Jarinya Khoontawad

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.

Protective effect of melatonin against Opisthorchis viverrini-induced oxidative and nitrosative DNA damage and liver injury in hamsters

Abstract:  The liver fluke, Opisthorchis viverrini, is the risk factor of cholangiocarcinoma, which is a major health problem in northeastern Thailand. Production of reactive oxygen and nitrogen species during the host’s response leads to oxidative and nitrosative stress contributing to carcinogenesis. We investigated the protective effect of melatonin against O. viverrini‐induced oxidative and nitrosative stress and liver injury. Hamsters were infected with O. viverrini followed by oral administration of various doses of melatonin (5, 10, and 20 mg/kg body weight) for 30 days. Uninfected hamsters served as controls. Compared to the levels in O. viverrini‐infected hamsters without melatonin treatment, the indoleamine decreased the formation of oxidative and nitrosative DNA lesions, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and 8‐nitroguanine, in the nucleus of bile duct epithelium and inflammatory cells, in parallel with a reduction in 3‐nitrotyrosine. Melatonin also reduced the expression of heme oxygenase‐1 and cytokeratin 19, nitrate/nitrite levels, and bile duct proliferation in the liver. Alanine transaminase activity and the levels of 8‐isoprostane and vitamin E were also dose dependently decreased in the plasma of melatonin‐treated hamsters. Melatonin reduced the mRNA expression of oxidant‐generating genes [inducible nitric oxide synthase, nuclear factor‐kappa B (NF‐κB), and cyclooxygenase‐2] and proinflammatory cytokines (TNF‐α and IL‐1β), accompanied by an increase in the expression of antioxidant genes [nuclear erythroid 2‐related factor 2 (Nrf2) and manganese superoxide dismutase]. Thus, melatonin may be an effective chemopreventive agent against O. viverrini‐induced cholangiocarcinoma by reducing oxidative and nitrosative DNA damage via induction of Nrf2 and inhibition of NF‐κB‐mediated pathways.

Proteomic identification of peroxiredoxin 6 for host defence against Opisthorchis viverrini infection.

Opisthorchis viverrini infection causes opisthorchiasis and is a risk factor for cholangiocarcinoma via chronic inflammation. To investigate the mechanism of O. viverrini ‐induced liver disease, we applied a proteomic approach to examine alterations in hepatic protein levels in O. viverrini ‐infected hamsters. Two‐dimensional gel electrophoresis (2DE) revealed that O. viverrini infection induced upregulation (1·5‐ to 4·3‐fold) of 25 proteins and downregulation (1·5 to 2·5‐fold) of 24 proteins compared with uninfected animals. Expression of proteins related to stress response, DNA replication and repair, and cell structure was significantly increased, whereas that of proteins associated with normal liver function, such as metabolism, blood volume maintenance and fatty acid cycle was decreased. Among the upregulated proteins, a 2·7‐fold increase in peroxiredoxin 6 (Prdx6), an antioxidant protein, was confirmed by 2DE and immunoblot analysis, Western blot and quantitative PCR. Immunohistochemical analysis showed that Prdx6 expression was observed mainly in the cytoplasm of inflammatory cells. These results suggest that Prdx6 is important for host defence against O. viverrini infection. This study provides basic information for Prdx6 as a potential biomarker and therapeutic target for opisthorchiasis.

Proteomic analysis to identify plasma orosomucoid 2 and kinesin 18A as potential biomarkers of cholangiocarcinoma

Proteomic analysis was performed to search for the diagnostic biomarkers of the early stage of cholangiocarcinoma (CCA). For this purpose, CCA was experimentally induced in hamsters by the combination of N-nitrosodimethylamine (NDMA) treatment and Opisthorchis viverrini (OV) infection. Pooled plasma of normal control, NDMA-treated, OV-infected and OV+NDMA (ON) treated group was separated by 1-D PAGE, and the trypsin-digested bands were analyzed with LC-MS/MS. Among 82 overexpressed proteins, the study focused on 26 proteins overexpressed in ON group because CCA development was almost exclusively found in this group. A further selection was made based on the protein overexpression on day 21, the precancerous stage. Orosomucoid 2 (Orm2) was overexpressed in OV and ON groups and kinesin 18A (KIF18A) was overexpressed in the ON group. The overexpression levels were verified by real-time RT-PCR and western blotting in the liver and plasma. The transcription and translation levels of these two candidate molecules increased significantly at 21 days post-treatment before tumor development. Immunohistochemistry revealed KIF18A was expressed in the epithelial cells of newly formed small bile ducts, some inflammatory cells and hepatocytes. These results suggest that Orm2 and KIF18A could be the potential biomarkers for early diagnosis of CCA.

Proteomic Identification of Plasma Protein Tyrosine Phosphatase Alpha and Fibronectin Associated with Liver Fluke, Opisthorchis viverrini, Infection

Opisthorchiasis caused by Opisthorchis viverrini induces periductal fibrosis via host immune/inflammatory responses. Plasma protein alteration during host-parasite interaction-mediated inflammation may provide potential diagnostic and/or prognostic biomarkers. To search for target protein changes in O. viverrini-infected hamsters, a 1-D PAGE gel band was trypsin-digested and analyzed by a LC-MS/MS-based proteomics approach in the plasma profile of infected hamsters, and applied to humans. Sixty seven proteins were selected for further analysis based on at least two unique tryptic peptides with protein ID score >10 and increased expression at least two times across time points. These proteins have not been previously identified in O. viverrini-associated infection. Among those, proteins involved in structural (19%), immune response (13%), cell cycle (10%) and transcription (10%) were highly expressed. Western blots revealed an expression level of protein tyrosine phosphatase alpha (PTPα) which reached a peak at 1 month and subsequently tended to decrease. Fibronectin significantly increased at 1 month and tended to increase with time, supporting proteomic analysis. PTPα was expressed in the cytoplasm of inflammatory cells, while fibronectin was observed mainly in the cytoplasm of fibroblasts and the extracellular matrix at periductal fibrosis areas. In addition, these protein levels significantly increased in the plasma of O. viverrini-infected patients compared to healthy individuals, and significantly decreased at 2-months post-treatment, indicating their potential as disease markers. In conclusion, our results suggest that plasma PTPα and fibronectin may be associated with opisthorchiasis and the hamster model provides the basis for development of novel diagnostic markers in the future.

Cytokine/Chemokine Secretion and Proteomic Identification of Upregulated Annexin A1 from Peripheral Blood Mononuclear Cells Cocultured with the Liver Fluke Opisthorchis viverrini

ABSTRACT We investigated the cytokine/chemokine secretions and alteration of protein expression from peripheral blood mononuclear cells (PBMCs) cocultured with adult liver flukes (Opisthorchis viverrini) for 6 to 24 h. PBMC-derived proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the cytokines/chemokines in the supernatant were assessed using a cytokine array. Exposure to O. viverrini induced increases in secretion of proinflammatory cytokines, costimulating protein, adhesion molecules, and chemotactic chemokines relative to untreated controls. In contrast, secretion of the CD40 ligand, interleukin 16, and macrophage inflammatory protein 1β decreased. Proteomic analysis revealed that expression of 48 proteins was significantly altered in PBMCs stimulated with O. viverrini. Annexin A1 (ANXA1) was selected for further study, and immunoblotting showed upregulation of ANXA1 expression in PBMCs after 12 and 24 h coculture with liver flukes. In an in vivo study, transcription and translation of ANXA1 significantly increased in livers of hamsters infected with O. viverrini at 21 days and from 3 months onwards compared to normal controls. Interestingly, immunohistochemistry revealed that ANXA1 was present not only in the cytoplasm of inflammatory cells but also in the cytoplasm of cholangiocytes, which are in close contact with the parasite and its excretory/secretory products in the biliary system. Expression of ANXA1 increased with time concomitant with bile duct enlargement, bile duct formation, and epithelial cell proliferation. In conclusion, several cytokines/chemokines secreted by PBMCs and upregulation of ANXA1 in PBMCs and biliary epithelial cells might have a role in host defense against O. viverrini infection and tissue resolution of inflammation.

α-Tocopherol and lipid profiles in plasma and the expression of α-tocopherol-related molecules in the liver of Opisthorchis viverrini-infected hamsters.

Opisthorchis viverrini infection induces inflammation-mediated oxidative stress and liver injury, which may alter α-tocopherol and lipid metabolism. We investigated plasma α-tocopherol and lipid profiles in hamsters infected with O. viverrini. Levels of α-tocopherol, cholesterol, and low-density lipoprotein increased in the acute phase of infection. In the chronic phase, α-tocopherol decreased, while triglyceride and very low-density lipoprotein increased. Notably, high-density lipoprotein decreased both in the acute and chronic phases. In the liver, cholesteryl oleate, triolein, and oleic acid decreased in the acute phase, and increased in the chronic phase. Such chronological changes were negatively correlated with the plasma α-tocopherol level. The expression of α-tocopherol-related molecules, ATP-binding cassette transporter A1 (ABCA1) and α-tocopherol transfer protein, increased throughout the experiment. These results suggest that O. viverrini infection profoundly affects on lipid and α-tocopherol metabolism in due course of infection.

Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma.

Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer.

The liver fluke Opisthorchis viverrini expresses nitric oxide synthase but not gelatinases

Host-parasite interaction during infection with the liver fluke Opisthorchis viverrini plays an important role in opisthorchiasis-associated cholangiocarcinoma via nitric oxide (NO) production. Host cells induce nitric oxide synthase (NOS)-dependent DNA damage and secrete Ras-related C3 botulinum toxin substrate (Rac)1, heme oxygenase (HO)-1, and gelatinases (matrix metalloproteinase (MMP)2 and MMP9). We evaluated whether these enzymes are expressed in O. viverrini. Colocalization of NOS and Rac1 was most prominently detected on day 30 post-infection (p.i.) in the gut, reproductive organ, eggs, acetabular and tegument. Expression of HO-1, an antioxidative enzyme, increased in a similar pattern to NOS, but was not present in the tegument. The levels of nitrate/nitrite, end products of NO, and ferric reducing antioxidant capacity, an indicator of antioxidant enzyme capacity, in parasite homogenates were highest on day 30 p.i. and then decreased on day 90 p.i. In contrast, zymography revealed that MMP2 and MMP9 were not present in parasite homogenates at all time points. In conclusion, O. viverrini induces NOS expression and NO production, but does not express gelatinases. The study may provide basic information and an insight into drug design for prevention and/or intervention approaches against O. viverrini infection.