Sodium-dependent bile acid transporter expression modulates deoxycholate-induced effects on hepatocellular carcinoma cells

Abstract

Advanced HCC has been a therapeutic challenge despite the advances in treatment modalities. In particular, hypoxia is the main obstacle to treat HCC because it induces aggressive growth of the tumor. Hydrophobic bile acid (BA), such as deoxycholate (DC), is accumulated in cholestatic patients and can induce hepatocytes apoptosis. Therefore, DC may also be applied to treat hepatocellular carcinoma (HCC). However, the roles of DC and its transporter are still not established in HCC cells. Thus, this study aimed to investigate DC-induced HCC cell growth alterations, particularly focused on the BAT expression. Four human HCC cell lines (Huh-BAT, Huh-7, SNU-761 and SNU-475) were used to determine whether those cells expressed BAT or not. Huh-BAT (BAT+ HCC cells) and Huh-7 cells which do not express BAT (BAT- HCC cells) were grown either in a normoxic or hypoxic condition with DC. Cell viability was assessed using the MTS assay. BAT expression and apoptotic signaling cascades activation were examined by immunoblot analyses. Wound healing and invasion assays were performed to evaluate migration and invasion ability of cells, respectively. Real-time PCR analysis was performed to measure IL-8 expression levels. Nuclear factor kappa B (NF-κB) activity was evaluated by ELISA method. HCC cells revealed various BAT expression levels. In BAT+ HCC cells, DC increased apoptosis in a dose dependent manner, and BAT mediated the DC-induced apoptosis. Moreover, the susceptibility to DC was markedly enhanced in hypoxia, which was dependent on enhanced ER stress following DC treatment. This enhanced ER stress in hypoxia accelerated apoptosis of BAT+ HCC cells via JNK activation. On the contrary, DC promoted migration and invasion of BAT- HCC cells and induced IL-8 overexpression. Particularly, DC-induced IL-8 overexpression was dependent on NF-κB/cyclooxygenase-2 (COX-2) activity. Wound healing and invasion assays revealed that IL-8 was responsible for this augmented cellular migration and invasion of BAT- HCC cells, and these were preventable by a COX-2 inhibitor. In summary, hydrophobic BA had different effects on HCC cells according to their BAT expression status. It induced apoptosis of BAT-containing HCC cells, especially in hypoxic condition. In HCC cells without BAT, hydrophobic BA and simultaneous blockage of COX-2 activity could markedly decrease aggressive cellular behaviors through the inhibition of NF-κB/COX-2/IL-8 pathway. Therefore, hydrophobic bile acid had a therapeutic potential for advanced HCC.