Induction of apurinic endonuclease 1 overexpression by endoplasmic reticulum stress in hepatoma cells

Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Previous studies have noted the induction of endoplasmic reticulum stress or apurinic endonuclease 1 (APE1) expression in many tumors. Therefore, the aim of this study was to investigate the relationship between endoplasmic reticulum (ER stress) and APE1 in hepatocellular carcinoma. Here we investigate the expression of APE1 during ER stress in HepG2 and Huh-7 cell lines. Tunicamycin or brefeldin A, two ER stress inducers, increased APE1 and GRP78, an ER stress marker, expression in HepG2 and Huh-7 cells. Induction of APE1 expression was observed through transcription level in response to ER stress. APE1 nuclear localization during ER stress was determined using immunofluorescence assays in HepG2 cells. Furthermore, expression of Hepatitis B virus pre-S2∆ large mutant surface protein (pre-S2∆), an ER stress-induced protein, also increased GRP78 and APE1 expression in the normal hepatocyte NeHepLxHT cell line. Similarly, tumor samples showed higher expression of APE1 in ER stress-correlated liver cancer tissue in vivo. Our results demonstrate that ER stress and HBV pre-S2∆ increased APE1 expression, which may play an important role in resistance to chemotherapeutic agents or tumor development. Therefore, these data provide an important chemotherapeutic strategy in ER stress and HBV pre-S2∆-associated tumors.

Figure 1

Elevated expression of APE1 in response to endoplasmic reticulum stress. (A) and (B) present the time- dependent effect of ER stress inducer, tunicamycin and brefeldin A, on APE1 expression. HepG2 and Hun-7 Cells were exposed to tunicamycin and brefeldin A in 10% FBS-supplemented DMEM for the times indicated. The cell lysates were analyzed by western blotting with antibodies for APE1, GRP78, tubulin, and β-actin. The expression levels of APE1 and GRP78 were quantified by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Figure 2

Induction of APE1 mRNA expression by tunicamycin (TM) was determined by real-time PCR in HepG2 cells (A). The cells were exposed with 2.5 μg/mL tunicamycin for times indicated. The total RNA was isolated and then subjected to real-time PCR analysis. Real-time PCR was performed as described in the Experimental Section. Columns, mean of three independent experiments; bars, SD (*, p < 0.05, Student’s t test); (B,C) Induction of APE1 expression by tunicamycin was inhibited by CHX in HepG2 cells. The cells were exposed with 2.5 μg/mL tunicamycin in the presence of CHX for as dose and time-indicated. The expression of APE1, GRP78 and β-actin were analyzed by Western blotting. The expression levels of APE1 and GRP78 were quantified by using ImageJ software; (D) HepG2 cell were treated with CHX for 6, 12, 24, and 36 h. The cell lysates were analyzed by western blotting with antibodies for APE1 and β-actin. The expression level of APE1 was quantified by using ImageJ software.

Figure 3

Nuclear localization of APE1 during ER stress. (A,B) Nuclear localization of APE1 in response to ER stress in HepG2 cells. The cells were treated with 2.5 μg/mL tunicamycin or 1 μg/mL BFA, and the localization of APE1 was determined by using immunofluorescence staining. Scale bar: 100 μm.

Figure 4

APE1 was involved in tunicamycin-induced cell death. (A) Down-regulation of APE1 expression by APE1 shRNA during ER stress. HepG2 cells were transfected with pLKO-APE1 shRNA plasmid, and then expression of APE1 was analyzed by immunoblotting; and (B) shRNA-mediated knockdown of APE1 increased HepG2 cells from tunicamycin-induced cell death. The cells were incubated with tunicamycin in 10% FBS-supplemented DMEM for 48 h. Cell viability was measured by the MTT assay. Columns, mean of three independent experiments; bars, SD (*, p < 0.05, Student’s t test).

Figure 5

Induction of APE1 expression by HBV large surface mutant protein in vitro and in vivo. (A) APE1 expression was induced by ER stress-associated protein, pre-S2Δ, in vitro. The NeHepLxHT cells and NeHepLxHT-pre-S2Δ cells were cultured in 10% FBS-supplemented DMEM. The total cell lysates were analyzed by Western blotting with antibodies for APE1, GRP78, and β-actin; (B) Elevated APE1 is associated with pre-S2∆ expression in vivo. The expression of APE1 and HBV surface proteins in liver tissues of control and pre-S2∆ transgenic mice was analyzed by immunohistochemical staining (×400). Scale bar: 50 μm; and (C) The expression levels of APE1 were quantified by using ImageJ software. The data represent the mean of APE1 protein expression level from three independent experiments. Columns, mean of three independent experiments; bars, SD (*, p < 0.05, Student’s t test).

Figure 6

Highly APE1 expression was observed in ER stress-correlated human liver tumor tissue. (A) Increased APE1 is associated with high GRP78 expression in human liver tumor tissues in vivo. The expression of APE1 and GRP78 in control liver tissue and tumor tissue were determined by Western blotting for six individual samples; (B) The expression levels of APE1 and GRP78 were quantified by using ImageJ software. The data represent the mean of APE1 protein expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (*, p < 0.05 and **, p < 0.01) between the control and experimental group are marked with an asterisk.