Identification of the Genes Chemosensitizing Hepatocellular Carcinoma Cells to Interferon-α/5-Fluorouracil and Their Clinical Significance

<div><p>The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. Interferon-alpha (IFN-α)/5-fluorouracil (5-FU) therapy is reportedly effective in some HCC patients. In the present study, to improve HCC prognosis, we identified the genes that are sensitizing to these agents. The screening strategy was dependent on the concentration of ribozymes that rendered HepG2 cells resistant to 5-FU by the repeated transfection of ribozymes into the cells. After 10 cycles of transfection, which was initiated by 5,902,875 sequences of a ribozyme library, three genes including protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (<em>PRKAG2</em>); transforming growth factor-beta receptor II (<em>TGFBR2</em>); and exostosin 1 (<em>EXT1</em>) were identified as 5-FU-sensitizing genes. Adenovirus-mediated transfer of <em>TGFBR2</em> and <em>EXT1</em> enhanced IFN-α/5-FU-induced cytotoxicity as well as 5-FU, although the overexpression of these genes in the absence of IFN-α/5-FU did not induce cell death. This effect was also observed in a tumor xenograft model. The mechanisms of <em>TGFBR2</em> and <em>EXT1</em> include activation of the TGF-β signal and induction of endoplasmic reticulum stress, resulting in apoptosis. In HCC patients treated with IFN-α/5-FU therapy, the <em>PRKAG2</em> mRNA level in HCC tissues was positively correlated with survival period, suggesting that <em>PRKAG2</em> enhances the effect of IFN-α/5-FU and serves as a prognostic marker for IFN-α/5-FU therapy. In conclusion, we identified three genes that chemosensitize the effects of 5-FU and IFN-α/5-FU on HCC cells and demonstrated that <em>PRKAG2</em> mRNA can serve as a prognostic marker for IFN-α/5-FU therapy.</p> </div

Enhancement of IFN-α/5-FU-induced apoptosis by <i>TGFBR2</i> and <i>EXT1</i> overexpression.

<p>(A) Evaluation of nuclear condensation. The arrows indicate cells with apoptosis-specific nuclear condensation and fragmentation. (B) Measurement of intracellular caspase 3/7 activity. After adenovirus infection, cells were treated with the indicated concentrations of IFN-α and 5-FU. Activity was expressed as the fold increase relative to that at 0 h. Data are expressed as mean ± standard deviation (<i>n = </i>3). Statistical significance was determined using Student’s <i>t</i>-test. *<i>P</i><0.05 compared to LacZ at 48 h.</p

Enhancing effects of genes on IFN-α/5-FU treatment.

<p>(A–C).Viability of HepG2 cells infected with adenovirus-carrying protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (<i>PRKAG2</i>) (A), transforming growth factor-beta receptor II (<i>TGFBR2</i>) (B), and exostosin 1 (<i>EXT1</i>) (C) with or without 5-FU. Adenovirus-carrying LacZ served as a negative control. Cell viability was determined using the WST assay at 72 h. *<i>P</i><0.05 and **<i>P</i><0.01, between the two groups. (D) Viability of HepG2, HuH7, and HLF cells infected with adenovirus-carrying <i>TGFBR2</i> and <i>EXT1</i> with or without agents. Data are expressed as mean ± standard deviation (SD) (<i>n = </i>3). Statistical significance was determined using Student’s <i>t</i>-test. *<i>P</i><0.05 between two groups. (E) In vivo studies using NOD/SCID mice subcutaneously transplanted with HepG2 cells. In adenoviruse-administered mice, tumor growth was assessed during IFN-α/5-FU treatment. Data are shown as means ± SD (<i>n = </i>4–7).</p

Effects of TGFBR2 on 5-FU- and IFN-α/5-FU-induced TGF-β signaling pathway.

<p>(A) 5-FU-induced <i>TGFB1</i> mRNA expression normalized to β-actin. Data are expressed as mean ± standard deviation (SD) (<i>n = </i>3). Statistical significance was determined using Student’s <i>t</i>-test. †<i>P</i><0.05 and ‡<i>P</i><0.001 compared to control. N.S., not significant. (B) Luciferase reporter assay for TGF-β signaling activation. Data are expressed as mean ± SD (<i>n = </i>3). Recombinant TGF-β was used as a positive control. The Y-axis is expressed as a logarithmic scale. (C) Western blot analysis of BAX, BCL-2, and BCL-xL in 5-FU and IFN-α/5-FU treatment with or without <i>TGFBR2</i> overexpression. Each band was quantified by Image J software and normalized to actin.</p

Clinical parameters of HCC patients.

<p>Parenthetical values in survival period showed minimum and maximum survival days.</p

Identification of 5-FU-sensitizing genes.

<p>(A) Determination of a 5-FU concentration sufficient to decrease cell viability in HepG2 cells, which were treated with various concentrations of 5-FU for 72 h. Data are shown as means ± standard deviation (SD) (<i>n</i> = 3). (B) An outline of the screening process. The cycle was repeated 10 times, and sequence analysis of ribozymes was performed for gene identification. (C) Assessment of successful screening. HepG2 cells transfected with plasmid DNA recovered from 6 (Rz-C6), 8 (Rz-C8), and 10 (Rz-C10) cycles of screening were treated with the indicated concentrations of 5-FU for 72 h. Data are expressed as mean ± SD. Statistical significance was determined by one-way analysis of variance and Tukey’s HSD method. *<i>P</i><0.05, compared to control. (D) Expression of the candidate genes in HepG2 cells. mRNA expression levels of each gene were normalized to β-actin. Data are expressed as mean ± SD (<i>n = </i>3). N.D., not detected. (E) Confirmation of the knockdown efficiencies of siRNA specific to protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (<i>PRKAG2</i>); transforming growth factor-beta receptor II (<i>TGFBR2</i>); and exostosin 1 (<i>EXT1</i>). The expression levels of each mRNA were normalized to β-actin expression. Data are expressed as mean ± SD (<i>n</i> = 3) (F) Acquisition of resistance to 5-FU by knockdown of the identified genes. HepG2 cells were transfected with siRNA targeting <i>PRKAG2</i>, <i>TGFBR2</i>, or <i>EXT1</i>, followed by treatment with the indicated concentrations of 5-FU for 72 h. Data are expressed as mean ± SD. Statistical significance was determined using Student’s <i>t</i>-test. *<i>P</i><0.05 and **<i>P</i><0.01 compared to control.</p

<i>In vivo</i> analysis of mice xenografted with MzChA-1 and MzChA-1_GR cells.

<p>NOD/SCID mice xenografted with MzChA-1 and MzChA-1_GR cells were employed as <i>in vivo</i> models. When the tumor volume was over 200 mm³, the mice were treated with the indicated drugs (GEM [125 mg/kg, once a week] and/or vorinostat [60 mg/kg, 5 consecutive days per week]). The prognostic value was evaluated by the Kaplan–Meier method and a log-rank test. (A) Survival curve for mice xenografted with MzChA-1 cells. (B) Survival curve for mice xenografted with MzChA-1_GR cells. (C) Immunohistochemistry for SMAD4 on the resected tumor specimens of the mice xenografted with MzChA-1_GR cells.</p

The mRNA expression of class I HDACs and HDAC activity in MzChA-1 and MzChA-1_GR cells.

<p>Values represent the mean ± S.D. *<i>P</i> < 0.05. All experiments were conducted at least three times. (A) Comparison of the expression of class I HDACs in MzChA-1 and MzChA-1_GR cells by qRT-PCR. (B) The effect of TGF-β1 on HDAC activity in MzChA-1 cells and the HDAC activity in MzChA-1_GR cells, activity was measured with the HDAC activity assay kit. (C) The changes in HDAC activity in MzChA-1 and MzChA-1_GR cells treated with TGF-β1 and vorinostat. In the experiments for panels (B) and (C), the cells were incubated with or without 5 ng/ml TGF-β1 or 100 nM vorinostat for 72 h.</p

The effect of TGF-β1 knockdown using TGF-β small interfering RNA (siRNA) on EMT and chemoresistance in MzChA-1_GR cells.

<p>MzChA-1_GR cells were transfected with scrambled oligonucleotide siRNA (negative control) or TGF-β1 siRNA. All experiments were conducted at least three times. Values represent the mean ± S.D. *<i>P</i> < 0.05. (A) Comparison of the expression of TGF-β1 in MzChA-1 and MzChA-1_GR cells. (B) The changes of EMT-related mRNA expression as a result of TGF-β siRNA transfection in MzChA-1_GR cells. (C) The effect of TGF-β siRNA transfection on chemoresistance in MzChA-1_GR cells. Growth inhibition assays were performed for transfected and non-transfected cells treated with GEM.</p

Inhibition of the nuclear translocation of SMAD4 by vorinostat in MzChA-1 cells.

<p>In each experiment, cells were incubated with or without 5 ng/ml of TGF-β1 and 100 nM of vorinostat for 72 h. Scale bars: 100 μm. All experiments were conducted at least three times. (A) The change in SMAD4 expression in whole cell lysates (left panel) and in the nucleus (right panel) caused by TGF-β and vorinostat. (B) ChIP assay showing expression of SNAI1, SNAI2, ZEB1, ZEB2, and TWIST, which are regulatory elements of CDH1. (C) Immunofluorescence of SMAD4 (green) was performed in MzChA-1 cells. The effect of TGF-β1 and vorinostat on SMAD4 nuclear translocation were investigated. Nuclear staining (blue) was performed with Hoechst.</p