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Altered gene expression in Opisthorchis viverrini‐associated cholangiocarcinoma in hamster model

Cholangiocarcinoma (CCA) induced by liver fluke (Opisthorchis viverrini, Ov) infection is one of the most common and serious disease in northeast Thailand. To elucidate the molecular mechanism of cholangiocarcinogenesis induced by Ov infection, we employed a hamster model of CCA induced by Ov and N‐nitrosodimethylamine and analyzed candidate genes involved in CCA using fluorescence differential display‐PCR. Of 149 differentially amplified bands we identified, the upregulation of 23 transcripts and downregulation of 1 transcript related to CCA hamsters were confirmed by a reverse northern macroarray blot. The upregulated genes include signal transduction protein kinase A regulatory subunit Iα (Prkar1a), myristoylated alanine‐rich protein kinase C substrate, transcriptional factor LIM‐4‐only domain, oxysterol‐binding protein involved in lipid metabolism, splicing regulatory protein 9, ubiquitin conjugating enzyme involved in protein degradation, β tubulin, β actin, and collagen type VI. Quantitative real‐time PCR confirmed that the expression of Prkar1a was significantly higher in CCA and its precursor lesion when compared with normal liver and normal gall bladder epithelia (P < 0.05). Prkar1a expression tended to increase along with the progression of biliary transformation from hyperplasia and precancerous lesions to carcinoma. These findings contribute to our understanding of the processes involved in the molecular carcinogenesis of CCA in order to provide a unique perspective on the development of new chemotherapeutics in future.

Mechanisms of oxysterol-induced carcinogenesis

Oxysterols are oxidation products of cholesterol that are generated by enzymatic reactions mediated by cytochrome P450 family enzymes or by non-enzymatic reactions involving reactive oxygen and nitrogen species. Oxysterols play various regulatory roles in normal cellular processes such as cholesterol homeostasis by acting as intermediates in cholesterol catabolism. Pathological effects of oxysterols have also been described, and various reports have implicated oxysterols in several disease states, including atherosclerosis, neurological disease, and cancer. Numerous studies show that oxysterols are associated with various types of cancer, including cancers of the colon, lung, skin, breast and bile ducts. The molecular mechanisms whereby oxysterols contribute to the initiation and progression of cancer are an area of active investigation. This review focuses on the current state of knowledge regarding the role of oxysterols in carcinogenesis. Mutagenicity of oxysterols has been described in both nuclear and mitochondrial DNA. Certain oxysterols such as cholesterol-epoxide and cholestanetriol have been shown to be mutagenic and genotoxic. Oxysterols possess pro-oxidative and pro-inflammatory properties that can contribute to carcinogenesis. Oxysterols can induce the production of inflammatory cytokines such as interleukin-8 and interleukin-1β. Certain oxysterols are also involved in the induction of cyclo-oxygenase-2 expression. Inflammatory effects can also be mediated through the activation of liver-X-receptor, a nuclear receptor for oxysterols. Thus, several distinct molecular mechanisms have been described showing that oxysterols contribute to the initiation and progression of cancers arising in various organ systems.

Myristoylated alanine-rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C-dependent pathway

(Cancer Sci 2010; 101: 658–665)

PRKAR1A is overexpressed and represents a possible therapeutic target in human cholangiocarcinoma

The protein kinase A regulatory subunit 1 alpha (PRKAR1A/PKAI) pathway is overexpressed in varieties of tumors and cancer cell lines including cholangiocarcinoma (CCA), although its role in CCA growth modulation is unclear. In our study, we evaluated the effect of PRKAR1A/PKAI targeting on CCA cell proliferation. Real‐time PCR demonstrated an increased mRNA expression of PRKAR1A/PKAI, whereas protein kinase A regulatory subunit 2 beta (PRKAR2B/PKAII) was downregulated in human CCA tissues and CCA cell lines. Immunohistochemistry of human CCA tissues revealed increased PRKAR1A with decreased PRKAR2B protein expression. Moreover, CCA cell lines showed abundantly expressed PRKAR1A, while lacking PRKAR2B expression. Silencing PRKAR1A expression induced growth inhibition and apoptosis of CCA cells, with an associated decrease in mitogen‐activated protein kinases, PI3K/Akt, JAK/STAT and Wnt/β‐catenin pathway signaling. The inhibition of PKA using a PKA inhibitor and cAMP analogs also led to a significant cell growth inhibition. In conclusion, our study reports the overexpression as well as molecular mechanisms by which PRKAR1A/PKA regulates human CCA cell growth. Importantly, abrogation of gene expression caused significant CCA cell growth inhibition, oncogenic signaling and coupled apoptosis induction, suggesting PRKAR1As potential as a drug target for CCA therapy.

Overexpression of microRNA-21 regulating PDCD4 during tumorigenesis of liver fluke-associated cholangiocarcinoma contributes to tumor growth and metastasis.

MicroRNA, an endogenous noncoding RNA modulating gene expression, is a key molecule that by its dysregulation plays roles in inflammatory-driven carcinogenesis. This study aimed to investigate the role of oncomiR miR-21 and its target, the programmed cell death 4 (PDCD4) in tumor growth and metastasis of the liver fluke Opisthorchis viverrini-associated cholangiocarcinoma (CCA). The expression levels of miR-21 and PDCD4 were analyzed using the TaqMan miRNA expression assay and immunohistochemistry in liver tissues of both O. viverrini plus N-nitrosodimethylamine (NDMA)-treated hamsters and human CCA samples (n = 23 cases). The functional assay for miR-21 was performed in CCA cell lines by the anti-miR-21 and pre-miR-21 transfection procedures. The peak of miR-21 levels were reached at 2 (hyperplastic lesions) and 6 (CCA) months of the O. viverrini plus NDMA-induced group and had a reverse response with its target PDCD4 proteins. In human CCA, miR-21 was overexpressed in tumor tissues when compared with nontumor tissues (P = 0.0034) and had a negative correlation with PDCD4 protein (P = 0.026). It was also found that high expression of miR-21 was significantly correlated with shorter survival (P < 0.05) and lymph node metastasis (P = 0.037) of CCA patients. Transient transfection of pre-miR-21 reduced the PDCD4 level and resulted in an increase of M213 CCA cell growth and wound-induced migration ability. These results indicated that miR-21 plays a role in the carcinogenesis and metastasis of O. viverrini-associated CCA by suppressing the function of PDCD4. Modulation of aberrantly expressed miR-21 may be a useful strategy to inhibit tumor cell phenotypes or improve response to chemotherapy.

Characterization of 5-Fluorouracil-Resistant Cholangiocarcinoma Cell Lines

Background: Although 5-fluorouracil (5-FU) is the drug of choice for the palliative treatment of cholangiocarcinoma (CCA), resistance to the drug is a therapeutic obstacle. The aim of this study was to explore the mechanisms underlying 5-FU resistance of CCA using cell lines derived from CCA associated with liver fluke infection. Methods: A stepwise exposure was used for inducing 5-FU-resistant CCA cell lines, and the expression of nine genes associated with 5-FU resistance was analyzed using real-time (RT)-PCR. Results: Altered expression of several genes involved in 5-FU resistance in CCA cell lines was observed. The expression levels of almost all target genes investigated including TP, DPD, ENT1, UNG1, TOP2A, BIRC5, TP73 and DeltaNp73 appeared to be significantly altered in these resistant strains. The expression of the TS gene tended to be increased but the fold change was not significantly different from their parental cell lines. UNG1 (a DNA repairing enzyme) and BIRC5 (an apoptotic inhibitor) expressions were increased whereas TP73 (a proapoptotic factor) expression levels decreased concomitantly. Conclusion: Our study showed that increases in UNG1 and BIRC5 expression and concomitant decreases in TP73 expression may be associated with development of acquired 5-FU resistance in CCA lines and their phenotypes.

Histological confirmation of periductal fibrosis from ultrasound diagnosis in cholangiocarcinoma patients

Cholangiocarcinoma (CCA) has no specific clinical signs and symptoms and non‐specific bio‐ and tumor‐markers in the early disease stage. Usually patients present to tertiary care with advanced disease stage. In order to detect early cases of CCA that may present as a mass, dilatation of intrahepatic duct or combination, ultrasonography is accepted as a powerful imaging tool. A smaller mass or bile duct segmental dilatation requires further imaging for characterization, including computerized tomography (CT) or magnetic resonance imaging (MRI). We examined whether liver echo pattern was correlated with high risk for CCA in an endemic area of Opisthorchis viverrini (Ov). Ov infestation caused chronic inflammation of the biliary tree by periductal fibrosis (PDF), which may subsequently lead to CCA development. In our study, a World Health Organization classification of pattern of increased periportal echo (IPE) for schistosomiasis was applied. Two CCA patients gave consent for operation. Histopathological diagnosis showed both had cholangiocarcinoma with periductal fibrosis of the non‐tumorous area of the liver. Ultrasonography was used to compare the non‐tumorous area with parenchymal echo pattern and was shown to have an early CCA detection role and a surveillance role in an endemic area of Ov by detection of PDF.

Tumor necrosis factor-α modulates epithelial mesenchymal transition mediators ZEB2 and S100A4 to promote cholangiocarcinoma progression

The epithelial‐mesenchymal transition (EMT) process strongly contributes to cancer metastasis. This study was to investigate the alteration of EMT‐related proteins (ZEB1, ZEB2 and S100A4) in cholangiocarcinoma (CCA) tissues. The effect of tumor necrosis factor‐α (TNF‐α) on the expression of those molecules in CCA cells was investigated.

Survey of activated kinase proteins reveals potential targets for cholangiocarcinoma treatment

Improving therapy for patients with cholangiocarcinoma (CCA) presents a significant challenge. This is made more difficult by a lack of a clear understanding of potential molecular targets, such as deregulated kinases. In this work, we profiled the activated kinases in CCA in order to apply them as the targets for CCA therapy. Human phospho-receptor tyrosine kinases (RTKs) and phospho-kinase array analyses revealed that multiple kinases are activated in both CCA cell lines and human CCA tissues that included cell growth, apoptosis, cell to cell interaction, movement, and angiogenesis RTKs. Predominately, the kinases activated downstream were those in the PI3K/Akt, Ras/MAPK, JAK/STAT, and Wnt/β-catenin signaling pathways. Western blot analysis confirms that Erk1/2 and Akt activation were increased in CCA tissues when compared with their normal adjacent tissue. The inhibition of kinase activation using multi-targeted kinase inhibitors, sorafenib and sunitinib led to significant cell growth inhibition and apoptosis induction via suppression of Erk1/2 and Akt activation, whereas drugs with specificity to a single kinase showed less potency. In conclusion, our study reveals the involvement of multiple kinase proteins in CCA growth that might serve as therapeutic targets for combined kinase inhibition.

A Comprehensive Public Health Conceptual Framework and Strategy to Effectively Combat Cholangiocarcinoma in Thailand

1 Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 2 Cholangiocarcinoma Screening and Care Program (CASCAP), Khon Kaen University, Khon Kaen, Thailand, 3 Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 4 Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 5 Department of Epidemiology and Biostatistics, Faculty of Public Health, Khon Kaen University, Khon Kaen, Thailand, 6 Department of Radiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand