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Background Cholangiocarcinoma (CCA), a common cancer in northeastern Thailand, is a severe disease with poor prognosis and short survival time following diagnosis. DNA damage in CCA is believed to be caused by liver fluke infection in combination with exposure to carcinogens. p53, a tumor suppressor, is the most mutated gene in human cancers including liver fluke-associated CCA. Hence, expression patterns of p53 and its target genes may be useful for diagnosis and/or prognosis of CCA patients. Methods Differential mRNA expression of p53 and its target genes, namely, FUCA1, ICAM2 MDM2, p21, PAI-1, S100A9, and WIP1 in CCA tissue samples (n = 30) relative to matched adjacent non-tumor tissues was determined by quantitative RT-PCR and compared to clinicopathological features. Level of p53 protein was determined by immunohistochemistry and correlated with the expression of its target genes. Results Immunohistochemistry showed elevation of p53 protein level in 77% of the cases, while RT-PCR showed downregulation of p53 mRNA and its seven target genes in 23% and 47-97% of the samples. PAI-1 was down-regulated in almost all CCA samples, thus highlighting it as a potential diagnostic marker for CCA. However, no significant clinical associations were found except for down-regulation of WIP1 that was significantly correlated with non-papillary type tissue (p-value = 0.001) and with high p53 protein level (p-value = 0.007). Conclusion Our results demonstrated statistically significant association between down-regulation of WIP1 with non-papillary type and with high p53 protein level, and PAI-1 was down-regulated in almost all CCA. Therefore, expression level of WIP1 and PAI-1 may be useful for predicting p53 functional status and as a potential diagnostic marker of CCA, respectively.

Intra-tumoral hypoxia is an environment that promotes tumor cell migration, angiogenesis and epithelial- mesenchymal transition that accounts for a major mechanism of metastasis. Chloroquine potentially offers a new therapeutic approach with an 'old' drug for effective and safe cancer therapies, as it exerts anti-metastatic activity. We investigated the inhibitory effect of chloroquine on cholangiocarcinoma (CCA) cell migration under cobalt chloride (CoCl2)-stimulated hypoxia. We showed that chloroquine suppressed CCA cell migration under hypoxic-mimicking conditions on exposure to 100 μM CoCl2. Moreover, chloroquine stabilized the protein level of prolyl hydroxylase domain proteins (PHD-2) but reduced the levels of hypoxic responsive proteins such as hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF). It also suppressed epithelial mesenchymal transition (EMT) by increasing the ratio of E-cadherin to N-cadherin under hypoxic conditions. In conclusion, chloroquine can inhibit hypoxia-stimulated metastasis via HIF-1α/VEGF/EMT which may serve as a useful additional strategy for CCA therapy.

Cytochrome P450 (CYP) enzymes are a large family of constitutive and inducible mono-oxygenase enzymes that play a central role in the oxidative metabolism of both xenobiotic and endogenous compounds. Several CYPs are involved in metabolism of oxysterols, which are cholesterol oxidation products whose expression may be dysregulated in inflammation-related diseases including cancer. This study focused on CYP39A1, which can metabolize 24-hydroxycholesterol (24-OH) that plays important roles in the inflammatory response and oxidative stress. We aimed to investigate the expression status of CYP39A1 and its transcription factor (RUNX2) in relation to clinical significance in cholangiocarcinoma (CCAs) and to determine whether 24-OH could induce oxidative stress in CCA cell lines. Immunohistochemistry showed that 70% and 30% of CCA patients had low and high expression of CYP39A1, respectively. Low expression of CYP39A1 demonstrated a significant correlation with metastasis. Our results also revealed that the expression of RUNX2 had a positive correlation with CYP39A1. Low expression of both CYP39A1 (70%) and RUNX2 (37%) was significantly related with poor prognosis of CCA patients. Interestingly, oxidized alpha-1 antitrypsin (ox-A1AT), an oxidative stress marker, was significantly increased in CCA tissues in which CYP39A1 and RUNX2 were down regulated. Additionally, immunocytochemistry showed that 24-OH could induce ox-A1AT in CCA cell lines. In conclusion, our study revealed putative roles of the CYP39A1 enzyme in prognostic determination of CCAs.

Hypoxia and autophagy are known to facilitate tumor progression. We here aimed to investigate the role of hypoxia-associated autophagy in cholangiocarcinoma (CCA) survival and metastasis. Immunostaining of hypoxic- responsive proteins (HIF-1α and BNIP3) and a key regulator of autophagy (PI3KC3) were examined in CCA tissues and their expression levels were compared with clinicopathological parameters. A hypoxia mimicking condition (CoCl2 treatment) was also tested regarding CCA cell functions. Our results showed that HIF-1α (66%), BNIP3 (44%) and PI3KC3 (46%) showed strong staining in human CCA tissues. Positive expression of HIF-1α (p=0.033), BNIP3 (p=0.040) and PI3KC3 (p=0.037) was significantly correlated with lymph node metastasis. HIF-1α was well associated with BNIP3 (r=0.3, p