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Introduction

Breast cancer is the most common cancer among women (1), representing ~25% of all cancer diagnoses (2). This disease and its associated social impacts have become a major health problem worldwide. Standard chemotherapy and radiotherapy protocols for breast cancer have been developed over the years; however, several subtypes of breast cancer have been identified that exhibit different responses to these therapeutic regimens, which further limits the agent and treatment options. In this scenario, the identification of more effective alternative therapies or novel drugs, targeting one or more tumor-specific biomarkers that define the more aggressive breast cancer subtypes, is urgently required.

Apoptosis, an energy-dependent process of programmed cell death undertaken by living cells, is associated with internucleosomal DNA fragmentation, chromatin condensation and cell shrinkage (3,4). Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways, including the death receptor-mediated pathways and the mitochondrial apoptosis (5,6). During mitochondrial apoptosis, the activation of the caspase family, including casapase-3, −8 and −9, serves a central role (7). For instance, the overaccumulation of intracellular reactive oxygen species (ROS) enhances the dissipation of the mitochondrial membrane potential (MMP; also known as ∆ψm), and the resulting increased permeability of the mitochondria further enhances the excessive release of ROS into the cytoplasm, as well as of cytochrome c, which triggers the activation of caspase-9 (8,9). In addition, the auto-catalytic activation of procasapase-8 can cleave Bid into truncated Bid, which initiates the mitochondrial apoptotic pathway (10,11). The activation of caspase-8 or −9 is followed by that of caspase-3, which initiates the cell death process and can serve as an index of cell apoptosis (12).

Recently, various natural compounds have been proven to be valuable sources of alternative antitumor agents, due to their greater efficacy and fewer adverse effects (13). One such compound is 18-β-glycyrrhetinic acid, which is mainly obtained from Glycyrrhiza plants and displays pro-apoptotic properties in pituitary adenoma cells via the regulation of ROS and mitogen-activated protein kinases (MAPKs) (14). It has also been reported that carnosic acid induces the apoptosis of hepatocellular carcinoma cells via an ROS-mediated mitochondrial apoptosis pathway (15). Astilbin is a flavonoid that is commonly found in various herbal medicines and foods, such as Smilax glabra Roxb., Sarcandra glabra (Thunb.) Nakai and grapes (16), and its structure is displayed in Fig. 1. According to previous studies, astilbin exhibits anti-arthritic (17), anti-inflammatory (18) and anti-oxidative (19) effects. Although the antitumor properties of astilbin have been described (20), its pro-apoptotic effect on breast cancer has not yet been reported.

Figure 1. Structural formula of astilbin.

In the present study, the pro-apoptotic effects of astilbin on breast cancer were investigated in MCF-7 and MDA-MB-231 cells, and in nude mice bearing MCF-7-xenografted tumors. Astilbin exhibited cytotoxicity, mainly through the modulation of the caspase-dependent mitochondrial apoptosis pathway.

Materials and methods

Cell culture

The breast cancer cell lines MDA-MB-231 (ATCC no. HTB-26) and MCF-7 (ATCC no. HTB-22) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) under a humidified atmosphere containing 5% CO2 and 95% air at 37°C. The culture medium was changed every other day.

Cell viability assay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) assay was applied to detect the cell viability (21). Briefly, samples of 100 µl breast carcinoma cells at a density of 50,000 cells/ml were seeded into 96-well plates. After 24- and 48-h treatment with astilbin (CAS no. 29838-67-3; obtained from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) at doses of 0–300 µM, MTT was added to each well (final concentration, 0.5 mg/ml). The cells were incubated for a further 4 h at 37°C in the dark, and then 100 µl dimethyl sulfoxide was added to solubilize the purple formazan crystals. A microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to detect the absorbance at a wavelength of 490 nm. The half maximal inhibitory concentration (IC50) values were calculated by the SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA).

Cell apoptosis and migration ability assays

In order to examine the cell apoptosis, samples of 2 ml breast carcinoma cells at a density of 2×105 cells/ml were seeded into 6-well plates. After 24-h exposure to astilbin at the doses of 50 and 200 µM, the cells were collected and stained with propidium iodide and Annexin V (Dead Cell Kit; EMD Millipore, Billerica, MA, USA) for 15 min at 25°C in the dark. A Muse™ Cell Analyzer from EMD Millipore was applied to analyze the fluorescence intensity.

For determination of the cell migration ability, a wound healing assay was performed. Briefly, the seeded cells were scraped with a p200 pipette tip, and then exposed to astilbin at the doses of 50 and 200 µM for 24 h. The distances traveled by the migrating cells were quantified using the ImageJ 1.48v software (National Institutes of Health, Bethesda, MD, USA; rsb.info.nih.gov/ij/download.html) to evaluate the cell migratory ability.

Assessment of intracellular ROS levels and MMP

For intracellular ROS level determination, samples of 2 ml breast carcinoma cells at a density of 2×105 cells/ml were seeded into 6-well plates, and exposed to astilbin at the doses of 50 and 200 µM for 12 h. Subsequently, cells were stained for 20 min with 2,7-dichlorofluorescein diacetate (Sigma-Aldrich; Merck KGaA) at 37°C in the dark. The changes in intracellular ROS levels were observed using fluorescent microscopy (magnification, ×20; CCD camera, Axio Observer Z1; Carl Zeiss AG, Oberkochen, Germany). The quantitative data were analyzed with the ImageJ software, and are expressed as the green fluorescence intensity.

For MMP determination, the treated cells were stained with 2 µM 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich; Merck KGaA) for 15 min at 37°C in the dark. The changes in fluorescence from red to green were detected using fluorescent microscopy (magnification, ×20; CCD camera, Axio Observer Z1; Carl Zeiss AG). The quantitative data were analyzed with the ImageJ software, and are expressed as the ratio of red to green fluorescence intensity.

MCF-7 ×enograft tumor model

The experimental animal study was approved by the Ethics Committee of Changchun Medical College (approval no. CCMC2016-1201; Changchun, China). In total, 10 male BALB/c athymic nude mice (5-week-old; SCXK 2012-0001) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). The mice were maintained on a 12-h light/dark cycle at 23±1°C with water and food available ad libitum. A sample of 0.15 ml MCF-7 cell suspension at a density of 2×108 cells/ml was subcutaneously injected into the right-side waist of each nude mouse. After 4 days, the mice were randomly divided into two groups (n=5 each), and intraperitoneally injected with astilbin (20 mg/kg; 0.3 ml/mouse) or normal saline (0.3 ml/mouse), respectively, every other day for 14 days. The tumor dimensions were monitored throughout the experiment. The tumor volume (mm3) was estimated using the following equation: Volume = length × (width)2 × 0.5. Finally, the mice were sacrificed by administration of 200 mg/kg pentobarbital. Liver, spleen, kidney and tumor tissues were carefully dissected from each mouse for western blot analysis. The protocol of the animal experiments was followed as described in previous studies (22–24).

Biochemical assays

Blood was collected from the caudal vein of each mouse prior to euthanasia. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were measured using commercial diagnostic kits (Nanjing Jiancheng Institute of Biotechnology Co., Ltd., Nanjing, China).

Histopathological examination

The liver, spleen and kidney tissues were fixed in 4% paraformaldehyde and subjected to histopathological examination using hematoxylin and eosin staining, as described in our previous study (15).

Western blot analysis

Samples of 4×105 cells per well were plated into 6-well plates, and treated with astilbin at the doses of 50 and 200 µM for 24 h. The treated cells and collected tumor tissues from the nude mice were lysed by radioimmunoprecipitation assay buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Subsequent to protein concentration detection, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the protein samples (40 µg), which were further transferred electrophoretically onto 0.45-µm nitrocellulose membranes (Bio Basic, Inc., Markham, ON, USA). The membranes were blotted with primary antibodies, including B-cell lymphoma 2 (Bcl-2; cat. no. 3498), Bcl-2-associated X protein (Bax; cat. no. 14796), cleaved caspase-3 (cat. no. 9661), −8 (cat. no. 8592) and −9 (cat. no. 9509) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. 5174) (all purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C for 12 h. The dilution of all primary antibodies was 1:2,000. After three washes with Tris-buffered saline/Tween-20 buffer, the membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. 7074 and 7076; Santa Cruz Biotechnology, Inc.) at a dilution of 1:3,000 for 4 h at room temperature. Chemiluminescence was performed using ECL detection kits (GE Healthcare Life Sciences, Little Chalfont, UK). ImageJ software was used to detect the intensity of the bands via scanning densitometry.

Statistical analysis

All experimental data in the present study are expressed as the mean ± standard deviation. The statistical data were analyzed using one-way analysis of variance, followed by post hoc multiple comparisons (Duncan's multiple range test) using the SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). A value of P<0.05 was considered to denote a statistically significant difference.

Results

Astilbin suppresses the proliferation and migration, and enhances the apoptosis of breast carcinoma cells

Astilbin reduced the viability of both MDA-MB-231 and MCF-7 cells in a dose-dependent manner (P<0.001 at 300 µM; Fig. 2A). The half maximal inhibitory concentration (IC50) values of astilbin for MDA-MB-231 and MCF-7 cells for a 24-h exposure were approximately 167.9 and 191.6 µM, respectively (Fig. 2A). The percentages of cell apoptosis after 24-h exposure to 50 µM astilbin were >19.1% (P<0.01) and >24.5% (P<0.001) in MCF-7 cells and MDA-MB-231, respectively (Fig. 2B). Furthermore, the effects of astilbin on the migration ability of the breast carcinoma cells were investigated using a wound healing assay. In contrast to the non-treated cells, the cells treated with 24-h exposure to astilbin at the doses of 50 and 200 µM exhibited significantly reduced migration into the wound area (Fig. 2C), indicating the suppressed migration abilities of the breast carcinoma cells.

Figure 2. Intracellular toxicity of astilbin in MDA-MB-231 and MCF-7 cells. (A) MTT assay demonstrated that astilbin dose-dependently suppressed the cell viability after 24-h exposure (n=10). (B) Fow cytometry assay demonstrated that astilbin at doses of 50 and 200 µM caused apoptosis in MDA-MB-231 and MCF-7 cells after 24-h exposure (n=6). (C) Astilbin inhibited the migration ability of MDA-MB-231 and MCF-7 cells after 24-h incubation, as observed via a wound healing assay (magnification, ×10; scale bar, 200 µm; n=6). The distances of migrating cells were quantified using ImageJ software. Data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001, vs. non-treated cells. CTRL, control.

Astilbin modulates mitochondrial function, and the expression levels of anti- and pro-apoptotic proteins

Intracellular levels of ROS not only influence mitochondrial function, but also serve an important role during cell apoptosis (14). Compared with the non-treated cells, the treated cells exhibited an enhanced green fluorescence intensity (Fig. 3A), suggesting the potential role of astilbin in promoting ROS production. In addition, treatment with astilbin at 200 µM caused a statistically significant accumulation of ROS by >5-fold in MDA-MB-231 and MCF-7 cells (P<0.001; Fig. 3A).

Figure 3. Astilbin caused the (A) accumulation of intracellular ROS levels and (B) dissipation of MMP in MCF-7 and MDA-MB-231 cells after 24-h exposure (magnification, ×20; scale bar, 100 µm). The quantification data for ROS and MMP were expressed as the green fluorescence intensity or the ratio of red to green fluorescence intensity, respectively. Data are expressed as the mean ± standard deviation (n=6). *P<0.05, **P<0.01 and ***P<0.001, vs. non-treated cells. ROS, reactive oxygen species; MMP, mitochondrial membrane potential; CTRL, control.

JC-1 staining was also performed to investigate the regulatory effect of astilbin on the MMP in the breast carcinoma cells, which is an indicator of mitochondrial function. Incubation with astilbin for 12 h strongly reduced the MMP in breast carcinoma cells, as indicated by the increased green fluorescence intensity and reduced red fluorescence intensity (Fig. 3B). Compared with the control cells, astilbin resulted in an approximately 50% reduction in the ratio of red/green fluorescence intensity in the treated breast carcinoma cells (P<0.001; Fig. 3B).

Members of the caspase family, which are considered as critical participants in intrinsic and extrinsic mitochondrial signaling, were analyzed in the present study via western blot assay. It was observed that astilbin significantly increased the expression levels of cleaved caspases-3, −8 and −9 in MDA-MB-231 and MCF-7 cells after 24-h exposure (P<0.05; Fig. 4). Furthermore, Bax and Bcl-2 levels were examined, since the ratio of Bax and Bcl-2 serves as an important index for mitochondrial function (15). Compared with the control cells, 24-h exposure to astilbin resulted in a significant increase in Bax expression levels and marked suppression of Bcl-2 expression levels (P<0.05; Fig. 4). All these data conclusively confirmed that astilbin induced intracellular toxicity in breast carcinoma cells through the modulation of mitochondrial function.

Figure 4. Astilbin enhanced the expression levels of Bax and cleaved caspase-3, −8 and −9, and reduced the expression levels of Bcl-2 in (A) MCF-7 and (B) MDA-MB-231 cells after 24-h exposure, as determined via western blot analysis. The protein expression levels were quantified by densitometry analysis and normalized to the corresponding GAPDH levels. Data are expressed as the mean ± standard deviation (n=6). *P<0.05, **P<0.01 and ***P<0.001, vs. non-treated cells. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; CTRL, control.

Astilbin suppresses MCF-7 ×enograft tumor growth in nude mice, and regulates the expression of anti- and pro-apoptotic proteins

In tumor xenograft male BALB/c nude mice, 20 mg/kg astilbin was intraperitoneally injected every other day for 14 days. The tumor growth was evidently suppressed by astilbin as compared with that observed in the control mice (Fig. 5A and B). The inhibitory activities of astilbin on tumor growth became apparent from day 7, and caused >6-fold inhibition of the MCF-7 ×enograft tumor growth by the day 15 (P<0.05; Fig. 5C). However, astilbin had no significant influence on the body weight of mice (Fig. 5D), serum levels of AST and ALT (Fig. 5E), or organ function, including the liver, spleen and kidneys of the mice, (Fig. 5F), suggesting its safety for mouse treatment.

Figure 5. Astilbin suppressed the MCF-7-xenografted tumor growth in nude mice without influencing their body weight and organ function following injection at 20 mg/kg every other day for 14 days. (A) Tumor-possessing nude mice, (B) tumor tissue specimens, and (C) tumor growth curves of MCF-7-xenografted nude mice in the control and astilbin-treated groups. Tumor sizes were measured every two days. (D) Mean body weight of the control and astilbin-treated mice (n=5). Data are expressed as the mean ± standard deviation (n=5). *P<0.05 and **P<0.01, vs. vehicle group. (E) Astilbin failed to influence the serum levels of AST and ALT in nude mice compared with those in the control group. (F) Organ function, including liver, spleen and kidney function, of the control and astilbin-treated mice was analyzed by hematoxylin and eosin staining (n=5). AST, aspartate aminotransferase; ALT, alanine aminotransferase; CTRL, control.

The expression levels of anti- and pro-apoptotic proteins in the tumor tissues were also measured. The results demonstrated that 14-day astilbin treatment significantly enhanced the expression levels of Bax and cleaved caspase-3, −8 and −9, and reduced the expression levels of Bcl-2 in the tumor tissues of nude mice (P<0.01; Fig. 6).

Figure 6. Astilbin enhanced the expression levels of Bax and cleaved caspase-3, −8 and −9, and reduced the expression levels of Bcl-2 in the MCF-7-xenografted tumor of nude mice (n=5). Protein expression levels were quantified by densitometry analysis and normalized to the corresponding GAPDH levels. Data are expressed as the mean ± standard deviation (n=5). **P<0.01 and ***P<0.001, vs. control group. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; CTRL, control.

Discussion

The anti-inflammatory and anti-oxidant properties of astilbin have been widely studied (16,18); however, its pro-apoptotic activities have not been fully elucidated. To the best of our knowledge, only one previous study examined the pro-apoptotic effect of Smilax glabra Roxb., which contains astilbin among its main chemical constituents, and reported that it induced apoptosis in hepatoma cell lines via modulation of the mitochondrial caspase-dependent apoptotic pathway (20). In the present study, the anti-breast cancer effects of astilbin were successfully confirmed in MDA-MB-231 and MCF-7 cells, and MCF-7-xenografted tumor nude mice. It was also verified that the caspase-dependent mitochondrial apoptotic pathway was involved in this process.

The safety of natural products has raised concerns among the medical community and the public (25). In the in vivo experiments conducted in the present study, astilbin did not influence the body weight of animals, serum levels of AST and ALT, or organ function (including the liver, spleen and kidney function), indicating its safety for use in animal experiments.

Astilbin also significantly reduced the viability, suppressed the migration ability, and increased the apoptosis rate of MDA-MB-231 and MCF-7 cells after 24-h exposure. Apoptosis, a physiological cell suicide mechanism, leads to cellular self-destruction characterized by chromatin condensation, distinct morphologic alterations and the formation of apoptotic bodies (26). The intrinsic apoptotic pathway is controlled by the mitochondria. The dissipation of MMP, which can serve as an index for mitochondrial apoptosis, is responsible for the loss of function in the mitochondria (27,28). Following this process, mitochondrial permeability transition pores (MPTPs) are opened; consequently, pro-apoptotic molecules are released from the mitochondria, and the caspase family and other catabolic enzymes are activated (29). In the current study, astilbin incubation resulted in a marked reduction of MMP and overaccumulation of intracellular ROS in MDA-MB-231 and MCF-7 cells. It has previously been confirmed that the overaccumulation of ROS is linked to mitochondrial function, involving a short feedback loop (6). Increased levels of intracellular ROS facilitate the opening of MPTPs, which leads to further ROS release from the mitochondria into the cytoplasm (30). In the present study, it was observed that astilbin reduced the expression levels of Bcl-2, and enhanced the expression levels of Bax, not only in human breast carcinoma cells, but also in MCF-7-xenografted tumor tissues. Bcl-2 and Bax are classic Bcl-2 family members, located in the outer mitochondrial membrane (31). The Bcl-2/Bax heterodimer that is formed by interaction between the two proteins is involved in regulating the MMP (32). Previously, liquiritigenin was demonstrated to induce apoptosis in hepatoma carcinoma cells via MAPK-mediated mitochondrial apoptosis, partly via regulation of the expression levels of Bcl-2 and Bax (33). This supports the findings of present study suggesting that astilbin exhibits anti-breast cancer properties via regulation of the mitochondrial or intrinsic apoptotic pathways.

Furthermore, astilbin was found to enhance the expression levels of cleaved caspase-3, −8 and −9 in MDA-MB-231 and MCF-7 cells, which is consistent with the earlier finding that mitochondrial function contributes to the activation of caspase (34). Caspase-8, mainly located in the mitochondria, can cleave itself to adopt the fully activated form, which results in the activation of Bid protein, leading to the dissipation of MMP (35–37). Consequently, cytochrome c, which is released from the mitochondria, helps to activate caspase-9 (38). Finally, caspase-3, the essential factor for the execution of the apoptotic program (39), is activated by cleaved caspase-8 and −9 via proteolytic cleavage (40). In fact, this process is also involved in the feedback loop between ROS accumulation and mitochondrial function. Taken together, the activation of caspases (caspase-3, −8 and −9) contributes to the astilbin-mediated apoptosis of breast carcinoma cells.

In the present study, male nude mice bearing MCF-7-xenografted tumors were used to assess the pro-apoptotic activity of astilbin. However, previous research suggests that it is better to develop this model in female nude mice, which can provide sufficient levels of estrogen to help the tumor growth (41). The present study only aimed to investigate the pro-apoptotic activities of astilbin, and therefore avoided the influence of estrogen hormones during this process, since another separated experiment found that astilbin exhibited regulatory activities on estrogen hormones in healthy female Balb/c mice (unpublished data).

In conclusion, the present experimental study confirmed the anti-cancer effects of astilbin in breast carcinoma cells and nude mice bearing MCF-7-xenografted tumors. Astilbin enhanced the activation of caspase-3, −8 and −9, and caused the overaccumulation of intracellular ROS and the dissipation of MMP, which led to apoptosis. The caspase-dependent mitochondrial pathway is, at least partially, involved in this process. These findings provide pharmacological support for astilbin as a candidate agent for breast cancer treatment.

Acknowledgements

Not applicable.

Funding

This study was supported by the Special Project of Industrial Technology Research and Development in Jilin Province (grant no. 2013-779).

Availability of data and materials

All data generated and analyzed during the present study are included in this published article.

Authors' contributions

SZ designed the experiments, wrote and revised the manuscript. XS drafted the manuscript and performed the experiments. HZ and YZ performed the experiments. QY analyzed the data. All authors read and approved the final manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

The experimental animal study was approved by the Changchun Medical College (approval no. CCMC2016-1201; Changchun, China) and the Second Hospital of Jilin University (Changchun, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References