Tet (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was dissolved as previously described (1). In brief, Tet powder was dissolved in one drop of 0.1 mM HCl and neutralized with NaHCO₃ solution to 7.4 pH Tet was subsequently diluted with phosphate-buffered saline (PBS) for a final concentration of 10 mM. Chloroquine (CQ) was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany) and dissolved in sterilized deionized water for a final concentration of 100 mM. Rabbit anti-LC3B antibody was purchased from Sigma-Aldrich, Merck Millipore. Mouse anti-β-actin antibody was purchased from Santa Cruz Biotechnology Inc. Other primary (β-actin; cat. no., 8457; dilution, 1:2,000; p62, cat. no., 8025; dilution, 1:1,000) and anti-rabbit secondary antibody (cat. no., 7074; dilution, 1:5,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Lipofectamine 3000 reagent and LysoSensor Yellow/Blue DND-160 were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The EGFP-LC3 plasmid was obtained from Osaka University, Osaka, Japan.

DU145 and PC-3 cells (National Platform of Experimental Cell Resources for Sci-Tech Beijing, China) were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 units/ml penicillin and 0.1 mg/ml streptomycin (Thermo Fisher Scientific, Inc.), and maintained in an atmosphere of 5% CO₂ at 37°C. The cells were digested with 0.25% of trypsin-EDTA solution for passage, and the cells were serum starved for 24 h prior to treatment.

For transfection, 1×10⁵/ml cells were seeded in a 6-well plate and transiently transfected with EGFP-LC3 plasmid at a final concentration of 50 nM by using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following 6 h of incubation at 37°C, the supernatant was replaced with RPMI-1640 medium containing 1% FBS and incubated at 37°C for 48 h.

Cells were digested and seeded (1×10⁴/well) onto a 96-well plate. Cell viability was detected with Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc. Kumamoto, Japan). Briefly, cells were treated with Tet or CQ and incubated at 37°C in RPMI-1640 medium containing 10% (v/v) CCK-8 for 2 h. Optical densities were read at 450 nm by using a microplate reader (Thermo Fisher Scientific, Inc.).

DU145 cells were transfected with EGFP-LC3 plasmid and cultured at 37°C for 48 h. The EGFP-LC3 puncta formation in each group were observed and counted using fluorescence microscopy (Leica Microsystems GmbH, Wetzlar, Germany) (13,14).

TEM was performed as previously described (1). In brief, the cells were digested, washed with pre-cold PBS, centrifuged at 1,000 × g for 5 min at 4°C and fixed with 2% glutaraldehyde at 4°C overnight. The cell pellets were post-fixed with 1% osmium tetroxide for 1 h. The samples were subsequently dehydrated and cut into ultrathin 40-nm sections. The subcellular structures of cells in each group were observed with an electron microscope (JEOL Ltd., Tokyo, Japan).

Lysosomal pH was quantified as previously described (1). A total of 1×10⁵/ml cells were seeded in a 96-well plate, treated with Tet and washed with PBS. The cells were subsequently incubated in 5 µM of LysoSensor Yellow/Blue DND-160 (Thermo Fisher Scientific, Inc.) at room temperature for 5 min. The supernatants were removed and replaced with 100 µl RPMI-1640 medium. Cells used for the standard curve were incubated at room temperature with 10 µM monensin and 20 µM nigericin in 20 mM 2-(N-morpholino) ethanesulfonic acid, 110 mM KCl and 20 mM NaCl at pH 3.6 to 6.9 for 10 min. Fluorescence in each well was assessed based on the ratio of light excited at 329 nm vs. 384 nm.

Total protein from each group was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 0.1% sodium dodecyl sulfate). The lysates were centrifuged at 8,000 × g at 4°C for 5 min and the supernatants were collected and subsequently quantified. The lysates in each group were diluted to the same concentration with RIPA lysis buffer, mixed with loading buffer and denatured at 95°C for 5 min. A total of 20 µg of the samples were loaded onto 12% SDS-PAGE and separated with electrophoresis. The proteins were then blotted onto a polyvinylidene difluoride membrane and blocked with 5% non-fat milk for 1 h at room temperature. The membrane was incubated with diluted primary antibodies at 4°C overnight. The membrane was washed with TBST and subsequently incubated with diluted secondary antibody for 1 h at room temperature. Following four washes, the blots were measured by using horseradish peroxidase substrate (Thermo Fisher Scientific, Inc.) and captured with a G:BOX Chemi Gel Documentation System (Syngene, Frederick, MD, USA). The relative density was calculated with Quantity One Software (v4.6.2, Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Student's t-test was performed to determine the statistical significance between two groups by using SPSS version 19.0 software (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference. Data are presented as the mean ± standard deviation.

The present authors have previously observed that DU145 cells are more sensitive to Tet treatment than PC-3 cells at 48 h post-incubation (1). In the present study, the DU145 and PC-3 cells were incubated with a series of Tet concentrations for 12, 24 and 48 h, respectively. It was observed that Tet treatment inhibited cell viability in the two cell lines in a dose- and time-dependent manner (Fig. 1). The half maximal inhibitory concentration (IC₅₀) values of Tet in DU145 cells were 24.0 (12 h), 10.2 (24 h) and 2.6 µM (48 h), whereas in PC-3 cells the values were 42.8 (12 h), 13.3 (24 h) and 9.5 µM (48 h), indicating that DU145 cells were more sensitive to Tet treatment compared with PC-3 cells (Fig. 1).

Autophagy has an important role in Tet-induced cell death (1,3). In order to explore the role of autophagy in Tet-induced cell death of DU145 cells, the LC3 conversion was measured using western blotting. The DU145 cells were treated with 10 µM Tet for 12 h. At 10 µM, Tet was able to induce a significant increase in LC3-II and EGFP-LC3 puncta formation in PC-3 cells (1). It was observed that in DU145 cells LC3-II expression was undetectable in baseline and Tet treatment conditions (Fig. 2A). This observation was not unexpected as the lack of one or two exons in ATG5 interferes with the formation of LC3-II in DU145 cells (12). Consistently, EGFP-LC3 puncta was not be observed in Tet-treated DU145 cells (Fig. 2B).

As autophagy mainly depends on the function of the lysosome, lysosomal pH of DU145 cells treated with a range of Tet concentrations (2.5 to 10 µM) was measured. The pH value increased in a concentration-dependent manner (Fig. 2C), indicating that Tet neutralized lysosomal acidity in DU145 cells.

De-acidulation of lysosomes results in the blockade of autophagic flux. As LC3 lipidation in DU145 cells is defective, assays based on LC3 turnover cannot be performed. Therefore, the expression of p62 (a protein that is degraded through autophagy and is upregulated when the autophagic flux is blocked) was analyzed. p62 protein in Tet-treated DU145 cells was upregulated, indicating that the autophagic flux may be blocked (Fig. 2D and E).

In order to investigate the role of autophagy in DU145 cells, the ultrastructural changes of DU145 cells upon Tet treatment for 12 h were observed. Notably, Tet triggered a large amount of autophagosome accumulation in the cytoplasm (Fig. 3). There were no significant differences in the shapes of the autophagosomes in DU145 cells compared with autophagosomes in other cell lines induced by Tet treatment, indicating that Tet may have triggered an alternative autophagy in DU145 cells.

The present authors have previously reported that Tet is a potent lysosomal inhibitor in a number of cell lines, which is closely associated with autophagy and its anti-tumor effects (1). In the present study, whether Tet-induced lysosomal de-acidulation contributed to its anti-tumor effects in DU145 cells was investigated. To reduce Tet-induced cell death, DU145 cells were treated with 2.5 µM Tet for 12 h. At 2.5 µM, Tet exhibited mild inhibitory effects on DU145 cells (Fig. 1), and the lysosomal pH was increased to 5.64. DU145 cells were subsequently co-treated with 8 µM CQ to increase the lysosomal pH to 6.78 (Fig. 2C). Treatment with 8 µM CQ alone for 12 h did not result in a significant change in viability of DU145 cells (Fig. 4). However, the presence of CQ synergized Tet-induced cell death (Fig. 4). These results indicated that high lysosomal pH is required in Tet-induced cell death of DU145 cells.