Roles of the MEK1/2 and AKT pathways in CXCL12/CXCR4 induced cholangiocarcinoma cell invasion

AIM： To evaluate the expression of C-X-C motif chemokine receptor 4 （CXCR4） and its signaling cascades, which were previously identified as a key factor for cancer cell progression and metastasis, in cholangiocarcinoma cell lines. METHODS： The expression of CXCR4 and its signaling cascades were determined in the cholangiocarcinoma cell lines （RMCCA1 and KKU100） by Western blotting. The invasion assays and the detection of actin polymerization were tested in these cholangiocarcinoma cells treated with CXC chemokine ligand -12 （CXCL12）. RESULTS： Expression of CXCR4 was detected in both cholangiocarcinoma cell lines and activation of CXCR4 with CXCL12 triggered the signaling via the extracellular signal-regulated kinase-1/2 （ERK1/2） and phosphoinositide 3-kinase （PI3K） and induction of cholangiocarcinoma cell invasion, and displayed high levels of actin polymerization. Addition of CXCR4 inhibitor （AMD3100） abrogated CXCL12-induced phosphorylation of MEKI/2 and Akt in these cells. Moreover, treatment with MEK1/2 inhibitor （U0126） or PI3K inhibitor （LY294002） also attenuated the effect of CXCL12- induced cholangiocarcinoma cell invasion. CONCLUSION： These results indicated that the activation of CXCR4 and its signaling pathways （MEK1/2 and Akt） are essential for CXCL12-induced cholangiocarcinoma cell invasion. This rises Implications on a potential role for the inhibition of CXCR4 or its signal cascades in the treatment of cholangiocarcinoma.

Prospective study of MMP7 serum levels in the diagnosis of cholangiocarcinoma

AIM: To determine whether the serum level of matrix metalloproteinase-7 (MMP7) has the potential to diagnosis cholangiocarcinoma from benign biliary tract diseases. METHODS: This study was performed according to the PRoBE (a prospective-specimen-collection, retrospectiveblinded-evaluation) design. A total of 187 patients with obstructive jaundice were consecutively enrolled. After the diagnostic status of these patients was ascertained, their levels of serum MMP7 were assayed and compared with serum carbohydrate antigen 19-9 (CA19-9). This was conducted in a blinded case (cholangiocarcinoma)control (benign biliary tract disease) setup. RESULTS: MMP7 and CA19-9 serum levels were significantly elevated in cholangiocarcinoma patients (P < 0.001). The area under the curve (AUC) from a receiver operating characteristic (ROC) curve analysis for thediagnosis of cholangiocarcinoma, using MMP7 was more accurate than CA19-9 (AUC = 0.84, 95% CI: 0.778-0.903 for MMP7 and AUC = 0.79, 95% CI: 0.708-0.868 for CA19-9). The sensitivity and specificity of serum MMP7 (cut-off value of 5.5 ng/mL) was 75% and 78%, respectively, while the sensitivity and specificity of serum CA19-9 (cut-off value of 100 U/mL) was 68% and 87%, respectively. CONCLUSION: Serum values of MMP7 and CA19-9 appear to be useful biomarkers for differentiating cholangiocarcinoma from benign biliary tract obstructive diseases.

Role of ciprofloxacin in patients with cholestasis after endoscopic retrograde cholangiopancreatography

AIM： To determine the role of ciprofloxacin in reducing cholangitis in cholestatic patients with adequate biliary drainage after endoscopic retrograde cholangiopancreatogra phy （ERCP）. METHODS： A randomized, controlled trial was performed in 48 cholestatic patients at Rajavithi Hospital （Tertiary Referral Center for ERCP： 600 cases per year）. All the 48 patients received 200 mg ciprofloxacin intravenous injection for 30 min before starting any procedures, and then were randomly divided in two groups. Twenty-two patients in study group continually received dprofloxacin until 48 h after ERCP. Causes of biliary obstruction, bacteriology of bile and blood （in cholangitis） and dinical cholangitis were recorded. RESULTS： Forty-eight patients were enrolled and divided into continuous ciprofloxacin treatment group （/7 = 22） and discontinuous ciprofloxacin treatment group （n = 26）. During ERCP, stones were found in 22 patients, malignant diseases in 24 patients and other pathologic lesions in 5 patients. One （4.5%） of the 22 patients who received ciprofloxacin and 2 （6.3%） of the 26 patients who discontinued ciprofloxacin after ERCP developed cholangitis （relative risk = 0.71; 95% CI = 0.14-3.65; P = 0.88）. Bacterobilia was found in 27 （56.3%） out of 48 patients. E. coli and Streptococcus viridans were the most common organisms. CONCLUSION： Continual use of ciprofloxacin in patients with cholestasis after adequate biliary drainage procedures plays no role in reducing cholangitis.

Establishment and characterization of a cholangiocarcinoma cell line (RMCCA-1) from a Thai patient

AIM: To establish and characterize a new cell line derived from peripheral cholangiocarcinoma of a Thai patient.METHODS: The peripheral cholangiocarcinoma specimen surgically obtained from the patient was aseptically processed by washing and mincing before culturing in Ham’s F12 medium containing 10% fetal bovine serum. After 3 mo, when the cell line has become homogeneous and stabilized, several features were investigated, including growth characteristics, immunofluorescence staining for cytokeratins, expression of tumor markers, chromosomal analysis by G-banding and multicolour fluorescence in situ hybridization (mFISH), in vitro migration and invasion characteristics. RESULTS: The RMCCA-1 cell line has been established. These cells proliferated as a monolayer with a population doubling time of 48 h. Immunofluorescence staining showed positive staining for human cytokeratin 7 and 19 verifying the biliary epithelial origin. RMCCA-1 secreted carbohydrate antigen 19-9 (CA19-9), but insignificant levels of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Chromosome analysis identified aneuploidy karyotypes with a modal chromosome number of 59. RMCCA-1 exhibited a low level of in vitro invasiveness, but a high degree of motility. The cell line exhibited a significant number of chromosomal aberrations as shown by mFISH and G-banding methods.CONCLUSION: A new cell line derived from peripheral cholangiocarcinoma of a Thai patient has been established. This cell line shows a low level of in vitro invasiveness, but a high degree of motility. It will serve as a valuable tool for further studies on tumor biology, molecular pathogenesis, metastatic mechanism and response to therapeutic drugs of cholangiocarcinoma.

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 malig- nant biliary tract diseases. Quantitative real-time re- verse 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 expres- sion, patient's age and serum bilirubin were each inde- pendently associated with overall survival. CONCLUSION: CK19 mRNA expression levels in pe- ripheral blood appear to provide a valuable marker to predict the overall survival of patients with advanced malignant biliary tract obstruction.

Antibiotics resistance rate of Helicobacter pylori in Bhutan

AIM:To survey the antibiotic resistance pattern of Helicobacter pylori(H.pylori)strains isolated from Bhutanese population.METHODS:We isolated 111 H.pylori strains from the gastric mucosa of H.pylori-infected patients in Bhutan in 2010.The Epsilometer test was used to determine the minimum inhibitory concentrations(MICs)of amoxicillin(AMX),clarithromycin(CLR),metronidazole(MNZ),levofloxacin(LVX),ciprofloxacin(CIP),and tetracycline(TET).RESULTS:Nineteen of the isolated H.pylori strains were susceptible to all antibiotics tested.The isolated strains showed the highest rate of antibiotic resistance to MNZ(92/111,82.9%).Among the 92 MNZresistant strains,74 strains(80.4%)showed high-level resistance(MIC≥256 g/mL).Three strains were resistance to LVX(2.7%).These strains were also resistance to CIP.None of the strains showed resistance to CLR,AMX and TET.CONCLUSION:CLR-based triple therapy is a more effective treatment approach over MNZ-based triple therapy for H.pylori infection in Bhutan.

Extremely high prevalence of Helicobacter pylori infection in Bhutan

AIM: To revealed the prevalence of Helicobacter pylori (H. pylori ) infection in the Bhutanese population. METHODS: We recruited a total of 372 volunteers (214 females and 158 males; mean age of 39.6 ± 14.9 years) from three Bhutanese cities (Thimphu, Punaka, and Wangdue). The status of H. pylori infection was determined based on five different tests: the rapid urease test (CLO test), culture, histology, immunohistochemistry (IHC), and serum anti H. pylori -antibody. RESULTS: The serological test showed a significantly higher positive rate compared with the CLO test, culture, histology and IHC (P < 0.001, P < 0.001, P=0.01, and P=0.01, respectively). When the subjects were considered to be H. pylori positive in the case of at least one test showing a positive result, the overall prevalence of H. pylori infection in Bhutan was 73.4%. The prevalence of H. pylori infection significantly decreased with age (P < 0.01). The prevalence of H. pylori infection was lower in Thimphu than in Punakha and Wangdue (P=0.001 and 0.06, respectively). The prevalence of H. pylori infection was significantly higher in patients with peptic ulcers than in those with gastritis (91.4% vs 71.3%, P=0.003). CONCLUSION: The high incidence of gastric cancer in Bhutan may be attributed to the high prevalence of H. pylori infection.

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.

New concept of ileocecal junction:Intussusception of the terminal ileum into the cecum

AIM: To prove that the terminal ileum is intussuscepted into the cecum creating the ileocecal junction,contrary to previous valvular concept which has been widely believed. METHODS: This study is based on gross and microscopic examinations of fresh specimens derived from colonic operations (right hemicolectomy or subtotal colectomy). Data compiled from observing and dissecting of specimens of seven patients are used to examine both gross and microscopic appearance of ileocecal junction. RESULTS: Intussusception of the terminal ileum was found in every specimen. However,the length of intussusception was different in each specimen. CONCLUSION: Gross and microscopic appearance studies suggest that the terminal ileum is intussuscepted into the cecum.

Circulating hTERT mRNA as a tumor marker in cholangiocarcinoma patients

AIM： To investigate human telomerase reverse transcriptase （hTERT） mRNA in the serum of cholangiocarcinoma patients. METHODS： The serum of thirty three cholangiocarcinoma patients, forty one benign biliary tract disease patients and ten healthy volunteers were collected and analyzed for the expression of hTERT mRNA by real-time reverse transcriptase-polymerase chain reaction （RT-PCR）. We then examined the correlation between values of serum hTERT mRNA and the pathological staging of cholangiocarcinoma. RESULTS： hTERT mRNA was detected in 28 of 33 （84.85%） of serum obtained from cholangiocarcinoma patients and 9 of 41 （21.9%） of serum obtained from benign biliary tract disease patients, hTERT mRNA was not detected in any serum obtained from healthy volunteers, on the other hand the common tumor marker, CA19-9 was detected in 20 of 33 （60.6%） of serum obtained from cholangiocarcinoma patients and 8 of 41 （19.5%） of serum obtained from benign biliary tract disease patients. However, no correlation was found between the present of serum hTERT mRNA and tumor staging. CONCLUSION： These results indicate that the detection of circulating hTERT mRNA was identified in almost all cholangiocarcinoma patients. It offers a novel tumor marker, which can be used as a complementary study for diagnosis of cholangiocarcinoma .