Siriluck Narong

Detection of serum MMP-7 and MMP-9 in cholangiocarcinoma patients: evaluation of diagnostic accuracy

BackgroundCholangiocarcinoma is an aggressive tumor with a tendency for local invasion and distant metastases. Timely diagnosis is very important because surgical resection (R0) remains the only hope for a cure. However, at present, there is no available tumor marker that can differentiate cholangiocarcinoma from benign bile duct disease. Previous studies have demonstrated that matrix metalloproteinase (MMP)-7 and MMP-9 are frequently expressed in cholangiocarcinoma specimens.MethodsThis study was designed to determine whether the serum levels of MMP-7 and MMP-9 can discriminate cholangiocarcinoma patients from benign biliary tract disease patients in comparison to carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). We measured the level of CEA, CA19-9, MMP-7 and MMP-9 in the serum of 44 cholangiocarcinoma and 36 benign biliary tract diseases patients.ResultsAmong the serum levels of CEA, CA19-9, MMP-7 and MMP-9, only the serum MMP-7 level was significantly higher in the patients with cholangiocarcinoma (8.9 ± 3.43 ng/ml) compared to benign biliary tract disease patients (5.9 ± 3.03 ng/ml) (p < 0.001). An receiver operating characteristic (ROC) curve analysis revealed that the detection of the serum MMP-7 level is reasonably accurate in differentiating cholangiocarcinoma from benign biliary tract disease patients (area under curve = 0.73; 95% CI = 0.614–0.848). While the areas under the curve of the ROC curves for CEA, CA19-9 and MMP-9 were 0.63 (95% CI = 0.501–0.760), 0.63 (95% CI = 0.491–0.761) and 0.59 (95% CI = 0.455–0.722), respectively.ConclusionSerum MMP-7 appears to be a valuable diagnostic marker in the discrimination of cholangiocarcinoma from benign biliary tract disease. Further prospective studies for serum MMP-7 measurement should be carried out to further investigate the potential of this molecule as a biomarker of cholangiocarcinoma.

Inhibition of PI3K increases oxaliplatin sensitivity in cholangiocarcinoma cells

BackgroundResistance of cholangiocarcinoma to chemotherapy is a major problem in cancer treatment. The mechanism of resistance is believed to involve phosphoinositide-3- kinase (PI3K)/Akt activation. Although the platinum-containing compound oxaliplatin has been extensively used in the treatment of several solid tumors, recent data regarding its use to treat cholangiocarcinoma are ambiguous. Oxaliplatin resistance in this disease could potentially involve PI3K pathways. We, therefore, examined the effects of PI3K pathways in cholangiocarcinoma cells in modulating oxaliplatin resistance.ResultsAfter exposing the cholangiocarcinoma cell lines RMCCA1 and KKU100 to oxaliplatin, the levels of Akt and mTOR phosphorylation increased, as shown by western blot analysis. The WST-1 cell proliferation assay showed increased inhibition of cell growth under high concentrations of oxaliplatin. The combination of oxaliplatin with LY294002, an inhibitor of PI3K, resulted in a remarkable arrest of cell proliferation. Deactivation of mTOR by RAD001 was also synergistic with oxaliplatin, although to a lesser extent. The combination of oxaliplatin and a PI3K inhibitor also resulted in a significant induction of apoptosis, as demonstrated by the TUNEL assay.ConclusionActivation of PI3K might protect cholangiocarcinoma cells from oxaliplatininduced cytotoxicity. Although the inhibition of PI3K and the inhibition of mTOR both enhance oxaliplatin-induced cytotoxicity, PI3K inhibition has a greater effect. Targeting the PI3K pathway may be a useful approach to improve the chemotherapeutic sensitivity of cholangiocarcinoma.

Expression of CD24 in cholangiocarcinoma cells is associated with disease progression and reduced patient survival

Cholangiocarcinoma is frequently found to invade local tissues and metastasize to distal organs. We investigated the expression of CD24 in cholangiocarcinoma samples and its prognostic significance. In addition, the cellular function of CD24 was studied in the RMCCA1 cholangiocarcinoma cell line. High CD24 expression significantly correlated with lymph node metastasis and positive surgical margins in cholangiocarcinoma patients. Univariate and multivariate analyses further demonstrated that CD24 expression was significantly associated with the overall survival of these patients (p=0.007 and p=0.040, respectively). For in vitro studies, the magnetic-activated cell sorting (MACS) system was used to isolate CD24+ and CD24- cell populations from RMCCA1 cells. CD24+ RMCCA1 cells had increased chemoresistance, adhesion (p=0.004), motility (p<0.001), migration (p<0.001) and invasion (p<0.001) capabilities when compared to CD24- cells. The matrix metalloproteinase (MMP)-7 was significantly elevated in CD24+ RMCCA1 cells (p=0.01). We found that inhibition of CD24 using siRNA silencing significantly decreased the invasive capacity of RMCCA1 cells. Both clinical and in vitro studies suggest that expression of CD24 is associated with cholangiocarcinoma disease progression. CD24 may thus serve as a new target for directed molecular therapy of cholangiocarcinoma.

Strong expression of CD133 is associated with increased cholangiocarcinoma progression

AIM To determine the role of CD133 in cholangiocarcinoma progression. METHODS CD133 protein expression was evaluated by immunohistochemistry in 34 cholangiocarcinoma specimens. In addition, proliferation, chemoresistance and invasive properties of CD133-enriched (CD133(+)) and CD133-depleted (CD133(-)) RMCCA1 cholangiocarcinoma cells were studied and compared. RESULTS Strong CD133 expression was observed in 67.6% (23/34) of the cholangiocarcinoma specimens. Strong expression of CD133 was significantly associated with nodal metastasis (P = 0.009) and positive surgical margin status (P = 0.011). In the in vitro study, both the CD133(+) and CD133(-) cells had similar proliferation abilities and resistance to chemotherapeutic drugs. However, the CD133(+) cells had a higher invasive ability compared with CD133(-) cells. CONCLUSION CD133+ cells play an important role in the invasiveness of cholangiocarcinoma. Targeting of the CD133+ cells may be a useful approach to improve treatment against cholangiocarcinoma.

Serum NGAL to Clinically Distinguish Cholangiocarcinoma from Benign Biliary Tract Diseases

Aim. To determine whether the serum level of NGAL can discriminate cholangiocarcinoma from benign biliary tract disease in patients. Methods. This study was performed according to a prospective-specimen-collection, retrospective-blinded-evaluation (PRoBE) design. A total of 50 cholangiocarcinoma and 50 benign biliary tract disease cases were randomly selected from a cohort of consecutive cases of biliary tract diseases. Their sera were measured for the levels of NGAL and the widely used serum cholangiocarcinoma marker, carbohydrate antigen 19-9 (CA19-9). Results. The serum CA19-9 and NGAL levels were significantly elevated in cholangiocarcinoma patients (CA19-9: P < .001, NGAL: P < .001). The area under the curve (AUC) of a receiver operating characteristic (ROC) curve analysis for the diagnosis of cholangiocarcinoma of CA19-9 and NGAL was 0.81 and 0.79, respectively. Conclusion. The diagnostic accuracy of serum NGAL and CA19-9 makes them good candidates for use as biomarkers to discriminate cholangiocarcinoma patients from benign biliary tract disease patients.

The Dual Effects of Δ9-Tetrahydrocannabinol on Cholangiocarcinoma Cells: Anti-Invasion Activity at Low Concentration and Apoptosis Induction at High Concentration

Currently, only gemcitabine plus platinum demonstrates the considerable activity for cholangiocarcinoma. The anticancer effect of Delta (9)-tetrahydrocannabinol (THC), the principal active component of cannabinoids has been demonstrated in various kinds of cancers. We therefore evaluate the antitumor effects of THC on cholangiocarcinoma cells. Both cholangiocarcinoma cell lines and surgical specimens from cholangiocarcinoma patients expressed cannabinoid receptors. THC inhibited cell proliferation, migration and invasion, and induced cell apoptosis. THC also decreased actin polymerization and reduced tumor cell survival in anoikis assay. pMEK1/2 and pAkt demonstrated the lower extent than untreated cells. Consequently, THC is potentially used to retard cholangiocarcinoma cell growth and metastasis.

Induction of MKP-1 prevents the cytotoxic effects of PI3K inhibition in hilar cholangiocarcinoma cells.

PurposeHilar cholangiocarcinoma (Klatskin tumor) is one of the most difficult cancers to treat. We demonstrate activation of phosphoinositide-3-kinase (PI3K)/Akt signaling, which is a critical pathway for cell survival, in hilar cholangiocarcinoma cells. However, inhibition of PI3K has little effect on hilar cholangiocarcinoma cell survival. In this study, we investigated the mechanism by which hilar cholangiocarcinoma cells resist PI3K inhibitors.MethodsHuman hilar cholangiocarcinoma cells KKU-100 were treated with PI3K inhibitors, and cell viability and apoptosis assays were performed. The expression of a MAPK phosphatase (MKP-1) that contributes to cancer cell survival in response to multiple stress stimuli was assayed by quantitative real-time RT-PCR and western blotting. In addition, the effects of the MKP-1 inhibitor were studied in KKU-100 cells treated with PI3K inhibitors.ResultsIncubation of KKU-100 cells with PI3K inhibitors resulted in increased expression of MKP-1. Furthermore, we found that inhibition of MKP-1 using siRNA silencing sensitized KKU-100 cells to PI3K inhibitor-induced apoptosis via increased phosphorylation of p38 MAPK.ConclusionsThese results indicate that concurrent inhibition of PI3K and MKP-1 induces apoptosis in KKU-100 cells. Simultaneous targeting of the PI3K pathway and MKP-1 may be a useful approach to improve therapies directed against hilar cholangiocarcinoma.

Proteomic Profiles of Mesenchymal Stem Cells Induced by a Liver Differentiation Protocol

The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs.

Prognostic relevance of circulating CK19 mRNA in advanced malignant biliary tract diseases

AIM To determine the role of circulating tumor cells (CTCs) in prediction of the overall survival of patients with advanced malignant biliary tract obstruction. METHODS We investigated the prognostic value of CTCs by examining two markers, cytokeratin (CK) 19 and human telomerase reverse transcriptase (hTERT) mRNA, in 40 patients diagnosed with advanced malignant biliary tract diseases. Quantitative real-time reverse transcription polymerase chain reaction was used to detect CK19 and hTERT mRNA in the peripheral blood of these patients. Overall survival was analyzed using the Kaplan-Meier method and Cox regression modeling. RESULTS Positive CK19 and hTERT mRNA expression was detected in 45% and 60%, respectively, of the 40 patients. Univariable analysis indicated that positive CK19 mRNA expression was significantly associated with worse overall survival (P = 0.009). Multivariable analysis determined that positive CK19 mRNA expression, patients age and serum bilirubin were each independently associated with overall survival. CONCLUSION CK19 mRNA expression levels in peripheral blood appear to provide a valuable marker to predict the overall survival of patients with advanced malignant biliary tract obstruction.