iNOS is associated with tumorigenicity as an independent prognosticator in human intrahepatic cholangiocarcinoma

Abstract

Background: Inducible nitric oxide synthase (iNOS) has supposed to implicate in inflammation, infection, liver cirrhosis, and neoplastic diseases. This study was designed to explore the biological and clinical function of iNOS in intrahepatic cholangiocarcinoma (ICC).

Methods: RT-PCR (Real-time quantitative PCR) and immunohistochemical staining were used to analyze the expression of iNOS in ICC and adjacent tissues. CCK8, transwell assays, flow cytometry were conducted to detect the proliferation, apoptosis, cell cycle. Western blotting was performed to detect the expression of target proteins. Multivariate analyses were conducted to analysis associates between clinicopathological values and survival.

Results: We found that levels of iNOS mRNA and protein were dramatically increased in ICC samples and positively correlated with complicated bile duct stone, differentiation, pathology T, pathology M, Wip1, MMP-2, and MMP-9. iNOS expression was significantly correlated with the poor survival of ICC patients. Furthermore, iNOS was high expression in ICC cell lines (QBC-939, ICC-9810, SSP-25) compare with human normal biliary epithelium cell line (HIBEpic); both iNOS knockdown and iNOS inhibitor (1400 W) suppressed cell proliferation, invasion, and migration though nitric oxide production in ICC cells. Down-regulation of iNOS also induced G0/G1 cell cycle arrest and ICC cell apoptosis. Moreover, iNOS knockdown treatment significantly decreased Wip1, MMP-9, and MMP-2 gene expression.

Conclusion: Lowly expressed iNOS-inhibited proliferation yet promoted apoptosis of ICC cells. Our data show targeted inhibition of iNOS in ICC may have therapeutic value.

Keywords: iNOS; intrahepatic cholangiocarcinoma; prognosis.

Figure 1

Expression of iNOS in ICC tissues and cell lines. Representative staining of iNOS in ICC tissues (A) and adjacent normal tissues (B) by immunohistochemistry (the same donor). (C) Expression of iNOS mRNA was frequently up-regulated in ICC tissues compared with adjacent normal samples according to quantitative real-time PCR analysis. (D) iNOS mRNA expression was significantly inversely associated with tumor differentiation according to quantitative real-time PCR analysis (upper panel): Representative staining of iNOS in tumor differentiation. (E) Increased iNOS mRNA expression in metastasis tumor compared with non-metastasis tumor was detected by real-time quantitative PCR (upper panel): Representative staining of iNOS in metastasis tumor or not. (F) iNOS mRNA expression in the human normal biliary epithelium cell line (HIBEpic) and three ICC cell lines (QBC-939, ICC-9810, and SSP-25) using qRT-PCR. Data are represented as the means ± SEM of three independent experiments (upper panel): iNOS protein expression in (HIBEpic, QBC-939, ICC-9810, and SSP-25) cells. *p<0.05. Abbreviations: iNOS, inducible nitric oxide synthase; ICC, intrahepatic cholangiocarcinoma.

Figure 2

iNOS is essential for ICC cell proliferation and invasion. (A) iNOS siRNA or non-T small interfering RNA was transfected into QBC-939 and ICC-9810 cells. Five groups of cells were used in the present study, including untransfected cells (“untreated”), cells transfected with non-T small interfering RNA (“Non-T siRNA”), and cells transfected with iNOS-siRNA (‘iNOS siRNA-1, siRNA-2, and siRNA-3ʹ). The mRNA levels of iNOS in the five groups of QBC-939 and ICC-9810 cells were assayed by quantitative real-time PCR. (B,C) The protein levels of iNOS in the five groups of cells were assayed by Western blotting, and β-Actin was used as an internal control. Bar graphs are derived from densitometric scanning of the blots. (D) Knockdown of iNOS-inhibited cell proliferation in both QBC-939 and ICC-9810 cells according to the CCK-8 assay. (E) Down-regulated iNOS-suppressed migration and invasion in both QBC-939 and ICC-9810 cells. (F) NO production and cell viability under knockdown of iNOS in QBC-939 cells. Data are represented as the means ± SEM of three independent experiments. *p<0.05, **p<0.01 (All were compared with the untreated control group). Abbreviations: iNOS, inducible nitric oxide synthase; ICC, intrahepatic cholangiocarcinoma.

Figure 3

iNOS knockdown induces G0/G1 cell cycle arrest and ICC cell apoptosis. (A, B) Down-regulation of iNOS-induced G0/G1 phase cell cycle arrest in QBC939 cells according to flow cytometry analysis. (C, D) Knockdown of iNOS expression increased apoptosis in QBC-939 cells by flow cytometry analysis. Data are presented as the means ± SEM of three independent experiments. *p<0.05. Abbreviations: iNOS, inducible nitric oxide synthase; ICC, intrahepatic cholangiocarcinoma.

Figure 4

iNOS inhibitor (1400 W) inhibits tumor growth and metastasis of ICC cells in vitro. (A) The effects of different concentrations of 1400 W on the NO content. (B) The influence of different concentrations of 1400 W on cell viability. (C) Scratch wound-healing assay. 1400 W-treated cells and control group cells at 2 time points (0 and 36 hrs) are shown. (E) Graphical presentation of the wound-healing rate was measured with the following formula: (0 hrs width-36 hrs width of wound)/(0 hrs width of wound). (D) The invasive properties of the cells were analyzed by an invasion assay using a Matrigel-coated plate. 1400 W suppressed migration and invasion in vitro in QBC939 cells. (F) Bar graphs are derived from the number of invasive cells in the 1400 W group and control group. Data are presented as the means ± SEM of three independent experiments. *p<0.05 (All were compared with the untreated control group), **P<0.01. Abbreviations: iNOS, inducible nitric oxide synthase; ICC, intrahepatic cholangiocarcinoma; NO, nitric oxide.

Figure 5

Gene expression results for iNOS down-regulation. (A, B), Wip1, MMP-2, MMP-9 were down expression in QBC-939 tumor cells following SiRNA-iNOS treatment. (C, D) Similar results were shown in ICC-9810 cells. The values are mean ± SD relative to the control values, and normalized based on GAPDH (n=3). The * indicates statistical significance at p<0.05 in comparison to control. Abbreviations: iNOS, inducible nitric oxide synthase; ICC, intrahepatic cholangiocarcinoma.

Figure 6

Kaplan–Meier survival curves for iNOS expression. (A) Overall survival (B) tumor-free survival. Patients with positive iNOS protein expression had a significantly worse outcome compared to patients with negative iNOS expression. (p=0.0232, log-rank test) and (p=0.0416, log-rank test). Abbreviation: iNOS, inducible nitric oxide synthase.