HBV suppresses thapsigargin-induced apoptosis via inhibiting CHOP expression in hepatocellular carcinoma cells

Abstract

Hepatocellular carcinoma (HCC) accounts for a proportion of cancer-associated mortalities worldwide. Hepatitis B virus (HBV) infection is a major cause of HCC in China. Thapsigargin (TG) is a potential antitumor prodrug, eliciting endoplasmic reticulum (ER) stress via the inhibition of the ER calcium pump, effectively inducing apoptosis. The present study therefore examined the role of HBV in TG-induced apoptosis using two HCC cell lines, HBV positive HepG2.2.15 and HBV negative HepG2. When these two cell lines were treated with TG, HepG2.2.15 was less susceptible to apoptosis than HepG2. This phenomenon was confirmed by an MTT assay and Annexin V-FITC/propidium iodide staining. Reverse transcription quantitative polymerase chain reaction and western blotting were used to detect the expression levels of genes in the ER stress pathway subsequent to treatment with TG. Notably, the mRNA and protein levels of the apoptosis factor DNA damage inducible transcript 3 (CHOP) increased significantly in the HepG2 cells compared with the HepG2.2.15 cells. Additionally, the HepG2.2.15 cells treated with interferon-α exhibited higher levels of CHOP compared with the untreated cells. The overexpression or knockdown of CHOP microRNA in HepG2.2.15 or HepG2 cells may reduce the difference in apoptosis status between the two cell lines. These results suggest that HBV may inhibit the apoptosis induced by ER stress. These findings may be useful in the development of selective therapies for patients with HBV-positive tumors.

Keywords: CHOP; HBV; apoptosis; endoplasmic reticulum stress; thapsigargin.

Figure 1.

Morphology of HepG2 and HepG2.2.15 cells viewed under a microscope. Magnification ×100. NC, negative control; TG, thapsigargin.

Figure 2.

MTT assay results for the HepG2 and HepG2.2.15 cells treated with thapsigargin (TG) or PBS. Final abundance was calculated as the ratio of absorbance at 490 nm for the cells treated with TG relative to the cells treated with PBS at each time point. Data are presented as mean ± standard deviation of three independent experiments. OD, optical density.

Figure 3.

(A) Apoptosis of the HepG2 and HepG2.2.15 cells detected by flow cytometry assay. (B) TG increased the apoptotic populations of HepG2 cells more significantly compared with HepG2.2.15 cells. The data are presented as the mean ± standard deviation from three independent experiments, and P-values were determined using an unpaired Student's t-test. ^#HepG2 cells treated with TG vs. HepG2.2.15 cells treated with TG (P<0.01). *HepG2.2.15 cells treated with IFNα-2A and TG vs. HepG2.2.15 cells treated with TG (P<0.05). NC, negative control; TG, thapsigargin; IFN, interferon.

Figure 4.

(A) Cell cycles of the HepG2 and HepG2.2.15 cells detected by flow cytometry assay. TG reduced the proportion of cells in G2 phase. Data are presented as the mean ± standard deviation. ^#HepG2 cells vs. HepG2 cells treated with TG (P<0.05). *HepG2.15 cells vs. HepG2.2.15 cells treated with TG (P<0.05). PI, propidium iodide; NC, negative control; TG, thapsigargin; IFN, interferon.

Figure 5.

mRNA expression levels of genes in the 3 UPR pathways. (A) mRNA expression levels of CHOP and GADD34 were tested by qPCR, and XBP1 mRNA splicing was tested by reverse transcription PCR. (B) mRNA expression levels of ATF6 and ATF4 were tested by qPCR. Final abundances were adjusted to yield an arbitrary value of 1 for each gene in the HepG2 cells. mRNA, micro RNA; negative control; TG, thapsigargin; IFN, interferon; CHOP, DNA damage inducible transcript 3; GADD34, protein phosphatase 1 regulatory subunit 15A; XBP1, X-box binding protein; ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; qPCR, quantitative polymerase chain reaction.

Figure 6.

Western blot analysis of CHOP and GAPDH. Western blotting of CHOP, measuring 30 kDa, and GAPDH, measuring 36 kDa, in HepG2.2.15 and HepG2 cells subsequent to treatment with thapsigargin at each time point. GAPDH served as an internal control. CHOP, DNA damage inducible transcript 3; GADPH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 7.

Cell proliferation measured by an MTT assay when CHOP was overexpressed in HepG2.2.15 cells or knocked down in HepG2 cells. Final abundances were calculated as the ratio of absorbance at 490 nm for cells overexpressing CHOP or with CHOP knockdown relative to cells treated with PBS at each time point. Data are presented as the mean ± standard deviation from three independent experiments, and P-values were determined using an unpaired Student's t-test. OD, optical density; CHOP, DNA damage inducible transcript 3.