Kawin Leelawat

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.

CD24 induces the invasion of cholangiocarcinoma cells by upregulating CXCR4 and increasing the phosphorylation of ERK1/2

Cholangiocarcinoma is a malignant biliary tract tumor with an extremely poor prognosis. CD24 expression has been linked to the aggressiveness of cholangiocarcinoma cells and the adverse prognosis of cholangiocarcinoma patients. In the present study, the underlying mechanism of aggressive CD24+ cholangiocarcinoma cell behavior was elucidated. The magnetic-activated cell sorting system was used to isolate CD24+ and CD24− cell populations from RMCCA1 cholangiocarcinoma cells. Using a human tumor metastasis PCR array, it was observed that numerous tumor-associated genes were upregulated in the CD24+ cells, including CXC chemokine receptor type 4 (CXCR4). In addition, an intracellular signaling array demonstrated the activation of extracellular signal-regulated kinase (ERK)1/2, which is downstream of the CXCR4 signaling cascade, in the CD24+ cells. Inhibition of CXCR4 or ERK1/2 significantly inhibited the motility and invasiveness of the CD24+ cells. The present study indicates that CXCR4 and ERK1/2 are induced by CD24 and that these proteins are associated with cholangiocarcinoma cell invasion.

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.

TaqMan real-time PCR assay for specific detection of Opisthorchis viverrini DNA in Thai patients with hepatocellular carcinoma and cholangiocarcinoma.

The aim of this study was to develop TaqMan real-time PCR assay that detected Opisthorchis viverrini DNA from 18 normal and 18 tumor tissue specimens from Thai patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), who underwent liver resection from October 2005 to May 2006. Control liver specimens were seven non-primary liver cancers. A conserved probe representing 100% sequence homology was used as a reference for O. viverrini-specific probe. Five of six tumors (83%) and all six normal tissues from CCA group; and seven of twelve tumors (58%) and ten of twelve normal tissues (83%) from HCC group were found to have O. viverrini DNA. The O. viverrini DNA detection among HCC and CCA patients were not associated (p=0.193; 90%CI). This RT-PCR will be a useful tool for investigating the relationship between cancer type and presence of the parasite and also for conducting epidemiological surveys.

Role of mTOR inhibitor in cholangiocarcinoma cell progression

Cholangiocarcinoma (CCA) is a lethal malignancy of the biliary epithelium. CCA is resistant to currently available chemotherapy; therefore, new drugs as well as new molecular targets must be identified for the development of an effective treatment for CCA. The present study showed that RAD001 (everolimus), a derivative of rapamycin and an orally bioavailable mammalian target of rapamycin (mTOR) inhibitor, exhibits cytotoxic and antimetastatic effects in a CCA cell line, RMCCA-1. Treatment with low concentrations of RAD001 resulted in a significant reduction of in vitro invasion and migration of RMCCA-1, concomitant with a reduction of filopodia and alteration of the actin cytoskeleton. Although, matrix metalloproteinase-9 and -14 activities were unaltered. However, at high concentrations, RAD001 exhibited cytotoxic effects, reducing cell proliferation and inducing apoptotic cell death. Overall, RAD001 exhibits multiple effects mediated by the inhibition of the mTOR, which may serve as a promising agent for the treatment of CCA.

Novel Serum Biomarkers to Differentiate Cholangiocarcinoma from Benign Biliary Tract Diseases Using a Proteomic Approach.

Background and Aim. Cholangiocarcinoma (CCA) is the most frequent biliary malignancy, which poses high mortality rate due to lack of early detection. Hence, most CCA cases are present at the advanced to late stages with local or distant metastasis at the time of diagnosis. Currently available tumor markers including CA19-9 and CEA are inefficient and of limited usage due to low sensitivity and specificity. Here, we attempt to identify serum tumor markers for CCA that can effectively distinguish CCA from benign biliary tract diseases (BBTDs). Methods. Serum samples from 19 CCA patients and 17 BBTDs were separated by SDS-PAGE followed with LC-MS/MS and were subjected to statistical analysis and cross-validation to identify proteins whose abundance was significantly elevated or suppressed in CCA samples compared to BBTDs. Results. In addition to identifying several proteins previously known to be differentially expressed in CCA and BBTDs, we also discovered a number of molecules that were previously not associated with CCA. These included FAM19A5, MAGED4B, KIAA0321, RBAK, and UPF3B. Conclusions. Novel serum biomarkers to distinguish CCA from BBTDs were identified using a proteomic approach. Further validation of these proteins has the potential to provide a biomarker for differentiating CCA from BBTDs.

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.

Hypoxia enhances cholangiocarcinoma invasion through activation of hepatocyte growth factor receptor and the extracellular signal‑regulated kinase signaling pathway

Hypoxia is associated with tumor progression and poor prognosis in several cancer types. The present study aimed to examine the contribution of hypoxia (1% O2) to cancer progression in a cholangiocarcinoma cell line, RMCCA-1. The molecular basis of the hypoxic response pathway was investigated. The results showed that hypoxia significantly accelerated cancer cell proliferation and enhanced cell invasion (P<0.05). By using receptor tyrosine kinase and intracellular signaling antibody array kits, an increased phosphorylation/activation of a number of signaling molecules, particularly hepatocyte growth factor receptor (Met) and extracellular signal-regulated kinase (ERK) 1/2, was identified. Inhibition of Met and ERK by small hairpin RNA and U0126, respectively, significantly inhibited hypoxia-induced the invasive potential of RMCCA-1 cells (P<0.05). However, according to immunohistochemical analysis, hypoxia-inducible factor-1α expression was not correlated with cancer staging or tumor differentiation in 44 samples of cholangicarcinoma cases. The findings of the present study emphasized the importance of Met/ERK pathway activation as a key molecular event that may be responsible for a more invasive phenotype in hypoxic tumors and suggest Met as a potential target for the treatment of cholangiocarcinoma.

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.