A Novel Serum Glycobiomarker for Diagnosis and Prognosis of Cholangiocarcinoma Detected by Butea monosperma Agglutinin.

Plant lectins are widely used in medical glycosciences and glycotechnology. Many lectin-based techniques have been applied for the detection of disease-associated glycans and glycoconjugates. In this study, Butea monosperma agglutinin (BMA), a lectin purified from seeds of the medicinal plant Butea monosperma, was used for the detection of cholangiocarcinoma (CCA)-associated glycans. Expression of BMA-binding N-acetyl galactosamine/galactose (GalNAc/Gal)-associated glycan (BMAG) in CCA tissues was determined using BMA lectin histochemistry; the results showed that BMAG was undetectable in normal bile ducts and drastically increased in preneoplastic bile ducts and CCA. The study in hamsters showed that an increase of BMAG was associated with carcinogenesis of CCA. Using an in-house double BMA sandwich enzyme-linked lectin assay, BMAG was highly detected in the sera of CCA patients. The level of serum BMAG in CCA patients (N = 83) was significantly higher than non-CCA controls (N = 287) and it was applicable for diagnosis of CCA with 55.4% sensitivity, 81.9% specificity, and 76.0% accuracy. A high level of serum BMAG (≥82.5 AU/mL) was associated with unfavorable survival of CCA patients; this information suggested the potential of serum BMAG as a poor prognostic indicator of CCA. In summary, BMAG was aberrantly expressed in preneoplastic bile ducts and CCA, it was also highly detected in patient serum which potentially used as a marker for diagnosis and prognostic prediction of CCA.

Discovering proteins for chemoprevention and chemotherapy by curcumin in liver fluke infection-induced bile duct cancer.

Modulation or prevention of protein changes during the cholangiocarcinoma (CCA) process induced by Opisthorchis viverrini (Ov) infection may become a key strategy for prevention and treatment of CCA. Monitoring of such changes could lead to discovery of protein targets for CCA treatment. Curcumin exerts anti-inflammatory and anti-CCA activities partly through its protein-modulatory ability. To support the potential use of curcumin and to discover novel target molecules for CCA treatment, we used a quantitative proteomic approach to investigate the effects of curcumin on protein changes in an Ov-induced CCA-harboring hamster model. Isobaric labelling and tandem mass spectrometry were used to compare the protein expression profiles of liver tissues from CCA hamsters with or without curcumin dietary supplementation. Among the dysregulated proteins, five were upregulated in liver tissues of CCA hamsters but markedly downregulated in the CCA hamsters supplemented with curcumin: S100A6, lumican, plastin-2, 14-3-3 zeta/delta and vimentin. Western blot and immunohistochemical analyses also showed similar expression patterns of these proteins in liver tissues of hamsters in the CCA and CCA + curcumin groups. Proteins such as clusterin and S100A10, involved in the NF-κB signaling pathway, an important signaling cascade involved in CCA genesis, were also upregulated in CCA hamsters and were then suppressed by curcumin treatment. Taken together, our results demonstrate the important changes in the proteome during the genesis of O. viverrini-induced CCA and provide an insight into the possible protein targets for prevention and treatment of this cancer.

Establishment of an allo-transplantable hamster cholangiocarcinoma cell line and its application for in vivo screening of anti-cancer drugs.

Opisthorchis viverrini (O. viverrini) is a well-known causative agent of cholangiocarcinoma (CCA) in humans. CCA is very resistant to chemotherapy and is frequently fatal. To understand the pathogenesis of CCA in humans, a rodent model was developed. However, the development of CCA in rodents is time-consuming and the xenograft-transplantation model of human CCA in immunodeficient mice is costly. Therefore, the establishment of an in vivo screening model for O. viverrini-associated CCA treatment was of interest. We developed a hamster CCA cell line, Ham-1, derived from the CCA tissue of O. viverrini-infected and N-nitrosodimethylamine-treated Syrian golden hamsters. Ham-1 has been maintained in Dulbecco's Modified Essential Medium supplemented with 10% fetal bovine serum for more than 30 subcultures. These cells are mostly diploid (2n=44) with some being polyploid. Tumorigenic properties of Ham-1 were demonstrated by allograft transplantation in hamsters. The transplanted tissues were highly proliferative and exhibited a glandular-like structure retaining a bile duct marker, cytokeratin 19. The usefulness of this for in vivo model was demonstrated by berberine treatment, a traditional medicine that is active against various cancers. Growth inhibitory effects of berberine, mainly by an induction of G1 cell cycle arrest, were observed in vitro and in vivo. In summary, we developed the allo-transplantable hamster CCA cell line, which can be used for chemotherapeutic drug testing in vitro and in vivo.

Serial analysis of gene expression reveals promising therapeutic targets for liver fluke-associated cholangiocarcinoma.

Cholangiocarcinoma (CCA) continues to be a serious health problem and is the most common fatal cancer in northeastern Thailand. Comprehensive gene expression analysis was here used to identify possible therapeutic targets for CCA treatment. We assessed liver fluke-associated CCA tissue using serial analysis of gene expression (SAGE) and compared the data to normal liver tissue as a part of the Cancer Genome Anatomy Project (CGAP). The analysis identified 509 differentially expressed genes. Of 142 up-regulated examples, we selected candidates including TMSB10, GAL3, VDR, CYPA and CD147 for further validation in CCA tissues by immunohistochemistry. VDR, CYPA and CD147 were confirmed to be consistently overexpressed in the samples tested. The therapeutic and diagnostic potential of these genes warrants further investigation.

22-oxa-1,25-dihydroxyvitamin D3 efficiently inhibits tumor growth in inoculated mice and primary histoculture of cholangiocarcinoma.

BACKGROUND: It is well known that 1α,25-Dihydroxyvitamin D3 (1,25[OH]2 D3) restrains cell proliferation and induces differentiation and apoptosis in normal and tumor cells. The authors of this report recently demonstrated that 1,25(OH)2 D3 effectively inhibits the proliferation of cholangiocarcinoma (CCA) cell lines. The antitumor activity and the underlying mechanism of 22-oxa-D3, an analog of vitamin D, in mice and in tissue cultures from patients with CCA were further explored in the current study. METHODS: Cell growth and cell cycle distribution were examined in CCA cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Mice were injected subcutaneously with 4×10(6) CCA cells at both flank sides and intraperitoneal injections with phosphate-buffered saline or 22-oxa-D3 (15 µg/kg/day) for 17 days thereafter. Tumors were removed the next day. The expression levels of cyclin D1 and the cyclin-dependent kinase inhibitor p21 were determined by Western blot analysis and immunohistochemistry. Growth inhibition of 22-oxa-D3 in fresh tissue samples from patients with CCA was analyzed by using a histodrug response assay. RESULTS: 22-oxa-D3 effectively suppressed the growth of CCA cell lines in a time-dependent and dose-dependent manner. 22-oxa-D3 arrested CCA cells at G1 phase to S phase by the suppression of cyclin D1 expression and the up-regulation of p21. Supplementation of 22-oxa-D3 to CCA-inoculated mice significantly inhibited tumor growth without hypercalcemia or serious side effects. The treatment also induced cellular apoptosis in tissue samples from patients with CCA. CONCLUSIONS: 22-oxa-D3 effectively suppressed tumor growth in CCA-inoculated mice and induced cellular apoptosis in tissue samples from patients with CCA. The current data encourage further investigation of 1,25(OH)2 D3 or its analogues as therapeutic agents in the treatment of patients with CCA.