Histone modification-mediated CYP2E1 gene expression and apoptosis of HepG2 cells

Abstract

The incidence of hepatocellular carcinoma is rising due to alcohol drinking, hepatitis C viral infection and metabolic syndrome. Differential expression of CYP2E1 may play a pleiotropic role in the multistep process of liver carcinogenesis. Considerable attention has focused on the antitumor effect of trichostatin A (TSA) as well as CYP2E1 expression-induced apoptosis of cancer cells. However, very few studies have examined the mechanisms by which TSA has an antitumor effect and its association to CYP2E1 expression. The current study examined the action of TSA on CYP2E1 expression and the role of CYP2E1 in inducing apoptosis of HepG2 cells. Our data showed that TSA selectively induced CYP2E1 in four studied human hepatocellular carcinoma (HCC) cell lines (Huh7, PLC/PRF/5, Hep3B and HepG2), but not in normal primary human hepatocytes. TSA-mediated up-regulation of CYP2E1 expression was associated with histone H3 acetylation and the recruitment of HNF-1 and HNF-3beta to the CYP2E1 promoter in HepG2 cells. siRNA-mediated knockdown experiments showed that TSA-induced caspase-3 cleavage was decreased due to reduced expression of CYP2E1 in HepG2 cells. Moreover, down-regulation of CYP2E1 was accompanied by decreased production of mitochondrial reactive oxygen species. These results suggest that histone modification is involved in CYP2E1 gene expression and that CYP2E1-dependent mitochondrial oxidative stress plays a role in TSA-induced apoptosis.

Figure 1

TSA induced CYP2E1 mRNA level in HCC cells, but not in primary human hepatocytes. HCC cells and primary human hepatocytes were treated with DMSO or TSA (1 µM) for 24 h. Realtime PCR was used to quantify CYP2E1 mRNA level. Data were expressed as mean ± SD from three independent experiments. *P < 0.05, **P < 0.001 versus DMSO treatment group. TSA, trichostatin A; HCC, hepatocellular carcinoma; DMSO, dimethyl sulfoxide

Figure 2

The induction of CYP2E1 mRNA levels in HepG2 cells is time dependent. Realtime PCR was used to quantify CYP2E1 mRNA level. Data were expressed as mean ± SD from three independent experiments. *P < 0.05, **P < 0.001 versus DMSO treatment group. DMSO, dimethyl sulfoxide; TSA, trichostatin A

Figure 3

Accumulation of acetylated histone H3 and H4 in HepG2 cells after TSA treatment. HepG2 cells were treated with TSA (1 µM) for 24 h and immunostained with antiacetylated histone H3 (a) and H4 (b) antibodies and imaged by confocal microscopy as described in the Materials and methods. TSA increased acetylated histone H3 and H4 in the nuclei. Nuclei are shown counterstained with DAPI. Representative images of three independent experiments are shown. *P < 0.01 versus DMSO treatment group. TSA, trichostatin A; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide (A color version of this figure is available in the online journal)

Figure 4

Induction of CYP2E1 by TSA is associated with the recruitment of acetylated H3 to the CYP2E1 promoter. HepG2 cells were treated with TSA (1 µM) for 24 h followed by chromatin immunoprecipitation (ChIP) assay as described in the Materials and methods. CYP2E1 promoter fragments were immunoprecipitated with antiacetylated H3 or antiacetylated H4 antibody and amplified by PCR. Results are representative of three independent experiments. TSA, trichostatin A; DMSO, dimethyl sulfoxide

Figure 5

TSA enhanced the recruitment of HNF-1 and HNF-3β to the −290 to −70 region of the CYP2E1 gene. HepG2 cells were treated by TSA (1 µM) for 24 h. ChIP assay was performed using anti-HNF-1 (a) and anti-HNF-3β (b) antibody as described in the Materials and methods. Results were representative of three independent experiments. *P < 0.05 versus DMSO treatment group. TSA, trichostatin A; DMSO, dimethyl sulfoxide

Figure 6

CYP2E1 siRNA blocked TSA-induced CYP2E1 expression and mitochondria ROS generation induced by TSA in HepG2 cells. (a) HepG2 cells were transfected with either scramble siRNA or CYP2E1 siRNA at concentrations of 30 and 60 nM for 48 h as described in the Materials and methods. Endogenous CYP2E1 mRNA level was quantified by realtime PCR and expressed as relative fold compared to cells transfected with scramble siRNA transfection. (b) HepG2 cells were transfected with either scramble siRNA or CYP2E1 siRNA (60 nm) for 48 h and then cells were treated with TSA (1 µM) for 24 h followed by immunofluorescence staining for CYP2E1 and nuclear counterstaining. Representative results are from at least two independent experiments. (c) CYP2E1 was knocked down by siRNA in HepG2 cells and then cells were treated with TSA (1 µM) for 12 h. ROS was detected by MitoSOX™ Red mitochondrial superoxide indicator for live-cell imaging staining. MitoSOX™ Red reagent is highly selective for detection of mitochondrial superoxide in live cells. Once the compound is oxidized by superoxide in the mitochondria, red fluorescence is visible under fluorescence. The positive cells were shown in red with MitoSOX™ Red mitochondrial superoxide indicator staining. Four random fields of more than 200 cells each were counted under a fluorescence microscope. The results were generated from three independent experiments. *P < 0.01 versus DMSO treatment group, #P < 0.05 versus TSA (scramble siRNA). TSA, trichostatin A; ROS, reactive oxygen species; DMSO, dimethyl sulfoxide (A color version of this figure is available in the online journal)

Figure 7

TSA-induced cleaved caspase-3 is mediated through CYP2E1 in HepG2 cells. CYP2E1 expression was knocked down by siRNA in HepG2 cells and then cells were treated with TSA (1 µM) for 24 h followed by immunofluorescence staining with a specific antibody against cleaved caspase-3 and nuclear counterstaining with DAPI and viewed by confocal microscopy. Representative images of three independent experiments are shown. *P < 0.01 versus DMSO treatment group, #P < 0.05 versus TSA (scramble siRNA). TSA, trichostatin A; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide (A color version of this figure is available in the online journal)