transforming-growth-factor-beta and Opisthorchiasis

Cholangiocarcinoma (CCA) is a highly aggressive tumor with a greater risk of distant metastasis. The promising anti-CCA activity and safety profile of Atractylodes lancea (AL) have previously been reported in a series of in vitro, in vivo and clinical studies. The present study investigated the effect of AL extract on apoptosis and metastasis signaling pathways in the Opisthorchis viverrini/dimethylnitrosamine (OV/DMN)-induced CCA hamster model.. Hamster liver tissues were obtained from the four groups (n = 5 per group), i.e., (i) 5-FU treated CCA (40 µg/mL); (ii) CCA; (iii) AL-treated CCA (5,000 mg/kg), and (iv) normal hamsters. Total RNA was isolated, and the expression levels of apoptosis-related and metastasis-related genes were determined by qRT-PCR analysis.. The expression levels of p16, caspase-3, caspase-8, caspase-9, Apaf-1, p53 and Eef1a1 were downregulated, while that of the remaining genes were upregulated in CCA hamsters compared with normal hamsters. AL treatment increased the expression of p16, caspase-9, caspase-3, Apaf-1, p53 and E-cadherin and decreased the expression of cyclin D1, cdk4, Bax, Akt/PKB, Bcl-2, Mfge-8, Lass4, S100A6, TGF-β, Smad-2, Smad-3, Smad-4, MMP-9, and N-cadherin. The expression of Eef1a1 was unchanged.. The anti-CCA activity of AL in OV/DMN-induced CCA hamsters could be due to the induction of cell cycle arrest at the G1 phase and activation of the apoptosis pathway, resulting in cancer cell death. The activation of the apoptosis pathway mainly involved the intrinsic pathway (activation of caspase-3 and caspase-9 through p53 and Mfge-8 modulation and downregulation of anti-apoptotic genes Akt and Bcl-2). In addition, AL could also inhibit the canonical TGF-β signaling pathway, MMP-9 and N-cadherin to suppress tumor metastasis.

Topics: Animals; Atractylodes; bcl-2-Associated X Protein; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cadherins; Caspase 3; Caspase 8; Caspase 9; Cholangiocarcinoma; Cricetinae; Cyclin D1; Dimethylnitrosamine; Fluorouracil; Humans; Matrix Metalloproteinase 9; Mesocricetus; Opisthorchiasis; Opisthorchis; Plant Extracts; Proto-Oncogene Proteins c-akt; RNA; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4

Topics: Animals; Antigens, Helminth; B7-1 Antigen; Dendritic Cells; Gene Expression Regulation; Genes, MHC Class II; Interleukin-10; Male; Mice; Mice, Inbred C57BL; Opisthorchiasis; Opisthorchis; Transforming Growth Factor beta; Up-Regulation

Opisthorchis viverrini infection is a significant health problem in Thailand and other countries in Southeast Asia. There is little known about the mechanisms of the immune response to O. viverrini in immunoprotection. However, it has been reported that this parasite can suppress both cell and antibody mediated immune responses. The TGF-β and IL-10 are immunosuppressive cytokines that play an important role in inhibition of host immune response leading to worm survival. In this study, we immunized hamsters to protect against O. viverrini infection and the IL-4, IL-10, TGF-β and IFN-γ expression in spleen was investigated by real time PCR analysis. An O. viverrini-crude somatic antigen preparation (CSAg) administered with complete Freund's adjuvant (CFA) or with alum was used to stimulate immune responses in O. viverrini-primed hamsters. The greatest percent protection (48.4%) was seen following immunization with CSAg plus alum. The mean number±SD of worms recovered in the PBS control, CFA alone, CSAg plus CFA, alum alone and CSAg plus alum was 17.4±2.3, 17.1±3.3, 14.5±3.8, 14.5±2.3 and 9±2.7, respectively. Significant protection correlated with the reduction of TGF-β and IL-10, but not IL-4, IFN-γ expressions. Since TGF-β expression is significantly increased in the spleens of hamsters with opisthorchiasis, stimulation of this cytokine by parasite antigens was confirmed by using CSAg and primary hamster spleen cells. Antigen fractions with molecular masses of 81-92, 64-72 and 19-21.4kDa were found to significantly induce TGF-β production. Our results suggested that TGF-β induction by O. viverrini may have an important role in parasite survival.

Topics: Animals; Cricetinae; Freund's Adjuvant; Immunization; Interferon-gamma; Interleukin-10; Interleukin-4; Male; Mesocricetus; Opisthorchiasis; Opisthorchis; Parasite Egg Count; Primary Cell Culture; Real-Time Polymerase Chain Reaction; RNA, Messenger; Spleen; Transforming Growth Factor beta

Opisthorchiasis is the major public health problem in the endemic areas of Thailand and Laos because Opisthorchis viverrini infection causes serious hepatobiliary diseases including CCA. The molecular mechanism of the CCA carcinogenesis induced by the infection remains obscure. To reveal the potential genes and signaling pathways to involve in the carcinogenesis, the present study investigated the expression of c-Ski, an oncogene, and two TGF-β signaling pathway relative genes, TGF-β and Smad4, during the development of CCA induced by O. viverrini infection in hamster model, and in human opisthorchiasis associated CCA. The results showed that the expression of c-Ski gene was greatly up-regulated during the carcinogenesis of CCA in hamster model. The overexpression of c-Ski was confirmed by immunohistological staining result which showed the increased expression of c-Ski protein in cytoplasm of the epithelial lining of hepatic bile ducts. Moreover, the immunohistological staining of the specimens of human opisthorchiasis associated CCA revealed the up-regulated expression of c-Ski and Smad4 proteins in the cytoplasm of the epithelial lining of hepatic bile ducts and stomal fibrosis respectively. The expression of TGF-β and Smad4 were up-regulated, which expression kinetics was time-dependent of CCA development. These results suggest that c-Ski is likely involved in the carcinogenesis of CCA induced by O. viverrini infection through regulating TGF-β signaling pathway.

Topics: Animals; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinogens; Cell Transformation, Neoplastic; Cholangiocarcinoma; Cricetinae; Dimethylnitrosamine; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Mesocricetus; Opisthorchiasis; Opisthorchis; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; Smad4 Protein; Transforming Growth Factor beta

Chronic infection with the liver fluke, Opisthorchis viverrini, induces advanced periductal fibrosis and is a relative risk factor for cholangiocarcinoma in Southeastern Asia. We examined the reducing effect of curcumin on hepatobiliary fibrosis using O. viverrini-infected hamsters supplemented with dietary 1% curcumin (w/w) as an animal model. The expression profile of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), cytokines, and collagens was assessed in relation to liver fibrosis. Histopathological studies revealed that curcumin had no effect on fibrosis at the short-term infection (21 days and 1 month); however, peribiliary fibrosis was significantly reduced after the long-term curcumin treatment for 3 months, compared to the untreated group. Expression of alpha-smooth muscle actin was associated with the reduction of liver fibrosis. A decrease in hepatic hydroxyproline level and mRNA expression of collagen I and III supported the reduction of fibrosis. The expression of TIMP-1, TIMP-2, and tumor necrosis factor-alpha genes was also decreased after curcumin treatment. In contrast, curcumin increased mRNA expression of MMP-13, MMP-7 (at 6 months), interleukin-1 beta, and transforming growth factor beta, implying that increased MMPs activity contributes to extracellular matrix degradation. These results suggest that curcumin reduces periductal fibrosis after long-term treatment by tissue resorption via inhibition of TIMPs expression and enhancement of MMPs expression mediated by cytokines. In conclusion, curcumin may serve as a promising nutraceutical agent exerting antifibrotic effect in O. viverrini-infected patients and contribute to cholangiocarcinoma prevention.

Topics: Actins; Animals; Collagen; Cricetinae; Curcumin; Drug Administration Schedule; Gene Expression Regulation; Hydroxyproline; Inflammation; Interleukin-1beta; Liver Cirrhosis; Liver Cirrhosis, Experimental; Male; Matrix Metalloproteinases; Opisthorchiasis; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Praziquantel has been used for the treatment of diseases caused by infection with various parasites. Praziquantel treatment in Opisthorchis viverrini-infected patients reduces the hepatobiliary fibrosis due to tissue resorption, however, some fibroses are irreversible. To clarify the effect of praziquantel treatment on hepatobiliary fibrosis, we examined the expression of matrix metalloproteinases (MMPs) in relation to fibrolysis on praziquantel-treated hamsters after O. viverrini acute infection (AI) for 21 days and chronic infection (CI) for 4 months. The reduction rate of hydroxyproline content in the livers of the AI group with 6-month praziquantel treatment (71%) was higher than that of corresponding praziquantel-treated CI group (13%), similar to the decrease in the thickness of peribiliary fibrosis. Hepatic mRNA levels of collagen I significantly decreased compared with untreated control after praziquantel treatment both in AI and CI groups. Expression of collagen III significantly decreased in the AI group but unchanged in the CI group. MMP-9 and MMP-13 (except 3 months in CI group) levels significantly decreased in both groups. Notably, expression of MMP-7 level increased at 1 and 6 months in both AI and CI groups, respectively. MMP-2 level did not change in both groups. Gelatinase activity of MMPs-2 and -9 was associated with their transcriptional levels. Tissue inhibitors of MMP (TIMP)-1 and TIMP-2 significantly decreased in the AI group, whereas TIMP-1 levels did not change and TIMP-2 level was significantly increased in CI group. Praziquantel treatment increased the expression of TNF-alpha in both groups, suggesting an association with MMP-7 expression. TGF-beta expression was significantly increased only in the CI group indicating its may involve in TIMPs expression. These results suggest that in animals with chronic O. viverrini infection, praziquantel treatment induces the expression of TGF-beta, and TIMPs and resultant inhibition of MMP activity, leading to slow resorption of hepatic fibrosis.

Topics: Animals; Collagen; Cricetinae; Gene Expression Profiling; Hydroxyproline; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinases; Opisthorchiasis; Opisthorchis; Praziquantel; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The cytokine mRNA expression of IL-12, IFN-gamma, TGF-beta, IL-4, and IL-10 were investigated in spleen, liver and mesenteric lymph nodes (MLN) in hamsters experimentally infected with Opisthorchis viverrini. Animals were infected with 5, 25 or 100 metacercariae (Mc) and examined by RT-PCR and real-time PCR at 2 weeks, 2 and 6 months after infection. The cytokine expression was compared using HPRT. The IL-12 was significantly expressed at 2 weeks in the liver of the 5- and 25-Mc-infected groups. It is correlated with the inflammation intensity found in the liver at the same time. The production of IFN-gamma was not increased. The significant increase in expression of IL-10 was observed in the 6-month group in the spleen, which may suppress the Th1 and lead to a Th2 response. The IL-4 and TGF-beta expressions in MLN were significantly increased, and correlated with the dose of infection, especially in the 6-month groups. The TGF-beta level in MLN was 15-fold higher than in the uninfected control, compared to a twofold increase in spleen and liver. Because this parasite resides in the bile duct, the regulatory cytokine levels of mucosal immunity were enhanced more than those in systemic immunity. These results indicate the predominance of Th2 responses in chronic O. viverrini infection, and the high level of TGF-beta may inhibit the immune functions, which allows the parasites to evade host immune response.

Topics: Animals; Concanavalin A; Cricetinae; Cytokines; DNA Primers; Enzyme-Linked Immunosorbent Assay; Immunity, Mucosal; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-4; Liver; Lymph Nodes; Opisthorchiasis; Opisthorchis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spleen; Transforming Growth Factor beta

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product.. NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR.. Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05).. O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antigens, Helminth; Cell Proliferation; Coculture Techniques; Cytoskeletal Proteins; DNA, Complementary; Epidermal Growth Factor; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Helminth Proteins; Liver Cirrhosis; Mice; NIH 3T3 Cells; Oligonucleotide Array Sequence Analysis; Opisthorchiasis; Opisthorchis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta