RESUMO
Aquaporin (AQP) 7 and AQP9 are membrane channel proteins called aquaglyceroporins and are related to glucose and lipid metabolism. AQP7 is mainly expressed in white adipose tissue (WAT) and is involved in releasing glycerol into the bloodstream. AQP9 is the glycerol channel in the liver that supplies glycerol to the hepatic cells. In this study, we investigated the relationship between the expression of aquaglyceroporins and lifestyle-related diseases, such as obesity and fatty liver, using 22-week-old db/db mice. Body weight, WAT, and liver weight showed increases in db/db mice. The levels of liver lipids, plasma lipids, insulin, and leptin were also increased in db/db mice. Gene expression related to fatty acid and triglyceride synthesis in the liver was enhanced in db/db mice. In addition, gene and protein expression of gluconeogenesis-related enzymes was increased. Conversely, lipolysis-related gene expression in WAT was reduced. In the db/db mice, AQP9 expression in the liver was raised; however, AQP7 expression in WAT was reduced. These results suggest that in db/db mice, enhanced hepatic AQP9 expression increased the supply of glycerol to the liver and induced fatty liver and hyperglycemia. Additionally, reduced AQP7 expression in WAT is associated with excessive lipid accumulation in adipocytes. Aquaglyceroporins are essential molecules for glucose and lipid metabolism, and may be potential target molecules for the treatment of obesity and lifestyle-related diseases.
Assuntos
Aquagliceroporinas , Aquaporinas , Fígado Gorduroso , Obesidade , Animais , Camundongos , Aquagliceroporinas/genética , Aquagliceroporinas/metabolismo , Aquaporinas/genética , Aquaporinas/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Lipídeos , Fígado/metabolismo , Obesidade/genética , Obesidade/metabolismoRESUMO
Gas samples were collected from the air surrounding single and mixed laboratory cultures, and preliminary data on human breath samples were also obtained. The infrared spectra of a variety of gasses were measured at high resolution (0.5 cm-1) to obtain information about the infrared absorption bands to be used as indicators. These key bands enable bacterial classification, and the discrimination rates can be improved by observing multiple infrared absorptions. The air around Pseudomonas aeruginosa was distinguished from the other gas samples by the infrared absorptions at wavenumbers of 778.4 cm-1 and 2213.2 cm-1. For Acinetobacter baumannii, infrared absorptions at 1215.0 cm-1 and 2982.3 cm-1 were used; furthermore, adding those at 4768.7 cm-1 and 5353.8 cm-1 was shown to improve identification.
Assuntos
Acinetobacter baumannii , Infecções por Pseudomonas , Antibacterianos/uso terapêutico , Gases , Humanos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , Espectrofotometria InfravermelhoRESUMO
In this study, eight types of bacteria were cultivated, including Staphylococcus aureus. The infrared absorption spectra of the gas surrounding cultured bacteria were recorded at a resolution of 0.5 cm-1 over the wavenumber range of 7500-500 cm-1. From these spectra, we searched for the infrared wavenumbers at which characteristic absorptions of the gas surrounding Staphylococcus aureus could be measured. This paper reports two wavenumber regions, 6516-6506 cm-1 and 2166-2158 cm-1. A decision tree-based machine learning algorithm was used to search for these wavenumber regions. The peak intensity or the absorbance difference was calculated for each region, and the ratio between them was obtained. When these ratios were used as training data, decision trees were created to classify the gas surrounding Staphylococcus aureus and the gas surrounding other bacteria into different groups. These decision trees show the potential effectiveness of using absorbance measurement at two wavenumber regions in finding Staphylococcus aureus.
Assuntos
Árvores de Decisões , Gases/química , Espectrofotometria Infravermelho/métodos , Staphylococcus aureus/isolamento & purificação , Algoritmos , Aprendizado de Máquina , OdorantesRESUMO
The aim of this study is to examine 1) muscle fiber type composition, 2) myofiber diameter, and 3) aquaporin (AQP) 7 and AQP 9 mRNA expressions by quantitative PCR in muscles of obese db/db mice. The myofiber type composition of skeletal muscle was not statistically significantly different between db/db mice and control mice; while the average myofiber diameter ratio showed a decrease in db/db mice. The expression of AQP7 but not AQP9 mRNA in the skeletal and cardiac muscles was significantly upregulated in db/db mice. Thus this study revealed quantitatively that type 2 myofiber atrophy was shown in the skeletal muscles of db/db mice. AQP7 mRNA expression was upregulated in the skeletal and cardiac muscles of db/db mice.
Assuntos
Fibras Musculares Esqueléticas , Doenças dos Roedores , Animais , Camundongos , Camundongos Endogâmicos , Miocárdio , Obesidade/genética , Obesidade/veterinária , RNA Mensageiro/genéticaRESUMO
Mice housed in an enriched environment (EE) have inhibited tumor development because of eustress (positive stress) stimulation. However, the mechanisms underlying increased cancer resistance in EEs remain unclear; this may be due to poor reproducibility of the results because of the complicated EE assembly requirements. In this study, we examined the effects of a simplified EE (sEE) model, consisting only of a nesting shelter and a running wheel, on cancer development in mice. We found that, similar to the complex EE, the sEE promoted motor function and alleviated anxiety in mice. Moreover, the mice housed in the sEE showed inhibited tumor growth and metastasis in addition to a higher average body temperature (especially at the point of transition from light to darkness). Furthermore, mice in the sEE had a decreased brown adipose tissue (BAT) mass, with a significant upregulation of the Ucp1 and Adrb3 genes (which encode uncoupling protein 1 and ß-adrenergic receptor, respectively) observed in the BAT at the point of transition from light to darkness. An antibody against the immune checkpoint protein programmed cell death 1 was also found to have an additive effect with the sEE against tumor development. Our findings indicate that the established sEE model may be a useful tool for studying the antitumor effects of eustress and can potentially introduce new avenues for cancer prevention and treatment.
Assuntos
Temperatura Corporal , Meio Ambiente , Neoplasias/prevenção & controle , Animais , CamundongosRESUMO
Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem-spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.5 s) of the rostral pFRG reset the respiratory rhythm. At the cellular level, photostimulation depolarised Phox2b-positive pre-inspiratory, inspiratory and respiratory-modulated tonic neurons and Phox2b-negative pre-inspiratory neurons. In contrast, changes in membrane potential of Phox2b-negative inspiratory and expiratory neurons varied depending on characteristics of ongoing synaptic connections in local respiratory networks in the rostral medulla. In the presence of tetrodotoxin, photostimulation depolarised Phox2b-positive cells, but caused no significant changes in membrane potential of Phox2b-negative cells. We concluded that depolarisation of Phox2b-positive neurons was due to cell-autonomous photo-activation and summation of excitatory postsynaptic potentials, whereas membrane potential changes of Phox2b-negative neurons depended on the network configuration. Our findings shed further light on local networks among respiratory-related neurons in the rostral ventrolateral medulla and emphasise the important role of pre-inspiratory neurons in respiratory rhythm generation in the neonatal rat en bloc preparation.
Assuntos
Channelrhodopsins/metabolismo , Proteínas de Homeodomínio/metabolismo , Bulbo/metabolismo , Neurônios/metabolismo , Centro Respiratório/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Tronco Encefálico/metabolismo , Feminino , Masculino , Potenciais da Membrana/fisiologia , Optogenética/métodos , Ratos , Respiração , Tetrodotoxina/metabolismoRESUMO
Pneumonitis is the leading cause of death associated with the use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) against non-small cell lung cancer (NSCLC). However, the risk factors and the mechanism underlying this toxicity have not been elucidated. Tumor necrosis factor (TNF) has been reported to transactivate EGFR in pulmonary epithelial cells. Hence, we aimed to test the hypothesis that EGFR tyrosine kinase activity regulates TNF-mediated bronchial epithelial cell survival, and that inhibition of EGFR activity increases TNF-induced lung epithelial cell apoptosis. We used surfactant protein C (SPC)-TNF transgenic (tg) mice which overexpress TNF in the lungs. In this model, gefitinib, an EGFR-TKI, enhanced lung epithelial cell apoptosis and lymphocytic inflammation, indicating that EGFR tyrosine kinase prevents TNF-induced lung injury. Furthermore, IL-17A was significantly upregulated by gefitinib in SPC-TNF tg mice and p38MAPK activation was observed, indicative of a pathway involved in lung epithelial cell apoptosis. Moreover, in lung epithelial cells, BEAS-2B, TNF stimulated EGFR transactivation via the TNF-α-converting enzyme in a manner that requires heparin binding (HB)-EGF and transforming growth factor (TGF)-α. These novel findings have significant implications in understanding the role of EGFR in maintaining human bronchial epithelial cell homeostasis and in NSCLC treatment.
Assuntos
Apoptose , Células Epiteliais/metabolismo , Gefitinibe/efeitos adversos , Lesão Pulmonar/metabolismo , Pneumonia/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína ADAM17/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Receptores ErbB/metabolismo , Gefitinibe/uso terapêutico , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/fisiologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/fisiopatologia , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Transgênicos , Modelos Animais , Pneumonia/induzido quimicamente , Pneumonia/fisiopatologia , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Fator de Crescimento Transformador alfa , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Pax6 is an important transcription factor expressed in several discrete domains of the developing central nervous system. It has been reported that Pax6 is involved in the specification of subtypes of hindbrain motor neurons. Pax6 homozygous mutant (rSey2/rSey2) rats die soon after birth, probably due to impaired respiratory movement. To determine whether the respiratory center in the medulla functions normally, we analyzed the histological and neurophysiological properties of the medulla and spinal cord in fetal rats with this mutation. First, the medulla of rSey2/rSey2 at embryonic (E) 21.5-E22.5 tended to be smaller than those from heterozygous mutant (rSey2/+) and wild-type (+/+) littermates. Through immunohistochemical analysis, we confirmed normal distribution of Phox2b-expressing cells in the parafacial respiratory group (pFRG) of rSey2/rSey2 rats. Expression of neurokinin-1 receptor (NK-1R) was weak and dispersed in rSey2/rSey2 rats. In addition, rSey2/rSey2 rats have a defect of the hypoglossal nerve root. Electrophysiological analysis using brainstem-spinal cord preparations (E21.5-E22.5) revealed that rSey2/rSey2 rats showed larger fluctuation of the amplitude of inspiratory activity monitored from the fourth cervical root although there was no significant difference in the respiratory rate among rSey2/rSey2, rSey2/+, and +/+ littermates. The response of respiratory rhythm to high CO2 was similar among all genotypes. Optical recordings of neuronal activity revealed that the activity of the pFRG tended to be weaker and inspiratory activity appeared in more scattered areas in the caudal ventral medulla in the rSey2/rSey2 rats. These results suggest that the basal activity of the respiratory system was preserved with mild impairment of the inspiratory activity in the rSey2/rSey2 rats and that the Pax6 gene is involved in the functional development of the neuronal system producing effective inspiratory motor outputs for survival.
Assuntos
Fator de Transcrição PAX6/genética , Sistema Respiratório/embriologia , Sistema Respiratório/inervação , Animais , Animais Recém-Nascidos , Tronco Encefálico/metabolismo , Feminino , Feto , Proteínas de Homeodomínio/metabolismo , Masculino , Bulbo/metabolismo , Neurônios Motores/metabolismo , Fator de Transcrição PAX6/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Respiração , Centro Respiratório/metabolismo , Medula Espinal/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Preinspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG) comprise one of the respiratory rhythm generators in the medulla of the neonatal rat. A subgroup of pFRG/Pre-I neurons expresses the transcription factor Phox2b. To further analyze detailed neuronal mechanisms of respiratory rhythm generation in the neonatal rat, we developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch). Brainstem-spinal cord preparations were isolated from 0-2-day-old Tg newborn rats and were superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25-26 °C. Inspiratory fourth cervical ventral root (C4) activity was monitored, and membrane potentials of neurons in the pFRG including Pre-I and inspiratory neurons were recorded. Phox2b-positive cells in the Tg rats were essentially positive for enhanced green fluorescent protein (EGFP) signals (reporter for Arch) in the pFRG. Continuous photo-stimulation of the rostral ventral medulla for up to 90 s by covering the pFRG with green laser light (532 nm) induced a decrease of respiratory rate measured at C4 accompanied by membrane hyperpolarization of Phox2b-positive pFRG/Pre-I neurons. In contrast, Phox2b-negative inspiratory neurons were not hyperpolarized during the photo-stimulation. Our findings showed that Phox2b-expressing pFRG/Pre-I neurons are involved in the maintenance of the basic respiratory rhythm in neonatal rat.
Assuntos
Proteínas Arqueais/biossíntese , Proteínas de Homeodomínio/metabolismo , Rede Nervosa/metabolismo , Centro Respiratório/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Proteínas Arqueais/genética , Tronco Encefálico/metabolismo , Proteínas de Homeodomínio/biossíntese , Masculino , Bulbo/metabolismo , Neurônios/metabolismo , Ratos , Ratos Transgênicos , Respiração , Centro Respiratório/metabolismo , Medula Espinal/fisiologia , Fatores de Transcrição/biossínteseRESUMO
The critical T790M mutation in EGFR, which mediates resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKI; gefitinib, erlotinib, and afatinib), has facilitated the development of third-generation mutation-selective EGFR TKIs (rociletinib and osimertinib). We previously reported heterogeneous afatinib-resistant mechanisms, including emergence of T790M-EGFR, and responses to third-generation EGFR TKIs. Here, we used afatinib-resistant lung adenocarcinoma cells [AfaR (formerly AFR3) cells], carrying exon 19 deletion/T790M in EGFR To identify the novel resistance mechanisms in post-afatinib treatment, RocR1/RocR2 and OsiR1/OsiR2 cells were established using increasing concentrations of rociletinib and osimertinib, respectively. Attenuation of exon 19 deletion and T790M was confirmed in both rociletinib-resistant cells; in addition, EGFR and KRAS amplification was observed in RocR1 and RocR2, respectively. Significant KRAS amplification was observed in the osimertinib-resistant cell lines, indicating a linear and reversible increase with increased osimertinib concentrations in OsiR1 and OsiR2 cells. OsiR1 cells maintained osimertinib resistance with KRAS amplification after osimertinib withdrawal for 2 months. OsiR2 cells exhibited KRAS attenuation, and osimertinib sensitivity was entirely recovered. Phospho-EGFR (Y1068) and growth factor receptor-bound protein 2 (GRB2)/son of sevenless homolog 1 (SOS1) complex was found to mediate osimertinib resistance in OsiR1 cells with sustained KRAS activation. After 2 months of osimertinib withdrawal, this complex was dissociated, and the EGFR signal, but not the GRB2/SOS1 signal, was activated. Concomitant inhibition of MAPK kinase and EGFR could overcome osimertinib resistance. Thus, we identified a heterogeneous acquired resistance mechanism for third-generation EGFR TKIs, providing insights into the development of novel treatment strategies.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Acrilamidas/farmacologia , Afatinib/farmacologia , Compostos de Anilina/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Amplificação de Genes , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Transplante de Neoplasias , Pirimidinas/farmacologia , Deleção de SequênciaRESUMO
Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.
Assuntos
Adenocarcinoma/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Gefitinibe , Humanos , Indóis/farmacologia , Neoplasias Pulmonares/patologia , Mutação , Proteínas Proto-Oncogênicas c-met/genética , Quinazolinas/farmacologia , Sulfonas/farmacologiaRESUMO
EGFR tyrosine kinase inhibitors (TKI) are associated with significant responses in non-small cell lung cancer (NSCLC) patients harboring EGFR-activating mutations. However, acquired resistance to reversible EGFR-TKIs remains a major obstacle. In particular, although the second-generation irreversible EGFR-TKI afatinib is currently used for treating NSCLC patients, the mechanisms underlying acquired afatinib resistance remain poorly understood. Here, heterogeneous mechanisms of acquired resistance were identified following long-term exposure to increasing doses of afatinib in EGFR-mutant lung adenocarcinoma PC-9 cells. Notably, three resistant cell lines, PC-9AFR1, PC-9AFR2, and PC-9AFR3 (AFR1, AFR2, and AFR3, respectively) employed distinct mechanisms for avoiding EGFR inhibition, with increased EGFR expression being detected in all resistant cell lines. Moreover, an activating EGFR mutation was partially lost in AFR1 and AFR2 cells. AFR1 cells exhibited afatinib resistance as a result of wild-type KRAS amplification and overexpression; however, these cells showed a progressive decrease and eventual loss of the acquired KRAS dependence, as well as resensitization to afatinib, following a drug holiday. Meanwhile, AFR2 cells exhibited increased expression of insulin-like growth factor-binding protein 3 (IGFBP3), which promoted insulin-like growth factor 1 receptor (IGF1R) activity and subsequent AKT phosphorylation, thereby indicating a potential bypass signaling pathway associated with IGFR1. Finally, AFR3 cells harbored the secondary EGFR mutation T790M. Our findings constitute the first report showing acquired wild-type KRAS overexpression and attenuation of afatinib resistance following a drug holiday.Implications: The heterogeneous mechanisms of afatinib resistance should facilitate the development of more effective therapeutic strategies for NSCLC patients. Mol Cancer Res; 15(7); 915-28. ©2017 AACR.
Assuntos
Adenocarcinoma/tratamento farmacológico , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinazolinas/administração & dosagem , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Afatinib , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Quinazolinas/efeitos adversos , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Met-amplified EGFR-tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) harboring an activating EGFR mutation is responsive to concurrent EGFR-TKI and Met-TKI treatment in a preclinical model. Here, we determined that Met-amplified gefitinib-resistant cells acquire dual resistance to inhibition of EGFR and Met tyrosine kinase activities. PC-9 lung adenocarcinoma cells harboring 15-bp deletions (Del E746_A750) in EGFR exon 19 were treated with increasing concentrations of the Met-TKI PHA665752 and 1 µmol/L gefitinib for 1 year; three resistant clones were established via Met amplification. The three dual-resistance cell lines (PC-9DR2, PC-9DR4, and PC-9DR6, designated as DR2, DR4, and DR6, respectively) exhibited different mechanisms for evading both EGFR and Met inhibition. None of the clones harbored a secondary mutation of EGFR T790M or a Met mutation. Insulin-like growth factor (IGF)/IGF1 receptor activation in DR2 and DR4 cells acted as a bypass signaling pathway. Met expression was attenuated to a greater extent in DR2 than in PC-9 cells, but was maintained in DR4 cells by overexpression of IGF-binding protein 3. In DR6 cells, Met was further amplified by association with HSP90, which protected Met from degradation and induced SET and MYND domain-containing 3 (SMYD3)-mediated Met transcription. This is the first report describing the acquisition of dual resistance mechanisms in NSCLC harboring an activating EGFR mutation to Met-TKI and EGFR-TKI following previous EGFR-TKI treatment. These results might inform the development of more effective therapeutic strategies for NSCLC treatment. Mol Cancer Ther; 15(12); 3040-54. ©2016 AACR.
Assuntos
Adenocarcinoma/genética , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Gefitinibe , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , Quinazolinas/farmacologia , Receptor IGF Tipo 1 , Receptores de Morte Celular/metabolismo , Receptores de Somatomedina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Somatomedinas/metabolismoRESUMO
AIMS: Cancer is a leading cause of morbidity and mortality worldwide; therefore, effective measures for cancer prevention and treatment are in constant demand. The extracts of Inonotus obliquus (Chaga mushroom) demonstrate potent anti-tumor activities and have been used to treat cancer in several countries; however, the actual effect and underlying mechanisms are still unclear. In the present study, we aimed to investigate the effects of continuous intake of aqueous extract from I. obliquus on tumor suppression. MAIN METHODS: Anticancer activity of the I. obliquus extract was examined in mouse models of Lewis lung carcinoma growth and spontaneous metastasis after 3 weeks of continuous extract intake at the dose of 6 mg/kg/day, which corresponded to that ingested daily with Chaga infusion in Japan. KEY FINDINGS: The extract of I. obliquus caused significant tumor suppressive effects in both models. Thus, in tumor-bearing mice, 60% tumor reduction was observed, while in metastatic mice, the number of nodules decreased by 25% compared to the control group. Moreover, I. obliquus extract-treated mice demonstrated the increase in tumor agglomeration and inhibition of vascularization. Interestingly, I. obliquus intake decreased body weight in middle-aged mice and increased body temperature in response to light-dark switching in mature adult mice. Furthermore, I. obliquus prevented temperature drop in mice after tumor implantation. SIGNIFICANCE: Our findings suggest that the I. obliquus extract could be used as a natural remedy for cancer suppression by promoting energy metabolism.
RESUMO
The dosage difference of X-linked genes between the sexes in mammals is compensated for by genetic inactivation of one of the X chromosomes in XX females. A noncoding RNA transcribed from the Xist gene at the onset of X chromosome inactivation coats the X chromosome in cis and induces chromosome-wide heterochromatinization. Here, we report a new Xist allele (Xist(CAG)) driven by a CAG promoter, which is known to be constitutively active in many types of cells. The paternal transmission of Xist(CAG) resulted in the preferential inactivation of the targeted paternal X (Xp) not only in the extra-embryonic but also the embryonic lineage, whereas maternal transmission ended with embryonic lethality at the early postimplantation stage with a phenotype that resembled mutant embryos carrying a maternal deficiency in Tsix, an antisense negative regulator of Xist, in both sexes. Interestingly, we found that the upregulation of Xist(CAG) in preimplantation embryos temporally differed depending on its parental origin: its expression started at the 4- to 8-cell stages when paternally inherited, and Xist(CAG) was upregulated at the blastocyst stage when maternally inherited. This might indicate that the Xist locus on Xp is permissive to transcription, but the Xist locus on the maternal X (Xm) is not. We extrapolated from these findings that the maternal Xist allele might manifest a chromatin structure inaccessible by transcription factors relative to the paternal allele. This might underlie the mechanism for the maternal repression of Xist at the early cleavage stage when Tsix expression has not yet occurred on Xm.
Assuntos
Alelos , Loci Gênicos , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Animais , Blastocisto/metabolismo , Metilação de DNA/genética , Regulação para Baixo/genética , Embrião de Mamíferos/metabolismo , Feminino , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Padrões de Herança/genética , Camundongos , Mutação/genética , Oogênese , Fenótipo , Regulação para Cima/genética , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.
Assuntos
Células Quimiorreceptoras , Cromossomos Artificiais Bacterianos , Proteínas de Homeodomínio , Troca Gasosa Pulmonar/fisiologia , Taxa Respiratória/fisiologia , Fatores de Transcrição , Corpo Trapezoide , Animais , Dióxido de Carbono/metabolismo , Células Quimiorreceptoras/citologia , Células Quimiorreceptoras/fisiologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Oxigênio/metabolismo , Ratos , Ratos Transgênicos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Corpo Trapezoide/citologia , Corpo Trapezoide/fisiologiaRESUMO
In embryos, neural crest cells emerge from the dorsal region of the fusing neural tube and migrate throughout tissues to differentiate into various types of cells including osteoblasts. In adults, subsets of neural crest-derived cells (NCDCs) reside as stem cells and are considered to be useful cell sources for regenerative medicine strategies. Numerous studies have suggested that stem cells with a neural crest origin persist into adulthood, especially those within the mammalian craniofacial compartment. However, their distribution as well as capacity to differentiate into osteoblasts in adults is not fully understood. To analyze the precise distribution and characteristics of NCDCs in adult oral tissues, we utilized an established line of double transgenic (P0-Cre/CAG-CAT-EGFP) mice in which NCDCs express green fluorescent protein (GFP) throughout their life. GFP-positive cells were scattered like islands throughout tissues of the palate, gingiva, tongue, and buccal mucosa in adult mice, with those isolated from the latter shown to form spheres, typical cell clusters composed of stem cells, under low-adherent conditions. Furthermore, GFP-positive cells had markedly increased alkaline phosphatase (a marker enzyme of osteoblast differentiation) activity and mineralization as shown by alizarin red staining, in the presence of bone morphogenetic protein (BMP)-2. These results suggest that NCDCs reside in various adult oral tissues and possess potential to differentiate into osteoblastic cells. NCDCs in adults may be a useful cell source for bone regeneration strategies.
Assuntos
Boca/citologia , Boca/fisiologia , Crista Neural/citologia , Crista Neural/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Envelhecimento/patologia , Animais , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Camundongos , Camundongos Transgênicos , Osteogênese/fisiologiaRESUMO
Although considerable progress has been made in understanding how the temporal and regional control of neural progenitor cells (NPCs) dictates their fate, their key regulators during neural development are still unknown. Ghrelin, which is isolated from porcine stomach extract, is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). The widespread expression of ghrelin and GHS-R in the central nervous system during development suggests that ghrelin may be involved in developmental neural growth. However, its role in regulating fetal NPCs is still unclear. In this study, we investigated the effects of ghrelin on primary cultured NPCs derived from fetal mouse telencephalon. The expressions of both ghrelin and its receptor were observed in NPCs using RT-PCR, immunoblotting and immunocytostaining. Interestingly, the exposure of fetal NPCs to ghrelin at concentrations of 10(-7) and 10(-9)M suppressed their proliferation, and caused them to differentiate into neurons and to extend neurites. These results strongly suggest that ghrelin plays an autocrine modulatory role in fetal neural development.
Assuntos
Diferenciação Celular/genética , Grelina/biossíntese , Células-Tronco Neurais/metabolismo , Receptores de Grelina/biossíntese , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Grelina/administração & dosagem , Camundongos , Neurogênese/genética , Receptores de Grelina/genética , SuínosRESUMO
RATIONALE: Janus kinase/signal transducer and activator of transcription (JAK/STAT) signals and their endogenous inhibitor, suppressor of cytokine signaling 3 (SOCS3), in vascular endothelial cells (ECs) reportedly dominate the pathological angiogenesis. However, how these inflammatory signals are potentiated during pathological angiogenesis has not been fully elucidated. We suspected that an intracellular protease calpain, which composes the multifunctional proteolytic systems together with its endogenous inhibitor calpastatin (CAST), contributes to the JAK/STAT regulations. OBJECTIVE: To specify the effect of EC calpain/CAST systems on JAK/STAT signals and their relationship with pathological angiogenesis. METHODS AND RESULTS: The loss of CAST, which is ensured by several growth factor classes, was detectable in neovessels in murine allograft tumors, some human malignant tissues, and oxygen-induced retinopathy lesions in mice. EC-specific transgenic introduction of CAST caused downregulation of JAK/STAT signals, upregulation of SOCS3 expression, and depletion of vascular endothelial growth factor (VEGF)-C, thereby counteracting unstable pathological neovessels and disease progression in tumors and oxygen-induced retinopathy lesions in mice. Neutralizing antibody against VEGF-C ameliorated pathological angiogenesis in oxygen-induced retinopathy lesions. Small interfering RNA-based silencing of endogenous CAST in cultured ECs facilitated µ-calpain-induced proteolytic degradation of SOCS3, leading to VEGF-C production through amplified interleukin-6-driven STAT3 signals. Interleukin-6-induced angiogenic tube formation in cultured ECs was accelerated by CAST silencing, which is suppressible by pharmacological inhibition of JAK/STAT signals, antibody-based blockage of VEGF-C, and transfection of calpain-resistant SOCS3, whereas transfection of wild-type SOCS3 exhibited modest angiostatic effects. CONCLUSIONS: Loss of CAST in angiogenic ECs facilitates µ-calpain-induced SOCS3 degradation, which amplifies pathological angiogenesis through interleukin-6/STAT3/VEGF-C axis.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Calpaína/metabolismo , Células Endoteliais/metabolismo , Neoplasias/irrigação sanguínea , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Adenocarcinoma/irrigação sanguínea , Sequência de Aminoácidos , Animais , Aorta , Proteínas de Ligação ao Cálcio/genética , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Células Cultivadas , Citocinas/fisiologia , Feminino , Glioblastoma/irrigação sanguínea , Humanos , Janus Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neovascularização Patológica/fisiopatologia , Proteínas Recombinantes de Fusão/metabolismo , Retinopatia da Prematuridade/fisiopatologia , Fatores de Transcrição STAT/fisiologia , Transdução de Sinais/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide considered to be a potent regulator of astrocytes. It has been reported that PACAP also affects astrocytoma cell properties, but the proliferative effects of this peptide in previous reports were inconsistent. The purpose of this study was to search for correlations between malignant potential, PACAP/PACAP receptor expression, and the proliferative potential of four astrocytoma cell lines (KNS-81, KINGS-1, SF-126, and YH-13). Immunohistochemical observations were performed using astrocyte lineage markers with a view to establishing malignant potential, which is inversely correlated to differentiation status in astrocytoma cells. YH-13 showed the most undifferentiated astrocyte-like status, and was immunopositive to a cancer stem cell marker, CD44. These observations suggest that YH-13 is the most malignant of the astrocytoma cell lines tested. Moreover, the strongest PAC1-R immunoreactivity was observed in YH-13 cells. Using real-time PCR analysis, no significant differences among cell lines were detected with respect to PACAP mRNA, but PAC1-R and VPAC1-R mRNA levels were significantly increased in YH-13 cells compared with the other cell lines. Furthermore, when cell lines were treated with PACAP (10(-11) M) for 3 days, the YH-13 cell line, but not of the other cell lines, exhibited a significantly increased cell number. These results suggest that PACAP receptor expression is correlated with the malignant and proliferative potential of astrocytoma cell lines.