Human PCa cells (DU145 and LNCaP; Korean Cell Line Bank) were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS). During experimentation, cells were grown in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) containing 1% FBS. Cells were grown at 37˚C and 5% CO₂ in a humidified incubator.

Neferine was acquired from Sigma-Aldrich; Merck KGaA, and TRAIL was acquired from Abfrontier.

Cytotoxicity is the amount of toxiciticy affecting cells. A cytotoxic agent can lead to a decrease in cell viability, the activation of apoptosis and the alteration of autophagy (20-22). Several methods for cytotoxicity assay, such as trypan blue stain, lactate dehydrogenase (LDH) assay, 3-(4,5-dimethyl-2-thiazoly)-2,5-dephenyl-2H-tetrazo-lium bromide (MTT) assay. In the present study, cell viability was assessed, and crystal violet and trypan blue staining was used to examine cells treated with a combination of neferine and TRAIL.

The DU145 and LNCaP cells seeded in 12-well plates were treated with 0, 5, 10, or 20 µM neferine for 18 h, cells were subseqently treated with 200 ng/ml TRAIL for 2 h and chloroquine (CQ; cat. no. c6628; Sigma-Aldrich; Merck KGaA) was treated 10 µM 1 h prior neferine or TRAIL treatment. In addition, the JNK inhibitor, SP600125 (cat. no. s5567; Sigma-Aldrich; Merck KGaA), was used to treat the cells at 1 µM for 1 h prior to neferine or TRAIL treatment. Cell morphology was evaluated using a light microscope (Nikon Corp.), and cell viability was evaluated using a crystal violet assay as previously described (23).

Cell viability was evaluated using a trypan blue exclusion assay. The DU145 and LNCap cells were seeded in a 24-well plate and following treatment, the cells were dissociated from plate with trypsin-EDTA and were then suspensed in 1 ml PBS per well followed by the addition of 1 ml trypan blue solution (Sigma-Aldrich; Merck KGaA). Follwing incubation for 5 min at 20˚C, the cells were counted using a hemocytometer (Marienfeld Corp.) and examined using a light microscope (Nikon Corp.). Each treatment was performed in triplicate, and the results are expressed as a percentage relative to the untreated controls.

To determine the effects of neferine on TRAIL-induced apoptosis, the levels of cleaved casapase-8 (Ac-cas8) and caspase-3 (Ac-cas3) were detected. In addition, Also, changes in the levels of p62 and LC-3 Ⅱ proteins were detected to monitor the autophagic flux. DU145 cells were cultured on poly-L-lysine-coated coverslips. Differentiated cells that had been treated were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were then incubated in blocking solution overnight at 4˚C and bathed in a solution containing anti-p62 (1:250; cat. no. PAS-20839; Invitrogen; Thermo Fisher Scientific, Inc.) and anti-p-JNK (1:500; cat. no. c.s 9255s; Cell Signaling Technology; Inc.) antibodies for 2-3 h at room temperature. After washing with PBS, the cells were incubated in the dark at 20˚C with secondary antibodies (Alexa Fluor^® 488-conjugated donkey polyclonal anti-rabbit; 1:500; cat. no. A-21206; Thermo Fisher Scientific, Inc. and Alexa FluorTM 546 goat anti-mouse IgG (1:500; cat. no. A-21206; Thermo Fisher Scientific, Inc.) for 2 h. A fluorescence microscope (Nikon Corp.) was used to visualize immunostaining.

Cells were bathed in a solution containing 2% glutaraldehyde, 2% paraformal-dehyde and 0.05 M sodium cacodylate (pH 7.2) (all from Electron Microscopy Sciences) for 2 h at 4˚C. The cells were then fixed by incubation with 1% osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4˚C, dehydrated with increasing concentrations of ethanol (25, 50, 70, 90 and 100%) for 5 min at each concentration, and embedded in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60˚C following the manufacturers' instructions. Ultrathin sections (60-nm-thick) were sliced using an LKB-III ultratome (Leica Microsystems GmbH). The slices were stained with 0.5% uranyl acetate (Electron Microscopy Sciences) for 20 min and with 0.1% lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Fluorescent images were recorded using the Hitachi H7650 electron microscope (Hitachi, Ltd.; magnification, ×10,000) located at the Center for University-Wide Research Facilities (CURF) at Jeonbuk National University.

Western blot analysis was performed as previously described (3). Total protein extraction was using immunoprecipitation assay buffer (Qiagen, Inc.). The supernatant was collected by centrifugation (11,200 × g); 4˚C; 10 min; the protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific; Inc.). Proteins (30 µg) were separated on 10% SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat dried milk at 25˚C for 1 h antibodies against the flowing proteins were used; β-actin (1:10,000; cat. no. A5441; Sigma-Aldrich; Merck KGaA), LC3A/B (1:1,000; cat. no. c.s 4108s; Cell Signaling Technology; Inc.), p62 (1:250; cat. no. PAS-20839; Invitrogen; Thermo Fisher Scientific, Inc.), ATG5 (1:1,000; cat. no. c.s 12994s; Cell Signaling Technology; Inc.), cleaved caspase-3 (1:1,000; cat. no. c.s 9661; Cell Signaling Technology; Inc.) (activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments, cleavage of caspase-3 requires aspartic acid at thep1 position), cleaved caspase-8 (1:1,000; cat. no. 551242; BD Pharmingen) (caspase-8 is produced as a proenzyme (55/50 kDa, doublet) it is cleaved into smaller subunits (40/36 kDa, doublet). and p-JNK (1:500; cat. no. c.s 9255s; Cell Signaling Technology; Inc.). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ADI-SAB-100 and ADI-SAB-300; 1:1,000; Enzo Life Sciences, Inc.) at 25˚C for 1 h. The immune reactive protein bands were visualized using enhanced chemiluminescence detection system (GE Healthcare Life Sciences) and detected with chemiluminescence imaging system (Fusion FX7; Viber Lourmat). The intensities of the protein bands were determined using Image J Java 1.8.0 software.xs.

Media RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Atlas Biologicals) was seeded with DU145 cells, and 24 h later, the cells were transfected with silencer-select small interfering RNA (ATG5 siRNA; oligo ID HSS114103; Sequence GGU UUG GAC GAA UUC CAA CUU GUU U; Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000 as per the manufacturer's recommendations. Simultaneously, a negative control (Invitrogen; Thermo Fisher Scientific, Inc.) was trans-fected with a non-targeting siRNA. The cells were incubated with ATG5 siRNA or negative control siRNA for 6 h and the medium was then changed to RPMI-1640 with 10% FBS for 24 h. The cells were then treated with neferine or neferine in combination with TRAIL.

All experiments were performed in triplicate, and the data are reported as the means ± standard error. One-way factorial analysis of variance (ANOVA), followed by Duncan's post-hoc test, was performed to evaluate the statistical significance of the differences between the treatment and control groups.

In the present study, in order to investigate the synergistic effects of combination treatment with neferine and TRAIL on PCa cells, the viability of neferine only-treated cells, TRAIL only-treated cells, and neferine and TRAIL combine-treated cells was compared. No significants changes were observed in the viability of the cells treated with neferine or TRAIL only; however, a significant decrease was detected in the viability of the cells treated with the combination of neferine and TRAIL (Fig. 1). To evaluate the effects of neferine treatment on PCa cell apoptosis, the DU145 and LNCaP cells were treated with various concentrations of neferine for 18 h and then exposed to TRAIL for a further 2 h. As shown in Fig. 1, treatment with TRAIL or neferine alone resulted in only marginal cell death and did not cause any novel morphological changes. By contrast, when cells were treated with both neferine and TRAIL, cell viability decreased significantly (Fig. 1). These data indicate that neferine treatment sensitizes human PCa cells to TRAIL-induced apoptosis.

In response to neferine treatment, the expression of LC3-II increased markedly in the DU145 cells, whereas p62 expression decreased significantly (Fig. 2A). The results obtained for p62 protein levels measured by immunofluorescence staining were consistent with those obtained by western blot analysis (Fig. 2B). TEM indicated that autophagic vacuoles were secreted by the neferine-treated cells (Fig. 2C). In addition, the cells treated with a combination of neferine and TRAIL exhibited higher expression levels of Ac-cas3 and Ac-cas8 (Fig. 2D). Caspase-8 activation also was detected in the cells treated with a combination of neferine and TRAIL, but not in the cells treated with neferine or TRAIL alone (Fig. 2D). Certain studies have compared the combination of two agents§ to either agent alone to investigate the synergistic effects of the two agents (24-26). These results suggest that neferine treatment can initiate autophagy in DU145 cells.

Cell morphological analysis revealed that treatment with chloroquine (CQ), an autophagy inhibitor, attenuated cellular apoptosis mediated by combined treatment with neferine and TRAIL (Fig. 3A). Co-treatment with neferine, TRAIL and CQ resulted in a distinct improvement in the viability of the DU145 cells (Fig. 3B-D). The autophagic flux activation by neferine was confirmed by the inspection of the autophagic flux following treatment with CQ as an autophagy inhibitor. CQ treatment led to the accumulation of membrane-bound LC3-II and an increase in p62 levels, these results indicated that CQ blocked neferine-induced autophagy (Fig. 4A). Immunofluorescence staining indicated that CQ treatment resulted in an increase in p62 protein levels (Fig. 4B). CQ also attenuated the upregulation of Ac-cas8 and Ac-cas3 that was observed following treatment with neferine and TRAIL (Fig. 4C). These results indicate that CQ modulates neferine-mediated, TRAIL-triggered apoptosis by inhibiting the autophagic flux.

Cell morphological analysis indicated that transfection with ATG5 siRNA (Fig. 5A). Co-treatment with neferine, TRAIL and ATG5 siRNA significantly attenuated cell death and markedly increased the viability of the DU145 cells (Fig. 5B-D). In the cells in which ATG5 was knocked down, the LC3-II protein levels were markedly decreased and p62 protein levels were markedly increased (Fig. 6A). The protein levels of p62 measured by immunofluorescence staining were similar to the levels measured by western blot analysis (Fig. 6B). Co-treatment of the cells with neferine, ATG5 siRNA and TRAIL resulted in a decrease in Ac-cas3 and Ac-cas8 expression (Fig. 6C). These results suggested that ATG5 siRNA blocked the synergistic cell death induced by neferine and TRAIL treatment.

As revealed by western blot analysis and immunofluorescence staining, neferine treatment of the DU145 cells resulted in a dose-dependent increase in the p-JNK protein expression levels (Fig. 7A and B). In the cells that were treated with 1 µM SP600125 for 1 h prior to neferine treatment, a decrease in neferine-induced p-JNK expression was observed. SP600125 treatment also increased the viability of the cells that had been treated with neferine and TRAIL (Fig. 7D and E). These results indicate that neferine treatment causes an increase in JNK expression that triggers TRAIL-mediated death of DU145 cells.