Decreased argininosuccinate synthetase expression in Thai patients with cholangiocarcinoma and the effects of ADI-PEG20 treatment in CCA cell lines

Abstract

Cholangiocarcinoma (CCA) is a severe cancer with poor prognosis. The aim of the present study was to explore the expression of argininosuccinate synthetase (ASS), as well as the possibility of using pegylated arginine deiminase (ADI-PEG20) for the treatment of CCA. ASS expression was determined in CCA specimens from 40 patients in Thailand. Immunohistochemical detection of ASS and determination of the proliferative index, Ki-67, were carried out in paraffin-embedded sections of these specimens, as well as in two CCA cell lines, HuCCA and RmCCA-1, derived from CCA samples from patients in Thailand. In total, ~45% of the CCA specimens had low ASS expression, and the level of expression was significantly negatively associated with cell differentiation (P<0.05) and Ki-67 expression (P<0.05). The level of ASS expression in tumor cells was significantly lower than that in non-tumor cells (1.3-fold, P<0.05). The HuCCA cell line had significantly lower levels (P<0.05) of ASS expression at the mRNA and protein levels relative to those of normal human immortalized fibroblast cells (BJ-1). By contrast, the RmCCA-1 cell line showed no significant difference. In addition, the effects of ADI-PEG20 on growth inhibition, apoptosis and cell cycle arrest were determined in HuCCA and RmCCA-1 cells. ADI-PEG20 treatment reduced cell viability and cell proliferation in the two CCA cell lines, though it had no effect in immortalized BJ-1 cells. Furthermore, ADI-PEG20 treatment significantly increased G0/G1 cell cycle arrest in HuCCA, though not in RmCCA-1 cells. ASS silencing in the RmCCA-1 cell line significantly enhanced its sensitivity to ADI-PEG20 treatment. Results from the in vitro study demonstrated that ADI-PEG20 has antitumor activity against CCA with low ASS expression.

Keywords: Ki-67; arginine deprivation; argininosuccinate synthetase; cholangiocarcinoma; pegylated arginine deiminase.

Figure 1.

Immunohistochemical detection of ASS expression in paraffin-embedded sections. Paraffin-embedded sections of samples from 40 CCA patients were subjected to immunohistochemical detection of ASS expression. The cytoplasmic staining of ASS was performed using 3,3′-diaminobenzidine in CCA specimens. (A) The intensity of ASS staining in tumor cells (indicated by T) was decreased when compared with non-tumor (normal) cells (indicated by NT). The enlarged images (magnification, ×100) of stained (B) non-tumor (ASS expression=44.23) and (C) tumor cells (ASS expression=33.56) are also presented. Levels of ASS expression in cells that are (D) well, (E) moderately and (F) poorly differentiated. The ASS expression was highest in well-differentiated CCA (ASS expression=37.56) followed by moderately-differentiated CCA (ASS expression=33.47) and poorly-differentiated CCA (ASS expression=28.70). Scale bars, 100 µm. CCA, cholangiocarcinoma; ASS, argininosuccinate synthetase.

Figure 2.

Expression of Ki-67 in CCA specimens stratified by TNM stage and its correlation with ASS expression. Paraffin-embedded sections of samples from CCA patients were subjected to immunohistochemical detection of proliferative activity by using the Ki-67 labeling index. The correlations are shown between: The intensity of Ki-67 and (A) the TNM stage of CCA in patients, and (C) ASS expression, as well as between the percentage of Ki-67 stained cells and (B) the TNM stage of CCA in patients and (D) ASS expression. CCA, cholangiocarcinoma; ASS, argininosuccinate synthetase; TNM, tumor-node-metastasis.

Figure 3.

Level of ASS expression in human CCA cell lines. ASS protein levels in HuCCA, RmCCA-1 and BJ-1 cells were determined by (A) western immunoblotting and (B) in situ immunocytochemistry. (C) The expression of ASS mRNA was determined by reverse transcription-quantitative polymerase chain reaction. For western blot analysis, the expression of actin was used as the loading control. Data are presented as the mean ± standard error of three independent experiments. *P<0.05 vs. BJ-1 cells. CCA, cholangiocarcinoma; ASS, argininosuccinate synthetase; DAB, 3,3′-diaminobenzidine.

Figure 4.

Effects of ADI-PEG20 treatment on cell death in human CCA cell lines. HuCCA, RmCCA-1 and BJ-1 cells were treated with ADI-PEG20 (0.05–1 µg/ml) for 3 days. The percentage of viable cells was determined by MTT assay. Data are presented as the mean ± standard error of three independent experiments. *P<0.05 vs. control. ADI-PEG20, pegylated arginine deiminase.

Figure 5.

Effect of ADI-PEG20 treatment on Ki-67 expression in human CCA cell lines. HuCCA and RmCCA-1 cells were treated with 0.1 or 1 µg/ml ADI-PEG20 for 3 days. The proliferation activity was determined using the Ki-67 labelling index. In situ immunocytochemical detection was used to determine Ki-67 intensity and the percentage of Ki-67 stained cells. Histogram plots present the (A) intensity of Ki-67 and (B) percentage of Ki-67 stained cells in either the control or ADI-PEG20-treated HuCCA and RmCCA-1 cells. The data are presented as the mean ± standard error of three independent experiments. *P<0.05 vs. control. CCA, cholangiocarcinoma; ADI-PEG20, pegylated arginine deiminase.

Figure 6.

Effect of ADI-PEG20 treatment on cell cycle arrest and induction of apoptosis in human CCA cell lines. HuCCA and RmCCA-1 cells were treated with 0.1 µg/ml ADI-PEG20 for 3 days. Fluorescence-activated cell sorting analysis was performed to determine the percentage of cells in each phase of the cell cycle (G0/G1, S and G2/M phase) in (A) HuCCA and (B) RmCCA-1 cells, and (C) the number of apoptotic cells in HuCCA and RmCCA-1 cells. The data are presented as the mean ± standard error of three independent experiments. *P<0.05 vs. control. CCA, cholangiocarcinoma; ADI-PEG20, pegylated arginine deiminase.

Figure 7.

Effect of arginine deprivation using ADI-PEG20 treatment or arginine-free medium in ASS knockdown human CCA cell lines. (A) Immunoblot analysis of ASS protein levels in HuCCA, RmCCA-1, RmCCA-1^siNT and RmCCA-1^siASS with or without treatment with 0.1 µg/ml ADI-PEG20 for 3 days. (B) The growth inhibitory effect of ADI-PEG20 using MTT assay in HuCCA, RmCCA-1, RmCCA-1^siNT and RmCCA-1^siASS cells. (C) The percentage of apoptotic cells as determined by fluorescence-activated cell sorting analysis following treatment with ADI-PEG20 for 3 days or culturing in arginine-free medium for 3 days in HuCCA, RmCCA-1, RmCCA-1^siNT and RmCCA-1^siASS cells. The data are presented as the mean ± standard error of three independent experiments. ***P<0.001 vs. control. CCA, cholangiocarcinoma; ASS, argininosuccinate synthetase; ADI-PEG20/ADI, pegylated arginine deiminase; si-, small interfering; NT, non-target scramble control.