Chawalit Pairojkul

Liver Fluke Induces Cholangiocarcinoma

The authors discuss the molecular pathogenesis of opisthorchiasis and associated cholangiocarcinogenesis, particularly nitrative and oxidative DNA damage and the clinical manifestations of cholangiocarcinoma.

Cholangiocarcinoma: Lessons from Thailand

Purpose of review To present the background of liver fluke-associated cholangiocarcinoma in Thailand focusing on recent epidemiological data and pathogenesis of this bile duct cancer. Recent findings More systematic tumor registration in Thailand nowadays uncovers new high-incidence areas that are confined to not only the northeastern part but also some provinces in northern Thailand. The link between the liver fluke, Opisthorchis viverrini, and cholangiocarcinoma, particularly in terms of cellular and molecular pathogenesis, is further elucidated. Summary Thailand is still the country with the highest incidence of cholangiocarcinoma in the world. Liver fluke induces chronic inflammation leading to oxidative DNA damage of the infected biliary epithelium and malignant transformation. Eradication of the fluke and identification of high-risk populations are urgently needed.

Exome sequencing of liver fluke-associated cholangiocarcinoma

Opisthorchis viverrini–related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini–related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8–3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini–related CCA.

Exome sequencing identifies distinct mutational patterns in liver fluke–related and non-infection-related bile duct cancers

The impact of different carcinogenic exposures on the specific patterns of somatic mutation in human tumors remains unclear. To address this issue, we profiled 209 cholangiocarcinomas (CCAs) from Asia and Europe, including 108 cases caused by infection with the liver fluke Opisthorchis viverrini and 101 cases caused by non–O. viverrini–related etiologies. Whole-exome sequencing (n = 15) and prevalence screening (n = 194) identified recurrent somatic mutations in BAP1 and ARID1A, neither of which, to our knowledge, has previously been reported to be mutated in CCA. Comparisons between intrahepatic O. viverrini–related and non–O. viverrini–related CCAs demonstrated statistically significant differences in mutation patterns: BAP1, IDH1 and IDH2 were more frequently mutated in non–O. viverrini CCAs, whereas TP53 mutations showed the reciprocal pattern. Functional studies demonstrated tumor suppressive functions for BAP1 and ARID1A, establishing the role of chromatin modulators in CCA pathogenesis. These findings indicate that different causative etiologies may induce distinct somatic alterations, even within the same tumor type.

Roles of liver fluke infection as risk factor for cholangiocarcinoma

Several factors are known to be associated with risk of cholangiocarcinoma (CCA) and infection with the liver flukes, Opisthorchis viverrini and Clonorchis sinensis, has often been singled out as the leading risk factor in east and southeast Asia. In this review, current knowledge of their biology, life cycle, and pathogenesis of O. viverrini, and its role as a carcinogenic parasite are presented. The trends of age‐specific incidence of liver cancer in Khon Kaen, northeast Thailand are considered and compared with the prevalence profiles of O. viverrini. Potential impacts of the liver fluke control program particularly by mass drug administration (MDA) and public health education in the past and a recent drop of incidence of CCA are discussed in relation to primary prevention and control of this fatal bile duct cancer.

Relationship between faecal egg count and worm burden of Opisthorchis viverrini in human autopsy cases

The relationship between faecal examination for egg output and worm burden of Opisthorchis viverrini in man of 181 autopsy cases from Northeast Thailand is described. Diagnosis of the parasite infection by stool examination for the presence of eggs was less sensitive than the worm recovery technique. Using Stolls dilution and formalin-ether technique, no eggs were detected in the faeces of 20 cases harbouring low worm burdens (less than 20 worms). The quantitative faecal egg count by Stolls dilution technique showed a strikingly close positive correlation with the number of worms recovered (r = 0.96, P less than 0.001) indicating a strong linear association between eggs per gram of faeces (epg) and worm burden. The number of epg per worm was inversely correlated to the worm burden (P less than 0.001), suggesting that density-dependent constraints on fecundity could operate to restrict egg output in heavy infections. The accuracy of egg counts for estimating worm burden and its relevance to parasite epidemiological research are discussed.

Comparison of gene expression profiles between Opisthorchis viverrini and non-Opisthorchis viverrini associated human intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary cancer in the liver, and its incidence is highest in the northeastern part of Thailand. ICCs in this region are known to be associated with infection with liver flukes, particularly Opisthorchis viverrini (OV), as well as nitrosamines from food. To clarify molecular mechanisms of ICC associated with or without liver flukes, we analyzed gene expression profiles of OV‐associated ICCs from 20 Thai patients and compared their profiles with those of 20 Japanese ICCs that were not associated with OV, by means of laser microbeam microdissection and a cDNA microarray containing 27,648 genes. We identified 77 commonly upregulated genes and 325 commonly downregulated genes in the two ICC groups. Unsupervised hierarchical cluster analysis separated the 40 ICCs into two major branches almost completely according to the fluke status. The putative signature of OV‐associated ICC exhibited elevated expression of genes involved in xenobiotic metabolism (UGT2B11, UGT1A10, CHST4, SULT1C1), whereas that of non–OV‐associated ICC represented enhanced expression of genes related to growth factor signaling (TGFBI, PGF, IGFBP1, IGFBP3). Additional random permutation tests identified a total of 49 genes whose expression levels were significantly different between the two groups. We also identified genes associated with macroscopic type of ICCs. In conclusion, these data may not only contribute to clarification of common and OV‐specific mechanisms underlying ICC, but also may serve as a starting point for the identification of novel diagnostic markers or therapeutic targets for the disease. (HEPATOLOGY 2006;44:1025–1038.)

Curcumin decreases cholangiocarcinogenesis in hamsters by suppressing inflammation-mediated molecular events related to multistep carcinogenesis

Cholangiocarcinoma (CCA) is a highly metastatic tumor linked to liver fluke infection and consumption of nitrosamine‐contaminated foods and is a major health problem especially in South‐Eastern Asia. In search for a suitable chemopreventive agents, we investigated the effect of curcumin, a traditional anti‐inflammatory agent derived from turmeric (Curcuma longa), on CCA development in an animal model by infection with the liver fluke Opisthorchis viverrini and administration of N‐nitrosodimethylamine and fed with curcumin‐supplemented diet. The effect of curcumin‐supplemented diet on histopathological changes and survival were assessed in relation to NF‐κB activation, and the expression of NF‐κB‐related gene products involved in inflammation, DNA damage, apoptosis, cell proliferation, angiogenesis and metastasis. Our results showed that dietary administration of this nutraceutical significantly reduced the incidence of CCA and increased the survival of animals. This correlated with the suppression of the activation of transcription factors including NF‐κB, AP‐1 and STAT‐3, and reduction in the expression of proinflammatory proteins such as COX‐2 and iNOS. The formation of iNOS‐dependent DNA lesions (8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine) was inhibited. Curcumin suppressed the expression of proteins related to cell survival (bcl‐2 and bcl‐xL), proliferation (cyclin D1 and c‐myc), tumor invasion (MMP‐9 and ICAM‐1) and angiogenesis (VEGF), and microvessel density. Induction of apoptotic events as indicated by caspase activation and PARP cleavage was also noted. Our results suggest that curcumin exhibits an anticarcinogenic potential via suppression of various events involved in multiple steps of carcinogenesis, which is accounted for by its ability to suppress proinflammatory pathways.

Time profiles of the expression of metalloproteinases, tissue inhibitors of metalloproteases, cytokines and collagens in hamsters infected with Opisthorchis viverrini with special reference to peribiliary fibrosis and liver injury.

The liver fluke Opisthorchis viverrini is endemic in southeastern Asia, and causes cholangiocarcinoma and liver fibrosis. We investigated the time profile of the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in relation to peribiliary fibrosis in O. viverrini-infected hamsters. Hepatic mRNA expression of MMPs, TIMPs, cytokines and collagens I and III was assessed by quantitative reverse transcription-PCR. Zymography and immunohistochemistry were also used to examine MMPs-2 and -9 expression. After infection, an increase of peribiliary fibrosis was time-dependent. Opisthorhis viverrini-induced gene expression in hamster liver, with increased mRNA expression levels of IL-1beta, TNF-alpha, TGF-beta, and collagens I and III, was observed at 21 days p.i. Expression of MMPs-2, -13 and -14 and TIMPs-1 and -3 genes, was significantly higher at 1 month, and maximal levels of most MMPs (MMPs-2, -9, -13 and -14) were observed at 2 months p.i. The cytoplasmic levels of MMP-2 and MMP-9 were similar to mRNA expression. Immunohistochemistry revealed that MMP-9 was expressed mainly in the cytoplasm of inflammatory cells at the invasive front of the fibrous area. In contrast, the highest levels of mRNA expression of TIMPs-2 and -3, and TGF-beta were observed 10 months p.i. Concentration of TIMP-2 protein in the plasma correlated with its transcriptional level (r=0.320, P=0.040). Peribiliary fibrosis correlated positively with liver hydroxyproline content (r=0.846, P<0.001), plasma hydroxyproline concentration (r=0.770, P<0.001), plasma TIMP-2 level (r=0.335, P=0.046), and mRNA expression levels of MMP-7 (r=0.511, P=0.006), TIMP-1 (r=0.320, P=0.040), TIMP-2 (r=0.428, P=0.026), and TIMP-3 (r=0.553, P=0.003). This study suggests that expression of MMPs is associated with an inflammatory reaction in the early phase and TIMPs expression at the late phase may contribute to both fibrosis and liver injury. MMPs and TIMPs may serve as diagnostic markers for the severity of O. viverrini-induced liver injury.

Involvement of MMP-9 in peribiliary fibrosis and cholangiocarcinogenesis via Rac1-dependent DNA damage in a hamster model

Peribiliary fibrosis caused by chronic infection with Opisthorchis viverrini (OV) is a risk factor of cholangiocarcinoma (CCA) in northeastern Thailand. Matrix metalloproteinases (MMPs) are enzymes capable of degrading and remodeling the extracellular matrix in the process of fibrosis and carcinogenesis. We examined MMPs expression and their role in fibrogenesis and cholangiocarcinogenesis in hamsters treated with OV and N‐nitrosodimethylamine (NDMA). We assessed the time profiles of MMPs, inducible nitric oxide synthase (iNOS), Rac1, α‐smooth muscle actin (α‐SMA) and DNA lesions (8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine, 8‐oxodG) in relation to fibrosis and CCA development. Histopathology revealed OV and NDMA synergistically induced peribiliary fibrosis time‐dependently, and CCA occurred at 3 months, whereas OV or NDMA alone induced less fibrosis. Hydroxyproline levels in the liver and plasma were positively associated with the expression of collagen I and α‐SMA. MMP‐9 expression was significantly increased and correlated with the accumulation of myofibroblast, fibrosis levels and cholangiocarcinogenesis. MMP‐9 activity was correlated with iNOS, and immunocolocalization was observed in inflammed tissues, early and invasive CCA. OV and NDMA synergistically induced MMP‐9 expression in association to Rac1. In addition, Rac1 was colocalized with iNOS, and 8‐nitroguanine, in inflammed tissues and CCA. Formation of 8‐nitroguanine and 8‐oxodG increased with tumor progression. The results suggest that MMP‐9 expression is associated with the accumulation of peribiliary fibrosis in conjunction to the induction of iNOS and Rac1 that may potentiate DNA damage and cholangiocarcinogenesis.