 137(11): 2589-606, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26061684

RESUMO

CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP-positive renal carcinogenesis. Genome (whole-exome and copy number), transcriptome and proteome (two-dimensional image converted analysis of liquid chromatography-mass spectrometry) analyses were performed using tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and corresponding specimens of non-cancerous renal cortex. Genes encoding microtubule-associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non-synonymous single-nucleotide mutations and insertions/deletions) in CIMP-positive RCCs, whereas CIMP-negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP-positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the "The metaphase checkpoint (p = 1.427 × 10(-6))," "Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10(-6))" and "Spindle assembly and chromosome separation (p = 9.260 × 10(-6))" pathways. Quantitative RT-PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP-positive than in CIMP-negative RCCs. All CIMP-positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP-positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP-positive RCCs.


Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Ilhas de CpG/genética , Metilação de DNA/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Pontos de Checagem da Fase M do Ciclo Celular/genética , Idoso , Aurora Quinases/genética , Carcinogênese/genética , Carcinogênese/patologia , Aberrações Cromossômicas , Feminino , Dosagem de Genes/genética , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Fenótipo , Proteoma/genética , Transcriptoma/genética
16.
Oncology ; 86(2): 94-103, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24457375

RESUMO

OBJECTIVE: Osteosarcoma (OS) is the most frequent primary malignant bone tumor in children and young adults. Although the introduction of combined neoadjuvant chemotherapy has significantly prolonged survival, the outcome for OS patients showing a poor response to chemotherapy is still unfavorable. In order to develop new therapeutic approaches, elucidation of the entire molecular pathway regulating OS cell proliferation would be desirable. METHODS: MicroRNA (miRNA) are highly conserved noncoding RNA that play important roles in the development and progression of various other cancers. Using miRNA microarrays capable of detecting a known number of 933 miRNA, 108 miRNA were found to be commonly expressed in 24 samples of OS tissue and subjected to a cell proliferation assay. RESULTS: We found that inhibition of 5 let-7 family miRNA (hsa-let-7a, b, f, g and i) significantly suppressed the proliferation of OS cells. Using a quantitative shotgun proteomics approach, we also found that the let-7 family miRNA regulated the expression of vimentin and serpin H1 proteins. CONCLUSIONS: Our present results indicate the involvement of let-7 family miRNA in regulation of the cell proliferation as well as epithelial-mesenchymal transition of OS. Thus, let-7 family miRNA may potentially provide novel targets for the development of therapeutic strategies against OS.


Assuntos
Neoplasias Ósseas/metabolismo , MicroRNAs/metabolismo , Osteossarcoma/metabolismo , Adolescente , Adulto , Sequência de Aminoácidos , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Proliferação de Células , Criança , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP47/química , Proteínas de Choque Térmico HSP47/genética , Proteínas de Choque Térmico HSP47/metabolismo , Humanos , Masculino , MicroRNAs/genética , Dados de Sequência Molecular , Osteossarcoma/genética , Interferência de RNA , Transcriptoma , Vimentina/química , Vimentina/genética , Vimentina/metabolismo , Adulto Jovem
17.
Insect Biochem Mol Biol ; 167: 104086, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38295885

RESUMO

Growth-blocking peptide (GBP), an insect cytokine, was first found in armyworm Mythimna separata. A functional analogue of GBP, stress-responsive peptide (SRP), was also identified in the same species. SRP gene expression has been demonstrated to be enhanced by GBP, indicating that both cytokines are organized within a hierarchical regulatory network. Although GBP1 (CG15917) and GBP2 (CG11395) have been identified in Drosophila melanogaster, immunological functions have only been characterized for GBP1. It is expected that the biological responses of two structurally similar peptides should be coordinated, but there is little information on this topic. Here, we demonstrate that GBP2 replicates the GBP1-mediated cellular immune response from Drosophila S2 cells. Moreover, the GBP2-induced response was silenced by pre-treatment with dsRNA targeting the GBP receptor gene, Mthl10. Furthermore, treatment of S2 cells with GBP2 enhanced GBP1 expression levels, but GBP1 did not affect GBP2 expression. GBP2 derived enhancement of GBP1 expression was not observed in the presence of GBP1, indicating that GBP2 is an upstream expressional regulator of a GBP1/GBP2 cytokine network. GBP2-induced enhancement of GBP1 expression was not observed in Mthl10 knockdown cells. Enhancement of GBP2 expression was observed in both Drosophila larvae and S2 cells under heat stress conditions; expressional enhancement of both GBP1 and GBP2 was eliminated in Mthl10 knockdown cells and larvae. Finally, Ca2+ mobilization assay in GCaMP3-expressing S2 cells demonstrated that GBP2 mobilizes Ca2+ upstream of Mthl10. Our finding revealed that Drosophila GBP1 and GBP2 control immune responses as well as their own expression levels through a hierarchical cytokine network, indicating that Drosophila GBP1/GBP2 system can be a simple model that is useful to investigate the detailed regulatory mechanism of related cytokine complexes.


Assuntos
Citocinas , Drosophila , Animais , Drosophila/metabolismo , Citocinas/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Transporte/metabolismo , Peptídeos/metabolismo , Imunidade
18.
Front Immunol ; 15: 1342625, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38449858

RESUMO

Introduction: Despite aggressive standard-of-care therapy, including surgery, radiation, and chemotherapy, glioblastoma recurrence is almost inevitable and uniformly lethal. Activation of glioma-intrinsic Wnt/ß-catenin signaling is associated with a poor prognosis and the proliferation of glioma stem-like cells, leading to malignant transformation and tumor progression. Impressive results in a subset of cancers have been obtained using immunotherapies including anti-CTLA4, anti-PD-1, and anti-PD-L1 or chimeric antigen receptor (CAR) T cell therapies. However, the heterogeneity of tumors, low mutational burden, single antigen targeting, and associated antigen escape contribute to non-responsiveness and potential tumor recurrence despite these therapeutic efforts. In the current study, we determined the effects of the small molecule, highly specific Wnt/CBP (CREB Binding Protein)/ß-catenin antagonist ICG-001, on glioma tumor cells and the tumor microenvironment (TME)-including its effect on immune cell infiltration, blood vessel decompression, and metabolic changes. Methods: Using multiple glioma patient-derived xenografts cell lines and murine tumors (GL261, K-Luc), we demonstrated in vitro cytostatic effects and a switch from proliferation to differentiation after treatment with ICG-001. Results: In these glioma cell lines, we further demonstrated that ICG-001 downregulated the CBP/ß-catenin target gene Survivin/BIRC5-a hallmark of Wnt/CBP/ß-catenin inhibition. We found that in a syngeneic mouse model of glioma (K-luc), ICG-001 treatment enhanced tumor infiltration by CD3+ and CD8+ cells with increased expression of the vascular endothelial marker CD31 (PECAM-1). We also observed differential gene expression and induced immune cell infiltration in tumors pretreated with ICG-001 and then treated with CAR T cells as compared with single treatment groups or when ICG-001 treatment was administered after CAR T cell therapy. Discussion: We conclude that specific Wnt/CBP/ß-catenin antagonism results in pleotropic changes in the glioma TME, including glioma stem cell differentiation, modulation of the stroma, and immune cell activation and recruitment, thereby suggesting a possible role for enhancing immunotherapy in glioma patients.


Assuntos
Glioma , beta Catenina , Humanos , Animais , Camundongos , Via de Sinalização Wnt , Recidiva Local de Neoplasia , Imunoterapia , Glioma/terapia , Microambiente Tumoral
19.
J Proteome Res ; 12(2): 753-62, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23298340

RESUMO

Pancreatic cancer is a devastating disease and early diagnosis and treatment are essential to improve the prognosis. We previously showed that α-fibrinogen containing hydroxylated proline residues at positions 530 and 565 is increased in plasma of pancreatic cancer patients. However, no antibody specific for hydroxylated proline-530 is available. Therefore, the purposes of this study were to develop a quantification method specific for both proline-hydroxylated α-fibrinogens by selected/multiple reaction monitoring (SRM/MRM), and to validate these modifications as pancreatic cancer markers. The target peptide for hydroxylated proline-530 contained methionine, and since variable partial oxidation of this residue would affect the quantification, hydrogen peroxide treatment was carried out to ensure complete oxidation. Quantification values of modified and unmodified α-fibrinogen were well correlated with those obtained by immunoblotting. Concentrations of modified and unmodified α-fibrinogen were quantified in 70 pancreatic cancer patients and 27 healthy controls. Percent hydroxylation of α-fibrinogen and concentration of hydroxylated α-fibrinogen were significantly greater in the plasma of patients. Furthermore, among 8 carbohydrate antigen 19-9 (CA19-9)-negative patients in stages I/II, 6 were positive for proline-hydroxylated α-fibrinogen. These results indicate that plasma concentration of proline-hydroxylated α-fibrinogen measured by SRM/MRM analysis may be a good pancreatic cancer marker, especially in CA19-9-negative patients.


Assuntos
Biomarcadores Tumorais/sangue , Fibrinogênio/análise , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/diagnóstico , Prolina/química , Idoso , Idoso de 80 Anos ou mais , Antígeno CA-19-9/sangue , Estudos de Casos e Controles , Feminino , Fibrinogênio/metabolismo , Humanos , Peróxido de Hidrogênio/química , Hidroxilação , Masculino , Metionina/química , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Oxirredução , Prognóstico , Proteômica
20.
Cancer Sci ; 104(8): 1045-51, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23648090

RESUMO

Preoperative chemoradiotherapy has been shown to improve the outcome of patients with esophageal cancer, but because response to this therapy varies, it is desirable to identify in advance individuals who would be unlikely to benefit, in order to avoid unnecessary adverse drug effects. The serum profiles of 84 cytokines and related proteins were determined in 37 patients with esophageal squamous cell carcinoma who received identical neoadjuvant preoperative chemoradiotherapy regimens and underwent surgical resection. Histological response to this therapy was assessed in surgically resected specimens. The serum soluble interleukin-6 receptor (sIL6R) level was significantly higher in 30 patients who failed to achieve a histological complete response (P = 0.005). Multivariate analysis revealed that the increased level of sIL6R was one of several significant independent predictors of an unfavorable outcome (hazard ratio, 2.87; P = 0.017). The increased level of this cytokine in patients who did not obtain a complete response was reproducibly observed in an independent cohort of 34 patients. Esophageal squamous cell carcinoma patients with an increased serum level of sIL6R are predicted to respond poorly to preoperative chemoradiotherapy, therefore, their exclusion from this treatment may be considered. Persistent systemic inflammation is implicated as a possible mechanism of resistance to this therapy.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/terapia , Neoplasias Esofágicas/sangue , Neoplasias Esofágicas/terapia , Receptores de Interleucina-6/sangue , Idoso , Carcinoma de Células Escamosas/patologia , Quimiorradioterapia/métodos , Estudos de Coortes , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Inflamação/sangue , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante/métodos , Estudos Retrospectivos
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