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Piperlongumine (PL) produces reactive oxygen species (ROS) and induces G2/M-phase arrest in cholangiocarcinoma (CCA) cells via the JNK/ERK pathway. A differential response to PL was observed among all CCA cell lines However, the underlying mechanisms have remained to be fully elucidated. The aim of the present study was to investigate the molecular mechanisms of PL-induced heme oxygenase-1 (HO-1) expression in CCA cell lines. The anti-proliferative action of PL in the CCA cell lines KKU-100 and KKU-213A was analyzed using sulforhodamine B assays. Reverse transcription-quantitative PCR and western blot analyses were used to examine mRNA and protein expression. HO-1 inhibition was achieved using the chemical inhibitor zinc protophoryn or specific small interfering RNA to HO-1. Intracellular ROS was detected using a 2,7-dichlorodihydrofluorescein diacetate fluorescence assay. High expression of phase-II detoxification enzymes, including NADPH quinone oxidoreductase-1, heme oxygenase-1, superoxide dismutases and aldo-keto reductase 1 subunits C-1 and 3, were detected in the KKU-100 cell line. Of the CCA cell lines tested, KKU-100 was the least sensitive to PL. Dose-dependent upregulation of HO-1 expression via PI3K/Akt activation was detected in PL-treated CCA cells. Inhibition of HO-1 eliminated the antioxidant defense mechanisms, leading to increased anti-cancer activity of PL in the CCA cell lines via an increase in intracellular ROS levels and apoptotic protein expression. These observations indicated that HO-1 inhibition had a chemosensitizing effect on CCA to PL. [ABSTRACT FROM AUTHOR]

Changes in protein glycosylation have been reported in various types of cancer, including cholangiocarcinoma (CCA). Nanospray ionization-linear ion trap mass spectrometry (NSI-MSn) was used in the present study to determine the comparative structural glycomics of the N-linked glycans in the serum of patients with CCA compared with healthy controls. A total of 5 high-mannose and 4 complex N-linked glycans were detected. Mannose7-N-acetyl-glucosamine2 was the most abundant structure among the high-mannose types (control 12.12±2.54 vs. CCA 9.27±2.66%), whereas NeuAc2H2N2M3N2 predominated the complex types (control 61.17±2.55 vs. CCA 64.68±4.23%). The expression of 3 different N-glycans differed significantly between the CCA cases and controls. These included mannose6-N-acetyl-glucosamine2 (P=0.044), mannose9-N-acetyl-glucosamine2 (Ρ=0.030) and NeuAc3H3N3M3N2F (Ρ=0.002). These three glycan structures may therefore be associated with tumor progression in CCA and may be useful for its diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: Infection by Opisthorchis viverrini (liver fluke) is a major public health problem in southeastern Asia, resulting in hepatobiliary disease and cholangiocarcinoma. Fluke surface glycoconjugates are prominently presented to the host, thereby constituting a crucial immunological interface that can determine the parasite’s success in establishing infection. Therefore, N- and O-linked glycoprotein glycan profiles of the infective metacercarial stage and of the mature adult were investigated by nanospray ionisation-linear ion trap mass spectrometry (NSI-MS n ). Glycan immunogenicity was investigated by immunoblotting with serum from infected humans. Metacercariae and adult parasites exhibit similar glycan diversity, although the prevalence of individual glycans and glycan classes varies by stage. The N-glycans of the metacercaria are mostly high mannose and monofucosylated, truncated-type oligosaccharides (62.7%), with the remainder processed to complex and hybrid type glycans (37.3%). The N-linked glycan profile of the adult is also dominated by high mannose and monofucosylated, truncated-type oligosaccharides (80.0%), with a smaller contribution from complex and hybrid type glycans (20.0%). At both stages, complex and hybrid type glycans are detected as mono-, bi-, tri-, or tetra-antennary structures. In metacercariae and adults, O-linked glycans are detected as mono- to pentasaccharides. The mucin type core 1 structure, Galβ1–3GalNAc, predominates in both stages but is less prevalent in the adult than in the metacercaria. Immunogenic recognition of liver fluke glycoproteins is reduced after deglycosylation but infected human serum was unable to recognise glycans released from peptides. Therefore, the most potent liver fluke antigenic epitopes are mixed determinants, comprised of glycan and polypeptide elements. [Copyright &y& Elsevier]

Cholangiocarcinoma (CCA) is an aggressive tumor of the biliary epithelium with poor survival that shows limited response to conventional chemotherapy. Increased expression of glucosylceramide synthase (GCS) contributes to drug resistance and the progression of various cancers; the expression profiles of GCS (UGCG) and the genes for glucocerebrosidases 1, 2, and 3 (GBA1, GBA2, and GBA3) were therefore studied in CCA. The biological functions of GCS for cell proliferation and cisplatin sensitivity in CCA were explored. GCS expression was higher in CCA tumor tissues than that of GBA1, GBA2, and GBA3. Verification of GCS expression in 29 paired frozen CCA tissues showed that 8 of 29 cases (27.6%) had high GCS expression. The expression of GCS and GBA2 was induced in CCA cell lines following low-dose cisplatin treatment. Suppression of GCS by either palmitoylamino-3-morpholino-1-propanol (PPMP), GCS knockdown or a combination of the two resulted in reduced cell proliferation. These treatments enhanced the effect of cisplatin-induced CCA cell death, increased the expression of apoptotic proteins and reduced phosphorylation of ERK upon cisplatin treatment. Taken together, inhibition of the GCS increased cisplatin-induced CCA apoptosis via the inhibition of the ERK signaling pathway. Thus, targeting GCS might be a strategy for CCA treatment. [ABSTRACT FROM AUTHOR]

RING finger protein 43 (RNF43) is a ubiquitin E3 ligase that negatively regulates Wnt/β-catenin signalling. Mutation, inactivation and downregulation of RNF43 in cholangiocarcinoma (CCA) are associated with a less favourable prognosis. Since the functional role of RNF43 in CCA has not yet been demonstrated, the present study aimed to assess the effect of its overexpression in mediating CCA suppression via Wnt/β-catenin signalling pathway inhibition. Accordingly, RNF43 was overexpressed, and various malignant phenotypic changes studied, including cell proliferation, cell migration, chemotherapeutic sensitivity and the expression of several Wnt/β-catenin target genes. Overexpression of RNF43 in the CCA cell-line KKU-213B hindered activation of Wnt/β-catenin signalling, evidenced by: i) Accumulation of β-catenin in the cytoplasmic fraction and downregulation of several known Wnt target genes at the mRNA level [AXIN2, survivin (BIRC5), CCND1, MMP-7, c-MYC and ABCB1 (MDR1)]; ii) a reduction of cell proliferation; iii) a significant decrease in KKU-213B cell migration with RNF43 overexpression via upregulation of E-cadherin (CDH1); and iv) a reduction in N-cadherin (CDH2), MMP-2, MMP-7 and MMP-9. In addition, overexpression of RNF43 increased 5-fluorouracil sensitivity and downregulation of ABC transporter genes [including ABCB1 and ABCC1 (MRP1)]. The current results demonstrate a functional role for RNF43 in CCA by: i) Blocking β-catenin nuclear translocation; and ii) the subsequent downregulation of Wnt/β-catenin target genes (the latter being involved in the progression of CCA and chemotherapeutic drug susceptibility). Therefore, the present findings suggest that RNF43 could serve a tumour suppressive role in CCA. [ABSTRACT FROM AUTHOR]

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov -M60-like-1 and Ov -M60-like-2. Ov -M60-like-1 was previously characterized. Here, we study the Ov -M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini -infected hamsters. Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov -M60-like-2 possess a mucinase activity, together with Ov -M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct. [Display omitted] • The carcinogenic liver fluke Opisthorchis viverrini releases Ov -M60-like-2 metallopeptidases. • Ov -M60-like-2 displays mucinase activity over a wide pH range (pH 2 to pH 10) against mucin substrate. • Ov -M60-like-2 digest mucin from the bile duct, allows the liver fluke colonize the bile duct of the host. [ABSTRACT FROM AUTHOR]

Alteration of mucin-type O-glycosylation is implicated in tumor progression and metastasis of cholangiocarcinoma (CCA). Core 1 β1-3 Galactosyltransferase (C1GALT1) is a primary enzyme that regulates the elongation of core 1-derived mucin-type O-glycans. Dysregulation of C1GALT1 has been documented in multiple cancers and is associated with aberrant core 1 O-glycosylation and cancer aggressiveness; however, the expression of C1GALT1 and its role in CCA progression remains unknown. Our study demonstrated that C1GALT1 was downregulated in CCA tissues at both the mRNA and protein levels. The biological function of C1GALT1 using siRNA demonstrated that suppression of C1GALT1 in the CCA cell lines (KKU-055 and KKU-100) increased CCA progression, evidenced by: (i) Induction of CCA cell proliferation and 5-fluorouracil resistance in a dose-dependent manner; (ii) up-regulation of growth-related genes, ABC transporter genes, and anti-apoptotic proteins; and (iii) an increase in the activation/phosphorylation of AKT and ERK in silencing C1GALT1 cells. We demonstrated that silencing C1GALT1 in CCA cell lines was associated with immature core 1 O-glycosylation, demonstrated by high expression of VVL-binding glycans and down-regulation of other main O-linked glycosyltransferases β1,3-N-acetylglucosaminyltransferase 6 (B3GNT6) and ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 (ST6GALNAC1) in C1GALT1 knockdown. Our findings demonstrate that down-regulation of C1GALT1 in CCA increases the expression of immature core 1 O-glycan, enhancing CCA progression, including growth and 5-fluorouracil resistance via the activation of the AKT/ERK signaling pathway. [ABSTRACT FROM AUTHOR]

Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation [ABSTRACT FROM AUTHOR]