The MCF-7 breast cancer cell line was obtained from American Type Culture Collection (Manassas, VA, USA). RPMI-1640 culture medium, bovine serum albumin (BSA) and fetal bovine serum (FBS) was obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). PF was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The Cell Counting kit (CCK)-8 assay was purchased from Peptide Institute, Inc. (Osaka, Japan), the transwell chamber system was obtained from Corning Incorporated (Corning, NY, USA), the Nanodrop2000 nucleic acid protein detector was purchased from Thermo Fisher Scientific, Inc., the RNA extraction reagent, TRIzol^®, was purchased from Invitrogen; Thermo Fisher Scientific, Inc., the SYBR Green Mixture was purchased from Toyobo Life Science (Osaka, Japan), and the real-time fluorescence quantitative reverse transcription-quantitative polymerase chain reaction (RT-qPCR) kit was obtained from Takara Bio, Inc., (Otsu, Japan). The gel imaging system and the ViiA7 Real-Time PCR System were obtained from Applied Biosystems; Thermo Fisher Scientific Inc., the total protein extraction kit was purchased from BestBio (Shanghai, China), and the Coomassie Brilliant Blue protein assay kit was obtained from Shanghai Meiji Biological Technology Co. Ltd. (Shanghai, China). In addition, the SDS-PAGE gel, phosphate-buffered saline plus 0.1% Tween-20 (PBST) solution (Nanjing Senbeijia Biological Technology Co., Ltd., Nanjing, China), vertical polyacrylamide electrophoresis apparatus and the GIS-2020D gel imaging analysis system were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Antibodies for NOTCH-1 (cat. no. ab52627), Hes family basic helix-loop helix transcription Factor (HES)-1 (cat. no. ab108937), β-actin (cat. no. ab8226) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. ab6721) were purchased from Abcam (Cambridge, MA, USA).

The MCF-7 cells were cultured in RPMI-1640 medium supplemented with streptomycin and penicillin (1,000 IU/l) and 10% FBS. The cells were then placed in an incubator at 37°C with 5% CO₂, and the cells were passaged once every 2–3 days. During passaging, the culture medium in the culture dish was discarded and the cells were washed with PBS twice. Following incubation of cells with trypsin for 1 min, the adherent cells were gently pipetted into single cell suspensions, which were then centrifuged at 500 × g at room temperature for 5 min. The supernatants were subsequently discarded and the cell pellet was re-suspended in 1 ml medium; 200 µl of cell suspension was used for subculture.

Cells growing in the logarithmic phase were digested and counted, then 1×10⁴ cells/well were transferred to a 96-well plate. The cells were allowed to adapt to the culture environment for 1 h, before they were treated with 0, 7.5, 15 and 30 µM PF for 24, 48 and 72 h. For each dose, there were five replicate wells, and a blank control group was included in each assay. Following the treatment intervention, the cells were washed with PBS three times before 100 µl CCK-8 mixture (1:10, CCK-8 reagent: medium) was added to the wells, which were then incubated in a 5% CO₂ incubator at 37°C for 2 h. The absorbance (A) values were measured at a wavelength of 450 nm using a microplate reader, and the rate of cell survival was calculated using the following formula: Cell survival rate=(A_{treated group}-A_{blank control group})/(A_{control group}-A_{blank control group}).

A total of 50 mg/l Matrigel was diluted in serum-free medium at a ratio of 1:8. The inner surface of the membrane at the bottom of the transwell chamber was coated with the Matrigel matrix mixture, and the chamber was placed in an incubator for 30 min at 37°C before it was transferred to a safety cabinet for air-drying overnight. The chamber was blocked with serum-free RPMI-1640 medium containing 10 g/l BSA for 30 min. MCF-7 cells were serum-starved for 24 h before they were digested, collected and centrifuged at 500 × g for 5 min at room temperature. The supernatant was discarded and the cell pellet was washed with PBS twice before the cells were re-suspended in serum-free RPMI-1640 medium containing 10 g/l BSA. The cell concentration was adjusted to 5×10⁷ cells/l. A total of 200 µl cell suspension was added into the lower transwell chamber, and 600 µl RPMI-1640 culture medium supplemented with 10% FBS was added into the lower chamber. Cells were incubated for 6 h to allow them to adapt to the culture conditions before PF was added to the lower chamber at concentrations of 0, 7.5, 15 and 30 µM. A total of 5 replicate wells for each group were analyzed, and each assay included a blank control. Following the addition of PF, the plates were incubated for 48 h at 37°C. Cells that remained in the upper chamber of the transwell invasion system were removed using a cotton swab, and the wells were washed with PBS twice. The cells on the underside of the upper chamber were fixed with methanol (100%) for 15 min at room temperature, stained with 1% crystal violet for 10 min at room temperature, and then observed under an inverted microscope for analysis and counting; for each group a total of five fields were analyzed under the 400-fold microscope. A total of three independent repeats of this assay were performed.

Following treatment of MCF-7 (5×10⁵) cells with 0, 7.5, 15 and 30 µM PF for 48 h, the cells were collected and RNA was extracted using TRIzol^® reagent according to the manufacturer's instructions. The purity and content of RNA was determined using the Nanodrop 2000 protein and nucleic acid analyzer. The integrity of RNA was detected via 1% agarose gel electrophoresis. A total of 1 µg RNA was reverse transcribed to cDNA using an RNA reverse transcription kit (Takara Bio, Inc.), according to the manufacturer's protocol. The reaction mixture for the quantitative (q)PCR reaction consisted of the following: 5 µl SYBR Green Mixture (2X), 0.5 µl cDNA, 0.5 µl forward primer (10 µM), 0.5 µl reverse primer (10 µM) and 3.5 µl ddH₂O. The thermocycling conditions for the qPCR reaction were as follows: Pre-denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing and extension at 60°C for 60 sec. The following primers were used: Notch-1, forward, 5′-CACTGTGGGCGGGTCC-3′ and reverse, 5′-GTTGTATTGGTTCGGCACCAT-3; HES-1, forward, 5′-TAGCTCGCGGCATTCCAAGC-3′ and reverse, 5′-GTGCTCAGCGCAGCCGTCATCT-3′; β-actin, forward, 5′-CCACACTGTGCCCATCTACG-3′ and reverse, 5′-AGGATCTTCATGAGGTAGTCAGTCAG-3′. This was performed using the ViiA7 Real-Time PCR System. Experiments were performed in triplicate for each group and β-actin was used as the internal reference gene. The relative expression level of mRNA was determine using the by 2^−∆∆Cq method (21).

Following treatment of MCF-7 (5×10⁵) cells with 0, 7.5, 15, and 30 µM PF for 48 h, the cells were lysed in lysis buffer (20 mM Tris-HCl, pH 8.0, 100 mM KCl and 0.2 mM EDTA) and centrifuged at 2,600 × g for 30 min at room temperature. Total proteins were then extracted from the supernatant using the BestBio kit. The protein concentration was determined using the Coomassie Brilliant Blue protein assay kit, and the buffers were added into protein solutions and boiled. Protein samples (40 µg) were loaded onto a 6–12% SDS-PAGE gel and electrophoresed. Following the addition of transfer buffers, the gel was transferred to a nitrocellulose membrane. The membrane was then blocked with PBST containing 5% skim milk powder and placed on a shaker at room temperature for 2 h. PBST was used to wash the membrane three times before the primary antibodies were added and incubated on a shaker in the refrigerator at 4°C overnight. The following primary antibodies were used: NOTCH-1 (1:1,000), HES-1 (1:2,000) and β-actin (1:2,000). The membrane was then washed with PBST for 30 min and incubated for 1 h at the room temperature with the HRP-conjugated goat anti-rabbit secondary antibody (1:10,000). Following washing with PBST three times, the electroluminescence detection reagent (Thermo Fisher Scientific, Inc.) was evenly spread on the nitrocellulose membrane. The optical densities of NOTCH-1, HES-1 and β-actin protein bands were analyzed using the GIS-2020D gel imaging analysis system, and the ratio of NOTCH-1 and HES-1 protein band optical densities were compared with the β-actin protein band optical density to determine the expression intensities of the NOTCH-1 and HES-1 proteins.

SPSS software (version, 13.0; SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis, and normally distributed data are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used to measure the effect of PF on cell invasion capacity and the mRNA expression levels of NOTCH1 and HES-1. Repeated measures ANOVA was used to analyze the effect of PF on the proliferation of MCF-7 cells at different time points, and Mauchly's sphericity test was used to validate the results. The post hoc Dunnett's test was used for multiple comparisons. GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to generate the graphs. P<0.05 was considered to indicate a statistically significant difference.

MCF-7 cells were treated with increasing concentrations of PF at various time points, and the effect of PF on the proliferation of MCF-7 cells was determined using the CCK-8 assay. The results of the repeated measures ANOVA test indicated that statistically significant differences among all time points were observed (F=600.02; P<0.05). In addition, it was revealed that PF significantly inhibited the proliferation of MCF-7 cells in a time- and concentration-dependent manner (F=100.93; P<0.05; Table I). PF was observed to inhibit the proliferation of MCF-7 cells in a dose- and time-dependent manner (Table I. The inhibitory effect of 30 µM PF on the proliferation of cells was most pronounced when the cells were treated for 72 h.

MCF-7 cells were treated for 48 h with 0, 7.5, 15 and 30 µM PF, and the transwell assay system was used to detect the invasion ability of the cells. The number of cells that traversed the membrane was 132.44±6.58 in the control group, whereas 102.56±5.74, 80.72±7.82 and 62.46±6.88 cells were observed to traverse the membrane following treatment with 7.5, 15 and 30 µM PF, respectively (Fig. 1). The results demonstrated that a statistically significant and dose-dependent reduction in cell invasion capabilities was observed in PF-treated cells when compared with control cells (F=99.51; P<0.05; Fig. 1).

The 0 µM PF-treated group served as a control, and the β-actin gene served as an internal reference gene for the analysis of NOTCH-1 and HES-1 mRNA levels. Following treatment with PF for 48 h, the mRNA levels of NOTCH-1 and HES-1 were determined using RT-qPCR. Different concentrations of PF significantly decreased NOTCH-1 and HES-1 mRNA expression (NOTCH-1, F=29.13, P<0.05; HES-1, F=29.97, P<0.05). When compared with the results of the control group, the mRNA expression levels NOTCH-1 and HES-1 decreased in a dose-dependent manner, and were significantly different in the 15 and 30 µM PF groups (P<0.05). No significant differences were observed between treatment with 7.5 µM PF and the control (0 µM PF) group (Fig. 2).

Following treatment of MCF-7 cells with 0, 7.5, 15 and 30 µM PF for 48 h, the protein expression levels of NOTCH-1 and HES-1 were detected via western blotting. The results are presented in Table II and Fig. 3. The pattern of NOTCH-1 and HES-1 protein expression following PF treatment was similar to the pattern of mRNA expression. The protein expression levels of NOTCH-1 and HES-1 decreased with the increasing concentrations of PF (Table II; Fig. 3). A statistically significant difference in NOTCH-1 (t=22.15; P<0.05) and HES-1 (t=19.81; P<0.05) protein expression was observed at all concentrations (7.5, 15 and 30 µM) of PF tested when compared with the untreated controls (0 µM PF; Table II).