Human breast cancer cell line MCF-7 was provided by the School of Pharmacy, Jilin University (Jilin, China). MCF-7 was incubated in RPMI 1640 medium, which contained 10% fetal bovine serum, 100 U/l penicillin and 100 mg/ml streptomycin, at 37°C in 5% CO₂. Groups of cells were treated with TPI (0, 10, 50, 100, 200, 400 nmol/l) for 12, 24 and 48 h. TPI was purchased from Preferred Biological Co. (16091402). All other reagents, including 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), were purchased from Sigma Chemical, Co. (St. Louis, MO, USA), unless otherwise indicated.

TPI was dissolved in DMSO to make a stock solution (40 mmol/l) and diluted to various concentrations with cell culture medium. Also used were: Caspase-3 antibody (p42574), Bcl-2-associated X protein (Bax) antibody (Q07812), phospho-p44/42 MAPK (P-Erk1/2) antibody (p27361), phospho-p38 MAPK antibody (Q16539), light chain-3B (LC3B) antibody (Q9GZQ8), SQSTM1/p62 antibody (Q13501), and Beclin-1 antibody (Q14457; all from Cell Signaling Technology, Inc., Danvers, MA, USA); B-cell lymphoma 2 (Bcl-2) antibody (AF0060; Beyotime Institute of Biotechnology, Haimen, China); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (2118; Cell Signaling Technology, Inc.); goat anti-rabbit IgG H&L (ab6720; Abcam, Cambridge, UK); Hoechasa Hoechst 33258 staining kit (C0003 Beyotime Institute of Biotechnology); and an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit apoptosis detection kit (C1062; Beyotime Institute of Biotechnology); 3-methyladenine and U0126 (T1879/T6223; Targeted Molecules Corp., San Diego, CA, USA).

Grouped according to the different culture periods, 4,000-6,000 MCF-7 cells in a logarithmic growth phase were seeded to 96-well plates. After incubation for 12 h, cell culture medium containing TPI was added to the cells, after which they were further treated for 12, 24 and 48 h, respectively. The control group was added to RMPI-1640 medium with 1% DMSO. MTT (100 µl of 5 mg/ml) was added to all wells, and cells were incubated for 4 h. Formazan was dissolved by addition of 150 µl DMSO. Absorption at 490 nm was measured using a microplate reader. The data were representative of at least three independent tests, and the cell viability of study groups was expressed relative to the control group (relative viability).

MCF-7 cells (1.1×10⁴) were seeded in a 6-well plate. After cell adherence and rapid growth, TPI was added in different concentrations for 24 h, after which cold phosphate-buffered saline (PBS) was used to stop each treatment. Cells were lysed using RIPA buffer, which contains 1% phenylmethanesulfonyl fluoride (PMSF). Lysed cells were placed on ice, and protein was collected by centrifugation at 4°C and 12,000 × g for 15 min.

Protein concentrations were determined by Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein (30 µg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 200 mA for 150 min and transferred on to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk in Tris-buffered saline with Tween-20-buffered solution (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% Tween-20) and probed with the primary antibodies overnight at 4°C. The corresponding horseradish peroxidase-conjugated secondary antibodies were combined with the primary antibodies for 1 h. The relative protein band intensities were detected using an enhanced chemiluminescence (ECL) reagent, quantified by Image J (National Institute of Health, Bethesda, MD, USA) and Quantity One software (Bio-Rad Laboratories, Inc.). The data were representative of at least three independent tests, and a representative immunoblot is shown.

MCF-7 cells (1.1×10⁴) were seeded in a 6-well plate. After cell adherence and rapid growth, cells were treated with TPI at different concentrations for 24 h, after which cold PBS was used to stop each treatment. Cells were added to a Hoechst 33258 staining kit and incubated at room temperature for 15 min in the dark. Fluorescence microscopy was used to determine nuclear condensation and chromatin fragmentation (Olympus IX81; Olympus, Tokyo, Japan). Annexin V-FITC and propidium iodide (PI) dye were used to confirm the results.

Dead and late-apoptosis cells, which had lost membrane integrity, were stained with PI, which appears as red fluorescence. Annexin V-FITC can enter the dead cells' cytoplasm and appears as green fluorescence. The data were representative of at least three independent tests, and a representative immunoblot is shown.

After treatment at different concentrations, cells were collected and suspended in PBS. Cells (5×10⁵) were taken into tubes and suspended in binding buffer, then stained using an Annexin V-FITC/PI apoptosis detection kit (Beyotime Institute of Biotechnology) and evaluated using a flow cytometer (FACS; BD Bioscience, Sparks, Maryland, USA).

Data were expressed as the mean ± standard deviation and analyzed via one-way analysis of variance among treatment groups with Tukey's post hoc test for multiple comparisons. All data in this study were processed in this way at least three times independently. For all statistical analyses, differences were considered significant at P<0.05. Analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA).

The effect of TPI on the viability of MCF-7 cells was investigated as a function of exposure time and drug concentration. TPI reduced the viability of MCF-7 breast cancer cells in a dose- and time-dependent manner compared with the vehicle control group (Fig. 1A). When cells were treated with 50 nmol/l TPI for 12 h, the cell viability was 70%, a significant decrease compared with the vehicle group. At the maximum concentration tested (400 nmol/l), cell viability decreased to 60, 50, and 25% when treated for 12, 24, and 48 h, respectively. Treatment with 400 nmol/l TPI for 48 h resulted in marked inhibitory effects on the cell viability compared with treatment at the same TPI concentration for 12 h, indicating that the anti-cancer activity of TPI occurred in a time-dependent manner.

Consistent with the MTT assay results, the proportion of apoptotic cells gradually increased when TPI concentration was increased, which was confirmed by the statistical data from flow cytometry analysis (Fig. 1B-E). The proportions of Annexin V-FITC-positive and PI-positive cells significantly increased after TPI treatment, compared with the untreated cells. Approximately 40% of MCF-7 cells were apoptotic at 100 nmol/l, a significant increase compared with the vehicle group, in which the early apoptotic cell proportion was 17%.

When cells undergo apoptosis, caspase-3 is activated and cleaved caspase-3 expression is increased, stimulating programmed cell death (21). To evaluate apoptotic cell death in MCF-7 cells comprehensively, we carried out a western blot with apoptosis-related proteins Bax, Bcl-2 and caspase-3 as apoptosis markers. Fig. 1F shows the changes in levels of Bax, Bcl-2 and caspase-3 with different TPI concentrations. Bax and cleaved caspase-3 were significantly increased when cells were incubated with 50 nmol/l TPI for 24 h. A low concentration of TPI (10 nmol/l) markedly increased the expression of Bax; however, the trend for Bcl-2 was unchanged. These results indicate a dose-dependent mechanism for the effects of TPI on cell survival.

Having determined that TPI has toxic effects on MCF-7 breast cancer cells, we further investigated the apoptotic effects by immunofluorescence assay. Cells were treated with TPI (0, 100 and 200 nmol/l) for 24 h. Fluorescence microscopy was performed following staining with Hoechst 33258 and Annexin V-FITC/PI. The proportion of cells undergoing positive or morphological changes such as membrane shrinking was significantly increased after TPI treatment, compared with the untreated cells. Fig. 2A shows the morphological changes in visible light. Nuclear condensation and chromatin fragmentation were increased after treatment with 200 nmol/l TPI for 24 h (Fig. 2B). During the apoptosis process, phosphatidylserine is translocated to the outer layer of the plasma membrane, where it is recognized and bound by Annexin V; the complex appears as green fluorescence after activation of apoptosis. PI can pass through disordered areas of the dead cell's membrane, where it intercalates with the DNA double helix. Thus, necrotic cells will appear red and green at the same time, as shown in Fig. 2C. The above results indicate that TPI inhibits the proliferation of MCF-7 cells by inducing apoptosis.

Autophagy is a conserved process aimed at maintaining cell homeostasis under normal and stress conditions (22). Above experiments showed that TPI induced MCF-7 cell apoptosis by Bax upregulation, as well as cleaved caspase-3 activation, which indicates that TPI induces cytotoxicity through the mitochondrial pathway. Moreover, TPI-induced cell variations, such as membrane damage, organelle aging and mutant protein accumulation, are also associated with triggering autophagy.

Here, induction of autophagy was evaluated by western blotting to detect p62 protein downregulation and increased Beclin-1 and LC3B protein levels. LC3B-II can interact with p62 allowing autophagosomal engulfment of mitochondria, and subsequent degradation via fusion with the lysosome (23). Treatment with TPI at different concentrations (0, 10, 20, 50 and 100 nmol/l) for 24 h led to increased LC3B-II and Beclin-1 accumulation (Fig. 3), indicating the conversion of LC3B-I to LC3B-II. The p62 protein level decreased. The results showed that TPI induced autophagy, and indicated that this took place in a time- and dose-dependent manner. MCF-7 breast cancer cell growth may be inhibited by TPI through simultaneous induction of autophagy and programmed cell death.

As well as the effects on LC3B, p62 and Beclin-1, our western blot results showed that TPI activated Erk1/2, mammalian target of rapamycin (mTOR) and p38 MAPK signaling in MCF-7 cells, which are also related to autophagy induction (Fig. 4) (24). Erk1/2 has a critical role in cell proliferation, differentiation, skeleton construction and apoptosis. The protein levels were quantified as p-p38/p38 and p-Erk/Erk expression ratios, which were increased with increasing drug concentration, with the most marked changes found in the 100 nmol/l groups. However, we found that the phosphorylation of mTOR was decreased, which indicated that the activation of mTOR suppressed by TPI.

After co-treatment with the Erk1/2 inhibitor U0126 and TPI, p62 expression levels were higher than with TPI alone and upregulation of Bax was inhibited, indicating that activation of Erk1/2 influenced autophagy induction, as pharmacological inhibition of Erk1/2 activation attenuated TPI-induced autophagy (Fig. 4). The results indicated that Bax and p62 protein expression were affected by Erk1/2 protein.