The human breast cancer (BC) cell lines MCF-7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). MCF-7 and MDA-MB-231 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C with 5% CO₂.

Cisplatin and hydroxychloroquine (HCQ) were purchased from Aladdin (Shanghai, China) and Selleck Chemicals (Houston, TX, USA), respectively. The MAPK inhibitor PD98059 and the JNK inhibitor SP600125 were purchased from Beyotime Biotechnology (Shanghai, China). The p38 inhibitor PD169316 was purchased from MedChem Express (Monmouth Junction, NJ, USA). Hoechst 33342 was purchased from Beijing Solarbio Science & Technology (Beijing, China). Cisplatin and HCQ were diluted in ultrapure water. Antibodies against β-actin and YAP were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against LC3, p62 and pJNK-Thr183/Tyr185 were purchased from the Novus Biologicals (Littleton, CO, USA), Abcam (Cambridge, MA, USA) and Bioworld Technology (St. Louis Park, MN, USA), respectively. Antibodies against p-YAP, caspase 3, p-p38, p38, pERK-Thr202/Tyr204, ERK and JNK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). The horseradish peroxidase (HRP)-conjugated goat anti-mouse, anti-rabbit IgG and FITC-conjugated anti-rabbit IgG secondary antibodies were purchased from Beyotime Biotechnology.

Human BC cells were lysed in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (both from Beyotime Biotechnology) as a protease inhibitor. Protein concentrations were assessed using bicinchoninic acid (BCA) protein assay reagent kit (Beyotime Biotechnology). A total of 40 µg of cell lysate was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h followed by incubation at 4°C overnight with the primary antibodies. HRP anti-mouse and anti-rabbit IgG (Beyotime Biotechnology) were used as the secondary antibodies. Immunoreactive bands were detected by ECL chemiluminescent substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The grey value of each band was calculated using ImageJ 1.50i (National Institute of Health, Bethesda, MD, USA) and normalized to the internal control β-actin.

Human BC cells were fixed for 10 min in 4% paraformaldehyde after being treated. The fixed cells were incubated in 0.25% TritonX-100 for 10 min, then incubated in Hoechst 33342 for 10 min. After being washed three times with phosphate-buffered saline (PBS), the cells were mounted with antifade reagent. Apoptotic cells were identified by the condensation and fragmentation of their nuclei, and were photographed using an Eclipse 80i microscope (Nikon, Tokyo, Japan).

Human BC cells were fixed for 20 min in 4% paraformaldehyde after being treated. The fixed cells were incubated in 0.25% Triton X-100 for 10 min, and then blocked for 1 h in 5% goat serum. The cells were stained with anti-LC3 antibodies (1:50) overnight at 4°C. After being washed three times with PBS, the cells were stained with appropriate secondary antibodies (1:200) for 1 h at room temperature. After being washed three times with PBS, the cells were incubated with 4,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Finally, after being washed another three times with PBS, the cells were mounted with antifade reagent. Fluorescence images were collected using an Eclipse 80i microscope.

BC cells were detached using EDTA-free trypsin and re-suspended in growth medium. After centrifugation, the cell precipitates were washed with cold PBS three times and resuspended in 1 ml PBS. To determine the rate of apoptosis, the cells were stained with 5 µl propidium iodide (PI) and 5 µl Annexin V-FITC for 15 min in the dark. Samples were then analyzed by flow cytometry on a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

For observation under transmission electron microscope (TEM), the cells were first washed with PBS and pelleted by centrifugation before fixing the cell pellet in 2.5% glutaric dialdehyde and 1% osmic acid for 2 h. After washing, the pellet was dehydrated and embedded in epoxy resin. Ultrathin sections (45 nm) were stained with lead acetate, and the stained sections were observed using a transmission electron microscope (H-7500; Hitachi, Tokyo, Japan).

MCF-7 and MDA-MB-231 cells were plated into 6-well plates at 2×10⁵ cells/well. Lentivirus-based short hairpin RNA (shRNA) vector and lentivirus-based cDNA targeting YAP were transfected into the cells according to the manufacturer's protocol. Cells were selected with puromycin for 10 days at 37°C. The effectiveness of transfection was ascertained by western blotting. The lentivirus-based shRNA vector and lentivirus-based cDNA targeting YAP were purchased from GenePharma (Shanghai, China). The sequence for YAP shRNA was as follows: YAP, 5′-CCGGGCCACCAAGCTAGATAAAGAACTCGAGTTCTTTATCTAGCTTGGTGGCTTTTTG-3′. The control shRNA was synthesized using a scrambled sequence.

Statistical analysis was performed using SPSS for Windows (version 17.0) (SPSS, Inc., Chicago, IL, USA). Student's t-test was used to compare groups. All quantitative results are displayed as the average value ± the standard deviation. P-values of <0.05, 0.01 or 0.001 were considered to indicate a statistically significant result.

To test the cytotoxic effect of cisplatin on BC cells, the human BC cell lines MCF-7 and MDA-MB-231 were treated with 0, 2, 4, 6, 8 and 10 µg/ml of cisplatin for 48 h. Cell viability was assessed by Cell Counting Kit-8 (CCK-8) cytotoxicity assay. The results revealed that cisplatin-mediated cytotoxicity was dose-dependent, and the half maximal inhibitory concentrations (IC₅₀) of cisplatin for MCF-7 and MDA-MB-231 were around 6 and 8 µg/ml, respectively (Fig. 1A). Therefore, cisplatin was applied at 6 and 8 µg/ml in subsequent experiments in MCF-7 and MDA-MB-231 cells, respectively. Next, apoptosis was detected by western blot analysis. The levels of cleaved-caspase 3 and cleaved-PARP increased in a dose-dependent manner (Fig. 1B). Compared with the control, the cells exposed to cisplatin had increased dose-dependent apoptosis, with the typical apoptosis morphological changes of condensed nuclear chromatin detected by Hoechst 33342 staining (Fig. 1C). Apoptosis was further evaluated using transmission electron microscopy. We observed cytoplasmic vacuolation, mitochondrion swelling, chromatin condensation, and apoptotic body formation in cisplatin-treated MCF-7 cells (Fig. 1D). These results clearly indicated that cisplatin significantly triggered apoptosis in BC cells.

Cisplatin is known to induce autophagy in cancer cells (22). To investigate whether cisplatin induced autophagy in BC cells, we examined autophagy markers LC3 and p62 by immunoblotting. Compared with that of the control, the expression levels of LC3-II increased, and the expression levels of p62 decreased in a dose-dependent manner in MCF-7 and MDA-MB-231 cells after 48 h of cisplatin treatment (Fig. 2A). To further confirm the role of cisplatin in inducing autophagy, fluorescence microscopy was used to examine the localization of LC3 punctate. Compared with that of the control, we observed increased specific punctate distribution of endogenous LC3 as punctate dots of green fluorescence after 48 h of cisplatin treatment in both BC cell lines (Fig. 2B). In addition, we examined autophagy using TEM. The control MCF-7 cells had obvious nuclei, and numerous mitochondria. In the cisplatin-treated MCF-7 cells, we observed several clearly visible double-membrane structures resembling autophagosomes, including non-degradable organelles and macromolecules could be observed (Fig. 2C). Collectively, the aforementioned results suggest that cisplatin can induce autophagy in BC cells. To further clarify the function of autophagy as a pro-death or pro-survival mechanism in cisplatin-induced apoptosis, we used the autophagy inhibitor HCQ to suppress the autophagy induced by cisplatin treatment. Immunoblotting assay was used to analyze the activation of cleaved-caspase 3 during the induction of apoptosis. When cells were exposed to cisplatin combined with HCQ, we observed significantly enhanced apoptosis-related changes (Fig. 2D). We then used flow cytometric analysis to confirm the role of autophagy induced by cisplatin treatment in BC cells. Cisplatin combined with HCQ treatment increased the apoptosis rate from 46.8 to 67.0%, and from 52.9 to 74.9% in MCF-7 and MDA-MB-231 cells, respectively (Fig. 2E). Collectively, these results indicate that inhibition of autophagy enhanced cisplatin-induced apoptosis in BC cells.

Accumulating evidence indicates that a role for all three MAPK subfamilies in autophagy (23). To investigate whether MAPKs contributed to cisplatin-induced autophagy in BC cells, we assessed the expression levels of phosphorylated ERK1/2, p38 and c-Jun N-terminal kinases (JNK) by immunoblotting. MDA-MB-231 cells were treated with 0, 2, 4, 6, 8 and 10 µg/ml of cisplatin for 48 h, and the levels of p-ERK1/2, p-p38 and p-JNK increased in a dose-dependent manner compared to that of the control, suggesting that cisplatin activated all three MAPK pathways in BC cells (Fig. 3A). To determine which MAPK is involved in cisplatin-induced autophagy, ERK inhibitor PD98059 (10 µmol/ml), p38 inhibitor PD169316 (10 µmol/ml) and JNK inhibitor SP600126 (10 µmol/ml) were used. MDA-MB-231 cells exposed to cisplatin combined with the ERK inhibitor, but not the p38 or JNK inhibitors, had decreased LC3-II when compared to cisplatin alone (Fig. 3B-D), suggesting that the ERK signaling pathway may be involved in cisplatin-induced autophagy.

The transcription co-activator YAP is thought to be involved in autophagy-dependent proliferation and invasion of BC cells (19). However, the role of YAP activation under cisplatin-induced autophagy remains unknown. The rate of phosphorylated YAP decreased with cisplatin treatment in a dose-dependent manner in MCF-7 and MDA-MB-231 cells (Fig. 4A). Consistent with this observation, immunoblotting of the nuclear and cytoplasmic fractions revealed increased accumulation of YAP protein in the nucleus of MCF-7 and MDA-MB-231 cells with cisplatin treatment (Fig. 4B). As YAP could be regulated by autophagy, we validated the impact of autophagy on YAP phosphorylation using the autophagy inhibitor HCQ. Compared with cisplatin alone, phosphorylation of YAP increased when treated with cisplatin and HCQ in MCF-7 and MDA-MB-231 cells (Fig. 4C). In addition, we found that cisplatin-induced autophagy increased the expression of CTGF, the target gene of YAP. These results indicate that cisplatin-induced autophagy had a significant impact on YAP activity.

Since cisplatin-induced autophagy has a significant impact on YAP activity, we next investigated the role of YAP in the apoptosis of cisplatin-treated cells. MCF-7 and MDA-MB-231 cells were transfected with either non-target (shNC) or YAP shRNAs (shYAP), and the expression of YAP was examined by western blotting. Compared with the control-transfected cells, the amount of YAP in cells transfected with YAP shRNA decreased significantly (Fig. 5A). Next, we used a CCK-8 assay to examine the cytotoxic effects of shNC and shYAP. Depletion of YAP significantly increased the cytotoxicity of cisplatin in MCF-7 and MDA-MB-231 cells (Fig. 5B). Consistent with this observation, we also found that the apoptotic marker cleaved-caspase 3 was increased in YAP knockdown cisplatin-treated cells, compared with that of their control shRNA-expressing counterparts (Fig. 5C). This data indicated that YAP inactivation was essential for the apoptotic effect of cisplatin in BC cells. Collectively, these results revealed that cisplatin induced autophagy, which functioned as a protective mechanism by regulating nuclear translocation of YAP, and subsequently impairing the apoptosis of BC cells (Fig. 5D).