Zileuton suppresses cholangiocarcinoma cell proliferation and migration through inhibition of the Akt signaling pathway

Sasikamon Khophai,1,2 Malinee Thanee,1,2 Anchalee Techasen,2,3 Nisana Namwat,1,2 Poramate Klanrit,1,2 Attapol Titapun,2,4 Apiwat Jarearnrat,2,4 Prakasit Sa-Ngiamwibool,2,5 Watcharin Loilome1,2 1Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 2Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand; 3Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand 4Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand 5Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand Background: Inflammatory lipid mediators play an important role in several cancer types. Leukotrienes (LTs), pro-inflammatory lipid mediators, are involved in chronic inflammation and cancer progression. They are derived from arachidonic acid by 5-lipoxygenase (5-LOX) activity. On the other hand, 15-lipoxygenase (15-LOX-1) converts LTs into lipoxins (LXs), pro-resolving lipid mediators. LXs are involved in the attenuation of inflammation and cancer development. Purpose: We aimed to investigate the lipid mediator pathways, especially the LTs and LXs pathways, by studying 5-LOX and 15-LOX-1 expression in human cholangiocarcinoma (CCA) tissue. We also investigated the efficiency of zileuton (5-LOX inhibitor) treatment and BML-111 (LXA4 analog) addition on CCA cell lines properties. Patients and methods: The expression of 5-LOX and 15-LOX-1 in fifty human cholangiocarcinoma (CCA) tissue was analyzed using immunohistochemical staining. In addition, the effect of zileuton and BML-111 on CCA cell growth and migration was demonstrated using a cell viability assay and wound-healing assay, respectively. Furthermore, the molecular mechanism by which zileuton inhibits CCA cell migration was revealed using immunofluorescent staining and western blot analysis, respectively.Results: We demonstrate that the upregulation of 5-LOX is significantly correlated with CCA recurrent status. A positive 15-LOX-1 signal was significantly associated with a longer survival time in CCA patients. We found that co-expression of 5-LOX and 15-LOX-1 resulted in a relatively good prognosis in CCA patients. In addition, zileuton could inhibit CCA cell migration as well as BML-111. Interestingly, zileuton treatment not only downregulated 5-LOX, but also upregulated 15-LOX-1, together with reversing the epithelial-mesenchymal transition to mesenchymal-epithelial transition phenotype as observed in EMT marker western blot. Conclusion: These findings suggest that 5-LOX and 15-LOX-1 play a key role in CCA and may serve as targets for CCA therapy. Keywords: cholangiocarcinoma, 5-LOX, 15-LOX-1, zileuton, EMT, Akt pathwa