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.

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.