Brief Article
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World J Gastroenterol. Jan 21, 2014; 20(3): 795-803
Published online Jan 21, 2014. doi: 10.3748/wjg.v20.i3.795
Bortezomib effect on E2F and cyclin family members in human hepatocellular carcinoma cell lines
Daniele Baiz, Barbara Dapas, Rossella Farra, Bruna Scaggiante, Gabriele Pozzato, Fabrizio Zanconati, Nicola Fiotti, Lara Consoloni, Sara Chiaretti, Gabriele Grassi
Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy
Daniele Baiz, Wake Forest School of Medicine, Department of Cancer Biology and Comprehensive Cancer Center, Medical Center Boulevard, Winston Salem, NC 27157, United States
Rossella Farra, Department of Industrial Engineering and Information Technology, University of Trieste, 34100 Trieste, Italy
Gabriele Pozzato, Fabrizio Zanconati, Nicola Fiotti, Lara Consoloni, Gabriele Grassi, Department of ‘‘Scienze Mediche, Chirurgiche e della Salute’’, University of Trieste, Cattinara Hospital, 34100 Trieste, Italy
Author contributions: Baiz D performed the research; Dapas B, Farra R and Consoloni L performed the research; Scaggiante B, Pozzato G, Zanconati F, Fiotti N and Grassi G designed the research and wrote the paper; Chiaretti S analyzed the data.
Supported by The “Fondazione Cassa di Risparmio of Trieste; the “Fondazione Benefica Kathleen Foreman Casali of Trieste”; the Italian Minister of Instruction, University and Research (MIUR), PRIN 2010-11, No. 20109PLMH2 (in part)
Correspondence to: Gabriele Grassi, MD, PhD, Department of Life Sciences, University Hospital of Cattinara, Strada di Fiume 447, 34100 Trieste, Italy. ggrassi@units.it
Telephone: 39-40-3996227 Fax: +39-40-3994593
Received: June 29, 2013
Revised: November 11, 2013
Accepted: December 3, 2013
Published online: January 21, 2014
Processing time: 235 Days and 23.6 Hours
Abstract

AIM: 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.

CONCLUSION: 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.

Keywords: Bortezomib; Cyclins; E2F family; Hepatocellular carcinoma; Liver; Microarray; 26S proteasome

Core tip: The 26S proteasome inhibitor bortezomib has been proposed as a novel therapeutic molecule for hepatocellular carcinoma (HCC), being able to reduce cell growth. Little information is available on the effect of bortezomib (BZB) on many of E2Fs, a family of transcription factors regulating normal and tumor cell proliferation. Our data show, for the first time, the BZB effect on expression of E2F family members in HCC cell lines is not limited to the most studied E2F1, but, it extends also to other E2F members, in particular E2F2, E2F8 and E2F6, and the effect is phenotypic dependent.