The cytotoxic effects of NaBu on PCa cells
To determine the cell proliferation rate of PCa cells following treatment with NaBu for 24 h, WST-1 analysis was conducted (Fig. 1). A dose-dependent significant decrease was detected in both PCa cells for 24-hour treatment period (p < 0.001). We determined that NaBu treatment more inhibited DU-145 cell viability at 1, 2.5, 5, and 10 mM (70.5 ± 0.9%, 52.3 ± 0.7%, 32.5 ± 0.8%, and 26.4 ± 0.4%, respectively) than LNCaP cells (67.4 ± 0.8%, 64.4 ± 0.7%, 41.4 ± 0.8%, and 29.7 ± 0.9%, respectively) (p < 0.001). Additionally, the inhibition of PCa cell viability was more profound at 1 and 5mM concentrations for 24 treatment period. Thus, NaBu exhibited greater anti-proliferative activity in both PCa cells at 1 and 5 mM concentrations for 24 h and we selected these concentrations of NaBu for further analysis.
The effects of NaBu on apoptotic cell death in PCa cells
To investigate the apoptotic effects of NaBu, we performed the Annexin-V assay (Fig. 2). According to the Annexin V assay results, 1 and 5 mM NaBu treatment more increased the percentage of late apoptotic cells than the percentage of early apoptotic cells in both DU-145 and LNCaP cells (Fig. 2A). Additionally, 1 and 5 mM NaBu treatment increased the percentage of total apoptotic cells (66.5 ± 0.7% and 62.3 ± 0.7%, respectively) in DU-145 cells compared to the control (6.9 ± 0.3%) (Fig. 2B, p < 0.01). On the other hand, 1 and 5 mM NaBu treatment significantly increased the percentage of total apoptotic cells (37.3 ± 0.4% and 49.6 ± 0.8%, respectively) in a dose-dependent manner in LNCaP cells compared to the control (1.3 ± 0.5%) (Fig. 2A, p < 0.01). Thus, NaBu treatment more induced apoptotic cell death in DU-145 cells than LNCaP cells.
The effects of NaBu on the cell morphology of PCa cells
To further explore the apoptotic effects of NaBu, AO/PI dual staining was conducted (Fig. 3). AO/PI dual staining results showed that NaBu treatment exhibited apoptotic morphological changes in both PCa cells. After treatment of 1 and 5 mM NaBu for 24 h, chromatin condensation, nuclear blebbing, rounded cells, and cytoplasmic shrinkage were observed. However, the apoptotic effect of NaBu was more obvious in DU-145 cells than LNCaP cells. Our results were consistent with the WST-1 and Annexin-V assay results.
The effects of NaBu on subcellular localization of TLR4 signaling pathway proteins in PCa cells
We next determined the subcellular localizations of TLR4, IRF3 and NF-κB as downstream proteins of TLR4 signaling pathways in PCa cells (Fig. 4). For this purpose, we performed immune fluorescence (IF) assay following administration of 1 and 5 mM of NaBu for 24 h in DU-145 and LNCaP cells. The obtained results demonstrated that treatment with 1 mM NaBu induced cytoplasmic expression of TLR4, IRF3 and NF-κB compared to control cells and higher expression of nuclear IRF3 was observed in DU-145 cells. However, both cytoplasmic and nuclear NF-κB expression was decreased in DU-145 cells following 5 mM NaBu treatment. On the other hand, treatment with 1 and 5 mM NaBu induced cytoplasmic expression of TLR4 compared with control in LNCaP cells. Additionally, we observed cytoplasmic IRF3 and NF-κB at 1 mM NaBu treatment. However, nuclear translocation of NF-κB and IRF3 was not observed after 5 mM NaBu treatment in LNCaP cells. Thus, NaBu treatment induced the cytoplasmic TLR4 and IRF3 expression in particularly DU-145 cells without affecting nuclear translocation of NF-κB in PCa cells.