Hinokiflavone induces apoptosis and inhibits migration of breast cancer cells via EMT signalling pathway

Hinokiflavone is a natural product, isolated from Selaginella P. Beauv, Juniperus phoenicea and Rhus succedanea. Even though hinokiflavone was reported to possess cytotoxicity to many cancer cells, and has potential in cancer treatment, the anti‐proliferation and anti‐metastasis efficacy of hinokiflavone on human breast cancer cells has not a further research. In this study, we investigated the anti‐cancer activity of hinokiflavone in human breast cancer cells in vitro and in vivo. Hinokiflavone exhibited a time‐ and dose‐dependent manner apoptosis induction by upregulating expression of Bax and downregulating Bcl‐2 in breast cancer cells. Furthermore, hinokiflavone significantly inhibited the migration and invasion of breast cancer cells by impairing the process of epithelial‐to‐mesenchymal transition. In addition, the tumour growth was distinctly inhibited by treatment of hinokiflavone in a xenograft tumour mouse model of MDA‐MB‐231 cells. Immunohistochemical analysis of tumour sections showed that MMP‐2+ cells and Ki‐67+ cells were remarkably decreased in tumour tissues of mice after treatment of hinokiflavone, indicating that hinokiflavone inhibits not only proliferation but also metastasis of breast cancer cells. Our study suggested that hinokiflavone can be a potential drug to breast cancer. Significance of the study Hinokiflavone significantly inhibited proliferation and induced apoptosis in breast cancer cells. In addition, hinokiflavone remarkably inhibited migration and invasion of breast cancer cells via EMT signalling pathway. It is worth noting that hinokiflavone possesses anti‐tumour effect in tumour mouse xenograft model of breast cancer. Overall, our results indicated that hinokiflavone may be a potential anticancer drug for breast cancer treatment.

still very serious, and particularly the patients who suffer from the 'triple-negative' breast cancer (TNBC), which is negative for the expressions of progesterone (PR), estrogen receptors (ER) and HER2. [3][4][5] Compared with non-TNBC, advanced TNBC has aggressive clinical progress and poor prognosis. 6 Moreover, highly malignant and metastatic of breast cancer is a principle cause of female mortality. 7 Unfortunately, there is no effective therapeutic to control the recurrence and metastasis of breast cancer. Thus, it is necessary to search for effective drug candidates with potential anti-tumour activity and low toxicity for metastatic breast cancer.
Natural products have been used to treat human diseases for thousands of years and are of great value in the discovery and development of drugs. 8,9 Many anti-cancer and anti-infectious agents are derived from natural products. 9 Furthermore, in recent decades, the rapid development of more effective drugs with fewer side effects has been a common goal for scientists and clinicians. 10 Because of its low side effects, it is crucial to identify natural disease-resistant plant compounds from medicinal plants and natural products. 11 Hinokiflavone (Figure 1), a natural product, derived from several plants such as Selaginella P. Beauv, Juniperus phoenicea and Rhus succedanea and so on, 12,13 has proved to have several biological activities, including anti-HIV-1, reverse transcriptase, 14 antiinfluenza virus sialic acid enzyme 15 and antioxidant activity. 16 Lin et al. reported that hinokiflavone exhibited antitumour activity in KB human oral cancer cells in vitro. 17 Sukesh Kalva et al. proved that hinokiflavone had a good suppressed activity against MMP2 and MMP9. 18 MMP2 and MMP9, affiliated to the matrix metalloproteinase (MMP) family, were regarded as attractive targets for various cancer therapies, which was involved in the tumour metastasis, growth and neovascularization. [19][20][21][22] However, the effects of hinokiflavone on inhibiting pulmonary metastasis of breast cancer and its related molecular mechanism have not yet been reported. Considering the effects of hinokiflavone on MMPs, we hypothesized that hinokiflavone might inhibit the migration and invasion of breast cancer. Therefore, the anti-proliferation and antimetastasis efficacies of hinokiflavone in vitro and in vivo were assessed in breast cancer cells in the current study. Furthermore, the antimetastasis mechanism of hinokiflavone was also explored.

| Reagents
Hinokiflavone as a 98% purity that detected by HPLC was bought from Chengdu Biopurify Phytochemicals Ltd (Chengdu, China).

| Cell proliferation assay
Cells were seeded in the 96-well plates and exposed with different

| Colony formation assay
Colony formation assay was conducted according to guideline. In short, MDA-MB-231 and 4T1 cells were seeded in six-well plates at 200-500 cells/well respectively. After 24 h incubation, cells were

| Western blot analysis
The western blot assay was conducted as follows. In brief, 4T1 and MDA-MB-231 cells which were treated with hinokiflavone in different concentrations for 48 h, were harvested, washed twice with cold PBS and lysed in RIPA buffer. The concentrations of total protein were measured by the Lowry method. Equal amounts of protein from each sample were loaded and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresed gels and transferred onto polyvinylidene difluoride membranes (Amersham Bioscience, Piscataway, NJ, USA). Then, the membranes were blocked by nonfat milk for 2 h at 37°C and incubated with specific primary antibodies overnight at 4°C. After incubation with the corresponding secondary antibodies, the targeted protein bands were visualized using an enhanced chemiluminescence kit (Amersham).

| Wound-healing migration assay
Wound-healing migration assay was performed as follows. Cancer cells which grew to about 90% confluence were scraped by sterile 0.1 ml pipette tips, and were cultured in fresh medium contains only 1% FBS and different concentrations of hinokiflavone. After 48 h incubation, cells were washed, fixed and photographed. Images were obtained using a microscope (Zeiss, Jena, Germany), and the inhibited percentage of migrated cells was showed using 100% as the specific value referred to untreated group.  The tumours were isolated, imaged, weighted and fixed with paraformaldehyde for further immunohistochemistry evaluation.  Colony formation assay was performed to further assess the antiproliferation effect of hinokiflavone. As shown in Figures 2C and 2D, the colony formation of melanoma cells was inhibited with the increase of hinokiflavone concentration after 24 hours treatment. In addition, the colony size was significantly smaller than that of the control group. Taken together, these results suggest that hinokiflavone could effectively suppress breast cancer cells proliferation.

| Hinokiflavone induces apoptosis in breast cancer cells
Cell apoptosis induction by hinokiflavone was evaluated by Annexin V-FITC/PI dual-labelling technique and the levels of apoptosis were investigated by FCM. As shown in Figures 3A and 3B, compared with control group, treatment of 40 μM of hinokiflavone significantly induced highest cell apoptotic rate (11.7± 3.2%, P<0.005). Importantly, we can find that the apoptotic rates in MDA-MB-231 cells were significantly increased as the concentration of hinokiflavone increased.
In addition, apoptosis-related proteins were analysed by western blot. The expression level of Bax and Bcl-2 is displayed in Figure 3C.
The expression level of Bcl-2, which is regarded as an anti-apoptotic protein, was dramatically inhibited while the expression level of Bax

| Hinokiflavone suppresses migration and invasion in breast cancer cells via EMT signalling pathway
The migration and invasion of tumour cells are critical for the process of tumour metastasis. 23 In addition, considerable metastatic capacity accounts for the high mortality of breast cancer. 24,25 Therefore, we exploited the wound healing assays and transwell assays to evaluate whether hinokiflavone possess a blocked effect on migration and invasion in breast cancer cells. As displayed in Figures 4A and 4B

| Antitumour efficacy of hinokiflavone in a tumour xenograft model of breast cancer cells
To explore whether the antitumour activity of hinokiflavone in vivo is consistent with its effects in vitro, MDA-MB-231 bearing mice were dosed daily at the designated doses (control, 20 and 40 mg kg −1 ) for 21 days. As a key indicator of health, the average body weight of the control (corn oil treated) and hinokiflavone-treated mice did not differ significantly throughout the experiment (data not shown). As displayed in Figure 5A, tumour growth was significantly inhibited by treatment of hinokiflavone. Hinokiflavone distinctly reduced tumour weight in a dose-dependent manner ( Figure 5B).
As evident from the results of immune histochemistry staining, less Ki67-positive and MMP-2-positive cells were observed in the tumours sections from hinokiflavone-treated mice compared with the control mice ( Figures 6A and 6B), indicating that hinokiflavone impedes human breast cancer cells growth in vivo, which is consistent with the findings in vitro.

| DISCUSSIONS
Breast cancer is the most prevalent cancer among women worldwide and the second most common cause of cancer death in women. 1 Even though dramatic advances have been made in screening methods and treatments of breast cancer over the last decade, the overall survival of patients with breast cancer is still quite low due to the high rate of vital organ metastasis. Therefore, it is of great need to develop novel diagnostic and therapeutic agents to improve the treatment of   Overall, our results indicated that hinokiflavone may be a potential anticancer drug for breast cancer treatment.

AVAILABILITY OF DATA AND MATERIALS
The data sets used or analysed in this study are available from the corresponding author on reasonable request.

AUTHORS' CONTRIBUTIONS
XY and ZG designed the study. XY and CL wrote the article. XY, CL, WX and CL collected and analysed the data. SL and ZG collected and analysed the data to revise the manuscript in accordance with reviewer's comments. Also, SS helped the authors write the revised version. All authors read and approved the final manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
All of the animal experiments in this study were performed according to the National Institutes of Health (Bethesda, MD, USA) guidelines and were approved by the Ethical Committee of First Affiliated Hospital of Gannan Medical University (Ganzhou, China).

CONSENT FOR PUBLICATION
Consent for the publication of the clinical and pathological data was obtained from all patients who were involved in this study.

FUNDING
No funding was received.
ACKNOWLEDGEMENT Not applicable.