Eeffacy Of Acyclic Retinoid On The Fibroblast Growth Factor Mediated Signaling Pathways And Analysis On Hepatocelluar Genes Of 19 Patients Infection With Hepatitis C Virus

Part Ⅰ Acyclic Retinoid Inhibits the Human Hepatoma Cell Growth by Suppressing Fibroblast Growth Factor Mediated Signaling PathwaysBackground and Aim: Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Its high mortality rate is mainly a result of high intrahepatic recurrence. Acyclic retinoid (ACR), a novel synthetic retinoid, was reported to prevent the recurrence of human HCC after surgical resection of primary tumors, but the molecular mechanisms are yet to be elucidated. The purpose of this study is to determine the molecular mechanisms of the preventive effects of HCC recurrence induced by ACR and to clarify its target molecule by comprehensive studies for the development of more effective therapeutics. Materials and Methods: Acyclic retinoid( NIK-333)(ACR), Anti-FGF receptor 3 were used. Human liver tumor cell lines (HepG2, HLF, and HuH7) were obtained from the Riken cell bank.(1).The inhibitory effects by ACR on hepatoma cell growth was examined using MTT assay.(2). The inhibitory effects by ACR on hepatoma cell motility was examined using invasion assay. (3).Intracellular signaling changes induced by ACR were comprehensively studied by a reporter assay[Serum response factor (SRF),nuclear factor-κB,??NF-κB),p53,(APC) / β-catenin,serum response element (SRE),c-fos] and a transcriptional factor array(54 kinds of transcriptional factors).(4).Gene expression changes by ACR were examined using a microarray assay.(5).Rho and Rac activation assays were using immunoprecipitates and Western blotted.(6). Fibroblast growth factor (FGF) receptor 3 expression changes by ACR were examined using RT-PCR(mRNA level) ,Western blotted( protein level) and reporter assay(To determine whether these expression changes induced by ACR are dependent on the transcriptional level or post-transcriptional level).(7). HepG2 stably over-expressing FGF receptor 3 cell line was constructed,Then,This cell line was using for cell proliferation assays.(8). To examine whether FGF receptor 3 mediated pathways play a major role in proliferation of HCC cells,cell proliferation assays (using MTT) were performed,after neutralizing the FGF receptor 3 by anti-FGF receptor 3 antibody and silencing FGF receptor 3 using siRNA.Results: (1). ACR exerted a dose-dependent inhibition of growth of the HLF,Huh7, and HepG2 human hepatoma cell lines, with IC50 values of 10μM, 45μM, and 45μM, in DMEM containing 10% FBS, respectively. The growth inhibitory effect was observed only from 48 hours after the addition of ACR.(2). Fifty four kinds of transcription factor activities were examined in parallel by protein/DNA array on the basis of array-based technology, and four pathways (NF-κB, p53, APC/β-catenin, and SRF) were examined using luciferase assay. Among them, only SRF pathway was significantly reduced by ACR.In addition, ACR markedly inhibited not only synthetic SRE driven promoter activity but also c-fos promoter activity.(3). Because SRF activation is known to be mediated by Rho family , we determined whether ACR affects on the Rho A and Rac activities. ACR inhibited the activation of Rho A .In contrast, Rac was not expression in HepG2.(4). we evaluated the function of ACR, which suppresses Rho activity, on liver tumor cell migration in vitro. The invasion of HepG2 cells through the Matrigel-coated membranes of Transwell culture chambers was inhibited by the treatment with ACR in a dose-dependent manner , indicating that ACR is a potent inhibitor of not only growth but liver tumor cell motility. This phenomenon was not dependent on the growth reduction because the assay was performed 24 hours after addition of ACR, when the growth inhibition was not yet apparent.(5). we performed microarray experiments for identifying cellular gene expression changes after the treatment of ACR. Of genes investigated, about 6% of genes were modulated their expression when the amount of expression change over two-fold or under half by ACR was determined as significant (Full microarray data are deposited...