Anlotinib inhibits the proliferation, migration and invasion, and induces apoptosis of breast cancer cells by downregulating TFAP2C

Cell culture and treatment

The human breast cancer cell line MCF-7 was purchased from the American Type Culture Collection and cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin at 37°C with 5% CO₂ (all from Gibco; Thermo Fisher Scientific, Inc.).

Cell transfection

The full length of the TFAP2C sequence was cloned into the pcDNA3.1 vector by Gene Script Biotech Co., Ltd. to construct 2 µg TFAP2C overexpression vector (Ov-TFAP2C). The empty vector pcDNA3.1 (2 µg) was used as a negative control (Ov-NC). Cells were cultured overnight to reach 60–70% confluence before transfection. The plasmid transfection was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cells were incubated with 5% CO₂ at 37°C and were used in subsequent experiments 48 h post-transfection.

Cell viability measurement

Cell viability was measured using Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) assays. Control or transfected MCF-7 cells were seeded in 96-well plates at 5×10³ cells/well and incubated at 37°C for 24 h. The cells were the treated with different concentrations of Anlotinib (0, 2, 4 and 6 µM; >99%; Selleck Chemicals) at 37°C for 24 h. A total of 10 µl CCK-8 solution was added to each well for 2 h of culture at 37°C. Cell viability was detected using a microplate reader (Bio-Rad Laboratories, Inc.) at the absorbance of 450 nm.

Colony formation assay

For colony formation assays, control or transfected MCF-7 cells (1×10³ cells) were resuspended in 1 ml medium, then cultured in 24-well plates overnight. The attached cells were treated with Anlotinib (0, 2, 4 and 6 µM) at 37°C for 24 h, then cultured in complete RPMI-1640 medium for an additional 2 weeks. After fixation with 20% methanol for 30 min at room temperature, 0.5% crystal violet was used to stain cells for 5 min at room temperature and the number of colonies with >50 cells was counted manually using a light microscope (magnification, ×100).

Wound healing assay

Control or transfected MCF-7 cells were seeded onto 6-well plates (4×10⁵ cells/well) and cultured in normal conditions to 70–80% confluence. The cell surface was scratched with a 100-µl pipette tip to create an artificial wound, and the medium was replaced with serum-free RPMI-1640 containing Anlotinib (0, 2, 4 and 6 µM) and culturing for 24 h at 37°C. The cell migration was observed with images captured in each group at 0 and 24 h using a light microscope (magnification, ×100; Olympus Corporation). The cell migration rate was calculated using the following equation: (Initial width at 0 h - final width at 24 h)/Initial width at 0 h. The relative migration rate was obtained by normalizing to the control group.

Transwell assay

For Transwell analysis, an 8-µm Transwell chamber (Corning, Inc.) was coated with 250 mg/ml Matrigel™ (BD Biosciences) at room temperature for 24 h (18–20), then placed into a 24-well plate. The upper chambers were seeded with control or transfected MCF-7 cells suspended in 200 µl serum-free DMEM at a density of 1×10⁵ following Anlotinib treatment. RPMI-1640 medium containing 10% FBS was added to the lower chamber for 24 h of incubation at 37°C. Finally, the invading cells into the bottom of chamber were fixed with 4% methanol at 37°C for 10 min, then stained with 0.1% crystal violet solution at 37°C for 15 min. The number of invasive cells in five random fields were counted using a light microscope (magnification, ×100).

TUNEL staining

Cell apoptosis was detected using the TUNEL assay kit (Abcam) following the manufacturer's instructions. The control or transfected MCF-7 cells were fixed with 1% paraformaldehyde at room temperature for 15 min after Anlotinib treatment, and then treated with 70% ethanol on ice. After being exposed to the TUNEL detection reagent for 1 h at 37°C, cells were treated with 10 µg/ml DAPI (Shanghai Haoyang Bio Technology Co., Ltd.) to stain the nucleus at 37°C for 2–3 min and mounted in an anti-fade reagent (Beijing Solarbio Science & Technology Co., Ltd.). In total, three fields of view were selected at random, each with ~300–500 cells and the images were captured using a fluorescence microscope (magnification, ×100; Olympus Corporation) and analyzed using the ImageJ software (version 1.48v; National Institutes of Health).

Western blot analysis

Cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) containing protease inhibitors (Roche Diagnostics), and protein concentration was quantified using a bicinchoninic-acid assay kit (Thermo Fisher Scientific, Inc.). Equal amount of protein samples (20 µg) were separated 12% SDS-PAGE on 12% gels, then transferred onto PVDF membranes (EMD Millipore). After being blocked with 5% non-fat milk for 2 h at room temperature (diluted with PBS-0.1% Tween-20), membranes were incubated overnight at 4°C with the primary antibodies against proliferating cell nuclear antigen (PCNA; 1:5,000, cat. no. ab29; Abcam), Ki-67 (1:1,000, cat. no. ab92742; Abcam), matrix metalloproteinase (MMP)2 (1:1,000, cat. no. ab92536; Abcam), MMP9 (1:1,000, cat. no. ab76003; Abcam), tissue inhibitor of metalloproteinase-1 (TIMP-1; 1:1,000, cat. no. ab211926; Abcam), Bcl-2 (1:2,000, cat. no. ab32124; Abcam), Bax (1:5,000, cat. no. ab32503; Abcam), cleaved caspase-3 (1:500 cat. no. ab32042; Abcam), caspase-3 (1:5,000, cat. no. ab32351; Abcam), TFAP2C (1:1,000, cat. no. ab218107; Abcam), GAPDH (1:5,000, cat. no. ab8245; Abcam) and the corresponding secondary HRP-conjugated goat anti-rabbit IgG (1:5,000, cat. no. 7074; Cell signaling technology) at room temperature for 2 h, followed by visualization with the Pierce™ enhanced chemiluminescence western blotting substrate (Thermo Fisher Scientific, Inc.) and quantitative analysis with the ImageJ software (version 1.48v; National Institutes of Health).

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers protocol. A total of 5 µg RNA was then reverse transcribed into cDNA using PrimerScript™ RT Master Mix (cat. no. RR036A; Takara Bio, Inc.). The temperature protocol was 37°C for 15 min and reaction at 85°C for 5 sec. The qPCR reactions were performed using TB Green^® Premix Ex Taq™ (cat. no. RR420A; Takara Bio, Inc.) on an ABI Prism 7500 sequence detector (Applied Biosystems, Inc.; Thermo Fisher Scientific, Inc.). The thermocycling conditions: Pre-denaturation at 95°C for 30 sec, followed by denaturation at 95°C for 10 sec and annealing at 55°C for 30 sec for 40 cycles. The specific primers used were as follows: TFAP2C forward, 5′-ATCGAAAAATGGAGGCCGGT-3′ and reverse, 5′-CGGCTTCACAGACATAGGCA-3′; PCNA forward, 5′-GTTACTGAGGGCGAGAAGCG-3′ and reverse, 5′-AAGTCTAGCTGGTTTCGGCT-3′; Ki-67 forward, 5′-CTGACCCTGATGAGAGTGAGGGA-3′ and reverse, 5′-GGACAGGTGGAGTGTGCATTA-3′ and GAPDH forward, 5′-GCAACCGGGAAGGAAATGAATG-3′ and reverse, 5′-CCCAATACGACCAAATCAGAGA-3′. Results were normalized to GAPDH expression, and the comparative Cq method (2^−ΔΔCq) was used to calculate the relative gene expression levels (21).

Statistical analysis

GraphPad Prism 8 (GraphPad Software, Inc.) was used for data analysis. Data were generated from three independent experimental repeats. The results are presented as the mean ± standard deviation. Data analysis was carried out using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Anlotinib inhibits the proliferation, migration and invasion of breast cancer cell line MCF-7 in a concentration-dependent manner

Different concentrations of Anlotinib (0, 2, 4 and 6 µM) were used to treat MCF-7 cells, and cell viability, colony formation and PCNA and Ki-67 protein expression were assessed. As shown in Fig. 1A, cell viability was significantly reduced following 2, 4 and 6 µM Anlotinib treatment. Moreover, the protein expression of PCNA and Ki-67 was significantly reduced by Anlotinib (Fig. 1B). Anlotinib also significantly reduced the number of colonies in MCF-7 cells (Fig. 1C). Furthermore, wound healing and Transwell assays were used to evaluate the effect of Anlotinib on cell migration and invasion, respectively. As demonstrated in Fig. 1D and E, both the cell migration and invasion rate were significantly inhibited by Anlotinib. Moreover, Anlotinib resulted in a significant decrease in MMP2 and MMP9 expression levels and a significant increase in TIMP-1 expression, further indicating an inhibitory effect on cell migration (Fig. 1F). Thus, Anlotinib exerted a concentration-dependent inhibitory effect on MCF-7 cell proliferation, migration and invasion.

Anlotinib promotes the apoptosis of MCF-7 breast cancer cells

Subsequently, the effect of Anlotinib on MCF-7 cells apoptosis was evaluated using TUNEL staining and western blot assays. As shown in Fig. 2A, cells exposed to 2, 4 and 6 µM Anlotinib exhibited a higher frequency of TUNEL-positive (apoptotic) cells compared with control (0 µM) cells, indicating that Anlotinib could promote apoptosis. This was further evidenced by the decrease in Bcl-2 expression, as well as the increase in Bax expression and cleaved-caspase-3/caspase-3 ratio following treatment with 2, 4 and 6 µM Anlotinib (Fig. 2B).

TFAP2C overexpression partially blocks the effect of Anlotinib on MCF-7 cell proliferation, migration and apoptosis

Next, the effect of Anlotinib on TFAP2C expression was determined. As shown in Fig. 3A and B, both the mRNA and protein expression levels of TFAPC2 were reduced in response to Anlotinib treatment, and the effect was concentration-dependent.

TFAP2C was overexpressed in MCF-7 cells (Fig. 3C and D) to explore the changes in the effect of Anlotinib on MCF-7 cells proliferation, migration and apoptosis. Cell viability was higher in Anlotinib + Ov-TFAPC2 group compared with that in the Anlotinib + Ov-NC group (Fig. 3E). In addition, Anlotinib inhibited the expression of PCNA and Ki-67, whereas TFAP2C overexpression partially rescued PCNA and Ki-67 expression (Fig. 3F and G). Similarly, the decreased number of colony-forming units caused by Anlotinib was also significantly increased by TFAP2C overexpression (Fig. 3H). These data revealed the protective effects of TFAP2C overexpression against Anlotinib-induced reduction in MCF-7 cell viability. Finally, TFAP2C-overexpressing MCF-7 cells were exposed to 4 µM Anlotinib, then cell migration, invasion and apoptosis were assessed. As shown in Fig. 4A and B, the reduced ability of migration and invasion caused by Anlotinib was reversed following TFAP2C overexpression. Consistently, TFAP2C overexpression also blocked the effect of Anlotinib on MMP2, MMP9 and TIMP-1 expression (Fig. 4C). Furthermore, TFAP2C overexpression significantly reversed the effect of Anlotinib on the frequency of apoptotic cells, as well as on the protein expression of Bcl-2, Bax and the cleaved-caspase-3/caspase-3 ratio (Fig. 5A and B).