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Mesenchymal stromal cells (MSCs) have recently been shown to play an important role in the growth and progression of many solid tumors, including cholangiocarcinoma (CCA). The human placental amniotic membrane (hPAM) is one of the most favorable sources of MSCs due to its availability and non-invasive harvesting procedure. However, the role of human placental amniotic membrane mesenchymal stromal cells (hPAMSCs) in the growth and progression of human CCA has not yet been determined. This study investigates the effects of conditioned medium derived from hPAMSCs (PA-CM) on the properties of three human CCA cell lines and explores possible mechanisms of action. Varying concentrations of PA-CM were used to treat CCA cells to determine their effects on the proliferation and apoptosis of CCA cells. The results showed that PA-CM inhibited the proliferation and colony-forming capacity of KKU100, KKU213A, and KKU213B cells. PA-CM also promoted the apoptosis of these CCA cells by causing the loss of mitochondrial membrane potential. Western Blotting confirmed that PA-CM induced CCA cell apoptosis by increasing the levels of the Bax/Bcl-2 ratio, cleaved caspase 3, and cleaved PARP, possibly by inhibiting the IL-6/JAK2/STAT3 signaling pathway. Moreover, our in vivo study also confirmed the suppressive effect of hPAMSCs on CCA cells by showing that PA-CM reduced tumor volume in nude mice transplanted with human CCA cells. Taken together, our results demonstrate that PA-CM has potent tumor-suppressive effects on human CCA cells and could potentially be used in combination with chemotherapy to develop a more effective treatment for CCA patients.

Cholangiocarcinoma (CCA) is one of the oxidative stress-driven carcinogenesis through chronic inflammation. Insulin receptor substrate 1 (IRS1), an adaptor protein of insulin signaling pathways, is associated with the progression of many inflammation-related cancers. This study hypothesized that oxidative stress regulates IRS1 expression and that up-regulation of IRS1 induces CCA progression. The localizations of IRS1 and an oxidative stress marker (8-oxodG) were detected in CCA tissues using immunohistochemistry (IHC). The presence of IRS1 in CCA tissues was confirmed using immortal cholangiocyte cells (MMNK1), a long-term oxidative-stress-induced cell line (ox-MMNK1-L), and five CCA cell lines as cell culture models. IRS1 was overexpressed in tumor cells and this was associated with a shorter patient survival time and an increase in 8-oxodG. IRS1 expression was higher in ox-MMNK1-L cells than in MMNK1 cells. Knockdown of IRS1 by siRNA in two CCA cell lines led to inhibition of proliferation, cell cycle progression, migration, invasion, stemness, and oxidative stress resistance properties. Moreover, a transcriptomics study demonstrated that suppressing IRS1 in the KKU-213B CCA cell line reduced the expression levels of several genes and pathways involved in the cellular functions. The findings indicate that IRS1 is a key molecule in the connection between oxidative stress and CCA progression. Therefore, IRS1 and its related genes can be used as prognostic markers and therapeutic targets for CCA therapy.

Cholangiocarcinoma (CCA), a malignancy of biliary epithelium, is related to liver stem cell deregulation. FoxAs are a group of transcription factors that play critical roles in liver stem cell differentiation. In this study, the expression levels of FoxAs (i.e., FoxA1, FoxA2 and FoxA3) were detected in intrahepatic CCA tissues and the functions of FoxAs were studied in CCA cell lines. FoxA1 and FoxA2 were mainly localized in the nuclei of normal bile duct (NBD) cells and some of the cancer cells. Low expression of FoxA1 in CCA tissues (72%) was significantly correlated with poor prognosis. FoxA3 expression of CCA cells was localized in the nucleus and cytoplasm, whereas it was slightly detected in NBDs. High expression of FoxA3 in cancer tissues (61%) was significantly related to high metastasis status. These findings suggest the opposing roles of FoxA1 and FoxA3 in CCA. Moreover, the FoxA1-over-expressing CCA cell line exhibited a significant reduction in proliferative and invasive activities compared to control cells. Knockdown of FoxA3 in CCA cells resulted in a significant decrease in proliferative and invasive activities compared with control cells. Taken together, in CCA, FoxA1 is down-regulated and has tumor suppressive roles, whereas FoxA3 is up-regulated and has oncogenic roles., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Zinc finger protein 423 (ZNF423) is a transcriptional factor involved in the development and progression of cancers but has not yet been examined in cholangiocarcinoma (CCA), an oxidative stress-driven cancer of biliary epithelium. In this study, we hypothesized that oxidative stress mediated ZNF423 expression regulates its downstream genes resulting in CCA genesis. ZNF423 protein expression patterns and 8-oxodG (an oxidative stress marker) formation in CCA tissues were investigated using immunohistochemical analysis. The results showed that ZNF423 was overexpressed in CCA cells compared to normal bile duct cells adjacent of the tumor. Notably, ZNF423 expression was positively correlated with 8-oxodG formation. Moreover, ZNF423 expression in an immortalized cholangiocyte cell line (MMNK1) was increased by hydrogen peroxide-treatment, suggesting that oxidative stress induces ZNF423 expression. To investigate the roles of ZNF423 in CCA progression, ZNF423 mRNA was silenced using specific siRNA in CCA cell lines, KKU-100 and KKU-213. Silencing of ZNF423 significantly inhibits cell proliferation and invasion of both CCA cell lines. Taking all these results together, the present study denoted that ZNF423 is an oxidative stress-responsive gene with an oncogenic property contributing to the regulation of CCA genesis.

Reactive oxygen and nitrogen species have been implicated in diverse pathophysiological conditions, including inflammation, neurodegenerative diseases and cancer. Accumulating evidence indicates that oxidative damage to biomolecules including lipids, proteins and DNA, contributes to these diseases. Previous studies suggest roles of lipid peroxidation and oxysterols in the development of neurodegenerative diseases and inflammation-related cancer. Our recent studies identifying and characterizing carbonylated proteins reveal oxidative damage to heat shock proteins in neurodegenerative disease models and inflammation-related cancer, suggesting dysfunction in their antioxidative properties. In neurodegenerative diseases, DNA damage may not only play a role in the induction of apoptosis, but also may inhibit cellular division via telomere shortening. Immunohistochemical analyses showed co-localization of oxidative/nitrative DNA lesions and stemness markers in the cells of inflammation-related cancers. Here, we review oxidative stress and its significant roles in neurodegenerative diseases and cancer.