Mouse mammary carcinoma 4T1 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Luciferase-labeled 4T1 cells (4T1-luc2) were provided by Caliper Life Sciences; PerkinElmer, Inc. (Waltham, MA, USA). Human breast cancer cells (MDA-MB-231 and MCF-7) and HUV-EC-C human umbilical vein endothelial cells were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Science (Beijing, China). RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin was used for culture of 4T1, 4T1-luc2 and MDA-MB-231 cells. MCF-7 and HUV-EC-C cells were cultured in Dulbecco's modified Eagle medium (Gibco; Thermo Fisher Scientific, Inc.). All cells were maintained in incubators at 37°C in an atmosphere of 5% CO₂ and 95% humidity. Fisetin (>98% purity), purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany), was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA), and storage solutions were prepared at a concentration of 80 mM. In all cell experiments, the final concentration of DMSO was controlled and limited to <0.1% (v/v).

Exponentially growing cells (4T1, MCF-7 and MDA-MB-231) were seeded into 96-well plates (1×10³ cells/well) and were routinely cultured for 24 h. Subsequently, 100 µl fisetin-containing medium was added to each well; the final concentrations of fisetin were adjusted to 0, 20, 40 and 80 µM. Negative groups consisting of fisetin+medium+MTT without cells and medium+MTT without cells were used as controls, to eliminate underestimation of the fisetin effect. Following incubation for 24 and 48 h, viable cells were measured via MTT assay (Sigma-Aldrich; Merck KGaA) using DMSO to dissolve the purple formazan, and optical density was measured at 570 nm using a microplate reader (Thermo Fisher Scientific, Inc.). The optical density of fisetin+medium+MTT was subtracted from values obtained when cells were present. Experiments were performed in triplicate.

ECIS is a research platform that allows real-time, quantitative, non-invasive monitoring of cell behavior. The ECIS system is composed of an eight-well cell culture array with gold-plated electrodes attached to the bottom of the wells and an impedance detection system. Due to the physical properties of ECIS, the number of attached cells is proportional to the change in resistance. Therefore, cellular behavior can be monitored in real time without interruption or labeling by recording cell-induced resistance (39,40). The ECIS array 8WCP (Applied Biophysics, Inc., Troy, NY, USA) was prepared, and electrodes were stabilized according to the manufacturer's protocol (39,40). Basic resistance data of the array were collected overnight in the Muti-Fre mode. After the curves were stable, the array was removed. Logarithmically growing 4T1 cells were exposed to fisetin in the array. The concentration of 4T1 cells was adjusted to 6×10⁴ cells/well. The final concentrations of fisetin were adjusted to 0, 20, 40 and 80 µM. Cell proliferation was determined by measuring the cell growth-induced resistance changes. Experiments were performed in triplicate.

A total of 1×10⁵ 4T1 cells/ml was added to each well of the 8W1E array (Applied Biophysics, Inc.). Once the resistance had reached a plateau, the medium was replaced with serum-free culture medium supplemented with fisetin (0, 2, 4 and 8 µM) for 6 h. Subsequently, 4T1 cells were subjected to electronic wounding, which resulted in cell death in the active electrode region and decreased cell impedance. Cells located outside of the electrode area migrated to the electrode region, which caused another rise in impedance. Wound-healing process was recorded by continuous impedance measurements for 10 h in the incubator at 37°C with 5% CO₂. The metastatic capability of the 4T1 cells was determined via an analysis of the cell growth-induced changes in resistance. Experiments were performed in triplicate.

HUV-EC-C suspension (1×10⁵ cells/well) was added to each well of the 8W1E array. Once the resistance value of HUV-EC-C reached a plateau, fisetin (0, 10, 20 and 40 µM)-treated 4T1 or fisetin (0, 20, 40 and 80 µM)-treated MDA-MB-231 cells were added to the array (1×10⁵ cells/well). HUV-EC-C cells were used to simulate an artificial vascular endothelial layer. The progress of tumor cell invasion with HUV-EC-C mimics tumor cell invasion of blood vessels in humans. Penetration of the HUV-EC-C barrier by 4T1 cells or MDA-MB-231 cells would lead to a decrease in resistance. The invasive capability of the 4T1 cells and MDA-MB-231 cells was determined by measuring the degree of decrease in the resistance value. Experiments were performed in triplicate.

Apoptosis was assessed using an Annexin V/Propidium Iodide (PI) Apoptosis Detection kit (cat. no. KGA 008; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Following treatment with fisetin (0, 20, 40 and 80 µM) for 24 h, 4T1 cells were harvested and resuspended in 500 µl binding buffer on ice. The cells were mixed thoroughly with 5 µl Annexin V/fluorescein isothiocyanate and then with 5 µl PI. Following incubation at room temperature for 15 min in the dark, apoptotic cells were examined using a flow cytometer (Beckman Coulter, Inc., Brea, CA, USA), and data was analyzed with EXPO32 Analysis Software (Version 1.1C; Beckman Coulter, Inc.). Experiments were performed in triplicate.

Following treatment of the 4T1 cells with fisetin (0, 20, 40 and 80 µM) for 24 h, proteins were extracted from the cells by lysis in radioimmunoprecipitation assay buffer (Applygen Technologies, Inc., Beijing, China) with protease inhibitor cOmplete tablets (Roche Applied Science, Penzberg, Germany) and PhosSTOP phosphatase inhibitor cocktail tablets (Roche Applied Science). Western blotting was performed as previously described (41,42). The primary antibodies, purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), were anti-mTOR (cat. no. 2983), anti-phosphorylated (p)-mTOR (cat. no. 5536), anti-Akt (cat. no. 9272), anti-p-Akt (cat. no. 9271), anti-PI3K (cat. no. 4263), anti-p-PI3K (cat. no. 13857), anti-B cell lymphoma (Bcl)-2 associated X protein (Bax; cat. no. 14796), anti-Bcl-extra large (Bcl-xL; cat. no. 2764), anti-P70 (cat. no. 2708), and anti-p-p70 (cat. no. 9234). Reference protein (β-actin) antibody (cat. no. E021020-01) was purchased from Beijing GuanXingYun Sci & Tech Co., Ltd. (Beijing, China). The membrane was incubated with above primary antibody in 5% BSA (Amresco, LLC, Solon, OH, USA) at 1:1,000, and then incubated with the Dylight™ 680- (cat. no. 072-06-15-06) or 800-labeled secondary antibody (cat. no. 072-07-18-06; KPL, Inc., Gaithersburg, MD, USA) at 1:8,000. Odyssey infrared imaging system V3.0 (LICOR Biosciences Company, USA) was used for membrane scanning. The signal intensity of each protein was measured with Image-Pro Plus Version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

All animal experiments were carried out in accordance with the National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals (43), and were approved by the Animal Ethics Committee of Beijing Hospital of Traditional Chinese affiliated with Medicine Capital Medical University (application no. 2017020201). A total of 30 female BALB/c mice (age, 6-8 weeks; weight, 18±2 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and housed under specific pathogen-free conditions at a constant temperature (20±2°C) and 40-50% humidity in a 12-h light/dark cycle with free access to food and water. The 4T1 orthotopic mammary tumor model was established as previously described (41,44). Briefly, 1×10⁴ 4T1 cells were injected into the left fourth mammary fat pad during isoflurane gas anesthesia. At 12 days following inoculation, tumor length (L) and width (W) were measured using electronic vernier caliper, and tumor volume (V) was calculated (V=LxW²/2). Mice were reordered and numbered according to tumor volume from small to large, and randomly divided into the following three groups (n=10/group) according to the block random sequence generated by SPSS 19.0 (IBM Corp., Armonk, NY, USA): Vehicle group, fisetin group and control group. The mice were administered an intraperitoneal injection of 30 µl vehicle alone (DMSO:polyethylene glycol 200, 1:4), fisetin (223 mg/kg), or normal saline every day for 3 successive weeks, respectively. At 33 d following inoculation, the number of photons emitted by the 4T1 orthotopic tumors was measured using an In Vivo Optical Imaging Spectrum system (Caliper Life Sciences; PerkinElmer, Inc.) as previously described followed the manufacturer's protocol (41,44,45). At 34 days, mice were sacrificed, and the tumors were collected and weighed.

Apoptosis was analyzed using an In Situ Cell Death Detection kit (Roche Applied Science). The 4T1 breast tumors, fixed in 4% paraformaldehyde at 4°C for 24 h, were paraffin-embedded and sectioned. Tissue sections were deparaffinized and rehydrated according to standard protocols, and then incubated for 15-30 min at room temperature with proteinase K working solution. Subsequently, the TUNEL reaction mixture was added to the tumor sections. Following incubation in a humidified container for 2 h, the sections were mounted using anti- fluorescence quenching agent (Beyotime Institute of Biotechnology, Haimen, China) and observed in five fields under a fluorescence microscope (BX-53; Olympus Corporation, Tokyo, Japan) at 200× magnification.

A blood sample (~0.8 ml) was harvested from the heart prior to sacrifice, serum was collected via centrifugation at 827 × g for 15 min at room temperature. Serum levels of alanine amino transferase (ALT), aspartate amino transferase (AST), blood urea nitrogen (BUN) and creatinine (CREA) were measured using assay kits (cat. nos. C009, C010, C013 and C011, respectively; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols.

Data were statistically analyzed using SPSS 19.0 (IBM Corp., Armonk, NY, USA) and expressed as the mean + standard deviation. Two-tailed Student's t-test was used to determine statistical differences between two groups. Comparisons among multiple groups were performed using one-way analysis of variance, with post hoc Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

To explore the anti-tumor potency of fisetin against breast cancer cells, the MTT assay was used to examine the effect of fisetin on the viability of breast cancer cells (4T1, MCF-7 and MDA-MB-231). The results demonstrated that fisetin significantly reduced the number of viable 4T1, MCF-7 and MDA-MB-231 breast carcinoma cells, compared with controls (Fig. 1). Furthermore, fisetin significantly inhibited the proliferation of 4T1 cells in a concentration- and time-dependent manner. Therefore, 4T1 cells were selected for further study.

The inhibitory effect of fisetin was measured by ECIS, cell proliferation was recorded by cell electric resistance for 48 h. Consistent with the results of the MTT assay, ECIS experiments demonstrated that fisetin significantly inhibited the proliferation of 4T1 cells in a concentration-dependent manner (Fig. 2A). The ECIS platform-based wound-healing assay allows for the assessment of metastatic capability. Following electrical wounding, the gradually increasing value of resistance reflects the dynamic migration of the healthy neighboring cells. The results demonstrated that the untreated 4T1 cells rapidly recovered to the state observed prior to electrical wounding. It was demonstrated that 4T1 cells that had been treated with 2 µM fisetin also recovered to the state prior to electrical wounding, but their recovery was less rapid than the control group. Cells that had been treated with 4 or 8 µM fisetin were unable to completely recover (Fig. 2B). These results indicate that fisetin inhibited the migration of 4T1 cells.

In the ECIS platform-based cell invasion method, HUV-EC-C cells were used to simulate the in vivo vascular endothelial layer. When tumor cells penetrated the HUV-EC-C barrier, the resistance value decreased. As the growth of the HUV-EC-C cells plateaued and a stable barrier layer was formed, the layer was challenged with 4T1 and MDA-MB-231 cells. Fig. 2C indicated that 4T1 cells were able to penetrate the HUV-EC-C barrier, which led to a decrease in resistance. 4T1 cells that had been treated with 10 or 20 µM fisetin influenced, but failed to breach, the HUV-EC-C junction. However, 4T1 cells that had been treated with 40 µM fisetin failed to induce any change in the HUV-EC-C resistance (Fig. 2C). These results suggest that fisetin reduced the invasive capability of 4T1 cells. This finding was also confirmed in MDA-MB-231 cells (Fig. 2D).

To examine the effect of fisetin on the apoptosis of 4T1 cells, cells were treated with fisetin for 24 h, followed by Annexin V/PI double staining. The apoptotic rates of 4T1 cells (early apoptosis + late apoptosis) were 10.82±4.73%, 24.28±7.92%, and 22.89±4.21% in the 20, 40, and 80 µM fisetin groups, respectively (Fig. 3A–E). Fisetin at 40 µM primarily induced early apoptosis, whereas fisetin at 80 µM primarily induced late apoptosis (apoptotic rate: 20.65±2.93%; Fig. 3F).

Western blotting was performed to examine the expression of PI3K, Akt, mTOR, P70, p-PI3K, p-Akt, p-mTOR, p-P70, Bax, and Bcl-xL proteins in 4T1 cells, in an attempt to explore the potential mechanism of fisetin intervention. The results demonstrated that treatment of 4T1 cells with fisetin significantly reduced the expression of Akt, P70, and mTOR. In addition, p-PI3K and p-PI3K/PI3K, p-Akt and p-Akt/Akt, p-P70 and p-P70/P-70 and p-mTOR were significantly decreased, Bax was significantly upregulated, and Bcl-xL was significantly downregulated following fisetin treatment in comparison with controls (Fig. 4). The very low expressions of p-mTOR and mTOR in the fisetin-treated groups may have contributed to the unexpected significant increase in the p-mTOR/mTOR ratio (Fig. 4D).

To further determine the effect of fisetin on 4T1 cells in vivo, 4T1-luc2 cells were used to establish an orthotopic-transplant model of mammary carcinoma. Significant differences were detected in tumor volume and tumor weight between the fisetin-treated and control groups, whereas vehicle demonstrated no inhibitory effect (Fig. 5A and B). Although the difference did not reach statistical significance, the number of photons emitted from the tumor was markedly reduced in the fisetin-treated group compared with the control group (Fig. 5C). The proportion of apoptotic cells was markedly increased in the fisetin-treated group (Fig. 5D). The results of in vivo experiments further demonstrated the inhibitory effect of fisetin on mammary carcinoma. However, the results also demonstrated that ALT and AST were elevated in the fisetin group (Fig. 5E and F), whereas no effect of fisetin on BUN and CREA in tumor-bearing mice was indicated (Fig. 5G and H).