The present study was performed by the recommendations in the Guide for the Care and Use of Laboratory Animals of China. All the animal's protocols were experimented in accordance with National Institutes of Health and approved by Committee on the Ethics of Animal Experiments Defence Research.

MCF-7 (ATCC^® no. HTB-22™) and SKBR3 (ATCC^® no. HTB-30™) cells were purchased from American Type Culture Collection (ATCC). The cells were cultured in DMEM (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were cultured in a 37°C humidified atmosphere of 5% CO₂.

MCF-7 and SKBR3 were incubated with Paclitaxel (0, 2, 4 or 6 mg/ml, CAS no. 33069-62-4, LGM Pharma) in 96-well plates for 24, 48 and 72 h in triplicate for each condition with PBS as control. After incubation, 20 µl of MTT (5 mg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) in PBS solution was added to each well, the plate was further incubated for 4 h. The majority of the medium was removed and 100 µl of DMSO was added into the wells to solubilize the crystals. The OD was measured using a BIO-RAD (ELISA) microplate reader at wavelength of 450 nm.

All siRNAs were synthesized by Invitrogen including Si-RNA-Aurora kinase (Si-AKN) or Si-RNA-vector (Si-vector). MCF-7 or SKBR3 cells (1×10⁶) were transfected with 150 pmol of Si-AKN targeting Aurora kinase (Applied Biosystems Life Technologies, Foster City, CA, USA) with Si-vector as control (Applied Biosystems Life Technologies) by using a Cell Line Nucleofector kit for cell Lines (VCA-1003; Lonza, Basel, Switzerland) (19). Si-AKN-transfected MCF-7 or SKBR3 cells were treated with Paclitaxel (4 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 24 h for further analysis.

MCF-7 or SKBR3 cells were cultured until 85% confluences and the media was then removed from culture plate and washed three times with PBS. MCF-7 or SKBR3 cells were transfected by plentivirus-Aurora kinase (pAKN) using lipofectamine 2000 (cat. no. 11668-027; Invitrogen). The vectors of pAKN and p-vector were synthesized in Invitrogen; Thermo Fisher Scientific, Inc. Sable Aurora kinase-overexpression MCF-7 or SKBR3 cells were treated by Paclitaxel (4 mg/ml; Sigma-Aldrich) for 24 h to analyze the purpose protein expression levels determined by western blot analysis.

Sable AKN-overexpressed, AKN-knockdown, pvector-treated or Si-vector-treated MCF-7 and SKBR3 cells were grown at 37°C with 5% CO₂ until 90% confluence was formatted. Apoptosis assay was assessed by incubation these cells with Paclitaxel (4 mg/ml) for 48 h. After incubation, the tumor cells were trypsinized and collected. The cells were then washed in cold PBS, adjusted to 1×10⁶ cells/ml with PBS, labeled with annexin V-FITC and PI (Annexin V-FITC kit; BD Biosciences, Franklin Lakes, NJ, USA), and analyzed with a FACScan flow cytometer (BD Biosciences). The treatments were performed in triplicate, and the percentage of labeled cells undergoing apoptosis in each group was determined and calculated.

MCF-7 and SKBR3 cells as well as tumor tissues were lysed in PBS. 1.0 mg homogenate was obtained and used to analyzed Aurora kinase and cofilin-1 activity referenced previously reports (20,21).

Sable AKN-overexpressed, AKN-knockdown, pvector-treated or Si-vector-treated MCF-7 and SKBR3 cells MCF-7 and SKBR3 cells were cultured with Paclitaxel (4 mg/ml) or PBS for 48 h. Migration and invasion of MCF-7 and SKBR3 cells was performed in a 6-well culture plate with chamber inserts (BD Biosciences). For migration assays, 1×10⁴/well concentration of the MCF-7 or SKBR3 cells were placed into the upper chamber. For invasion assays, MCF-7 or SKBR3 cells (1×10⁴/well) were placed into the upper chamber with the Matrigel-coated membrane. Migration and invasion of MCF-7 or SKBR3 cells were counted in at least three randomly stain-field microscope every membrane.

MCF-7 or SKBR3 cells were harvested by scraping and lysed in RIPA buffer followed homogenized at 4°C for 10 min. Protein were analyzed by SDS-PAGE assays followed transfer membrane. Protein were incubated with rabbit anti-mouse Aurora kinase (ab2245), cofilin-1 (ab131519) or β-actin (ab5694) (Abcam, Shanghai, China) for 2 h at 37°C and then incubated with goat anti-rabbit horseradish peroxidase-conjugated anti-rabbit IgG antibodies for 24 h at 4°C. The results were visualized by using chemi-luminescence detection system (Amersham Biosciences, Piscataway, NJ, USA).

For the colony formation assay, 1×10² MCF-7 or SKBR3 cells with paclitaxel (4 mg/ml) or PBS were seeded and cultured in six-well plates for 7 days until visible colonies were formed. Numbers of colonies were calculated by the crystal violet.

Specific pathogen-free (SPF) female Balb/c mice (6–8 weeks old, body weight: 25–32 g) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. (Shanghai, China). All mice were housed at preference temperature with a 12 h light/dark cycle and fed ad libitum. Mouse breeding and experiments were carried out under IACUC approved protocols of Ethics Committee of Library Animals. Mice were subcutaneously implanted MCF-7 cells (1×10⁷ cells) and were divided into two groups (n=20). Tumor treatments were initiated on day 5 after tumor implantation (diameter: 5–6 mm). Tumor-bearing mice were received intratumoral injection of Paclitaxel (4 mg/ml) or PBS. The tumor volumes were calculated according to previous study (22). The treatment was continued 7 times once a day.

Tumor tissues from xenografted mice were fixed by using 10% formaldehyde and embedded in paraffin wax. Tumor tissues were cut into serial sections of 4-µm thickness. Tumor samples were fabricated to tumor sections and antigen retrieval was also performed in tumor sections. Tumor sections were incubated with rabbit anti-mouse Aurora kinase (ab2245) or cofilin-1 (ab131519) antibody for 24 h at 4°C. Tumor tissues were washed with PBS three times and incubated with goat anti-rabbit horseradish peroxidase-conjugated anti-rabbit IgG antibodies for 24 h at 4°C. The results were visualized by using chemi-luminescence detection system (Amersham Biosciences).

All data were expressed as mean ± SD of triplicate dependent experiments and analyzed by using Student's t-test or one-way ANOVA (Tukey HSD test). Multiple group comparisons were made using Kruskal-Wallis test. All data were analyzed using SPSS Statistics 19.0 and Graphpad Prism v.5.0 with the help of Microsoft Excel. P<0.05 and P<0.01 were considered to indicate a statistically significant difference.

Paclitaxel is an efficient anticancer agent for breast cancer cells. We investigated the inhibitory effects of Paclitaxel on breast cancer cells growth and efficacy of inducing cell apoptosis in vitro. As shown in Fig. 1A-D, Paclitaxel treatment significantly suppressed breast cancer cells growth in dose- and time-dependent manner. Results showed that Paclitaxel could induce apoptosis of MCF-7 and SKBR3 cells (Fig. 1E). We also observed that Paclitaxel treatment markedly inhibited tumor colony forming units (Fig. 1F). These results indicate that Paclitaxel treatment could inhibit breast cancer cells growth and promotes apoptosis of breast cancer cells.

Migration and invasion of breast cancer cells was analyzed after Paclitaxel treatment. As shown in Fig. 2A, Paclitaxel treatment significantly suppressed migration of MCF-7 and SKBR3 cells compared to control. Our results demonstrated that Paclitaxel treatment inhibited invasion of MCF-7 and SKBR3 cells compared to control (Fig. 2B). These results suggest that Paclitaxel treatment could significantly suppress migration and invasion of breast cancer cells.

Paclitaxel treatment significantly suppresses Aurora kinase and cofilin-1 expression levels and activity in breast cancer cells. Aurora kinase and cofilin-1 activity is associated with tumor cells growth (23,24). Therefore, we analyzed Aurora kinase and cofilin-1 expression levels and activity in breast cancer cells. As shown in Fig. 3A and B, Paclitaxel treatment significantly suppressed Aurora kinase and cofilin-1 expression levels in MCF-7 and SKBR3 cells. We also found that Paclitaxel treatment significantly downregulated Aurora kinase and cofilin-1 activity in MCF-7 and SKBR3 cells (Fig. 3C and D). These results indicate that Paclitaxel treatment could significantly suppress Aurora kinase and cofilin-1 expression levels and activity in breast cancer cells.

Paclitaxel treatment regulates growth and aggressiveness of breast cancer cells through Aurora kinase-mediated cofilin-1 activity. In order to analyze the possible signal pathway of Paclitaxel-inhibited growth and aggressiveness, we investigated Aurora kinase-mediated cofilin-1 activity in MCF-7 and SKBR3 cells. As shown in Fig. 4A and B, knockdown of Aurora kinase (Si-AKN) suppressed Aaurora kinase expression and overexpression of Aurora kinase (pAKN) promoted Aurora kinase expression in MCF-7 and SKBR3 cells. We observed that Si-AKN decreased cofilin-1 expression, while pAKN promoted cofilin-1 expression in MCF-7 and SKBR3 cells (Fig. 4C and D). The results demonstrated that knockdown of AKN promoted the paclitaxel-mediated inhibition of MCF-7 and SKBR3 cells, whereas overexpression of AKN reversed the paclitaxel-mediated growth inhibition of these cells (Fig. 4E and F). In addition, knockdown of AKN significantly increased the paclitaxel-mediated inhibition of MCF-7 and SKBR3 cell migration and invasion, whereas AKN overexpression reversed the paclitaxel-mediated inhibition of MCF-7 and SKBR3 migration and invasion (Fig. 4G-J). These results suggest that Paclitaxel treatment regulates growth and aggressiveness of breast cancer cells through Aurora kinase-mediated cofilin-1 activity.

We further investigated in vivo efficient of Paclitaxel in a model of MCF-7-bearing mice. As shown in Fig. 5A, Paclitaxel treatment significantly inhibited breast tumor growth compared to control group. Results demonstrated that numbers of apoptotic cells were significantly increased in tumor tissues treated by Paclitaxel compared to control (Fig. 5B). Immunohistochemistry showed that expression levels of Aurora kinase and cofilin-1 were significantly downregulated by Paclitaxel treatment in tumor tissues (Fig. 5C). Notably, results also demonstrated that Paclitaxel treatment also downregulated Aurora and cofilin-1 activity in tumor tissues (Fig. 5D). These results suggest that Paclitaxel treatment could inhibit breast cancer cells growth in MCF-bearing mice in vivo.