Significance of S100P as a biomarker in diagnosis, prognosis and therapy of opisthorchiasis-associated cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a malignancy of bile duct with the difficulty in early diagnosis, poor prognosis and less alternation in therapy. S100P is a member of S100 family proteins and plays important roles in cancers. We investigated the S100P expression and its correlation with clinicopathology in 78 cases of opisthorchiasis-associated CCA, and the effects of S100P knockdown with shRNA interference on the proliferation, cell cycle, migration, apoptosis and sensitivity to anti-cancer drug. Extremely high expression of S100P mRNA was detected in the CCA tumor tissues. The increased S100P protein expression was immunohistochemically confirmed and localized in the CCA cytoplasm and/or nuclei as well as in the hyperneoplasia and dysplasia bile ducts, but not in normal bile ducts. The intensity of immunostaining was correlated with survival, tumor stage and metastasis, and the high expression could be an independent prognostic factor. High levels of S100P were detected in the serum and bile fluid of CCA patients. The shRNA-mediated knockdown of S100P expression inhibited the proliferation in vitro and in vivo, and migration of CCA cells, arrested cell cycle with the up-regulated expression of cell cycle arrest related factors, p21, p27, GADD45A, and 14-3-3 zeta. S100P knockdown also promoted CCA cell apoptosis by up-regulating expression of apoptosis related factors, DR5, TRADD, caspase 3 and BAX, and increased the sensitivity of CCA cells to the chemotherapeutic agents sunitinib and apigenin. Taken together, this study indicates that S100P might be a promising biomarker for the diagnosis, prognosis and therapy of CCA.

Milk fat globule epidermal growth factor 8 serves a novel biomarker of opisthorchiasis-associated cholangiocarcinoma.

Milk fat globule epidermal growth factor 8 (MFG-E8) is a pleiotropic secreted glycoprotein to play roles in mediating immune tolerance and homeostasis maintenance and enhancing angiogenesis. To evaluate its value as a biomarker in opisthorchiasis-associated cholangiocarcinoma (CCA), the present study investigated MFG-E8 expression kinetics during the tumorigenesis in Opisthorchis viverrini infection-induced CCA, and demonstrated its expression in the tumor tissues of CCA patients and its serum level among them. During the tumorigenesis of CCA, MFG-E8 expression was increased in a time-dependent manner with the pathological processes. Absolutely higher expression of MFG-E8 messenger RNA was detected in the tumor tissues from CCA patients, compared with those in adjacent tissues. Immunobiochemical analysis showed that more than 90% CCA cases were positive and the positive reaction located in the membrane and cytoplasm of the tumor cells. Moreover, the average serum level in the CCA patients was significantly higher than that in healthy individuals and those with O. viverrini infection or other parasitosis. Correlation analysis of MFG-E8 expression with CCA clinicopathology revealed that a high expression of MFG-E8 protein was significantly bound with a poor differentiation, pathological advanced stage, and metastasis of CCA. The multivariation analysis indicated that MFG-E8 was an independent prognostic factor. In addition, short hairpin RNA-mediated MFG-E8 knockdown in CCA cell line obviously suppressed the cell proliferation. Our results strongly suggested that MFG-E8 is a promising biomarker for the diagnosis, prognosis, and therapy target of opisthorchiasis-associated CCA.

Platelet-derived growth factor may be a potential diagnostic and prognostic marker for cholangiocarcinoma.

Our previous report showed that platelet-derived growth factor (PDGF) and related genes were upregulated in a Syrian hamster model and could be detected in all human cholangiocarcinoma (CCA) tissues. We therefore hoped that PDGF could be used as a diagnostic and prognostic marker. We analyzed 78 samples of human CCA and adjacent tissues for PDGF and related gene expression, and localized PDGF protein expression. The mechanism of anti-cancer drugs on PDGF and related genes or proteins in CCA cell lines (OCA17, M156, and KKU100) was studied through MTT cell viability assay, quantitative real-time PCR, and immunoblotting. Mutagenesis of the PDGFRA coding region was analyzed. Moreover, the PDGFRA in sera of CCA patients and healthy controls was investigated. PDGFA was found to be upregulated in CCA tissue (84.6 %). Positive PDGFA immunohistochemical staining was significantly correlated with status (P = 0.000), stage of CCA (P = 0.013), metastasis (P = 0.017), and short survival rate (P = 0.005), and the multivariate analysis confirmed that PDGFA positive immunostaining had a higher likelihood of the risk of death (HR = 2.907, P = 0.016). For DNA point mutation of the PDGFRA sequence, silent mutations were found at tyrosine kinase 2 V824V (exon 18) and A603A (exon 13), and a missense mutation in S478P (exon 10); there was only a missense mutation in S478P (29 %) that has significant correlation with the histopathological grading (P = 0.037) and positive immunoreactive PDGFA (P = 0.021). In vitro cell line study by immunowestern blotting found that sunitinib malate had an inhibitory effect on the PDGFA pathway by decreasing p-PDGFRA, AKT, and p-AKT expression. The serum level of PDGFA in CCA patients was significantly higher than those of healthy control by 1.4-fold (P = 0.014). The present results suggest that PDGFA and PDGFRA may be used for CCA prognosis and/or as diagnostic candidate markers.