Introduction

Parasites are infamous infectious sources for humans and other mammals. In particular, the infection of helminths caused by Opisthorchis viverrini and Clonorchis sinensis remains a major public health problem in the Southeast and East Asia. This kind of infection leads to chronic inflammation in the host’s biliary tract or even more severe problems (Silakit et al. 2015). Epidemiological and experimental evidence strongly indicate that C. sinensis and O. viverrini are the etiological agents of cholangiocarcinoma (CCA) (Yothaisong et al. 2015). CCA, the second largest contributor to primary liver cancer, is a devastating cancer arising from bile duct epithelial cells; CCA has been characterized as having very poor prognosis and poor response to current therapies (Mihalache et al. 2010; Sithithaworn et al. 2014). In order to gain some insight into these two worms and explore the cause of liver fluke related cancer, we review basic information related to C. sinensis and O. viverrini, and further discuss genome, and transcriptome research and specific carcinogenic mechanisms.

Characteristics of O. viverrini and C. sinensis

In 1915, the first O. viverrini infection case was reported in Thailand (Leiper 1915). Later, opisthorchias became endemic Southeast Asia countries, including Laos, Cambodia, Thailand, Vietnam, and with some reported cases in Malaysia, Singapore, and the Philippines (Sripa et al. 2010). Over 10 million people in Southeast Asian countries were infected (Sripa et al. 2010) with about 6 million Thais infected (Jongsuksuntigul and Imsomboon 2003). As for C. sinensis, it was first found in the bile duct of a young Chinese man through autopsy at the Medical College Hospital in Calcutta, India in 1874, more than 140 years ago (McConnell 1875; Qian et al. 2016). It infects more than 35 million individuals in a much wider area than O. viverrini, including Korea, China, Taiwan, Vietnam, and Russia, and almost half of them are Chinese (Keiser and Utzinger 2009). The major endemic regions of China are Guangdong, Guangxi, and Heilongjiang (Wu et al. 2012). In 1994, the International Agency for Research on Cancer (IARC) classified O. viverrini as a group 1 agent inducing cholangiocarcinoma in endemic regions while C. sinensis was classified as a group 2A agent as a probable carcinogen (1994). After collecting sufficient evidence of C. sinensis’ involvement in the etiology of CCA, C. sinensis was reclassified as a definite carcinogen in group 1 in 2009 (Bouvard et al. 2009; Qian et al. 2016).

As food-borne trematodes, O. viverrini and C. sinensis have similar life cycles. People that have a habit of eating uncooked fresh water fish are prone to infection. The definitive hosts, humans and other piscivorous mammals, pass parasite eggs in their feces into natural water reservoirs, where intermediate host snails, such as the prosobranch gastropod genus Bithynia, ingest these eggs, as the first intermediate host (Brandt 1974). Within the snail, the eggs metamorphose into sporocysts and cercariae are subsequently released into the water. Then cercariae live in their next intermediate host. Many species of freshwater fish, mostly cyprinoids, serve as the second intermediate host (Komiya 1966). In the fish muscle, cercariae encyst as metacercariae, which can infect humans who eat raw or undercooked infected fish. During digestion, the metacercariae excyst in the duodenum and migrate though the ampulla of Vater into the bile duct. Then, they mature into adult worms. The adult flukes inhabit the biliary tract, generally localizing within the intrahepatic bile ducts. As reported, adult C. sinensis and O. viverrini flukes can live up to 25 years in a human host (Kaewpitoon et al. 2008).

Genome and transcriptome of O. viverrini and C. sinensis

In recent years, related research has paid more attention to the genetic field, i.e., finding genes of certain traits to explore and identify their corresponding products, and proteins, and mining the carcinogenic mechanisms of CCA caused by infection of liver flukes. Via effective strategies and methods such as genomic and proteomic technology, the nucleic constitution and protein pattern become gradually clear. In this review, the whole genome of O. viverrini and C. sinensis will be laid out, followed by specific introduction and explanation of some carcinogenic-related proteins of these two species.

Generally, both genomes of these two kinds of liver flukes share many things in common while some discrepancies have been noticed as well. O. viverrini contains 620.45 Mb, outsizing C. sinensis (536.8Mbp). The guanine(G)-cytosine(C) pairs account for 43.8 % in all base pairs in the former and 0.3 % less than the GC% in the latter. There are 16,356 genes in a cell of O. viverrini with products, namely proteins, of the same number. In contrast, there are 13,634 genes and corresponding proteins in C. sinensis. When focusing on their circular mitochondrial DNA (mtDNA), the sizes are 13,510 and 13,875 bp in O. viverrini and C. sinensis, respectively. The former one contains 2 genes for rRNA, 12 genes for protein production, and 22 for tRNA transcription, which is very similar to C. sinensis, except that 2 more mitochondrial genes are needed in C. sinensis for tRNA (Cai et al. 2012) (http://www.ncbi.nlm.nih.gov/genome/?term=Opisthorchis+viverrini), (http://www.ncbi.nlm.nih.gov/genome/?term=clonorchis+sinensis).

In terms of gene structure, the genome of O. viverrini is strikingly different from that of C. sinensis (Huang et al. 2013). Just 22 % of the scaffold of O. viverrini is in line with 26 % of that of C. sinensis at the nucleotide level in regard to discrepant karyocyte (12 chromosomes for O. viverrini (2n) while 14 chromosomes are present for C. sinensis (2n)) (Kaewkong et al. 2012; Zadesenets et al. 2012).

In a few previous studies, about 50,000 distinguished sequences have been identified among O. viverrini and C. sinensis (Young et al. 2010a, b). According to comparative analysis, a majority of sequences (>85 %) derived from each species are novel (Cho et al. 2006; Laha et al. 2007; Cho et al. 2008). Proteins translated by adult O. viverrini and C. sinensis in metabolic, genetic and environmental data/information and cellular processing pathways are stable and conserved among eukaryotes while others (up to 90 %) cannot fall into any pathways (Young et al. 2010c). This blank space forms great potential for researchers to detect completely new biological mechanisms, pathways, and processes. Moreover, compared with nematodes and other trematodes, these two kinds of adult liver flukes can secrete more proteins, which are also more homologous to human beings. This fact indirectly testifies that carcinogenic liver flukes are capable of regulating host (human) response at biochemical and molecular levels (Young et al. 2010a, c).

The present pattern for liver fluke-associated CCA suggests ES products are a key factor in induction of cell malignant proliferation (Thuwajit et al. 2004; Smout et al. 2009). To date, there are several kinds of ES products that trigger development of CCA. In addition to Ov-GRN-1 specific for O. viverrini (Smout et al. 2009; Young et al. 2010a), growth factor receptor binding protein 2 (GRB2), Ras-related C3 botulinum toxin substrate 1 RAC1, v-Ha-ras Harvey rat sarcoma viral oncogene GTPase HRas, V-RAF murine sarcoma viral oncogene homologue B1 (B-RAF), and cyclin-dependent kinase regulatory subunit, CKS1 (Jordan et al. 1999; Giubellino et al. 2008; Lan et al. 2008; Oikonomou et al. 2009; Suda et al. 2010) (these are shared by both of O. viverrini and C. sinensis), are numerous novel molecules that have been identified recently.

Mechanisms of carcinogenesis

Researchers conjecture that there are three main mechanisms, along with several minor counterparts, by which liver flukes generate tumor microenvironments resulting in CCA: (i) mechanical damage caused by the activities of the parasites; (ii) effects of parasite excretory/secretory (ES) molecules; (iii) immunopathology due to infection-related inflammation (Sripa et al. 2012a). However, the detailed mechanisms of C. sinensis are less clear than those of O. viverrini.

Mechanical damage

Mechanical injuries from the activities of feeding and migrating flukes contribute to bile duct damage in the human host. The suckers of O. viverrini hook onto the bile duct epithelium, causing tissue damage early in infection, which can be seen in experimental hamster infections and human cases with bile duct obstruction (Bhamarapravati et al. 1978; Lee et al. 1993). When the fluke matures, the lesion becomes more pronounced and ulcerates. Fluke eggs become encircled in the periductal tissue because of the ulcer and induce granulomatous inflammation (Sripa 2003). C. sinensis is bigger than the bile duct lumen, which causes partial obstruction of the duct and bile stasis, increased biliary pressure, and enhanced egg entrapment (Rim 1986). In contrast, Opisthorchis is unlikely to cause obstruction because of its small body form.

Metabolic products

The proteins and other mediators are excreted and secreted from the tegument, oral openings, or guts of O. viverrini and C. sinensis into the bile or culture medium in vitro, which can induce chronic irritation and prolonged inflammation (Wongratanacheewin et al. 1988, 2003; Chaiyadet et al. 2016). This can also induce collagen turnover and remodel the extracellular matrix by regulating matrix metalloproteinases (MMPs) and their inhibitors like the tissue inhibitors of MMPs (TIMPs) to advance fibrosis (Prakobwong et al. 2009). They can also mediate infection-related pathogenesis, promote cell proliferation and create a tumorigenic environment (Thuwajit et al. 2006; Prakobwong et al. 2009; Mulvenna et al. 2010), as shown in an experiment where proliferation of NIH-3T3, a kind of murine fibroblast, increased more than fourfold despite there being no direct contact between the cell line and the parasite secreting excretory/secretory products (ESP) (Thuwajit et al. 2004). It has been demonstrated that ESP is responsible for the currency of cholangiocarcinoma. Furthermore, in some cases, homologues in the human host induce over-expression of some mRNAs encoding growth-promoting and have been associated with cancers, including proteases, protease inhibitors, orthologues of mammalian growth factors, and anti-apoptotic proteins (Mulvenna et al. 2010; Sripa et al. 2012a). After screening 300 ESP proteome (Mulvenna et al. 2010), Smout found that Ov-GRN-1, the fluke-derived gene encoding the granulin-like growth factor, is an essential growth factor for adult O. viverrini to survive in vitro (Smout et al. 2009; Papatpremsiri et al. 2015). Ov-GRN-1 is the homologue of human granulin, whose precursor could stimulate angiogenesis, suppress apoptosis, and promote tumor invasion and anchorage independence. The above experiments indicate that Ov-GRN-1 could drive the hyper-proliferation of cultured human cholangiocarcinoma cell lines KKU-100 and mouse cells NIH-3T3 in vitro. Under suppression of the expression of Ov-GRN-1, the hyper-proliferation of the co-culture human cholangiocyte (H-69) or human cholangiocarcinoma (KKU-M214) cell lines are also retarded (Sawanyawisuth et al. 2015).

The ESP of C. sinensis also plays an important role in promoting cell proliferation and carcinogenesis. A vitro study proved that the ESP of C. sinensis can induce transcription factor E2F1 expression to stimulate distinctive proliferation of the human embryonic kidney epithelial cell line (HEK293) (Kim et al. 2008). The transcription factor COX-2 could also be induced to promote the proliferation and S to G2/M transition of cholangiocarcinoma cells HuCCT1 in vitro and counteract the anticancer effects of parthenolide. In addition to the transcription factor, many proteins involved in the signal transduction, immunity, cell proliferation, and apoptosis can also be regulated by the ESP of C. sinensis (Pak et al. 2009). For example, adhesion proteins help maintain cell aggregates and MMPs promote three-dimensional invasion into ECM. The induced expression of microRNA may play other important roles of ESP in cholangiocarcinogenesis such as DNA methylation, migration, invasion, inflammation, and downregulation of tumor suppressor genes, like miR-24, miR-31, and miR-181 (Pak et al. 2014).

In addition, scientist have focused on several other proteins in the past 2 years. 14–3–3 eta is a kind of protein family that appears in both O. viverrini and C. sinensis. This protein is similar, to a high extent, to orthologs in mice, rats, and bovines and mediate signal transduction through binding to phosphoserine-containing proteins as a highly conserved family of proteins. Researchers have recognized seven isoforms (beta, gamma, epsilon, eta, sigma, theta, and zeta) of 14–3–3 in mammalian cells (Kilani et al. 2008; Haonon et al. 2016). 14–3–3 eta is engaged in carcinogenesis enhanced expression of which can depress the function of the transcription factor of Myc-interacting zinc finger protein 1 (Miz1). Miz1 is required for DNA damage-induced cell-cycle arrest. The whole inhibiting process is a key event linking chronic OV infection to CCA genesis (Wanzel et al. 2005; Pinlaor et al. 2006; Prakobwong et al. 2010).

Ov-DM9-1 is an unprecedented 16.5-kDa tegumental protein of O. viverrini (OvDM9-1) that can also be found in ESP. It belongs to a family of proteins consisting of the novel DM9 repeat (Labbunruang et al. 2016). The protein relocalizes intracellularly during invasion towards the cell membrane in mid gut epithelium and to vesicular structures in salivary gland cells (Chertemps et al. 2010). Several data sources indicate that DM9 proteins of trematodes are capable of oligomerization and their predicted β-rich secondary structure supports the fact that they are derived from the same ancestor as nematode MFP2 (major sperm protein filament protein 2) which can interact and promote polymerization of MSP (major sperm protein), which forms a unique cytoskeleton with sperm motility function in nematodes (Buttery et al. 2003; Grant et al. 2005; Labbunruang et al. 2016).

Thioredoxin (Trx) is an inflammation-inducible oxidoreductase. Produced by Ov-Trx-1 of O. viverrini, this 12-kDa protein takes part in many metabolic processes. On the one hand, it can protect normal cells and tissue from oxidative stress. Additionally, it enhances cell and tissue growth as a cytokine and growth factor extracellularly. On the other hand, encoded by proto-oncogene, it can be carcinogenic once it surpasses its normal level in and out of cells (Nakamura et al. 1997; Carvalho et al. 2006; Powis and Kirkpatrick 2007; Holmgren and Lu 2010). Ov-Trx-1 is identified in all developmental stages of the parasite especially in adult worms. And Ov-Trx-1 is prevalently expressed in tissues and organs of adult flukes (Matchimakul et al. 2015). Apoptosis signal-regulating kinase-1 (ASK-1) can be bound by thioredoxin with its anti-apoptotic activities (Zhang et al. 2004). Some researchers have extrapolated that Ov-Trx-1 antagonizes human-ASK-1 via the MAPK cascade, MAP3K-ASK-1/MAP2K-3,4,6,7/MAPK-JNK, P38K, and, thus, may accelerate infection-induced carcinogenesis in the biliary tract (Matchimakul et al. 2015).

Fructose-1,6-bisphosphate aldolase (FBPA) is a key enzyme in the glycolytic pathway of O. viverrini. Sequence analysis demonstrates that the amino acid sequence of Ov-FBPA-1 is almost identical (98 % similarity) to FBPA isoform 1 of C. sinensis which was classified as class-I FBPA based on highly conserved active residues (Prompipak et al. 2016). So far, the main function of this enzyme is to catalyze the reversible conversion of fructose-1,6-bisphosphate (FBP) to glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) (Rose et al. 1965). A highly conserved amino acid sequence takes part in actin binding. Many parasite FBPAs, however, have diverse roles including host invasion, not just glycolytic metabolism (Ramajo-Hernandez et al. 2007; Starnes et al. 2009). The function of the actin binding site found in liver fluke FBPA proteins is still evasive (Prompipak et al. 2016).

Immunopathology

The inflammation, usually chronic inflammation, induced by liver flukes mediates the hepatobiliary or hepatic abnormality. As a precursor of CCA (Riganti et al. 1989), many data imply that advanced periductal fibrosis is associated with the cytokine response to ES products, particularly the parasite-specific interleukin-6 (IL-6). A case–control study of O. viverrini infection showed the average level of IL-6 among participants with advanced periductal fibrosis was higher than that of O. viverrini-infected individuals without advanced periductal fibrosis. The higher the IL-6, the greater the risk of advanced periductal fibrosis. Or rather, a dose-dependent relationship exists between the plasma levels of IL-6 and periductal fibrosis or CCA from chronic Opisthorchis infection (Sripa et al. 2009; Fried et al. 2011; Sripa et al. 2012b). Another in vitro study incubated normal human cholangiocytes (H69) and human cholangiocarcinoma cells (KKU-100, KKU-M156) with ES products. They found that the IL-6 secretion by H69 was promoted and the secretion was significantly reduced by endocytosis inhibitors (Chaiyadet et al. 2015). Therefore, IL-6 is very likely to be a pro-inflammatory cytokine associated with advanced periductal fibrosis in O. viverrini-infected patients driven by ES products. In patients infected with C. sinensis, scholars have also found that the serum level of IL-6 is higher in the process of liver damage induced by clonorchiasis along with higher intercellular adhesion molecule-1 (ICAM-1), lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) in serum. And patients with more serious liver function injury have a higher level of sICAM-1, LPS, IL-6, and TNF-alpha according to the Child-Pugh classification (Cai et al. 2013). Thus, cytokines secreted during infection have an impact on liver function, usually a bad impact, during chronic inflammation.

Toll-like receptors are cell membrane receptors that actively participate in the immune response. In order to address the details of host immune/inflammatory responses, a normal human cholangiocytes (H69) was stimulated with ES products from O. viverrini, demonstrating the activation of TLRs and their downstream signaling pathway. Upregulated expression of TLR4 mRNA by ES products induces IkB-degradation in a MyD88-dependent manner and nuclear factor-kappa B (NF-kB) nuclear translocation. Once the NF-kB is triggered, the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are stimulated (Ninlawan et al. 2010; Sripa et al. 2012a). However, in a RAW 264.7 macrophage cell line treated with antigen from O. viverrini, the expression of Toll-like receptor-2 (TLR2), not TLR4, was upregulated and triggered NF-kB signaling. And TLR2, NF-kB, iNOS, and COX-2 reacted in a dose-dependent fashion (Pinlaor et al. 2005; Fried et al. 2011). As to whether the TLRs react in the same way in clonorchiasis, there is little evidence because only upregulated expression of TLR2 and TLR4 has been proven in a mouse model infected by C. sinensis (Yan et al. 2015). Whereas, ES products, such as C. sinensis ferritin heavy chain (CsFHC), were found to trigger free radical-mediated NF-kB signaling in chronic inflammation caused by C. sinensis infection (Nam et al. 2012; Mao et al. 2015).

Although iNOS inextricably kills O. viverrini through immune response, its excessive formation can lead to disaster. Endogenous NO, an indispensable particle derived from iNOS, can form 8-oxo-7, 8-hydro-2′-deoxiguanine (8-oxodG) and 8-nitroguanine, which lead to oxidative and nitrative DNA damage (Pinlaor et al. 2003; Yongvanit et al. 2012a). Research shows that 8-nitroguanine and 8-OxodG can give rise to apurinic sites in DNA, resulting in G:C to T:A transversions (Loeb and Preston 1986; Yermilov et al. 1995; Bruner et al. 2000; Pinlaor et al. 2003). And the accumulation of DNA base-pair mutation eventually leads to malignant tumors. Meanwhile, many studies have stated that oxidative stress-mediated carcinogenesis also occurs in clonorchiasis. In C. sinensis-infected mouse liver tissues, lipid peroxidation (LPO) products like 4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA) and 8-oxodG accumulate, with higher expression of LPO-related proteins COX-2 and 5-lipoxygenase (5-LOX) and pro-inflammatory cytokines. Moreover, the expression levels of LPO-related proteins COX-2 and 5-lipoxygenase (5-LOX) in human CCA cells (HuCCT1) treated with C. sinensis ESPs have also been found to be elevated (Maeng et al. 2016). Endogenous LPO products can modify DNA bases, forming various exocyclic DNA adducts such as malondialdehyde deoxyguanosine (M1dG) when DNA bases react with MDA and etheno-propano-DNA adducts when DNA bases react with HNE (Nair et al. 2007; Maeng et al. 2016).

Other possible pathways

In addition to the three major mechanisms mentioned above, several potential pathways of O. viverrini ought to be identified as well, including microbes dysbiosis and the influence of drug-processing enzymes in the liver.

Microbes dysbiosis

Gastrointestinal (GI)—tract microbes are necessary for the development of a healthy immune response. Novel findings show that infection with O. viverrini leads to changes in the microbial communities of the digestive tract, including the presence of microbes in the biliary system (O’Hara et al. 2013). Therefore, researchers postulate microbes and liver flukes together result in inflammatory reactions and the causative microbes in the biliary system come from the external environment. Microbial dysbiosis represents another dimension of liver fluke-induced CCA if this phenomenon reported in hamsters infected with O. viverrini also occurs in chronically infected people (Plieskatt et al. 2013). Another study suggested that O. viverrini is a reservoir for species of Helicobacter and the association between them may be an obligatory mutualism. Helicobacter pylori and other Helicobacter-like bacteria were found in worm guts. The prevalence of H. pylori infection in O. viverrini-infected hamsters was higher than that of O. viverrini-uninfected hamsters. However, when O. viverrini-infected hamsters were treated with antibiotics and praziquantel to remove the flukes, the frequency of H. pylori was lower (Deenonpoe et al. 2015). The latest article revealed Helicobacter species, including H. pylori, Helicobacter bilis, and Helicobacter hepaticus, as a possible risk of CCA, especially since former two are very common (Kaewpitoon et al. 2016). Thus, we believe that O. viverrini brings Helicobacter into the biliary duct, disturbing microbial homeostasis, and then creates a cancer-causing environment.

Influence of drug-processing enzymes in the liver

Some research has suggested that in male hamsters infected by O. viverrini, activities of the hepatic cytochrome P450 (CYP) isoenzymes (particularly CYP2E1 and CYP2A6) have been shown to be higher than those of controls, and the highest levels of these two enzymes occur in hepatocytes immediately adjacent to the area of inflammation (Kirby et al. 1994). The liver enzyme is induced by opisthorchiasis and will subside under effective treatment by antihelmintic such as praziquantel (Satarug et al. 1996). Some products of endogenous nitrosation like N-nitrosodimethylamine (NDMA) can be converted by CYP2E1 and CYP2A6 into toxic substance, methylating DNA, which does most harm in proliferating epithelial cells in the bile duct (Watanapa and Watanapa 2002). Also, steroid hormones originate from O. viverrini (estrone/estradiol). These hormones can be transformed, via P450, to 2-hydroxyestrogen and 4-hydroxyestrogen, which are further oxidized to 2,3-catechol estrogen quinone or 3,4-catechol estrogen quinone, respectively. The latter two substances can directly or indirectly react with DNA, which may result in a carcinogen (Correia Da Costa et al. 2014). Other research, however, found that the increment of CYP2A6 diminished, rather than increased, when CYP2E1 was involved in the progression of O. viverrini-related CCA. Nevertheless, the mechanisms by which inflammation in hepatic tissues surrounding CCA suppress CYP2E1 activity remain unknown (Yongvanit et al. 2012b). Also, the roles of the CYP39A1 enzyme serve as a prognostic determination of CCA in that this enzyme can metabolize 24-hydroxycholesterol (24-OH), which plays important roles in the inflammatory response and oxidative stress in the CCA cell line (Khenjanta et al. 2014).

Perspectives

“Omic” studies

Although various kinds of drugs are being used in parasitological infections, drug resistance and re-infection may occur. Consequently, basic molecular parasitological research must development new methods of diagnosis, treatment, and control. The advancement of genomics, transcriptomics, and proteomics will expedite many research fields. Through screening of particular genes and proteins, we can harness RNA interference technology (RNAi) to inhibit transcription and translation of certain genes, which can prevent the occurrence of malignancies (Sripa et al. 2011). In addition, “omics” can evaluate whether certain subspecies of O. viverrini and C. sinensis are carcinogenic or not. This can facilitate the classification of their subpopulation. Thus, researchers can effectively target those whose pathogens can lead to CCA and this will be helpful for disease control and prophylaxis (Sithithaworn et al. 2012). Moreover, on the basis of genomics, population genetic studies of O. viverrini and C. sinensis are available. Via this process, scientists can further sequence and analyze genomic DNAs from huge quantities of individual flukes without high cost. This will pave the way for comprehensively uncovering genetic relationships both within and among distinct populations of O. viverrini and C. sinensis (Gasser et al. 2016). Moreover, once we localize and identify the ribonucleic acid sequences and their corresponding proteins, it will be expedient for researchers to mine the natural relationship and interaction between the molecules from the worm and cell and tissue in the human body. Subsequently, new drugs and vaccines will be used to fight CCA (Laha et al. 2007). Nevertheless, it is necessary for researchers to explore other specific and sensitive molecular markers. Such research will contribute to population screening and disease diagnosis (Silsirivanit et al. 2014). What is more, because the majority of proteins work in unknown metabolic pathways, the functions of certain genes and proteins ought to be discovered.

Mechanisms of carcinogenesis

In this review, we tend to divide all mechanisms into two parts: traditional ones, which include mechanical damage, metabolic products, and immunopathology, and novel ones (other pathways). Though many possible mechanisms concerning O. viverrini-related CCA have been proposed, we need to further explore other roles of O. viverrini in the carcinogenesis of CCA. Compared to O. viverrini, the mechanisms of C. sinensis are similar to those of its counterpart except that the toll-like receptor hypothesis is immature and the results of other mechanisms are insufficient. For instance, the involved proteins and cytokines are scant in contrast with O. viverrini. Furthermore, both of the worms lack other novel carcinogenic pathways, especially the C. sinensis, and the result of this review should be further investigated.

In addition to serving scientific research, this review also aims to remind and caution corresponding physicians that whenever they encounter patients from Southeast Asia or returnees from there, especially those who suffer from bile duct diseases, or even CCA, they should not rule out the possibility of parasitic infections. If diagnosis of O. viverrini and C. sinensis related CCA is confirmed, combination of traditional and parasitological treatment for this kind of CCA is more effective. To conclude, despite creating a more difficult task, the involvement of parasites can expand our strategy of prevention and treatment and boost the growth of a new subject, parasitological oncology, as well.