Abstract
Lithocholic acid (LCA) is a secondary bile acid that is selectively toxic to human neuroblastoma, breast and prostate cancer cells, whilst sparing normal cells. We previously reported that LCA inhibited cell viability and proliferation and induced apoptosis and necrosis of androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress, autophagy and mitochondrial dysfunction in the toxicity of LCA in PC-3 and autophagy deficient, androgen-independent DU-145 cells. LCA induced ER stress-related proteins, such as CCAAT-enhancer-binding protein homologous protein (CHOP), and the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2α) and c-Jun N-terminal kinases (p-JNK) in both cancer cell-types. The p53 upregulated modulator of apoptosis (PUMA) and B cell lymphoma-like protein 11 (BIM) levels were decreased at overtly toxic LCA concentrations, although PUMA levels increased at lower LCA concentrations in both cell lines. LCA induced autophagy-related conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3BI–LC3BII), and autophagy-related protein ATG5 in PC-3 cells, but not in autophagy-deficient DU-145 cells. LCA (>10 µM) increased levels of reactive oxygen species (ROS) concentration-dependently in PC-3 cells, whereas ROS levels were not affected in DU-145 cells. Salubrinal, an inhibitor of eIF2α dephosphorylation and ER stress, reduced LCA-induced CHOP levels slightly in PC-3, but not DU-145 cells. Salubrinal pre-treatment increased the cytotoxicity of LCA in PC-3 and DU-145 cells and resulted in a statistically significant loss of cell viability at normally non-toxic concentrations of LCA. The late-stage autophagy inhibitor bafilomycin A1 exacerbated LCA toxicity at subtoxic LCA concentrations in PC-3 cells. The antioxidant α-tocotrienol strongly inhibited the toxicity of LCA in PC-3 cells, but not in DU-145 cells. Collectively, although LCA induces autophagy and ER stress in PC-3 cells, these processes appear to be initially of protective nature and subsequently consequential to, but not critical for the ROS-mediated mitochondrial dysfunction and cytotoxicity of LCA. The full mechanism of LCA-induced mitochondrial dysfunction and cytotoxicity in the similarly sensitive DU-145 cells remains to be elucidated.
Highlights
Prostate cancer is the second most common cancer worldwide in males and the fourth most common cancer overall, with more than 1,112,000 new cases diagnosed in 2012, representing 15% of male cancer cases and 8% of all cancers (Ferlay et al, 2015)
Hoechst 33342 and propidium iodide-staining of PC-3 and DU-145 cells exposed for 24 h to Lithocholic acid (LCA) showed a significant concentration-dependent increase in staining, with both necrotic and early-apoptotic cells starting to appear at a concentration at or above 3 μM (Fig. 2)
To determine whether the endoplasmic reticulum (ER) stress pathway was involved in LCA-induced prostate cancer cell death, we determined the concentration- and time-dependent effects of LCA on p-JNK, JNK, p-eIF2α, eIF2α and CCAAT-enhancer-binding protein homologous protein (CHOP) protein levels, as well as on levels of BIM and p53 upregulated modulator of apoptosis (PUMA) in PC-3 and DU-145 cells exposed for 24 h to sub-cytotoxic (3 and 10 μM) and overtly cytotoxic (30 and 50 μM) concentrations of LCA
Summary
Prostate cancer is the second most common cancer worldwide in males and the fourth most common cancer overall, with more than 1,112,000 new cases diagnosed in 2012, representing 15% of male cancer cases and 8% of all cancers (Ferlay et al, 2015). Standard treatment of prostate cancer consists of surgery (prostatectomy), antihormonal therapy and radiotherapy. These treatments are successful for early-stage prostate cancer, they each have potentially serious side-effects (Martin & D’Amico, 2014; Nguyen et al, 2015), among which some that last a life-time (Sanda et al, 2008). Androgendeprivation therapy uses drugs that blocking the action of male sex hormones either through androgen receptor antagonism (bicalutamide, hydroxyflutamide) or inhibition of androgen biosynthesis (finasteride, abiraterone). These treatments are initially effective in controlling androgen-dependent prostate tumor growth, side-effects include increased insulin-resistance, bone density loss, hypogonadism, gynecomastia, muscle mass loss and fatigue (Conde & Aronson, 2003; Nguyen et al, 2015). The limitations of current standard treatments of prostate cancer has encouraged the search for safer and more effective molecules based on naturally occurring compounds
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