Attapol Titapun

Urinary microRNA-192 and microRNA-21 as potential indicators for liver fluke-associated cholangiocarcinoma risk group.

Opisthorchis viverrini infection induces chronic inflammation in the bile ducts, leading to periductal fibrosis (PDF), which possibly associates to cholangiocarcinoma (CCA). Patients with CCA have a poor prognosis, which is linked to asymptomatic disease and late diagnosis. Hence, detecting early stage CCA is essential. Secretory miRNAs have been promoted as biomarkers for pathological changes associated with parasitic infections, fibrosis and/or cancer. We aimed to determine levels of miR-192 and miR-21 in the urine of O. viverrini infected, periductal fibrosis (PDF) and CCA groups using qRT-PCR. We found that miR-192 was significantly higher in O. viverrini infected, PDF and also CCA groups (p<0.05) than in healthy controls. By utilizing the Receiver Operation Characteristics (ROC) analysis, miR-192 differentiated patients with opisthorchiasis (the area under the curve; AUC=0.766), PDF subjects (AUC=0.781) and CCA patients (AUC=0.682) from healthy controls. MiR-21 was significantly higher in PDF and CCA groups (p<0.05) than in healthy controls. MiR-21 discriminated PDF subjects (AUC=0.735) and CCA patients (AUC=0.682) from healthy controls. Combined levels of these two miRNAs revealed an increased AUC of 0.812 for separating opisthorchiasis, AUC of 0.815 in discriminating PDF subjects, and AUC of 0.849 in differentiating CCA from healthy controls. Odds ratios (OR) indicated high levels of miR-192/miR-21 as risk predictors for opisthorchiasis, PDF and CCA. Levels of these miRNAs declined significantly for patients following praziquantel treatment. In conclusion, urinary miR-192/miR-21 have potential as risk indicators for opisthorchiasis and PDF-associated CCA in the endemic region.

Surgical management of perihilar cholangiocarcinoma: a Khon Kaen experience

Cholangiocarcinoma is the most common cancer in the northeast of Thailand. Most of the patients present when the disease is in an advanced stage. Improvement of preoperative diagnoses and surgical techniques provide more satisfactory results. Herein we reviewed our 30‐year experience in management of perihilar cholangiocarcinoma in Khon Kaen northeast Thailand. Between 1982 and 2012 we reviewed four specific studies of perihilar cholangiocarcinoma in Srinagarind Hospital, Khon Kaen, Thailand. The first study focused on advanced surgical pathology and palliative surgery, which were used to treat obstructive jaundice cholangiocarcinoma patients. Long‐term survival in this study was rare with a one‐year survival of just 15%. The second study was conducted on 30 consecutive cases of perihilar cholangiocarcinoma who presented with obstructive jaundice without preoperative biliary drainage. All the patients underwent major liver resection with bilio‐enteric reconstruction. Perioperative mortality was 6.7% without a 5‐year survival. The third study aimed to analyze the survival rates and factors affecting survival in extrahepatic CCA patients following surgical treatment at Srinagarind Hospital and concluded that resection margins are an important prognostic factor. The last study objective was the analysis of curative surgical attempt in 99 consecutive perihilar cholangiocarcinoma and results showed that R0 resection could improve long‐term survival. We evaluated four studies of perihilar cholangiocarcinoma in Srinagarind Hospital, Khon Kaen, Thailand from 1982–2012. Viewed chronologically there has been a progressive improvement of diagnosis and surgical treatment during the past 30 years. Despite these advances the 5‐year survival rate remains unsatisfactorily low. Future improvement of patient selection and surgical techniques can lead to a greater survival rate for patients.

Outcome of curative resection for perihilar cholangiocarcinoma in Northeast Thailand

AIMnTo examine survival outcomes of perihilar cholangiocarcinoma (PCCA) resection including mortality, morbidity and prognostic factors.nnnMETHODSnMultivariate analyses were carried out based on the survival data of all patients with histologically confirmed PCCA who underwent curative resection at Srinagarind Hospital from January 2006 to December 2011.nnnRESULTSnThere were 29 (19%) cases of intrahepatic CCA that involved hilar and 124 (81%) with hilar bile-duct cancer. R0 resection was carried out on 66 (43.1%) patients of whom 50 (32.7%) also had lymph node metastasis. The other patients underwent R1 resection. The overall 5-year survival rate was 20.6% (95%CI: 13.8-28.4) and median survival time was 19.9 mo. Postoperative mortality was 2%, and 30% of patients had complications. Patients without lymph node metastasis were 60% less likely to die than those with metastasis. Achieving R0 led to a 58% reduction in the chance of mortality as compared to R1.nnnCONCLUSIONnTo achieve a better survival outcome, focus should center on performing radical surgery and detection of patients with early stage cancer.

Upregulation of transferrin receptor-1 induces cholangiocarcinoma progression via induction of labile iron pool

Labile iron pool is a cellular source of ions available for Fenton reactions resulting in oxidative stress. Living organisms avoid an excess of free irons by a tight control of iron homeostasis. We investigated the altered expression of iron regulatory proteins and iron discrimination in the development of liver fluke–associated cholangiocarcinoma. Additionally, the levels of labile iron pool and the functions of transferrin receptor-1 on cholangiocarcinoma development were also identified. Iron deposition was determined using the Prussian blue staining method in human cholangiocarcinoma tissues. We investigated the alteration of iron regulatory proteins including transferrin, transferrin receptor-1, ferritin, ferroportin, hepcidin, and divalent metal transporter-1 in cholangiocarcinoma tissues using immunohistochemistry. The clinicopathological data of cholangiocarcinoma patients and the expressions of proteins were analyzed. Moreover, the level of intracellular labile iron pool in cholangiocarcinoma cell lines was identified by the RhoNox-1 staining method. We further demonstrated transferrin receptor-1 functions on cell proliferation and migration upon small interfering RNA for human transferrin receptor 1 transfection. Results show that Iron was strongly stained in tumor tissues, whereas negative staining was observed in normal bile ducts of healthy donors. Interestingly, high iron accumulation was significantly correlated with poor prognosis of cholangiocarcinoma patients. The expressions of iron regulatory proteins in human cholangiocarcinoma tissues and normal liver from cadaveric donors revealed that transferrin receptor-1 expression was increased in the cancer cells of cholangiocarcinoma tissues when compared with the adjacent normal bile ducts and was significantly correlated with cholangiocarcinoma metastasis. Labile iron pool level and transferrin receptor-1 expression were significantly increased in KKU-214 and KKU-213 when compared with cholangiocyte cells (MMNK1). Additionally, the suppression of transferrin receptor-1 expression significantly decreased intracellular labile iron pool, cholangiocarcinoma migration, and cell proliferation when compared with control media and control small interfering RNA. In Conclusion, high expression of transferrin receptor-1 resulting in iron uptake contributes to increase in the labile iron pool which plays roles in cholangiocarcinoma progression with aggressive clinical outcomes.

Progranulin modulates cholangiocarcinoma cell proliferation, apoptosis, and motility via the PI3K/pAkt pathway

Progranulin (PGRN) is a growth factor normally expressed in rapidly cycling epithelial cells for growth, differentiation, and motility. Several studies have shown the association of PGRN overexpression with the progression of numerous malignancies, including cholangiocarcinoma (CCA). However, the underlying mechanisms on how PGRN modulates CCA cell proliferation and motility is not clear. In this study, we investigated the prognostic significance of PGRN expression in human CCA tissue and the mechanisms of PGRN modulation of CCA cell proliferation and motility. We found that CCA tissues with high PGRN expression were correlated with poor prognosis and likelihood of metastasis. PGRN knockdown KKU-100 and KKU-213 cells demonstrated a reduced rate of proliferation and colony formation and decreased levels of phosphatidyl inositol-3-kinase (PI3K) and phosphorylated Akt (pAkt) proteins. Accumulation of cells at the G1 phase was observed and was accompanied by a reduction of cyclin D1 and CDK4 protein levels. Knockdown cells also induced apoptosis by increasing the Bax-to-Bcl-2 ratio. Increased cell apoptosis was confirmed by annexin V-FITC/PI staining. Moreover, suppression of PGRN reduced CCA cell migration and invasion in vitro. Investigating the biomarkers in epithelial–mesenchymal transition (EMT) revealed a decrease in the expression of vimentin, snail, and metalloproteinase-9. In conclusion, our findings imply that PGRN modulates cell proliferation by dysregulating the G1 phase, inhibiting apoptosis, and that it plays a role in the EMT affecting CCA cell motility, possibly via the PI3K/pAkt pathway.

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.

Inhibitory effect of NVP‑BKM120 on cholangiocarcinoma cell growth

Abnormal activation of the phosphatidylinositol 3-kinase (PI3K) pathway has been demonstrated in certain types of cancer, including cholangiocarcinoma (CCA). This pathway may therefore be a promising target for CCA treatment. The present study assessed the inhibitory effect of NVP-BKM120, a pan-class I PI3K inhibitor, on CCA cell growth. This inhibitory effect was determined using CCA cell lines and in CCA-inoculated mice. The result from sulforhodamine B (SRB) assay demonstrated that NVP-BKM120 treatment inhibited CCA cell growth in a dose-dependent manner, even at the lowest tested concentration. The in vivo study revealed that oral administration of NVP-BKM120 (10 or 30 mg/kg) to CCA-inoculated nude mice led to a reduction in tumor growth when compared with controls, which was indicated by an immunohistochemical assay for Ki67 expression. In addition, the result from TUNEL assay demonstrated that NVP-BKM120 induced cancer cell death without any signs of toxicity, which indicated by the body weight of mice (data not shown). Western blot analysis demonstrated that NVP-BKM120 inhibited CCA cell growth by suppressing RAC serine/threonine protein kinase/mechanistic target of rapamycin activation and inhibiting the phosphorylation of phosphatase and tensin homolog, which is the inactivation form of the negative regulator of this pathway. Therefore, the results of the present study indicated that NVP-BKM120 should be considered as a therapeutic agent against CCA that could be used to improve treatment.

Zileuton suppresses cholangiocarcinoma cell proliferation and migration through inhibition of the Akt signaling pathway

Background Inflammatory lipid mediators play an important role in several cancer types. Leukotrienes (LTs), pro-inflammatory lipid mediators, are involved in chronic inflammation and cancer progression. They are derived from arachidonic acid by 5-lipoxygenase (5-LOX) activity. On the other hand, 15-lipoxygenase (15-LOX-1) converts LTs into lipoxins (LXs), pro-resolving lipid mediators. LXs are involved in the attenuation of inflammation and cancer development. Purpose We aimed to investigate the lipid mediator pathways, especially the LTs and LXs pathways, by studying 5-LOX and 15-LOX-1 expression in human cholangiocarcinoma (CCA) tissue. We also investigated the efficiency of zileuton (5-LOX inhibitor) treatment and BML-111 (LXA4 analog) addition on CCA cell lines properties. Patients and methods The expression of 5-LOX and 15-LOX-1 in fifty human cholangiocarcinoma (CCA) tissue was analyzed using immunohistochemical staining. In addition, the effect of zileuton and BML-111 on CCA cell growth and migration was demonstrated using a cell viability assay and wound-healing assay, respectively. Furthermore, the molecular mechanism by which zileuton inhibits CCA cell migration was revealed using immunofluorescent staining and western blot analysis, respectively. Results We demonstrate that the upregulation of 5-LOX is significantly correlated with CCA recurrent status. A positive 15-LOX-1 signal was significantly associated with a longer survival time in CCA patients. We found that co-expression of 5-LOX and 15-LOX-1 resulted in a relatively good prognosis in CCA patients. In addition, zileuton could inhibit CCA cell migration as well as BML-111. Interestingly, zileuton treatment not only downregulated 5-LOX, but also upregulated 15-LOX-1, together with reversing the epithelial-mesenchymal transition to mesenchymal-epithelial transition phenotype as observed in EMT marker western blot. Conclusion These findings suggest that 5-LOX and 15-LOX-1 play a key role in CCA and may serve as targets for CCA therapy.

Current Perspectives on Opisthorchiasis Control and Cholangiocarcinoma Detection in Southeast Asia

Similar to bile duct cancer or cholangiocarcinoma (CCA) in the western world, opisthorchiasis-associated CCA in Southeast Asia is an aggressive cancer with high mortality rates. It is known to cause a significant health burden in the opisthorchiasis region in Thailand and possibly throughout mainland Southeast. To reduce this health burden, a comprehensive prevention and control program for opisthorchiasis, as well as CCA, is required. In this review, our aim is to provide a brief update of the current situation regarding the natural history of opisthorchiasis and health burden of CCA in Southeast Asia. A comprehensive approach to tackling these issues being implemented in Thailand under the “Cholangiocarcinoma Screening and Care Program” is described. This comprehensive program consists of a three stage prevention and patient care program. The primary prevention component involves opisthorchiasis screening using a new and sensitive urine assay. The secondary prevention component involves screening for CCA and periductal fibrosis, with suspected CCA patients following the protocol for confirmation and appropriate treatment. Due to the eco-epidemiology of opisthorchiasis-induced CCA, the anticipated impacts and outcomes of the program include short-, medium-, and the long-term goals for the reduction of CCA incidence. To achieve long-term sustainable impacts, concerted efforts to raise social awareness and participating action by general public, non-government organizations, and government agencies are necessary. The strategic plans developed for this program can be expanded for use in other endemic areas as well as being a model for use in other chronic diseases.

Inhibition of endothelial nitric oxide synthase in cholangiocarcinoma cell lines – a new strategy for therapy

The isoform of nitric oxide synthase (NOS) found in endothelial cells (eNOS) plays a crucial role in vasodilation. We recently reported the activation of eNOS in cholangiocarcinoma (CCA) tissues and cell lines. Moreover, we also reported that the abundance of eNOS and phosphorylated eNOS (p‐eNOS), as well as its upstream regulator proteins, is significantly associated with the metastatic status of CCA patients. However, the function of eNOS in CCA progression has not been addressed. Therefore, the present study aimed to investigate the function of eNOS involved in the migration and invasion ability of CCA cell lines. The results reveal that eNOS activation significantly increases migration and invasion ability of CCA cells via the up‐regulation of phosphorylated vasodilator‐stimulated protein (p‐VASP). A combination treatment with recombinant human vascular endothelial growth factor C and eNOS inhibitor (Nω‐nitro‐l‐arginine methyl ester hydrochloride) resulted in the down‐regulation of p‐VASP, as well as a decreased migration and invasion ability of the CCA cell line. Thus, this work suggests that eNOS can serve as an attractive target to inhibit the progression of CCA.