Siriporn Proungvitaya

Serum α1β-glycoprotein and afamin ratio as potential diagnostic and prognostic markers in cholangiocarcinoma

Cholangiocarcinoma (CCA) affects the intra- and extrahepatic bile ducts and is commonly burdened by a late presentation and resulting high mortality rate. Accordingly, finding non-invasive biomarkers with adequate diagnostic/prognostic values is a priority in high-risk populations. In this study, we analyzed proteomes of serum samples from six CCA cases and ten healthy subjects using two-dimensional polyacrylamide gel electrophoresis to identify CCA-associated spots. Thirty-six CCA-associated proteins found in sera were identified by mass spectrometry. α1β-Glycoprotein (A1BG) and afamin (AFM) were detected consistently at different degrees in CCA sera compared with controls and were validated for their diagnostic and prognostic potential in a larger cohort of 64 patients with CCA, 4 with benign biliary diseases and 20 healthy subjects and compared between pre- and postsurgery serum samples from 26 CCA patients to ascertain a prognostic correlation. A single blot test developed to assess the serum A1BG/AFM ratio could detect CCA cases with 87.5% specificity, 84.4% sensitivity and the levels were significantly higher in CCA compared with controls. A high level of postoperative serum A1BG/AFM ratio was associated with worse outcomes and the infiltration of resection margins. The A1BG/AFM ratio may constitute a novel non-invasive candidate marker to diagnose CCA and its outcomes with high specificity and sensitivity. Prospective studies are awaited to demonstrate the clinical value of this observation.

Serum Cathepsin B to Cystatin C Ratio as a Potential Marker for the Diagnosis of Cholangiocarcinoma

Cholangiocarcinoma (CCA) is a cancer of the bile duct epithelial cells. The highest incidence rate of CCA with a poor prognosis and poor response to chemotherapy is found in Southeast Asian countries, especially in northeastern Thailand and Lao PDR. Cathepsin B is a lysosomal cysteine protease which is regulated by cysteine proteinase inhibitors such as cystatin C. Elevation of cathepsin B levels in biological fluid has been observed in patients with inflammatory diseases and many cancers. We aimed to investigate the serum cathepsin B and cystatin C levels of CCA patients to evaluate the feasibility of using cathepsin B and cystatin C as markers for the diagnosis of CCA. Fifty-six sera from CCA patients, 17 with benign biliary diseases (BBD) and 13 from controls were collected and the cathepsin B and cystatin C levels were determined. In addition, cathepsin B expression was investigated immunohistochemically for 9 matched-pairs of cancerous and adjacent tissues of CCA patients. Serum cathepsin B, but not cystatin C, was significantly higher in CCA and BBD patient groups compared to that in the control group. Consistently, all cancerous tissues strongly expressed cathepsin B while adjacent tissues were negative in 7 out of 9 cases. In contrast, serum cystatin C levels were comparable between CCA and control groups, although serum cystatin C levels in the BBD group was higher than that in the control or CCA groups. When the serum cathepsin B to cystatin C ratio was calculated, that of the CCA group was significantly higher than that of the control group, and, although statistically not significant, the ratio of CCA group showed a trend to be higher than that of the BBD group. Thus, the cathepsin B to cystatin C ratio might be used as an alternative marker for aiding diagnosis of CCA.

High expression of apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) in human cholangiocarcinoma

Prognosis of cholangiocarcinoma (CCA) patients is absolutely poor in spite of extensive efforts for the development of chemotherapy. Mitochondrial proteins play key roles in carcinogenesis of various cancers. Therefore, mitochondria are considered as the target organelles for chemotherapy of several cancers including CCA. The purpose of this study is to identify potential candidate proteins for chemotherapy using mitochondrial proteome analysis for CCA tissues. A shotgun proteomic approach using SDS-PAGE coupled with LC-MS/MS was applied to compare the expression of mitochondrial proteins in CCA and the adjacent non-cancerous tissues. Using the proteomic analysis for the pooled mitochondrial proteins purified from three each of papillary and non-papillary types of CCA and their adjacent tissues, 281 proteins were identified as mitochondrial proteins, and 105 of them have significantly different expression levels compared with the corresponding counterparts. Among the 105 proteins, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) was a unique protein commonly over-expressed in both papillary and non-papillary types of CCA tissues but not in the adjacent non-cancerous tissues. In conclusion, AIFM3 was aberrantly expressed only in the mitochondria of CCA tissues. This finding suggests that AIFM3 could be a potential target molecule for CCA chemotherapy.

Total Serum Bile Acid as a Potential Marker for the Diagnosis of Cholangiocarcinoma without Jaundice

Diagnosis of cholangiocarcinoma (CCA) is difficult when patients do not show jaundice. The aim of this study was to examine the feasibility of using the total serum bile acid (TSBA) level as an aid for the diagnosis of CCA in patients without jaundice. For this purpose, TSBA of the following groups were measured using a Beckman Synchron CX4 clinical chemistry analyzer: 60 cases of CCA with total serum bilirubin ≤2 mg/dL (low total bilirubin group, LTB); 32 cases of CCA with total serum bilirubin >2 mg/dL (high total bilirubin group, HTB); and 115 healthy controls. Liver function parameters such as serum cholesterol, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were also examined. The results showed that the TSBA of both LTB and HTB groups of the CCA patients were significantly higher than that of the healthy controls. Also, significant correlation was observed between TSBA and total bilirubin levels in the HTB group of CCA patients. However, no such correlation was seen in the LTB group. The cut-off value of TSBA was determined for the LTB group of CCA patients using the receiver operating characteristic curve analysis, and it was 6.05 μmol/L with the sensitivity and specificity of 46.7% and 84.4%, respectively. In addition, the ALP level was correlated well with the TSBA level and ALP in HTB group was significantly higher than that of LTB group. Moreover, the combination of high TSBA and high ALP levels gave higher specificity up to 97.4%. TSBA might be useful for the diagnosis of CCA patients without jaundice.

Diagnostic value of serum bile acid composition patterns and serum glycocholic acid levels in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a cancer of biliary epithelial cell origin, which is prevalent in northeastern Thailand. The majority of patients with CCA are diagnosed at the advanced-stage of the disease. Although the early detection and diagnosis of CCA is critical to improve the prognosis of patients, there are presently no specific tumor markers for CCA. A previous study demonstrated that the total serum bile acid (TSBA) levels of patients with CCA are significantly increased, compared with those of healthy controls. In addition, although statistically insignificant, the TSBA levels in the sera of patients with CCA tended to be increased, as compared with the sera of patients with benign biliary disease (BBD). In the present study, the high performance liquid chromatographic (HPLC) patterns of bile acid composition were compared in the sera of patients with CCA, patients with BBD and normal controls. The results revealed that serum bile acid patterns in patients with CCA varied, compared with those in patients with BBD and normal controls. As hypothesized, glycocholic acid (GCA) levels in the sera of patients with CCA and BBD were high, compared with those in healthy controls. In addition, GCA levels in the sera of patients with CCA tended to be higher, as compared with patients with BBD; however, this result was not statistically significant. Therefore, determination of the bile acid patterns and GCA levels in sera using HPLC is feasible, and may aid the diagnosis of CCA.

High expression of CCDC25 in cholangiocarcinoma tissue samples

Cholangiocarcinoma (CCA) is a malignant transformation of biliary epithelial cells. It is a slow growing tumor, but is also highly metastatic with a poor prognosis. Bile acids are known to transactivate the epidermal growth factor receptor (EGFR) in cholangiocytes and induce cyclooxygenase-2 expression. The protein expression profiles of bile acid-treated CCA cells were studied using a proteomic approach. To elucidate the possible mechanisms involved in the bile acid-mediated enhancement of CCA cell migration, the effects of six bile acids, including cholic, deoxycholic, taurocholic, taurodeoxycholic, glycocholic and glycodeoxycholic acid, on the migration of CCA cells were examined in vitro using wound healing assays. Subsequently, the possible proteins involved in enhanced CCA cell migration were investigated using a proteomic approach. Changes to the protein expression profiles of CCA cells following bile acid treatment was examined using two-dimensional electrophoresis and mass spectrometry. The results demonstrated that cholic and deoxycholic acid significantly enhanced the migration of CCA cells, compared with the treated MMNK-1 control cells. CCA cells had 77 overexpressed protein spots following cholic acid treatment, and 50 protein spots following deoxycholic acid treatment, compared with the treated MMNK-1 control cells. Liquid chromatography tandem-mass spectrometry analysis revealed that coiled-coil domain containing 25 (CCDC25) was significantly overexpressed in cholic acid-treated CCA cells compared with in cholic acid-treated control cells. When the expression levels of CCDC25 were investigated using western blot analysis, CCDC25 was demonstrated to be highly expressed in CCA tissues, but not in the adjacent non-cancerous tissue samples. The identified proteins were further analyzed for protein-chemical interactions using STITCH version 3.1 software. CCDC25 protein was identified to be associated with Son of sevenless homolog 1 and growth factor receptor-bound protein 2, which are involved in EGFR signaling. The results of the present study demonstrated that following cholic acid treatment, CCDC25 is overexpressed in CCA cells, which is associated with significantly enhanced cell migration. This suggests that CCDC25 is a potential therapeutic target for the treatment of patients with CCA.