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Liu S.-C.,Chang Gung University | Tsang N.-M.,Chang Gung Memorial Hospital at Lin Kou | Chiang W.-C.,Chang Gung University | Chang K.-P.,Head and Neck Surgery | And 5 more authors.
Journal of Clinical Investigation

Radioresistance of EBV-associated nasopharyngeal carcinoma (NPC) is associated with poor prognosis for patients with this form of cancer. Here, we found that NPC patients had increased serum levels of leukemia inhibitory factor (LIF) and that higher LIF levels correlated with local tumor recurrence. Furthermore, in vitro studies with NPC cells and in vivo xenograft mouse studies demonstrated that LIF critically contributes to NPC tumor growth and radioresistance. Using these model systems, we found that LIF treatment activated the mTORC1/p70S6K signaling pathway, enhanced tumor growth, inhibited DNA damage responses, and enhanced radioresistance. Treatment with either soluble LIF receptor (sLIFR), a LIF antagonist, or the mTOR inhibitor rapamycin reversed LIF-mediated effects, resulting in growth arrest and increased sensitivity to γ irradiation. Immunohistochemical (IHC) analyses of human NPC biopsies revealed that LIF and LIFR were overexpressed in tumor cells and that LIF expression correlated with the presence of the activated p-p70S6K. Finally, we found that the EBV-encoded protein latent membrane protein 1 (LMP1) enhances LIF production. Together, our findings indicate that LIF promotes NPC tumorigenesis and suggest that serum LIF levels may predict local recurrence and radiosensitivity in NPC patients. Source

Lee T.-H.,Graduate Institute of Biomedical science | Wu T.-S.,Graduate Institute of Biomedical science | Tseng C.-P.,Graduate Institute of Biomedical science | Tseng C.-P.,Chang Gung University | Qiu J.T.,Graduate Institute of Biomedical science

Background: Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes. Methods: Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates. Results: A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215-221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established. Conclusions: This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner. © 2012 Lee et al. Source

Wu C.-J.,Graduate Institute of Biomedical science | Yang C.-Y.,Graduate Institute of Biomedical science | Chen Y.-H.,Graduate Institute of Biomedical science | Chen C.-M.,Graduate Institute of Biomedical science | And 3 more authors.
International Archives of Allergy and Immunology

Background: Asthma is characterized as a chronic inflammatory disorder of the airways associated with an enhanced TH2 response to inhaled allergens. CD4+ T regulatory (Treg) cells are controlled by the master transcription factor FoxP3 and strictly maintain peripheral immunotolerance. Epigenetic regulation of FoxP3 by DNA methyltransferase inhibitors, such as 5-azacytidine (Aza), can generate a steady supply of functional Treg cells. Therefore, we propose that Aza can augment Treg cells in vivo to prevent the pathogenesis of asthma. Methods: BALB/c mice were sensitized with chicken ovalbumin (OVA) and treated with different doses of Aza. Airway hyperresponsiveness to methacholine, eosinophilia in bronchoalveolar lavage fluid, circulating titers of OVA-specific IgG1 and IgE, and stimulating levels of TH2 cytokines from splenocytes were then determined. Cellular populations were examined by flow cytometry. PC61 antibody, which depletes CD25+ cells, was used to verify the role of CD25+ cells in Aza-induced tolerance. Results: Administration of Aza to OVA-sensitized mice diminished airway hyperreactivity, pulmonary eosinophilia, levels of OVA-specific IgG1 and IgE in serum, and secretion of TH2 cytokines from OVA-stimulated splenocytes in a dose-dependent manner. Percentages of CD25+ and FoxP3+ cells in the CD4+ cell population were notably increased in Aza-treated mice compared to sensitized control mice. Furthermore, the major symptoms of asthma were exacerbated by depleting CD25+ cells in Aza-treated mice. Conclusions: Aza may have applications as a novel clinical strategy to increase the production of Treg cells in order to modulate the airway inflammation associated with asthma. Copyright © 2012 S. Karger AG, Basel. Source

Wu C.-P.,Graduate Institute of Biomedical science | Wu C.-P.,Chang Gung University | Hsiao S.-H.,Graduate Institute of Biomedical science | Su C.-Y.,Graduate Institute of Biomedical science | And 5 more authors.
Biochemical Pharmacology

CUDC-101 is the first small-molecule inhibitor designed to simultaneously inhibit epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) and histone deacetylase (HDAC) in cancer cells. Recently, in its first in human phase I study, CUDC-101 showed promising single agent activity against advanced solid tumors and favorable pharmacodynamic profile. However, the risk of developing drug resistance to CUDC-101 can still present a significant therapeutic challenge to clinicians in the future. One of the most common mechanisms of developing multidrug resistance (MDR) in cancer is associated with the overexpression of ATP-binding cassette (ABC) drug transporters ABCB1 and ABCG2. Together, they are able to reduce the efficacy and modify the pharmacological properties of anti-cancer agents, including many small molecule tyrosine kinase inhibitors (TKIs). Here, we have investigated the impact of ABCB1 and ABCG2 on the efficacy of CUDC-101 in human cancer cells. We revealed that although CUDC-101 has potent antiproliferative and proapoptotic activities against most cancer cell lines, the overexpression of ABCB1 or ABCG2 in cancer cells significantly reduced the activity of CUDC-101 against HDAC, EGFR and HER2, as well as its cytotoxicity and proapoptotic activity. Moreover, we showed that CUDC-101 modulated the function of both transporters without affecting the protein expression of either ABCB1 or ABCG2. More importantly, our study provides support for the rationale of combining CUDC-101 with modulators of ABC drug transporters to improve drug efficacy and overcome multidrug resistance associated with the overexpression of ABCB1 and ABCG2. © 2014 Elsevier Inc. All rights reserved. Source

Han F.,Jimei University | Zhang Y.,Jimei University | Zhang D.,Jimei University | Liu L.,Jimei University | And 3 more authors.
Fish and Shellfish Immunology

Rab GTPases, members of the Ras superfamily, encode monomeric G-proteins. Rab proteins regulate key steps in membrane traffic transport and endocytic pathway of host immune responses. Rab5A is involved in immune regulation, particularly in T cell migration and macrophage endocytosis in higher vertebrates. However, little is known of the molecular structure of Rab5A gene in marine teleost fish species and its expression profile during the parasite infection. In this study, the full-length cDNA sequence and genomic structure of Rab5A gene of the large yellow croaker (Larimichthys crocea) (LycRab5A), one of the most economical marine fishes, were identified and characterized. The LycRab5A protein, containing the ATPase/GTPase binding motifs and the effector molecules binding motifs, was highly homologous to that of other animals. The expression plasmid containing LycRab5A cDNA fused with GST was engineered and transformed into Escherichia coli to produce recombinant protein GST-LycRab5A, which was purified to prepare a polyclonal antibody specifically against LycRab5A. Subcellular localization revealed that LycRab5A expressed in the membrane and cytoplasm. Based on real-time PCR and Western blot analysis, we found that both mRNA and protein of LycRab5A were expressed in all tissues we examined; especially it was highly expressed in blood and gill. Interestingly, both mRNA and protein of LycRab5A were substantially up-regulated when parasitic ciliate protozoan (Cryptocaryon irritans) was infected. The expression of LycRab5A was reached to the maximal level at 24 h after infection. The line of evidence suggested that LycRab5A might play an important role in large yellow croaker defense against parasite infection. Moreover, on the basis of protein interaction, it was found that the LycRab5A interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. This discovery might contribute better understanding to the molecular events involved in fish immune responses. © 2016 Elsevier Ltd. Source

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