Established Hs578T, MDA-MB-231, ZR-75-1, MCF7 and T47D human breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained in RPMI-1640 medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific Inc.), 100 units of penicillin and 100 μl/ml streptomycin. All cells were cultured in a 5% CO₂ incubator at 37°C. SH003 was extracted from Am, Ag or Tk, which were provided by Dr S.G. Ko (College of Korean Medicine, University of Kyung Hee, Seoul, Korea) as previously described (23).

Cells (2×10⁵ cells per plate) were seeded in a 60-mm plate and treated with various concentrations of SH003 (50, 100 or 200 μg/ml) for 48 h. Cell viability and cell death were assessed using a trypan blue exclusion method. Cell pellet was harvested and resuspend in 1 ml of phosphate-buffered saline (PBS). A total of 10 μl 0.4% trypan blue was gently mixed with 10 μl cell suspension. The mixture was applied to a hemocytometer and the number of trypan blue stained and non-stained cells were counted under a light microscope. The percentage of viable cells was calculated.

Cells were seeded at a density of 3×10² cells per well in a 6-well plate and were treated with various concentrations of SH003 (50, 100 or 200 μg/ml) for 24 h. The cells were cultured for 14 days and colonies were fixed with 4% paraformaldehyde and stained with a 0.01% crystal violet. Colony counts were performed manually using a light microscope and images of each plate were obtained.

Cells were transiently transfected with small interfering (si)RNA using the Lipofectamine RNAi MAX reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. The siRNA sequence for transfection was p73-siRNA, 5′-GCAAUAAUCUCUCGCAGUAUU-3′ and scramble-siRNA, 5′-GGACUCUCGGAUUGUAAGAUU-3′

Cell lysates were prepared with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.5; 50 mM NaCl, 1 μM EGTA and 1% Triton X-100) containing a protease inhibitor cocktail. Protein concentrations in extracts were determined using a Bradford assay (Bio Rad Laboratories, Inc., Hercules, CA, USA). Total cellular proteins (20 μg) were subjected to 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20 (TBST) buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) and probed with anti-poly ADP ribose polymerase (PARP; cat. no. 9542; 1:1,000), anti-p73 (cat. no. 14620; 1:1,000), anti-caspase 3 (cat. no. 9661; 1:1000; Cell Signaling Technology, Beverly, MA, USA) or anti-β-actin (cat. no. sc-47778; 1:2,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA) primary antibodies at 4°C overnight. Subsequently, the membranes were washed three times with TBST. Primary antibodies were detected following 2 h incubation at room temperature with a horseradish peroxidase-conjugated anti-mouse (cat. no. 7076; 1:2,000) or anti-rabbit secondary antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology, Danvers, MA, USA). Blots were developed with an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, UK).

In total, 3×10² cells harvested by trypsinization were fixed in 1 ml of cold 70% ethanol for 24 h at −20°C. After washing cell pellets with 1 ml PBS, pellets were centrifuged at 300 × g for 5 min, discarded supernatant, resuspended in 1 ml staining solution (50 μg/ml propidium iodide, 50 μg/ml RNase and 0.1% Triton X 100 in citrate buffer, pH 7.8), incubated for 30 min and washed with PBS. Cell cycle distribution was analyzed using a FACSCalibur fluorescence-activated cell sorter and CellQuest version 3.0 software (BD Biosciences, San Jose, CA, USA).

SPSS version 22.0 (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis. Data are presented as the mean ± standard deviation and multiple comparisons were conducted using one-way analysis of variance followed by Newman-Keuls post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

The viability of two breast cancer cell types, TNBC (Hs578T, MDA-MB-231 and ZR-75-1) and non-TNBC (MCF7 and T47D) was determined following exposure to SH003. Cells were treated with various concentrations (50, 100 and 200 μg/ml) of SH003. A significant decrease in cell viability was observed in TNBC cells (P<0.05 for the 100 μg/ml group and P<0.01 for the 200 μg/ml group compared with the untreated cells) but not in non-TNBC cells (Fig. 1A). MDA-MB-231 cells were further used as the cells showed the most effectively reduced cell viability in a dose-dependent manner. Additionally, colony formation analyses revealed a significant decrease in the number of MDA-MB-231 (TNBC) cells treated with SH003 but not MCF-7 (non-TNBC) cells (Fig. 1B; P<0.005 compared with the untreated cells). These results indicate that SH003 selectively decreases TNBC cell viability.

The effect of SH003 on cell death in MDA-MB-231 cells was determined using flow cytometry. A significant increase in the number of cells at the sub-G1 phase was observed following SH003 treatment (50, 100 and 200 μg/ml) (Fig. 2A) (P<0.05 and P<0.01 compared with the untreated cells). Apoptotic cell death and PARP cleavage in response to SH003 treatment were assessed in MDA-MB-231 cells using western blot analysis. The expression levels of cleaved PARP increased significantly in a dose-dependent manner compared with the untreated cells (P<0.05; Fig. 2B). SH003-induced apoptosis after pre-treatment with a pan-caspase inhibitor, Z-VAD was then examined. Pre-treatment with Z-VAD partially decreased the MDA-MB-231 cell death and levels of cleaved PARP induced by SH003 (Fig. 2C; P<0.05 compared with cells treated with SH003 only). Thus, SH003-induced cell death is partially caspase-dependent in TNBC cells.

A previous study indicated that p73 expression may prevent drug resistance and toxicity in p53-mutant TNBC (8). It was demonstrated that SH003 induced p73-mediated apoptosis in p53 mutant MDA-MB-231 cells. p73 expression in MDA-MB-231 cells was observed following treatment with SH003 using western blot analysis. The p73 protein levels in MDA-MB-231 cells treated with SH003 increased in a dose-dependent manner (Fig. 3A). To confirm that MDA-MB-231 cell death induced by SH003 was correlated with p73, the effect of knockdown of endogenous p73 using small interfering RNAs in MDA-MB-231 cells was examined. Cells were transfected with scrambled siRNA or p73 siRNA, followed by treatment with SH003. Transfected p73 siRNA decreased cell death and PARP cleavage compared with scrambled siRNA treatment (Fig. 3B; P<0.05 compared with SH003 single-treated scramble cells). Additionally, cell death was confirmed using flow cytometric analysis. The number of cells in the sub-G1 phase following SH003 treatment was decreased in the p73 siRNA-transfected cell line compared with the scrambled siRNA-transfected cell line (Fig. 3C; P<0.05 compared with SH003 single-treated scramble cells). These results indicated that the induction of p73 expression by SH003 leads to the apoptosis of MDA-MB-231 cells.

Paclitaxel (taxane) is a commonly used treatment in conjunction with other anticancer agents for TNBC; however, paclitaxel treatment occasionally fails due to drug resistance. It was demonstrated that SH003 in combination with paclitaxel synergistically increases cell death in TNBC cell compared with individual treatments (Fig. 4A; P<0.05 compared with cells treated with SH003 or paclitaxel only). Western blot analyses indicated that SH003 in combination with paclitaxel increased the levels of cleaved caspase-3 in MDA-MB-231 cells but did not alter p73 expression (Fig. 4B). These results indicate that SH003 in combination with paclitaxel synergistically enhances apoptosis in TNBC cells.