HM (Fig. 1) was separated from the seeds of Peganum nigellastrum Bunge by column chromatography on AB-8 Macroporous Resin eluted with gradient ethyl alcohol and water. Furthermore, HM was isolated by preparative high-performance liquid chromatography (Fig. S1). Structure identification and the purity (>98%) of this compound (Figs. S2-8) were identified by spectroscopic methods and compared with the reported spectral data.

The MDA-MB-231 and MCF-7 human breast cancer cell lines were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The cells were cultured in DMEM (cat. no. C11965500BT; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (cat. no. 10100154; Gibco; Thermo Fisher Scientific, Inc.) and 100 μg/ml streptomycin at 37°C in an incubator with 5% CO₂. A 200-mM stock solution of HM was prepared in DMSO (cat. no. V900090; Merck KGaA, Darmstadt, Germany) and diluted in culture media as necessary, to achieve the final desired experimental concentrations.

The pCMV6-TAZ expression vector and pCMV6-control vector were purchased from OriGene Technologies, Inc. (Rockville, MD, USA; cat. no. Rc204082). MDA-MB-231 and MCF-7 cells were cultured in 96-well plates at a density of 5×103 cells per well for 24 h at 37°C, and then transfected with pCMV6-TAZ or pCMV6-control using Lipofectamine 3000 (cat. no. L3000-015; Thermo Fisher Scientific, Inc.) for 2 days. The transfected cells were visualized using a fluorescence microscope.

The cells were plated in 96-well plates at a density of 5×10³ cells per well. Following 24 h of incubation, the cells were left untreated or treated with HM at 50, 100 or 150 μM for 24, 48 or 72 h, followed by the addition of 10 μl CCK-8 to each well. The absorbance was measured at 450 nm using EnSpire Multimode plate reader (PerkinElmer, Inc., Waltham, MA, USA). The optical density (OD) was calculated for cell viability assay. Cell viability (%) = OD (treated) / OD (control) ×100%.

Cell migration was measured using a wound healing assay. The MDA-MB-231 and MCF7 cells were cultured in 6-well plates. A clean pipette tip was used to inflict a ‘wound’ when cells formed a confluent monolayer, and the cultured cells in DMEM were supplemented with 2% FBS. The cells were left untreated or treated with HM at 50, 100 or 150 μM. Images of the wound margins were captured using an inverted light microscope (Olympus Corporation, Tokyo, Japan) formed 0 h at this time point. Following incubation for 24 h, images of the same region of cells were captured for measurement. The wound healing rate was calculated using the following formula: (average wound margin in 0 h - average wound margin in 24 h) / average wound margin in 0 h.

The cells were left untreated or treated with HM at 50, 100 or 150 μM for 24 h, washed twice with phosphate buffer saline (PBS), fixed in methanol for 10 min and finally stained with DAPI (cat. no. C1002; Beyotime Institute of Biotechnology, Haimen, China) for 10 min at room temperature. Images were then captured by fluorescent microscopy (Olympus Corporation).

Cell apoptosis was analyzed using a PE Annexin V apoptosis detection kit І (cat. no. 559763; BD Biosciences, USA). The cells were seeded in 6-well plates with 50, 100 and 150 μM HM for 24 h, and stained according to the manufacturer's instructions. The apoptotic cells were immediately detected by a FACS Caliber II Sorter and the Cell Quest FACS system (BD Biosciences). Data were analyzed using FlowJo software (version 7.6.5; Tree Star, Inc., Ashland, OR, USA).

The cells were lysed in cell lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and centrifuged (12,000 × g, 10 min, 4°C) to collect the supernatants following treatment with HM (50, 100 or 150 μM) for 24 h. The protein concentration was measured using a BCA Protein Assay kit (cat. no. CW0014S; CWBio, Beijing, China), and an equal quantity (20 μg per well) of protein was separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked using 5% nonfat skim milk for 1 h at room temperature and then incubated with primary antibodies overnight for 4°C. The primary antibodies included anti-TAZ (1:1,000; cat. no. 70148; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-Bax (1:1,000; cat. no. ab-32503; Abcam, Cambridge, MA, USA), anti-Bcl-2 (1:1,000; cat. no. ab-32124; Abcam), anti-Erk (1:500; cat. no. sc-514302; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-phosphorylated (p-)Erk (1:500; cat. no. sc-81492; Santa Cruz Biotechnology, Inc.), anti-Akt (1:500; cat. no. sc-24500; Santa Cruz Biotechnology, Inc.), anti-p-Akt (1:500; cat. no. sc-7985; Santa Cruz Biotechnology, Inc.) and anti-β-actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.). Following washing with TBST three times, for 10 min each time, goat anti-rabbit IgG (1:10,000; cat. no. sc-2040; Santa Cruz Biotechnology, Inc.) or goat anti-mouse IgG antibody (1:10,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) conjugated with horseradish peroxidase was incubated for 1 h at room temperature. The protein bands were visualized using the enhanced ECL Select Western Blotting Detection Reagent (cat. no. RPN2235; GE Healthcare Life Sciences, Chalfont, UK).

Total RNA was extracted from untreated and treated cells using TRIzol reagent (cat. no. 15596018; Thermo Fisher Scientific, Inc.) and cDNA was synthesized using the ReverTra Ace qPCR RT kit (cat. no. FSQ-101; Toyobo Life Science, Osaka, Japan). The qPCR was performed using UltraSYBR mixture (cat. no. CW-2601; CWBio) on an ABI StepOne Plus QPCR system (Thermo Fisher Scientific, Inc.). The thermocycling steps were as follows: Initial denaturation at 95°C for 10min, followed by 40 cycles at 95°C for 15 sec, 58°C for 1 min, and a final extension step at 72°C for 5 min. The 2^-ΔΔCq method was used to calculate changes in relative mRNA expression levels (35). The primer sequences used were as follows: TAZ, forward 5'-GTCACCAACAGTAGCTCAGATC-3' and reverse 5'-AGT GATTACAGCCAGGTTAGAAAG-3'; β-actin, forward 5'-AC TCTTCCAGCCTTCCTTCC-3' and reverse 5'-CGTCATACT CCTGCTTGCTG-3'.

A total of 20 six-week-old male athymic nude BALB/c mice (weight, 16-18 g) purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China) were housed under specific pathogen-free conditions (24-26°C; 12-h light/dark cycle; free access to food and water). The mice were subcutaneously inoculated with MCF-7 cells (1×10⁷) resuspended in 0.1 ml DMEM. When the tumor size reached 100 mm³, the mice were randomly divided into four groups (n=5/group) to receive one of the following treatments: Normal saline (NS; intraperitoneal injection) or 20, 40 or 80 mg/kg/day of HM (5 days per week for 2 weeks, intraperitoneal injection). Tumor length (L) and width (W) were measured at 2-day intervals using a Vernier caliper, and tumor volume (V) was calculated using the following formula: V (mm³) = L (mm) × W² (mm²) × 0.5. After 4 weeks injection, all mice were sacrificed and tumor tissues were removed. Each mouse bore a single tumor, the maximum diameter of a single subcutaneous tumor reached 15.98 mm, the maximum tumor volume reached 1,189 mm³. Tumor tissues from the mice were dissected for western blotting, RT-qPCR analysis and immunohistochemistry (IHC). The animal experiments were approved by the Committee on the Ethics of Animal Experiments of the Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China; IACUC no. 837).

IHC was performed to detect the levels of TAZ, Bcl-2, Bax, p-Erk and p-Akt in mouse tumor tissues. The samples were embedded in paraffin and sliced into thin sections (5 μm). The sections were dewaxed in xylene and sequentially rehydrated with 100, 95, 80 and 75% ethanol. The slides were blocked with BSA for 2 h at room temperature. The slides were incubated with primary antibodies anti-TAZ, Bcl-2, Bax, p-Erk and p-Akt (1:200) overnight at 4°C. Following washing three times with PBS for 10 min each time, biotinylated goat anti-rabbit secondary antibody (1:200; cat. no. A0279; Beyotime Institute of Biotechnology) was incubated for 1 h at 37°C, and diaminobenzidine tetrachloride (cat. no. P0203; Beyotime Institute of Biotechnology) was used for staining at 37°C for 10 min. Data were analyzed using a light microscope (Olympus Corporation).

Statistical analysis was performed by Student's t-test for comparison between two groups or one-way ANOVA for comparison between more than two groups. The least-significant difference post hoc test was used following ANOVA. All statistical analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The anti-proliferative effect of HM on breast cancer cells was evaluated via CCK-8 assay. The MDA-MB-231 and MCF-7 cells were treated with different concentrations of HM for 24, 48 and 72 h, respectively. As shown in Fig. 2A, HM inhibited cell proliferation in a dose- and time-dependent manner. In addition to inhibiting cell proliferation, HM suppressed the migration of breast cancer cells. The MDA-MB-231 and MCF-7 cells were exposed to different concentrations of HM for 24 h, as shown in Fig. 2B, and the results revealed a dose-dependent inhibition of cell migration. Apoptotic morphological changes were determined by DAPI staining. Chromatin condensation and nuclear fragmentation, which are characteristics of apoptosis, were evident in the MDA-MB-231 and MCF-7 cells treated with HM (50, 100 or 150 μM) for 24 h. As shown in Fig. 2C, a significant increase of chromatin condensation and nuclear fragmentation was observed in the HM-treated cells, when compared with that of control (no HM added) cells. To further confirm the pro-apoptotic effect of HM, cell apoptosis was determined by flow cytometry using a Annexin V apoptosis detection kit. Following treatment of the MDA-MB-231 cells with HM at 50, 100 or 150 μM for 24 h, the apoptotic population increased from 5.7 to 26.7% in a dose-dependent manner. Similar results were observed in MCF-7 cells (Fig. 2D). In combination, these results indicated that HM had an anticancer effect on the breast cancer cell lines.

Increasing studies have suggested that TAZ is an oncogene in human cancer cells and serves an important role in the development of tumors (17). It has been reported that TAZ is expressed at high levels in breast cancer cell lines, promotes cell proliferation, migration and tumorigenesis, and inhibits apoptosis in breast cancer (29,30,34). To further understand the mechanism underlying the anticancer activities induced by HM, the present study examined whether TAZ was involved in the anticancer mechanism. The relative mRNA expression of TAZ was significantly decreased, compared with that in the control group (Fig. 3A). Following 48 h of HM treatment (50, 100 or 150 μM), the protein expression of TAZ was reduced in the MDA-MB-231 and MCF-7 cells. The activation of MAPK kinase/ERK and PI3K/AKT has been reported to regulate cancer cell proliferation and migration through various pathways (36,37). In the present study, proliferation- and migration-related proteins, including p-Erk and p-Akt, were decreased, whereas anti-apoptotic protein Bcl-2 was decreased and pro-apoptotic Bax was increased (Fig. 3B).

In order to validate that HM-induced anticancer activity via targeting TAZ, the effect of increased TAZ on HM-mediated anticancer activity was measured. The MDA-MB-231 and MCF-7 cells were transfected with either a TAZ-expressing plasmid (pCMV6-TAZ) or a control plasmid (pCMV6-control). At 2 days post-transfection, western blotting was performed to check the transfection efficiency of TAZ, the expression of TAZ was increased in the TAZ overexpression group (Fig. 4A). The expression level of TAZ in the TAZ overexpression group under HM treatment (50, 100 or 150 μM) was decreased in a dose-dependent manner (Fig. 4B). The transfected cells were treated with HM (50, 100 or 150 μM) for 48 h. When compared with negative control cells, the overexpression of TAZ in breast cancer cells was found to prevent the HM-induced inhibition of cell proliferation and migration (Fig. 4C and D). HM-induced cell apoptosis was also inhibited by the overexpression of TAZ (Fig. 4E).

The MCF-7 cells were subcutaneously injected into athymic nude mice to establish a breast xenograft tumor model, and tumor growth was recorded. Different doses of HM (20, 40 or 80 mg/kg/day) were administered, and HM was shown to reduce tumor volume and weight in a dose- and time- dependent manner (Fig. 5A-D). Tumor tissues were used to evaluate the level of TAZ by IHC, RT-qPCR and western blot analyses. It was found that HM decreased the level of TAZ, compared with that in the control group (Fig. 5E-G). The IHC results showed that the proliferation and expression of metastasis-related proteins, including p-Akt and p-Erk, were decreased compared with the control group (Fig. 5H). These results indicated that HM inhibited the development of breast cancer in vivo.