br Western blot analysis br The cells were
2.9. Western blot analysis
The cells were seeded at a density of 5 × 106 cells in a 100 mm dish, and then cultured for 24 h in DMEM. After culturing, the cells were treated with the indicated concentrations of AC and TRAIL for 24 h, followed by centrifugation. The resulting pellets were lysed by lysis buﬀer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 1 mM PMSF, and 2 μg/mL of aprotinin) for 30 min on ice. The protein content of the supernatant was measured using a BCA protein kit (Pierce, Rockford, IL, USA). The protein samples were then loaded at 10 μg of protein/lane and then separated by 12% SDS-PAGE at 100 V of constant voltage/slab for 1.5 h. Following electrophoresis, the proteins were transferred onto nitrocellulose membranes. After blocking with 2.5% and 5% bovine serum albumin (BSA) for 1 h at 37 °C, the membranes were incubated with primary antibody at 4 °C overnight. Finally, the membranes were treated with horseradish per-oxidase-coupled secondary cck8 for 1 h at 4 °C. The membranes were then washed with T-TBS after each antibody binding reaction. Detection of each protein was performed using an ECL kit (Santa Cruz, CA, USA). All protein bands were analyzed 3 times, and the intensity of each band was quantified by Image studio™ Lite software (LI-COR Inc., NE, USA), and the fold of increase was presented comparing with β-
Fig. 3. Combined treatment with AC and
TRAIL induces caspase-dependent apop-
tosis in RC-58T/h/SA#4 cells. Cells were treated with 5 μM AC and/or 100 ng/mL of TRAIL for 24 h. (A) After treatment with AC and/or TRAIL for 24 h, total cell lysates were subjected to detect expression levels of proteins. Expression of caspase-9, -8, -3 and -10, Bax, Bcl-2, and PARP in RC-58T/h/ SA#4 cells were analyzed by Western blot-ting. (B) Cells were pretreated with 10 μM z-vad-fmk, a caspase inhibitor, for 2 h and then incubated with AC and/or TRAIL for 24 h. Cell viability was evaluated by SRB assay. Significant diﬀerences were com-pared with the control at *p < 0.05 and **p < 0.01 using one-way ANOVA.
2.10. Statistical analysis
The statistical analyses were evaluated by one-way analysis of variance (ANOVA), with diﬀerences analyzed using the Duncan's new multiple-range test. Levels of *p < 0.05, **p < 0.01, and ***p < 0.001 were regarded as statistically significant.
3.1. AC sensitizes TRAIL-resistant RC-58T/h/SA#4 cells to TRAIL-induced cell death
In order to determine the inhibitory eﬀect of combined treatment with AC and TRAIL on proliferation of prostate cancer cells, both RC-58T/h/SA#4 and RWPE-1 cells were treated with various doses of AC with or without TRAIL (Fig. 1). AC and TRAIL-induced suppression of cell viability was confirmed by SRB assay. RC-58T/h/SA#4 cells were moderately resistant to 2.5–10 μM AC and 50–200 ng/mL of TRAIL (Fig. 1A). However, co-treatment with AC (5–10 μM) and TRAIL (50–200 ng/mL) significantly reduced viability of RC-58T/h/ SA#4 cells, suggesting that TRAIL resistance in RC-58T/h/SA#4 cells could be overcome by addition of AC at low concentration (Fig. 1A, C). Unlike primary prostate cancer cells (i.e. RC-58T/h/SA#4 cells), pro-liferation of human normal prostate epithelial cells (RWPE-1) was in-hibited by a high dose of AC single treatment (7.5–10 μM) (Fig. 1B). However, single or combined treatment with low-dose AC (2.5–5 μM) and TRAIL (50–200 ng/mL) did not have significant cytotoxicity in RWPE-1 cells (Fig. 1B and C). Therefore, these results suggest that combined treatment with non-cytotoxic concentrations of AC (5 μM) and TRAIL (100 ng/mL) exerted a synergistic inhibitory eﬀect against
primary cancer cell proliferation.
3.2. AC sensitizes TRAIL-mediated apoptosis in RC-58T/h/SA#4 cells
After binding of TRAIL to extracellular DR4 and DR5, an apoptotic cascade of downstream proteins is activated during assembly of death-inducing signaling complex (DISC) at their intracellular death domains (Bertsch et al., 2014). To determine whether or not AC could sensitize RC-58T/h/SA#4 cells to TRAIL-mediated apoptosis, cells were treated with AC alone or in combination with TRAIL for 24 h. As shown in Fig. 2A, the percentage of apoptotic cells was significantly higher upon co-treatment with AC and TRAIL compared to the control. Furthermore, biological markers of apoptosis such as DNA fragmentation, con-densation of chromatin, and expression of apoptotic bodies were sig-nificantly enhanced upon combined treatment with AC and TRAIL treatment (Fig. 2B and C). These results show that co-treatment with non-cytotoxic concentrations of AC and TRAIL significantly elevated apoptosis in TRAIL-resistant RC-58T/h/SA#4 cells.