title: Bortezomib effect on E2F and cyclin family members in human hepatocellular carcinoma cell lines.
abstract: OBJECTIVE
  To evaluate the effects of the proteasome inhibitor bortezomib (BZB) on E2Fs and related genes in hepatocellular carcinoma (HCC) cells.
  
  
  METHODS
  The mRNA levels of the E2F family members (pro-proliferative: E2F1-3 and anti-proliferative: E2F4-8) and of their related genes cyclins and cyclin-dependent kinases (cdks) were evaluated in two HCC cell lines following a single BZB administration. mRNA levels of the epithelial-mesenchymal transition (EMT) genes were also measured in both cell lines after BZB treatment. The BZB concentration (40 nmol/L) used was chosen to stay well below the maximal amount/cm² recommended for in vivo application, and 2 d incubation was chosen as this time point has been found optimal to detect BZB effects in our previous studies. The HCC cell lines, HepG2 and JHH6, were chosen as they display different phenotypes, hepatocyte-like for HepG2 and undifferentiated for JHH6, thus representing an in vitro model of low and high aggressive forms of HCC, respectively. The mRNA levels of the target genes were measured by two-color microarray-based gene expression analysis, performed according to Agilent Technologies protocol and using an Agilent Scan B. For the E2F family members, mRNA levels were quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). Using small interfering RNA's, the effects of E2F8 depletion on cell number was also evaluated.
  
  
  RESULTS
  After BZB treatment, microarray analysis of the undifferentiated JHH6 revealed a significant decrease in the expression of the pro-proliferative E2F member E2F2. Quantitative RT-PCR data were in keeping with the microarray analysis, and showed a significant increase and decrease in E2F8 and E2F2 mRNA levels, respectively. In contrast, BZB treatment of the hepatocyte-like HCC cell line HepG2 had a significant impact on mRNA levels of 5 of the 8 E2F members. In particular, mRNA levels of the pro-proliferative E2F members E2F1, E2F2, and of the anti-proliferative member E2F8, decreased over 80%. Notably, a reduction in E2F8 expression in HepG2 and JHH6 cells following siRNA treatment had no impact on cell proliferation. As observed with JHH6, BZB treatment of HepG2 cells induced a significant increase in mRNA levels of an anti-proliferative E2F member, E2F6 in this case. As was observed with E2F's, more dramatic changes in mRNA levels of the E2F related genes cyclins and Cdks and EMT genes were observed after BZB treatment of HepG2 compared to JHH6.
  
  
  CONCLUSIONS
  The differential expression of E2Fs and related genes induced by BZB in diverse HCC cell phenotypes contribute to bortezomib's mechanism of action in hepatocellular carcinoma.
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Baiz
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Dapas
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Farra
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Bruna
authors.initials: B
authors.lastname: Scaggiante
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pozzato
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Zanconati
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Fiotti
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Lara
authors.initials: L
authors.lastname: Consoloni
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Chiaretti
authors.affiliation: Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Grassi
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D050684:E2F Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D011719:Pyrazines / Q000494:pharmacology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; D016213:Cyclins; D050684:E2F Transcription Factors; D061988:Proteasome Inhibitors; D011719:Pyrazines; D012333:RNA, Messenger; D000069286:Bortezomib
keywords: 26S proteasome; Bortezomib; Cyclins; E2F family; Hepatocellular carcinoma; Liver; Microarray