Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells

信州大学博士（医学）・学位論文・平成24年6月11日授与（甲第929号）・桂安萍Background: Since cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR)-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling. Methods: We evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS), and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling. Results: Upon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045), were also observed in RBE cells. Conclusion: In RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma.ArticleBMC Cancer. 2012, 12:179journal articl

A Hybrid Lesion of Lung Cancer and Aspergillosis

A 74-year-old man presented with gradual wall thickening of a cystic lung lesion. Serologic tests indicated Aspergillus infection, but neither fungal organisms nor evidence of malignant disease were recovered from repeated sputum collections, a bronchoscopic lung biopsy specimen, or bronchial washings. Treatment with antifungal agents did not result in clinical improvement. Surgical resection of the lesion demonstrated both squamous cell carcinoma and aspergillosis. These distinct disorders share common radiologic manifestations that can present a diagnostic challenge, as in the present case

Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells

Abstract Background Since cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR)-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling. Methods We evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS), and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling. Results Upon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045), were also observed in RBE cells. Conclusion In RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma.</p

Evaluation of Stability and Cytotoxicity of Self-Doped PEDOT Nanosheets in a Quasi-Biological Environment for Bioelectrodes

PEDOT:PSS is the most extensively used organic conductive material for flexible sensors; however, its long-term use in wet biological environments is difficult due to the swelling of PSS and the leaching of additives introduced by secondary interactions. Self-doped PEDOT (S-PEDOT) containing sulfonic acid groups covalently bonded to the PEDOT backbone has been applied to evaluate the stability of its performance as a conductive nanosheet and cytotoxicity. The S-PEDOT nanosheets and the control PEDOT:PSS nanosheets were prepared with a spin-coating method and immersed in a quasi-biological environment (PBS or culture medium, 37 °C, 5% CO2). Approximately 60 days of immersion increased the resistance of PEDOT:PSS 6-fold, while the resistance of S-PEDOT remained 3-fold. The difference would be caused by the crystal structure of the PEDOT chains being disrupted by the replacement of protons of the sulfonic acid groups in PSS with Na+ ions, swelling of PEDOT:PSS, and elution of the surfactant Zonyl, while the crystal structure of the main chain of S-PEDOT was maintained even after immersion, although the replacement by Na+ ions occurred. In addition, cytotoxicity was observed in the extract of the PEDOT:PSS nanosheet due to the elution of Zonyl, which is highly cytotoxic, while S-PEDOT showed low cytotoxicity because the additive containing Si was covalently bonded to the main chain. Thus, it was suggested that S-PEDOT nanosheets can function as minimally invasive bioelectrodes that can be used in the physiological environment

Intra-abdominal fat accumulation is a hypertension risk factor in young adulthood: A cross-sectional study

Accumulation of intra-abdominal fat is related to hypertension. Despite this, a relationship between hypertension and intra-abdominal fat in young adulthood is not clear. In this study, we verify whether intra-abdominal fat accumulation increases a hypertension risk in young adult subjects. In a cross-sectional study, intra-abdominal fat area was measured using a dual bioelectrical impedance analysis instrument in 697 university students (20.3±0.7 years, 425 men). Blood pressure and anthropometric factors were measured. Lifestyle variables including smoking, drinking, physical activity, and eating behavior were assessed with questionnaire. High blood pressure risk (systolic blood pressure ?130mm Hg and/or diastolic blood pressure ?85mm Hg) with increasing intra-abdominal fat area was evaluated. Participants were divided into 5 groups according to their intra-abdominal fat area (?24.9, 25-49.9, 50-74.9, 75-99.9, and ?100 cm2). As compared with the values of the smallest intra-abdominal fat area group, the crude and lifestyle-adjusted odds ratios (ORs) were elevated in larger intra-abdominal fat area groups [OR 1.31, 95% confidence interval (CI) 0.66-2.80; OR 3.38, 95% CI 1.60-7.57; OR 7.71, 95% CI 2.75-22.22; OR 18.74, 95% CI 3.93-105.64, respectively). The risk increase was observed only in men. Intra-abdominal fat accumulation is related to high blood pressure in men around 20 years of age. These results indicate the importance of evaluation and reduction of intra-abdominal fat to prevent hypertension