Antitumor Effects of Delta (9)-Tetrahydrocannabinol and Cannabinol on Cholangiocarcinoma Cells and Xenograft Mouse Models.

Cholangiocarcinoma (CCA) is a very aggressive tumor. The development of a new therapeutic drug for CCA is required. This study aims to evaluate the antitumor effect of ∆9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana (Cannabis sativa), and cannabinol (CBN), a minor, low-psychoactive cannabinoid, on CCA cells and xenograft mice. THC and CBN were isolated, and their identities were confirmed by comparing 1H- and 13C-NMR spectra and mass spectra with a database. Cell proliferation, cell migration, and cell apoptosis assays were performed in HuCCT1 human CCA cells treated with THC or CBN. The phosphorylation of signaling molecules in HuCCT1 cells was detected. To determine the effects of THC and CBN in an animal model, HuCCT1 cells were inoculated subcutaneously into nude mice. After the tumors reached an appropriate size, the mice were treated with THC or CBN for 21 days. Tumor volumes were monitored and calculated. The 1H- and 13C-NMR data of THC and CBN were almost identical to those reported in the literature. THC and CBN significantly inhibited cell proliferation and migration and induced apoptosis in HuCCT1 cells. The phosphorylation of AKT, GSK-3α/ß, and ERK1/2 decreased in HuCCT1 cells treated with THC or CBN. CCA xenograft mice treated with THC showed significantly slower tumor progression and smaller tumor volumes than control mice. THC and CBN induced apoptosis in CCA by inhibiting the AKT and MAPK pathways. These findings provide a strong rationale for THC and CBN as therapeutic options for CCA.

Cytokine Secretion of Peripheral Blood Mononuclear Cells by Hydnocarpus anthelminthicus Seeds.

BACKGROUND: Hydnocarpus anthelminthicus is primarily used as a traditional medicine for the treatment of leprosy. Previous studies demonstrated that the clinical course of leprosy and the susceptibility to mycobacteria are recognized by the immune response of the host. The current study aims to investigate the effect of H. anthelminthicus seed oil and extracts on the secretion of cytokines from PBMCs involved in immune regulation. METHODS: PBMCs from healthy volunteers were cultured and treated with LPS and H. anthelminthicus seed oil or extracts. Cell viability was detected with WST-1 cell proliferation assay reagent. Proinflammatory cytokines were quantified using ELISA with a specific antibody. RESULTS: LPS-treated PBMCs significantly increased IL6 and TNF-α secretion. H. anthelminthicus seed oil had a synergistic effect with LPS on TNF-α secretion. The aqueous extract of H. anthelminthicus seed kernels and hulls significantly induced IL6 and TNF-α secretion. However, the ethanol extract of H. anthelminthicus seed kernels and hulls significantly decreased IL6, IL8, and TNF-α secretion in LPS-treated PBMCs. CONCLUSIONS: Extracts of H. anthelminthicus seeds demonstrated various effects on the proinflammatory cytokine secretion of PBMCs. The application of these extracts should depend on the immune response of the host, which determines the manifestation of the disease.

Novel Potential Biomarkers for Opisthorchis viverrini Infection and Associated Cholangiocarcinoma.

BACKGROUND/AIM: Early detection of disease is a pivotal factor for determining prognosis and clinical outcome of patients with cancer. As cholangiocarcinoma (CCA) is currently difficult to detect and most cases of such cancer present with late-stage disease at the time of initial diagnosis, we employed proteomic analysis of the bile to identify potential candidate biomarkers for Opisthorchis viverrini (OV)-associated CCA. MATERIALS AND METHODS: Proteins in pooled bile samples from patients with CCA and OV infection, with CCA without OV infection, with OV infection but no CCA, and with neither OV infection nor CCA were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel trypsin digestion and analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: According to our analysis, three proteins, namely aristaless-like homeobox1 isoform X1 (ALX1), major histocompatibility complex polypeptide-related sequence A (MICA), and uncharacterized protein C14orf105 isoform X12 were found to be potential markers for OV infection, as they were predominantly found in all OV-infected groups. Although these proteins were detected in both OV-infected patients with and without CCA, their abundance was 2.90-, 7.06-and 3.65-fold higher, respectively, in those with CCA. In patients with CCA, potential novel biomarkers wre immunoglobulin heavy chain, translocated in liposarcoma (TLS), visual system homeobox 2 (VSX2) and an unnamed protein product. CONCLUSION: We provided novel information regarding potential biomarkers for OV infection and CCA. These two protein profiles could benefit diagnosis as well as monitoring of CCA.

Gadd45ß silencing impaired viability and metastatic phenotypes in cholangiocarcinoma cells by modulating the EMT pathway.

Growth arrest and DNA damage-inducible-ß (Gadd45ß) is a stress-response protein involved in a number of processes, including cell cycle control, DNA repair, survival and death control, and stress signaling, depending on its interactions. Gadd45ß expression is dysregulated in numerous types of cancer, functioning as either a tumor promoter or a tumor suppressor. However, the functions of Gadd45ß in cholangiocarcinoma (CCA), particularly in metastasis, has not been studied. The immunohistochemical analysis of Gadd45ß expression revealed that 75% of histological specimens from patients with CCA expressed high levels of Gadd45ß, and that high Gadd45ß expression was associated with metastasis. The role of Gadd45ß in CCA was examined using siRNA-mediated gene knockdown in HuCCA-1, a human CCA cell line established from a Thai patient. The effects of Gadd45ß downregulation upon cell viability and death, invasion, migration, matrix metalloproteinase (MMP) activity and epithelial-mesenchymal transition (EMT) marker expression were investigated. Gadd45ß knockdown impaired cell viability, which was associated with the induction of apoptosis. In addition, there was a marked reduction in invasion and migration, although MMP activity was unaffected. Impairment of these metastatic properties was accompanied by the decreased expression of EMT markers, including Slug, vimentin, claudin-1 and zona occludens protein 1, whereas E-cadherin expression was increased. The present study suggests that Gadd45ß is involved in regulating the viability and the metastatic potential of CCA cells, which may be mediated by the modulation of the EMT pathway.

Molecular mechanisms of cholangiocarcinoma cell inhibition by medicinal plants.

Cholangiocarcinoma (CCA) is one of the most common causes of cancer-associated mortality in Thailand. Certain phytochemicals have been demonstrated to modulate apoptotic signaling pathways, which may be targeted for the prevention and treatment of cancer. Therefore, the aim of the present study was to investigate the effect of specific medicinal plants on the inhibition of CCA cell proliferation, and to identify the molecular mechanisms underlying this. A WST-1 cell proliferation assay was performed using an RMCCA1 cell line, and apoptotic signaling pathways were also investigated using a PathScan Stress and Apoptosis Signaling Antibody Array Kit. The cell proliferation assay indicated that extracts from the Phyllanthus emblica fruit pulp (PEf), Phyllanthus emblica seed (PEs), Terminalia chebula fruit pulp (TCf), Terminalia chebula seed (TCs), Areca catechu seed (ACs), Curcuma longa (CL) and Moringa oleifera seed (MOs) exerted anti-proliferative activity in RMCCA1 cells. In addition, the PathScan assay revealed that certain pro-apoptotic molecules, including caspase-3, poly (ADP-ribose) polymerase, checkpoint kinase 2 and tumor protein 53, exhibited increased activity in RMCCA1 cells treated with the aforementioned selected plant extracts, with the exception of PEf. The mitogen-activated protein kinase (MAPK) pathways (including ERK1/2 and p38 MAPK) expression level was significantly increased in RMCCA1 cells pre-treated with extracts of PEs, TCf, CL and MOs. The activation of protein kinase B (Akt) was significantly demonstrated in RMCCA1 cells pre-treated with extracts of TCf, ACs and MOs. In summary, the present study demonstrated that extracts of PEs, TCf, TCs, ACs, CL and MOs exhibited anti-proliferative effects in CCA cells by inducing pro-apoptotic signals and modulating signal transduction molecules. Further studies in vivo are required to demonstrate the potential applications of specific plant extracts for the treatment of human cancer.

RED BLOOD CELL DUFFY ANTIGEN RECEPTOR FOR CHEMOKINES AND SUSCEPTIBILITY TO PLASMODIUM VIVAX INFECTION IN THAIS.

Plasmodium vivax is the most prevalent malaria infection in Thailand. P. vivax uses Duffy Antigen Receptor for Chemokines (DARC) or Duffy antigen (Fy) as a receptor for entry into reticulocytes. Polymorphism of DARC exon 2 gene (FYA/FYB) in 40 P. vivax-infected subjects were investigated using nested PCR of blood samples spotted on filter paper collected during August 2013 to November 2013 from various malaria clinics in Thailand. Distribution of DARC genotypes was FYA 62.5%, FYB 20% and FYAB 17.5%, consistent with that of Hardy-Weinberg equation. Mutation G17A was found in both FYA and FYB alleles, resulting in Gyl48 and Asp48 of Fya and Fyb antigen, respectively. Mean parasitemia among the three groups is not statistically different. To the best of our knowledge, this is the first such study in Thailand.

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.

Hypoxia enhances cholangiocarcinoma invasion through activation of hepatocyte growth factor receptor and the extracellular signal­regulated 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, RMCCA­1. 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 signal­regulated kinase (ERK) 1/2, was identified. Inhibition of Met and ERK by small hairpin RNA and U0126, respectively, significantly inhibited hypoxia­induced the invasive potential of RMCCA­1 cells (P<0.05). However, according to immunohistochemical analysis, hypoxia­inducible factor­1α 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.

High Expression of Matrix Metalloproteinase-11 indicates Poor Prognosis in Human Cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CHCA) is serious public health problem in Thailand, especially in the northeastern and northern regions. CHCA is known as one of the most aggressive malignant tumors associated with local invasion and a high rate of metastasis. A crucial step in the invasion process is the proteolytic degradation of the extracellular matrix (ECM) and basal membranes, for which several studies have shown a critical role played by matrix metalloproteinase-11 (MMP-11). OBJECTIVE: This study aim to detect MMP-11 expression in CHCA specimens and any correlation with survival time. MATERIALS AND METHODS: A retrospective analysis was conducted of 30 patients with CHCA in Rajvithi hospital, who had undergone immunohistochemical staining of MMP-11. Relationships between clinicopathological data and MMP-11 expression in CHCA specimens were analyzed by the χ2 test or Fisher's exact test. The estimated survival and the survival differences were analyzed by the Kaplan-Meier method and the log-rank test, respectively. RESULTS: MMP-11 expression was found in 15 specimens (50%). The overall mean survival time is 237.0 days (95% CI 135.4-338.5, SD 271.9). Specimens with a positive MMP-11 had an average survival time of 136.7 days (95%CI 50.3-223.1, SD 156.0). Survival differences was signficant for the positive and negative MMP-11(p=0.022), but not well differentiated tumor and moderate to poor differentiated tumor (p=0.755), CA19-9 level of >1,000 and <1,000 (p=0.488), and between advanced and non-advanced staging (p=0.388). CONCLUSIONS: The positive MMP-11 expression indicates poor prognosis in CHCA specimens.

Matrigel induces L-plastin expression and promotes L-plastin-dependent invasion in human cholangiocarcinoma cells.

The function of the extracellular matrix (ECM) in the tumor microenvironment is not limited to forming a barrier against tumor invasion. As demonstrated in pathological specimens, cholangiocarcinoma samples exhibit an enrichment of the ECM surrounding the tumor cells. In this study, we examined involvement of the ECM in the regulation of the invasiveness of cholangiocarcinoma cells. The RMCCA1 cholangiocarcinoma cell line was cultured in culture plates either with or without a coating of reconstituted ECM basement membrane preparation (BD Matrigel matrix). In vitro invasion assays were then performed. In addition, the protein expression profile of the cell line was examined using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. The proteins expressed and their functional associations with cancer progression were determined. Culturing the RMCCA1 cell line in the BD Matrigel matrix induced cell invasion. Numerous proteins were induced by culturing the RMCCA1 cells in the matrix gel. The expression of L-plastin, an actin-binding protein, was significantly upregulated. The knockdown of L-plastin expression by siRNA silencing significantly suppressed the cellular response to matrix gel-stimulated cancer cell invasion. The ECM promotes the invasiveness of cholangiocarcinoma cells by upregulating L-plastin. These findings suggest the potential exploitation of this mechanism as a means of inhibiting the invasiveness of cholangiocarcinoma cells.

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.

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.

Long-term outcome of patients undergoing liver transplantation at Rajavithi Hospital, Thailand.

OBJECTIVE: Examine long-term outcome and survival of patients with liver transplantation at Rajavithi Hospital, a small-volume transplant center in Bangkok, Thailand. MATERIAL AND METHOD: Between May 1996 and December 2010, 21 liver transplantations were performed Piggyback technique and portal vein flushing with one liter of cold normal saline was used to prevent reperfusion injury. Color Doppler ultrasound was performed routinely. Data collection included demographic data, complications, operation time, ischemic time, duration of stay in intensive care unit (ICU), hospitalization period, and survival. RESULTS: There were two cases withdrawn from immunosuppressant drugs due to loss of follow-up and recidivism. Late death in three patients was from bleeding after hemiarthroplasty, chronic rejection, and lymphoma. Overall, 5-year and 10-year survival were 62% and 42% respectively Biliary complication rate was 9.5%. Two cases were under early reoperation due to bleeding from hepatic artery and retrohepatic vein. Hepatic vein occlusion was found in one case that had underlying Budd Chiari. One case with hepatocellular carcinoma, 10 nodules in both lobes of liver had survived more than three years after transplantation. CONCLUSION: Liver transplantation is a high-cost procedure. Good long-term results depend on expensive drugs, skilled surgeons, state-of-the-art equipment, and good team work. Policy and support from the government play an important role for successful transplantation, especially in developing countries.

Validation of appropriate reference genes for gene expression studies in human thyroid gland using real-time RT-PCR.

BACKGROUND: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (qRT-PCR) becoming the method of choice. The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. However there has not been any study that compares the stability of these reference genes in human thyroid specimens. Therefore, the authors studied the stability values and the appropriate reference genes expressed in thyroid specimens. MATERIAL AND METHOD: 25 human thyroid specimens were prospectively collected and extracted for their RNA. The candidate reference genes (hypoxanthine phosphoribosyl-transferase1 (HPRT1), ribosomal protein LI3a (RPLIA), beta-2-micro-globulin (B2M), beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPD)) were amplified from these thyroid specimens using real-time RT-PCR. The stabilities of these candidate reference genes were analyzed using Normfinder software. RESULTS: The authors found that HPRT1 has the highest stability value (40.38 x 10(9)) while GAPD has the lowest stability value (85.46 x 10(7)). Therefore GAPD is the most stably expressed gene in thyroid specimens. CONCLUSION: Of the 5 genes studied, GAPD was found to be the best reference gene for gene expression studies in the thyroid gland. The present results may facilitate the choice of reference genes for expression studies in thyroid diseases.

Evaluation of the white test for the intraoperative detection of bile leakage.

We assess whether the White test is better than the conventional bile leakage test for the intraoperative detection of bile leakage in hepatectomized patients. This study included 30 patients who received elective liver resection. Both the conventional bile leakage test (injecting an isotonic sodium chloride solution through the cystic duct) and the White test (injecting a fat emulsion solution through the cystic duct) were carried out in the same patients. The detection of bile leakage was compared between the conventional method and the White test. A bile leak was demonstrated in 8 patients (26.7%) by the conventional method and in 19 patients (63.3%) by the White test. In addition, the White test detected a significantly higher number of bile leakage sites compared with the conventional method (Wilcoxon signed-rank test; P < 0.001). The White test is better than the conventional test for the intraoperative detection of bile leakage. Based on our study, we recommend that surgeons investigating bile leakage sites during liver resections should use the White test instead of the conventional bile leakage test.

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, patient's 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.

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.

Serum AFP and AFP-L3 in clinically distinguished hepatocellular carcinoma from patients with liver masses.

OBJECTIVE: To determine the ability of alpha fetoprotein (AFP) and AFP-L3% serum level in discriminating hepatocellular carcinoma (HCC) from other types of liver mass. MATERIAL AND METHOD: This study was performed according to a prospective-specimen-collection, retrospective-blinded-evaluation (PRoBE) design. A total of 109 HCC patients and 51 patients with other types of liver mass were consecutively selected. The levels of AFP and AFP-L3% in their sera were measured. RESULTS: AFP levels in serum significantly elevated while AFP-L3% levels significantly decreased in HCC patients (AFP: p < 0.001, AFP-L3%: p < 0.001). The area under the curve (AUC) of a receiver operating characteristic (ROC) curve analysis for the diagnosis of HCC of AFP and AFP-L3% was 0.71 and 0.67, respectively. In addition, the serum level of AFP-L3% was significantly different between the small (mass occupying lesser than 50% of liver volume) and large (mass occupying more than 50% of liver volume) HCC (p = 0.040). CONCLUSION: The diagnostic accuracy of serum AFP and AFP-L3% could provide them as candidate biomarkers to discriminate patients with HCC from patients with other types of liver mass. Serum AFP-L3% as a prognostic factor for HCC should be further evaluated in more details.

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.