Silencing of APE1 Enhances Sensitivity of Human Hepatocellular Carcinoma Cells to Radiotherapy <em>In Vitro</em> and in a Xenograft Model

<div><p>Resistance to radiotherapy is a key limitation for the treatment of human hepatocellular carcinoma (HCC). To overcome this problem, we investigated the correlation between radioresistance and the human apurinic/apyrimidinic endonuclease (APE1), a bifunctional protein, which plays an important role in DNA repair and redox regulation activity of transcription factors. In the present study, we examined the radiosensitivity profiles of three human HCC cell lines, HepG2, Hep3B, and MHCC97L, using the adenoviral vector Ad5/F35-mediated APE1 siRNA (Ad5/F35-siAPE1). The p53 mutant cell lines MHCC97L showed radioresistance, compared with HepG2 and Hep3B cells. APE1 was strongly expressed in MHCC97L cells and was induced by irradiation in a dose-dependent manner, and Ad5/F35-siAPE1 effectively inhibited irradiation-induced APE1 and p53 expression. Moreover, silencing of APE1 significantly potentiated the growth inhibition and apoptosis induction by irradiation in all tested human HCC cell lines. In addition, Ad5/F35-siAPE1 significantly enhanced inhibition of tumor growth and potentiated cell apoptosis by irradiation both in HepG2 and MHCC97L xenografts. In conclusion, down regulation of APE1 could enhance sensitivity of human HCC cells to radiotherapy <em>in vitro</em> and <em>in vivo</em>.</p> </div

Dose-dependent growth inhibition of three human HCC cells with different p53 status.

<p>Cell survival following exposure to various doses of X-ray was evaluated by MTT assay (A) and colony formation assay (B). The values are expressed as the mean±standard deviation from three independent experiments.</p

Ad5/F35-siAPE1 inhibits irradiation-induced APE1 expression.

<p>(A) MHCC97L cells were treated with Ad5/F35-siAPE1 or Ad5/F35-EGFP; cells were irradiated with 6 Gy of X-ray at 48 h post-infection, and the APE1, p53 and p21 protein expressions were determined at 48 h post irradiation by western blot. Normalized APE1, P53 and P21 protein levels after adjusting for loading. (B) Quantitative RT-PCR reaction target gene analysis was similarly performed in MHCC97L cells. Each data point represents the mean ± standard deviation of three independent determinations.^*P<0.01 vs Ad5/F35-EGFP; ^#P<0.01 vs Ad5/F35-EGFP+IR.</p

Cell apoptosis following combined treatment with Ad5/F35-siAPE1 and irradiation <i>in vitro</i>.

<p>HepG2, Hep3B and MHCC97L cells were treated with Ad5/F35-EGFP or Ad5/F35-siAPE1.Samples were collected at 48 h for a range of X-ray irradiation (0, 4 and 10 Gy). At another 48 h post-irradiation, the cells were harvested and then stained with annexin V-FITC and PI. Bar graphs represent the mean values of triplicate determinations ± standard deviation. *P<0.01 vs Ad5/F35-EGFP.</p

Combined treatment with Ad5/F35-siAPE1 and irradiation induces apoptosis <i>in vivo</i>.

<p>HepG2 or MHCC97L cells were treated with Ad5/F35-siAPE1 or Ad5/F35-EGFP; 48 h after infection, cells were irradiated (6 Gy), and apoptosis was determined at 24 h post irradiation by terminal dUTP nick end labeling (TUNEL) staining. (A) TUNEL staining of HepG2 xenograft. (B) TUNEL staining of MHCC97L xenograft. Data are expressed as percentage of apoptosis-positive cells examined with TUNEL. Bar graphs represent the mean values of triplicate determinations ± standard deviation. Lane 1, Ad5/F35-EGFP; lane 2, Ad5/F35-siAPE1; lane 3,Ad5/F35-EGFP+IR; lane 4, Ad5/F35-siAPE1+ IR.^*P<0.01 vs Ad5/F35-EGFP; ^#P<0.01 vs Ad5/F35-siAPE1; ^$P<0.01 vs Ad5/F35-EGFP+IR.</p

Tumor growth after treatment with single factor (Ad5/F35-siAPE1 or irradiation) or combination on hepatoma tumor model.

<p>IR = irradiation; N = number of mice in the experimental group; DT = tumor doubling time; SGD = specific growth delay.</p

Ad5/F35-siAPE1 enhances cell killing following irradiation in human HCC cell lines.

<p>Cells were infected with Ad5/F35-EGFP or Ad5/F35-siAPE1. At 48 h post-infection, samples were treated with different doses of irradiation. Cell survival following exposure to various doses of irradiation was evaluated by MTT (A) and colony formation assay (B). Each data point represents the mean±standard deviation of three independent determinations. Significant differences existed at all doses levels at the P<0.05 level.</p

APE1 is induced by X-ray radiation in a dose-dependent manner in MHCC97L cells.

<p>Western blot analysis of cell lysates for the protein expression of APE1 at 48 h post irradiation. Normalized APE1 protein levels after adjusting for loading. *P<0.05, **P<0.01 vs control.</p

Dose-dependent apoptosis induction in three human HCC cell lines irradiated with X-rays.

<p>Each data point represents the mean±standard deviation of three independent determinations. ^*P<0.05, ^**P<0.01 vs HepG2 cells; ^#P<0.05 vs Hep3B cells.</p

<i>In vivo</i> evaluation of tumor growth in human hepatoma wtp53 and mutp53 xenografts.

<p>Tumor-bearing mice were injected directly into the tumors with Ad5/F35-siAPE1 or Ad5/F35-EGFP at the 1,6,11th day. Two days later, tumors were irradiated with 6 Gy of X-ray. Tumors were assessed for growth by measuring the volume of xenografts. (A) Tumor volume of HepG2 xenograft. (B) Tumor volume of MHCC97L xenograft.</p