The human prostate PC-3 cell line, which was provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Science (Shanghai, China), was maintained in Dulbecco’s modified Eagle’s medium-F12 (Gibco Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal calf serum, at 37°C and under a mixture of 95% air and 5% CO₂. To investigate amino acid starvation-induced autophagy, the cells were cultured for 12 h in Earle’s balanced salt solution (EBSS) medium at 37°C, in a 95% air and 5% CO₂ atmosphere, to induce autophagy as previously described (16). The study was approved the ethics committee of Jilin Medical College (Jilin, China).

The pSilencer-shCD147 plasmid, which produces CD147 hairpin small interfering RNA, was provided by Dr Liguo Wang (Affiliated Hospital of Jilin Medical College, Jilin, China). The PC-3 cells were seeded in six-well culture plates at a concentration of 5×10⁵ cells/ml for 24 h. Transient transfections were performed in a six-well plate containing serum-free medium, using Lipofectamine™ 2000 reagent (Gibco Life Technologies) and 2 μg of plasmid DNA, according to the manufacturer’s instructions. The G418 antibiotic (1,000 μg/ml) was used to screen for positive clones. The cells that demonstrated low expression of CD147 were termed PC-3/shCD147. PC-3/Scramble negative control cells were prepared by transfecting the pSilencer-scramble plasmid into PC-3 cells as previously described (12).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). A one-step RT-PCR was performed for the CD147 gene using a kit from Qiagen GmbH (Hilden, Germany). β-actin was amplified as an internal control. The PCR primers used were: CD147 forward, 5′-AAGGTGGACTCCGACGACCAGTGG-3′ and reverse, 5′-CTTCCGGCGCTTCTCGTAGATGAAG-3′; and β-actin forward, 5′-ATCATGTTTGAGACCTTCAACA-3′ and reverse, 5′-CATCTCTTGCTCGAAGTCCA-3′. The amplified products were separated on a 1% agarose gel for 30 min, followed by ethidium bromide staining.

The cells were washed twice with phosphate-buffered saline (PBS). Subsequently, the cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). The protein concentrations were determined using a bicinchoninic acid kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of the total protein were separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, US). The membranes were subsequently immunoblotted with the appropriate primary antibody diluted in Tris-buffered saline, containing 0.05% Tween-20 and 5% skimmed dry milk, at 4°C overnight. The following primary antibodies were used: anti-CD147 (cat. no. 3212) and anti-β-actin (cat. no. 1854) rabbit IgG monoclonal antibodies (Epitomics, Burlingame, CA, USA); and anti-light chain 3 (LC3; cat. no. 3868), p-Akt (cat. no. 4060) and p-mTOR (cat. no. 2971) rabbit IgG monoclonal antibodies (Cell Signaling Technology, Inc, Danvers, MA, USA). The membranes were washed and incubated using appropriate secondary horseradish peroxidase-conjugated goat anti-rabbit (1:5,000) antibody (Beyotime Institute of Biotechnology). β-actin was used to confirm equal protein loading. The band density was evaluated by Bio-Rad Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

For the quantitative analysis of autophagy, GFP-LC3 puncta formation was quantified as previously described (17). The cells were cultured in six-well plates and transfected with the GFP-LC3 plasmid, using Lipofectamine 2000, following the manufacturer’s protocol. At 24 h post-transfection, the cells were cultured with EBSS medium for 12 h, and the cells were subsequently examined under Nikon fluorescence microscopy (magnification, ×100; 90i; Nikon Corporation, Tokyo, Japan). For each condition, three slides were used and 30 cells were counted per slide.

Starvation-induced cell death was evaluated by a trypan blue exclusion assay. In brief, subsequent to PC-3/shCD147 or PC-3/Scramble being cultured in EBSS medium for 12 h, adherent and non-adherent cells were harvested, and resuspended in 100 μl PBS. Subsequent to mixing with 100 μl of 0.8% trypan blue, the cells were counted using a hemocytometer (Beyotime Institute of Biotechnology). The number of dead cells with disrupted membranes, stained blue, out of a total 200 cells was counted in three replicates. Cell death was calculated by the mean percentage of blue cells/total cells.

The data were calculated as the mean ± standard deviation of the mean. One-way analysis of variance was used to compare the significant difference in means between all treatment groups, and a two-sided Student’s t-test was used to compare the means of individual treatments when the primary outcome was statistically significant. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA).

To evaluate the expression of CD147 under starvation conditions, autophagy was induced in the human prostate cancer PC-3 cell line by culturing cells in amino acid-free EBSS buffer for 0, 3, 6, 9, 12 and 24 h. CD147 protein expression was determined by western blot analysis. The results revealed that CD147 expression gradually increased at 3, 6, 9, 12 and 24 h (Fig. 1). These data demonstrated that CD147 was involved in starvation-induced autophagy in PC-3 cells.

PC-3 cells stably expressing CD147 or control hairpin RNA were generated by transfecting the cells with the pSilencer-shCD147 plasmid or control pSilencer-scramble plasmid, respectively. Clones positive for the plasmid were screened for using the G418 antibody. The expression of CD147 was evaluated by RT-PCR (Fig. 2A) and western blot analysis (Fig. 2B). The results confirmed that the expression of CD147 was significantly downregulated in the PC-3/shCD147 cells at the mRNA and protein levels.

To confirm the modulation of autophagy by CD147, the autophagosome formation was evaluated using two assays. LC3 is essential for final autophagosome formation and thus serves as an autophagosome marker. First, each experimental stable cell line was transfected with a GFP-LC3 plasmid and the cells were cultured in EBSS medium for 12 h. The formation of autophagosomes was detected by the presence of GFP-LC3 punctate dots. The results revealed that PC-3/shCD147 cells exhibited a higher number of punctate structures compared with PC-3/Scramble control cells (P<0.01) (Fig. 3A).

The endogenous levels of LC3-II most likely indicated the level of autophagy. Therefore, western blot analysis was performed to detect LC3-II expression in PC-3/Scramble and PC-3/shCD147 cells. Notably, during the induction of starvation, increased LC3-II was observed in PC-3/shCD147 cells compared with the control PC-3/Scramble cells (P<0.05), further indicating that autophagy was induced to higher levels in the absence of CD147 (Fig. 3B).

To investigate the effect of the PI3K/Akt/mTOR pathway in the regulation of autophagy induced by CD147, the expression levels of p-Akt, p-mTOR and LC3-II were examined in the transfected PC-3 cells that were cultured with or without 20 μM LY294002, a specific inhibitor of Class I PI3K. It was found that the expression level of p-Akt and p-mTOR notably decreased in PC-3/shCD147 cells compared with PC-3/Scramble cells cultured without LY294002. In addition, compared with control shRNA-transfected PC-3 cells, PC-3 cells transfected with CD147 shRNA exhibited a significantly decreased level of p-Akt and p-mTOR subsequent to culturing with LY294002. To further assess the effect of the PI3K/Akt/mTOR pathway on autophagy, the levels of LC3-II in transfected PC-3 cells were examined subsequent to culturing with or without LY294002, using western blot analysis. The present results revealed that the inhibition of PI3K activity by LY294002 further promoted the autophagic levels in PC-3/shCD147 cells compared with PC-3/Scramble cells (Fig. 4). These results indicate that the PI3K/Akt/mTOR pathway is involved in the autophagy inhibition caused by CD147.

Trypan blue exclusion was used to investigate the starvation-induced cell death in transfected PC-3 cells cultured in EBSS medium for 12 h. The present data indicated that starvation-induced cell death was increased in PC-3/shCD147 cells compared with control PC-3/Scramble cells (37.7±6.4 vs. 21.7±5.5%; P<0.05). These findings indicate that CD147 may enhance the survival of PC-3 cells by inhibiting starvation-induced autophagy.