Antibodies against phosphorylated (p)-mTOR (cat. no. 2971; dilution 1:500), m-TOR (cat. no. 2972; dilution 1:1,000), serne/threonine protein kinase ULK1 (ULK1; cat. no. 8054S; dilution 1:1,000), p-ULK1 (cat. no. 6888; dilution 1:500), p-p38 (cat. no. 9216, dilution 1:500), p-ERK (cat. no. 4376; dilution 1:1,000), ERK (cat. no. 9102; dilution 1:1,000), autophagy-related protein (ATG)5 (cat. no. 12994S; dilution 1:1,000), ATG7 (cat. no. 8558; dilution 1:1,000), p62 (cat. no. 5114; dilution 1:500), JNK (cat. no. 9252; dilution 1:1,000), c-caspase-3 (cat. no. 9661S; dilution 1:200), cathepsin D (cat. no. 2284; dilution 1:200), p-JNK (cat. no. 9251; dilution 1:500) and p38 (cat. no. 9212; dilution 1:1,000) were purchased from Cell Signaling Technology, Inc.; antibodies against lysosome-associated membrane glycoprotein (LAMP)1 (cat. no. sc-20011; dilution 1:1,000), LAMP2 (cat. no. sc-18822; dilution 1:1,000) and cathepsin B (cat. no. sc-13985; dilution 1:500) were obtained from Santa Cruz Biotechnology, Inc.; anti-cleaved (c)-poly (ADP-ribose) polymerase (c-PARP; cat. no. 380374; dilution 1:500) was from Zen-Bio, Inc.; anti-GAPDH (cat. no. AG019; dilution 1:1,000) was purchased from Beyotime Institute of Biotechnology; anti-microtubule-associated protein 1 light chain 3 beta (LC3B; cat. no. L7543; dilution 1:1,000) and anti-Beclin 1 (cat. no. B6186; dilution 1:500) were obtained from Sigma-Aldrich; Merck KGaA; and horseradish peroxidase-conjugated secondary antibodies (cat. nos. 074-1802, 074-1516 or 074-1802) were purchased from KPL, Inc. The following reagents were used in the present study: SSD (cat. no. A0259; Chengdu Must Bio-Technology Co. Ltd.), bafilomycin A1 (Baf; cat. no. 11038; Cayman Chemical Company), rapamycin (Rapa; cat. no. S1039; Selleck Chemicals), SB230580 (p38 MAPK inhibitor; cat. no. S1076; Selleck Chemicals), LysoTracker™ Red DND-99 (cat. no. L7528; Thermo Fisher Scientific, Inc.) DAPI (cat. no. C1006; Beyotime Institute of Biotechnology) and DMSO (as solvent and placebo; cat. no. D2650; Sigma-Aldrich; Merck KGaA).

MDA-MB-231 cells were purchased from the American Type Culture Collection. The MDA-MB-231 cells were cultured in DMEM (cat. no. C11995500bt; Gibco, Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (cat. no. 6140071; Gibco, Thermo Fisher Scientific, Inc.), and maintained in a humidified atmosphere at 37°C and 5% CO₂.

A total of 5×10³ cells/well were cultured at 37°C and 5% CO₂ for 24 h in 96-well plates. Following incubation with SSD (0, 2, 4, 6, 10, 12 or 15 µM) for 24 h, 20 µl 5% MTT solution was added to each well and incubated at 37°C and 5% CO₂ for 4 h. Subsequently, the medium was discarded and the purple formazan was dissolved in 150 µl DMSO/well. The absorbance was detected using a microplate reader (iMark™, Bio-Rad Laboratories, Inc.) at 495 nm. The half maximal inhibitory concentration (IC₅₀) was calculated using SPSS 17.0 software (SPSS, Inc.).

A total of 1×10⁵ cells/well were cultured in 6-well plates at 37°C and 5% CO₂ for 24 h. Following incubation with SSD (8 or 10 µM) for 24 h or pretreated with 10 µM SB203580 for 2 h and then exposed to 10 µM SSD at 37°C for 24 h, the cells were collected by centrifuging at 600 × g for 5 min at 4°C, then the cells pellet were resuspended in 1 ml PBS and centrifuged at 600 × g for 5 min at 4°C again. Subsequently the cells pellet were stained with 10 µl propidium iodide (PI; cat. no. P4170; Sigma-Aldrich; Merck KGaA; 5.0 µg/ml) and 5 µl Annexin V-FITC (cat. no. 556419; BD Pharmingen). Apoptotic cells were subsequently detected by flow cytometry (Accuri C6; BD Diagnostics; Becton, Dickinson & Company) and analyzed by FlowJo 7.6.1 software (FlowJo LLC).

A total of 5×10³ cells/well were cultured in 24-well plates at 37°C and 5% CO₂ for 24 h, then transfected with 500 ng plasmids for 24 h before the subsequent experimentation using Lipofectamine^® 3000 reagent (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The plasmids include LAMP1-mGFP plasmid (cat. no. 34831; Addgene, Inc.), tfLC3 plasmid (a tandem reporter construct carrying both EGFP-LC3 and mRFP-LC3; cat. no. 21074; Addgene, Inc. and mRFP-LC3 plasmid (cat. no. 21075; Addgene, Inc.). The short hairpin (sh)RNA plasmids of target ATG5 (shATG5; 5′-TTTCATTCAGAAGCTGTTT-3′) and non-specific control (shCon; 5′-TTCTCCGAACGTGTCACGT-3′) were purchased from Gene Chem Co. Ltd. (Shanghai, China). A total of 1×10⁶ 293FT cells cultured in 60 mm plate were co-transfected with lentiviral packaging vectors (cat. no. A43237, Invitrogen; Thermo Fisher Scientific, Inc.) along with 1.5 µg shATG5 plasmin or 1.5 µg shCon plasmid using Lipofectamine^® 3000 (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Supernatant containing the lentivirus was harvested after 48 h. A total of 1.5×10⁵ MDA-MB-231 cells cultured in 6-well were infected with 1 ml lentivirus for 24 h, subsequently knockdown stable cell lines were selected using 4 µg/ml puromycin (cat. no. P9620; Sigma-Aldrich; Merck KGaA).

Cells (5×10³ cells/well) transfected with LAMP1-mGFP, tfLC3 or mRFP-LC3 were treated with 8 µM SSD, 20 nM Baf or 0.25 µM Rapa for 24 h. Lysosomes were stained with 75 nM LysoTracker™ Red DND-99 at 37°C for 2 h. The fluorescence in the cells was detected using a Carl Zeiss LSM 780NLO confocal microscopy with a 63× oil objective.

Total protein was extracted from cells using RIPA lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology). The concentration of protein was determined by the BCA method and 15–60 µg proteins were separated by 10–12% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes and blocked with 5% milk at room temperature for 30 min. The membranes were incubated with the aforementioned primary antibodies at 4°C overnight. Following the primary antibody incubation, the membranes were incubated with the secondary antibodies (1:10,000) at room temperature for 2 h. The membrane was washed three times for 30 min each and total protein was visualized using an ECL substrate (cat. no. 170–5060; Bio-Rad Laboratories, Inc.). The densitometric analysis was performed by ImageJ 1.37C software (National Institutes of Health).

Data were expressed as the means ± standard deviation from three independent experiments. Statistical analysis was performed using SPSS 17.0 software (SPSS, Inc.). Statistical differences among multiple groups were compared using a one-way ANOVA and Scheffe post hoc test. The statistical differences among two groups were determined using a Student's t-test if the data followed a normal distribution or with a U-Mann Whitney test otherwise. P<0.05 was considered to indicate a statistically significant difference.

The cytotoxic effects of SSD were tested in MDA-MB-231 cells using an MTT assay. 6–15 µM SSD significantly inhibited the viability of MDA-MB-231 cells in a dose dependent manner (Fig. 1A); IC₅₀ was found to be 9.9±0.24 µM. Flow cytometric analysis of apoptosis also demonstrated that 8–10 µM SSD significantly increased the rate of apoptosis in MDA-MB-231 cells (Fig. 1B and C). Consistent with these findings, 8–10 µM SSD was also revealed to promote the cleavage of caspase-3 and PARP (Fig. 1D and E).

The MAPK cascade is an important signaling pathway involved in the apoptotic process (17). JNK, ERK and p38 MAPK all belong to MAPK signaling pathways (18). To understand whether these MAPKs were involved in SSD-mediated apoptosis, the protein expression levels of p-JNK, JNK, p-ERK1/2, ERK1/2, p-p38 and p38 were detected using western blotting. SSD promoted a significant increase in p-p38 expression levels; however, it did not affect the expression levels of p-JNK or p-ERK (Fig. 2A and B). These results indicated that p38 MAPK may be involved in SSD-mediated apoptosis.

The co-administration of SB203580, a p38-MAPK inhibitor, was found to significantly attenuate the apoptotic rate induced by SSD (Fig. 2C and D), reduce the upregulation of c-caspase-3 and c-PARP expression levels mediated by SSD (Fig. 2E and F), and weaken the inhibitory effect on cell viability mediated by SSD in MDA-MB-231 cells (Fig. 2G). These results further suggested that SSD may induce apoptosis through the p38 MAPK signaling pathway.

ATGs are important for autophagy (19). The treatment of cells with SSD promoted the accumulation of LC3-II and p62; however, it did not affect the expression levels of Beclin 1, ATG5, ATG7, p-ULK1, ULK1, p-mTOR or mTOR (Fig. 3A and B). These findings indicated that SSD may not promote autophagosome formation but inhibit autophagic degradation (20). To further confirm these observations, tfLC3 plasmid (a tandem reporter construct carrying both EGFP-LC3 and mRFP-LC3) was applied (21). Cells transfected with tfLC3 were treated with Baf, Rapa and SSD and the fluorescence of EGFP-LC3 and mRFP-LC3 puncta were detected using confocal laser microscopy. Both Baf and SSD treatment induced the formation of a large number of both red and green puncta in cells, which displayed a yellow overlay (Fig. 3C). However, only red puncta were detected in cells treated with Rapa. These findings indicated that SSD blocked the process of autophagic degradation.

LAMP1 and LAMP2, major lysosomal membrane proteins, are important for maintaining lysosomal function and assisting in autophagosome-lysosome fusion (22). Cathepsin B and D, types of lysosomal cathepsins, are also important for maintaining lysosomal function and the loss of lysosomal function is often accompanied by a decrease in cathepsin expression (23). The expression levels of LAMP1 and LAMP2 were significantly upregulated by SSD treatment compared with untreated cells; however, the expression levels of cathepsin B and D were not affected, suggesting that SSD may not impair lysosomal function (Fig. 4A and B).

The intralysosomal acidic environment is another important factor for lysosomal functions (24). The intralysosomal acidic environment can be detected using LysoTracker™ Red DND-99, an acidic organelle tracer that can emit deep red fluorescence (25). The red fluorescence was observed in cells treated with SSD, Rapa or the placebo, but not in cells treated with Baf. These results indicated that SSD may not affect the intralysosomal acidic environment (Fig. 4C).

Detecting the colocalization of LAMP1 and LC3 can be used to confirm autophagosome-lysosome fusion (26). There was no overlap between the green fluorescence of LAMP1-mGFP and the red fluorescence of mRFP-LC3 in cells treated with SSD or Baf; however, a yellow overlap could be observed in cells treated with Rapa (Fig. 4D), suggesting that SSD may impair the autophagosome-lysosome fusion, resulting in the inhibition of autolysosome formation, which eventually leads to the blockage of autophagic degradation.

To further understand whether the excessive accumulation of autophagosomes contributed to the SSD-mediated apoptosis, the expression levels of LC3B-II were reduced following the stable knockdown of ATG5 expression levels and the treatment with SSD (Fig. 5A and B). Knocking down ATG5 expression levels did not affect the apoptotic rate of SDD-treated cells (Fig. 5C and D) or the inhibitory effect of SDD on cell proliferation (Fig. 5E) in MDA-MB-231 cells. In addition, the knockdown of ATG5 did not alter the SSD-mediated activation of c-caspase-3, c-PARP or p-p38 (Fig. 5A and B). These results indicated that the excessive accumulation of autophagosomes may not contribute to the SSD-mediated apoptosis in MDA-MB-231 cells.