Untangling the Complexity of Liver Fluke Infection and Cholangiocarcinoma in NE Thailand Through Transdisciplinary Learning.

This study demonstrates how a transdisciplinary learning approach provided new insights for explaining persistent Opisthorchis viverrini infection in northern Thailand, as well as elucidating problems of focusing solely on the parasite as a means of addressing high prevalence of cholangiocarcinoma. Researchers from diverse backgrounds collaborated to design an investigative homestay program for 72 Singaporean and Thai university students in five northeast Thai villages. The students explored how liver fluke infection and potential cholangiocarcinoma development are influenced by local landscape dynamics, aquatic ecology, livelihoods, food culture and health education. Qualitative fieldwork was guided daily by the researchers in a collaborative, co-learning process that led to viewing this health issue as a complex system, influenced by interlinked multidimensional factors. Our transdisciplinary experience has led us to believe that an incomplete understanding of these linkages may reduce the efficacy of interventions. Further, viewing liver fluke infection and cholangiocarcinoma as the same issue is inadvisable. Although O. viverrini infection is an established risk factor for the development of cholangiocarcinoma, multiple factors are known to influence the likelihood of acquiring either. Understanding the importance of the current livelihood transition, landscape modification and the resulting mismatch between local cultures and new socio-ecological settings on cholangiocarcinoma initiation and liver fluke transmission is of critical importance as it may help readjust our view of the respective role of O. viverrini and other socioeconomic risk factors in cholangiocarcinoma etiology and refine intervention strategies. As demonstrated in this study, transdisciplinary approaches have the potential to yield more nuanced perspectives to complex diseases than research that focuses on specific aspects of their epidemiology. They may therefore be valuable when designing effective solutions to context-sensitive diseases such as liver fluke infection and cholangiocarcinoma.

Haem oxygenase 1 expression is associated with prognosis in cholangiocarcinoma patients and with drug sensitivity in xenografted mice.

OBJECTIVE: Haem oxygenase-1 (HO-1) plays important roles in cytoprotection and tumour growth. Cholangiocarcinoma (CCA) is a deadly malignancy with very poor prognosis. The role of HO-1 in tumour progression in CCA up to now has been relatively unexplored, thus, its possible therapeutic implications in CCA have been investigated here. MATERIALS AND METHODS: HO-1 expression in tumour tissues from 50 CCA patients was determined by immunohistochemical analysis and its association with survival time was evaluated using the Kaplan-Meier method. Its role in CCA cells in vitro was evaluated by transwell and wound healing assays and suppression of HO-1 expression by siRNA. Effects of HO-1 inhibition on gemicitabine (GEM)-mediated tumour suppression was evaluated in nude mice xenografted with CCA cells. RESULTS: HO-1 expression was inversely associated with median overall survival time. Hazard ratio of patients with high HO-1 expression was 2.42 (95% CI: 1.16-5.08) with reference to low expression and HO-1 knock-down expression inhibited transwell cell migration. Suppression of HO-1 by Zn-protoporphyrin (ZnPP) enhanced cytotoxicity to GEM in CCA cells, validated in CCA xenografts. Treatment with GEM and ZnPP almost completely arrested tumour growth, whereas treatment with only a single reagent, retarded it. Tumour inhibition was associated with reduction in expression of Ki-67 and microvascular density, and enhanced p53 and p21 immunohistochemical staining. CONCLUSION: High HO-1 expression was associated with poor prognosis of CCA. Synergistic role of HO-1 inhibition in chemotherapy of CCA is a promising insight for treatment of this tumour and warrants further investigation.

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.

Expression profiles of oncomir miR-21 and tumor suppressor let-7a in the progression of opisthorchiasis-associated cholangiocarcinoma.

Altered miRNA expression could be a determinant of cancer development and/or progression. We aimed to study the role of oncomir miR-21 and tumor suppressor let-7a in the genesis of Opisthorchiasis-associated cholangiocarcinoma (CCA). The results showed that miR-21 was up-regulated while let-7a was down-regulated during cholangiocarcinogenesis in the hamster model and also in human CCA samples. The expression level of miR-21 had an inverse correlation with the mRNA level of its target RECK, a metastasis suppressor, in human CCA. Knockdown of miR-21 of KKU100 CCA cells significantly increased the mRNA level of RECK and suppressed the wound-induced migration of CCA cells. Our data suggest that miR-21 is one key molecule playing crucial roles in the CCA growth and metastasis. Manipulation of miRNA expression offers a potential avenue of CCA therapy.

Downregulation of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is associated with enhanced expression of matrix metalloproteinases and cholangiocarcinoma metastases.

BACKGROUND: Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) has been implicated in the attenuation of tumor metastasis by negatively regulating metalloproteinase (MMP) levels. RECK gene expression is downregulated in many solid tumors, with this downregulation being associated with poor prognosis. This study evaluated the role of RECK in cholangiocarcinoma (CCA). METHODS: The expression of RECK, MMP-2, and MMP-9 in paraffin sections of hamster and human CCA specimens was analyzed by immunohistochemistry. Functional analysis of RECK was performed in RECK small interfering (si) RNA knockdown CCA cell lines. The effect of aspirin on RECK status and function was evaluated using Western blotting, gelatin zymography, invasion and proliferation assays, and PhosphoELISArray analysis of Ras downstream mediators. RESULTS: Hamster tissues showed high RECK expression in hyperplastic biliary duct epithelia, low RECK expression in precancerous lesions, and no RECK expression in CCA. In human specimens, RECK was highly expressed in normal biliary cells, whereas intrahepatic CCA showed low levels of expression. Downregulation of RECK was correlated with tumor metastasis (P < 0.01) and shorter patient survival (P < 0.02). RECK expression levels were inversely correlated with MMP-2 and MMP-9 expression (P < 0.05). SiRNA RECK-depleted M139 CCA cells exhibited increased MMP-2/-9 gelatinase activities and invasiveness. Aspirin (500 µM) demonstrated myriad effects in human CCA cell lines, including growth suppression, reduced phosphorylation of Akt/Erk/c-Jun, elevation of RECK expression, inhibition of MMP-2/MMP-9 activity, and enhanced invasiveness. CONCLUSIONS: RECK functions as a metastasis suppressor in CCA; upregulation of RECK expression could provide a potential therapy to improve the prognosis of this type of cancer.

Circulating CD14(+) CD16(+) monocyte levels predict tissue invasive character of cholangiocarcinoma.

Chronic inflammation as a risk factor for cancer development is driven in part by monocyte/macrophages, which in many cancers exhibit pro-tumorigenic activity. In this study we identified elevation in CD14(+) CD16(+) , a minor blood monocyte subpopulation in cholangiocarcinoma (CCA) patients, compared to normal and biliary disease patient specimens. Tumour association was suggested by the observation that this elevated level decreased to normal after tumour resection. Moreover, the elevated level of CD14(+) CD16(+) monocytes in CCA patient blood correlated with degree of MAC387-positive (recent blood-derived macrophage migrant-specific marker) tumour-associated macrophage infiltration as determined by immunohistochemistry. These CD14(+) CD16(+) monocytes were suggested to enhance tumour progression as this subpopulation possesses (i) high expression of adhesion molecules (CD11c, CD49d, and CD54) and scavenger receptor (CD163), which enable them to adhere strongly to endothelial cells, and (ii) that peripheral blood monocytes from CCA patients express high levels of growth and angiogenic factor-related genes (epiregulin, VEGF-A and CXCL3). Elevation of peripheral CD14(+) CD16(+) monocyte levels was associated with features associated with poor prognosis CCA parameters (non-papillary type and high number of tissue macrophages). These data indicate that the CD14(+) CD16(+) monocytes from CCA patients with pro-tumorigenic characteristics may associate with rapid tumour progression and poor patient outcome. If confirmed in subsequent studies, the level of CD14(+) CD16(+) monocytes may serve as a marker for disease activity in CCA patients and serve as a target for pathogenic macrophage specific drug development.