Paclitaxel inhibits breast cancer metastasis via suppression of Aurora kinase-mediated cofilin-1 activity.

The main problem in breast cancer treatment is the recurrence of tumor growth and metastases. Previous studies have suggested that Paclitaxel is widely used to treat various cancers. The present study analyzed the potential signaling pathway of Paclitaxel-inhibited breast cancer metastasis. It was demonstrated that Paclitaxel treatment significantly inhibited growth of breast cancer cell lines including MCF-7 and SKBR3 cells. Results demonstrated that Paclitaxel significantly inhibited breast cancer cell migration and invasion. Results additionally demonstrated that Paclitaxel treatment suppressed Aurora kinase and cofilin-1 activity in breast cancer cells. The potential mechanism indicated that activation of Aurora kinase activity stimulated cofilin-1 activity, which canceled Paclitaxel-inhibited growth and aggressiveness of breast cancer cells. An in vivo assay revealed that Paclitaxel treatment significantly inhibited breast cancer growth. Immunohistochemistry demonstrated that Paclitaxel treatment increased apoptosis of tumor cells in tumor tissue. Notably, Aurora kinase and cofilin-1 activity were downregulated by Paclitaxel in tumor tissues. In conclusion, these results indicated that Paclitaxel inhibited breast cancer cell growth and metastasis via suppression of Aurora kinase-mediated cofilin-1 activity, suggesting Paclitaxel may be an efficient anticancer agent for the treatment of this disease.

Introduction

Metastatic breast cancer is one of the most common metastatic tumors, which is threaten to women' health in the world (1). A systematic review has highlighted the relationship between migration and female breast cancer in across races and ethnicities (2). Genetic counseling and testing for inherited gene mutations in newly diagnosed patients with breast cancer has been also researched in a review of the existing literature and a proposed research agenda (3). Clinical investigations have indicated that the 5-year overall survival rate of patients with breast cancer remains poor (4–28%), and the proportion of young patients with breast cancer is increasing; a number of these patients present with metastatic disease at the time of diagnosis (4,5). Human breast carcinoma remains higher mortality rate due to local invasion and distant metastasis and more than 30% breast carcinoma patients will develop metastasis during the evolution of their disease (4,5). Therefore, efficient treatments for breast cancer are essential for patients in clinic.

Combinations of active antineoplastic agents have been the most effective treatment for metastatic breast cancer. Paclitaxel is a tricyclic diterpene compound that has been regarded as efficient and safety neoadjuvant chemotherapy for human breast cancer (6,7). Paclitaxel is used to treat breast cancer since 1994 (8). Reports have indicated that paclitaxel presents better therapeutic effects for human cancer by inhibiting tumor growth and inducing apoptosis (9–11). Combination therapy nanoparticle albumin-bound paclitaxel and cyclophosphamide presented therapeutic effects for patients with metastatic or recurrent breast cancer (12). In recent years, overexpression of cofilin-1 suppressed growth and invasion of lung and prostate cancer cells (13,14). In addition, previous study proposed that Aurora kinase may be used as a target for the treatment of human breast cancer (15). However, the relationships among paclitaxel, cofilin-1 and Aurora kinase have not been investigated in human breast cancer cells.

In this study, we investigated the inhibitory effects of Paclitaxel for growth and aggressiveness of MCF-7 and SKBR3 cells. Although previous reports have showed the efficacy of Paclitaxel strategy for breast cancer treatment (16–18), the importance of Aurora kinase in Paclitaxel-inhibited growth of breast cancer cells have not been completely reported yet. Findings in this study suggest that Paclitaxel could suppress growth and aggressiveness of breast cancer cells through regulation of Aurora kinase-mediated cofilin-1 activity.

Materials and methods

Ethics statement

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.

Cells culture

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.

MTT assay

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.

Transfection of small interference RNA (Si-RNA)

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.

Overexpression of Aurora kinase

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.

Apoptosis assay

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.

Aurora kinase and cofilin-1 activity

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).

Cell invasion and migration assays

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.

Western blot analysis

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).

Colony formation assay

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.

Animal study

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.

Immunohistochemistry

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).

Statistical analysis

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.

Results

Paclitaxel treatment inhibits breast cancer cells growth and promotes apoptosis of breast cancer cells

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.

Figure 1.

Paclitaxel treatment significantly inhibits growth and promotes apoptosis of breast cancer cells. (A and B) Paclitaxel treatment significantly suppressed MCF-7 (A) and SKBR3 (B) cells breast cancer cells growth in dose-dependent manner (0. 2, 4, 6 mg/ml). (C and D) Paclitaxel treatment significantly suppressed MCF-7 (C) and SKBR3 (D) cells breast cancer cells growth in time-dependent manner (24, 48 and 72 h). (E) Paclitaxel induces apoptosis of MCF-7 and SKBR3 cells. (F) Paclitaxel treatment markedly inhibits tumor colony forming units. All data were expressed as mean ± SD of three independent experiments using Student's t-test, one-way ANOVA or Kruskal-Wallis test. **P<0.01 vs. control group. ##P<0.01 4 mg/ml vs. 2 mg/ml; $$P<0.01 6 mg/ml vs. 2 mg/ml; ^^P<0.01 48 h vs. 24 h post-treatment; &&P<0.01 72 h vs. 48 h post-treatment.

Paclitaxel treatment significantly suppresses aggressiveness 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.

Figure 2.

Paclitaxel treatment suppresses aggressiveness of breast cancer cells. (A) Paclitaxel treatment suppresses migration of MCF-7 and SKBR3 cells. (B) Paclitaxel treatment suppresses invasion of MCF-7 and SKBR3 cells. All data were expressed as mean ± SD of three independent experiments using Student's t-test or one-way ANOVA or Kruskal-Wallis test. **P<0.01 vs. control group.

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.

Figure 3.

Paclitaxel treatment significantly suppresses Aurora kinase and cofilin-1 expression levels and activity in breast cancer cells. (A and B) Paclitaxel treatment significantly suppresses Aurora kinase and cofilin-1 expression levels in MCF-7 (A) and SKBR3 (B) cells. (C and D) Paclitaxel treatment downregulates Aurora kinase (C) and cofilin-1 (D) activity in MCF-7 and SKBR3 cells. All data were expressed as mean ± SD of three independent experiments using Student's t-test or one-way ANOVA or Kruskal-Wallis test. **P<0.01 vs. control group.

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.

Figure 4.

Paclitaxel treatment regulates growth and aggressiveness of breast cancer cells through Aurora kinase-mediated cofilin-1 activity. (A) Knockdown of Aurora kinase (Si-AKN) suppresses Aaurora kinase expression in MCF-7 and SKBR3 cells. (B) Overexpression of Aurora kinase (pAKN) promotes Aaurora kinase expression in MCF-7 and SKBR3 cells. (C) Knockdown of Aurora kinase (Si-AKN) decreases cofilin-1 expression in MCF-7 and SKBR3 cells. (D) Overexpression of Aurora kinase (pAKN) promotes cofilin-1 expression in MCF-7 and SKBR3 cells. (E) Si-AKN enhances the inhibitory effects of Paclitaxel on growth of MCF-7 and SKBR3 cells. (F) pAKN cancels Paclitaxel-inhibited growth of MCF-7 and SKBR3 cells. (G and H) Si-AKN significantly inhibits migration (G) and invasion (H) of MCF-7 and SKBR3 cells. (I and J) pAKN transfection cancels Paclitaxel-inhibited migration (I) and invasion (J) of MCF-7 and SKBR3 cells. All data were expressed as mean ± SD of three independent experiments using Student's t-test or one-way ANOVA or Kruskal-Wallis test. *P<0.05, **P<0.01 vs. control group. NS, no significant difference.

Paclitaxel treatment inhibits breast cancer cells growth in MCF-bearing mice in vivo

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.

Figure 5.

Paclitaxel treatment inhibits breast cancer cells growth in MCF-bearing mice in vivo. (A) Paclitaxel treatment significantly inhibits breast tumor growth compared to control group. (B) Paclitaxel treatment significantly increases numbers of apoptotic cells in tumor tissues. (C) Paclitaxel treatment downregulates expression levels Aurora and cofilin-1 in tumor tissues. (D) Paclitaxel treatment decreases Aurora and cofilin-1 activity in tumor tissues. All data were expressed as mean ± SD of three independent experiments using Student's t-test or one-way ANOVA or Kruskal-Wallis test. **P<0.01 vs control group.

Discussion

More and more women were diagnosed with breast cancer each year, who will receive breast-conserving surgery followed by adjuvant radiotherapy, chemotherapy and/or comprehensive treatments. It is crucial to analyze the potential mechanism(s) that are associated with the local invasion and distant metastasis to improve 5-year overall survival and the prognosis of breast carcinoma patients (25,26). Many reports have indicated that Paclitaxel can inhibit breast tumor cells growth, proliferation and metastasis (27–29). In this study, inhibitory effects of Paclitaxel for breast cancer cells were investigated both in vitro and in vivo. We conducted a series of experiments to analyze Paclitaxel-mediated inhibition of breast cancer cells growth and aggressiveness. Findings in this study indicated that Paclitaxel treatment inhibited growth and aggressiveness of breast cancer cells through Aurora kinase-mediated cofilin-1 activity.

Paclitaxel is an efficient anticancer chemical drug. Paclitaxel-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood (30). We found that Paclitaxel could significantly induced apoptosis of breast cancer cell line MCF-7 and SKBR3. Anti-tumor efficiency of paclitaxel and DNA when co-delivered by pH responsive ligand modified nanocarriers for breast cancer treatment has been investigated in previous report (31). Woo et al (32) demonstrated that paclitaxel can induce apoptotic cells death in human glioblastoma U87MG cells via regulation of apoptotic functions of p53 and c-Jun N-terminal kinase (JNK). Further, the combination of thymoquinone and paclitaxel shows antitumor activity through the interplay with apoptosis network in triple-negative breast cancer, which presented Pac-mediated cytotoxicity for breast cancer cells (33). Outcomes have demonstrated that Paclitaxel could significantly inhibit growth and aggressiveness of breast cancer cell line MCF-7 and SKBR3 in dose-dependent manner (<4 mg/ml), which could achieve the maximum cytotoxicity for breast cancer cells after 72-h incubation.

Aurora kinase mediates growth of breast cancer cells. Long et al (34) reported that adenovirus-mediated aurora A shRNA driven by stathmin promoter suppressed tumor growth and enhanced paclitaxel chemotherapy sensitivity of human breast carcinoma cells. Inhibiting Aurora kinases contributes to inhibition of tumor growth and suppression of tumor recurrence after chemotherapy in patient-derived triple-negative breast cancer xenografts (35). The results in the present study showed that Paclitaxel decreased expression and activity of Aurora kinase, which provided additional insights into the mechanism responsible for Paclitaxel-inhibited breast cancer cells growth and aggressiveness. However, further study need to be investigated the potential mechanism(s) mediated by Paclitaxel in various tumor cells.

Tahtamouni et al (36) have showed that non-overlapping activities of ADF and cofilin-1 were observed during the migration of metastasis of breast tumor cells. In addition, researches have suggested that cofilin-1 and EGFR pathways involve in cisplatin resistance of non-small-cell lung cancer and over-expression of cofilin-1 could suppress growth and invasion of cancer cells (13,14,37). Our study demonstrated the importance of including Aurora kinase-mediated cofilin-1 in the progression of breast cancer. Knockdown of Aurora kinase expression decreased cofilin-1 activity that further enhanced Paclitaxel-inhibited breast cancer cells growth and aggressiveness, presumably led to in vivo tumor growth inhibition in tumor-bearing mice. However, more histological analysis should be performed in breast tumor tissues our future work.

In summary, the current study observed that Paclitaxel significantly inhibited growth and aggressiveness as well as promoted apoptosis of breast cancer cell line MCF-7 and SKBR3. Notably, this study found that Paclitaxel treatment could regulate growth and aggressiveness of breast cancer cells through downregulation of Aurora kinase-mediated cofilin-1 activity. These findings indicate that Paclitaxel is an efficient anticancer agent, suggesting Aurora kinase-mediated cofilin-1 may be potential target for the treatment of breast cancer.