In many countries, breast cancer is the most frequent type of malignancy among women,1,2 and it is believed to be the second leading cause of cancer death.3 The incidence of breast cancer in women is reported to be more than 100 times higher than in men.4 Despite significant advances in the treatment of breast cancer, such as hormonal therapy, chemotherapy, radiotherapy, and surgery, the incidence and mortality rates of the disease continue to rise, owing to drug resistance and metastases.5,6 Unfortunately, breast cancer is highly resistant to chemotherapy; therefore, there is an urgent need to seek new therapeutic strategies and develop novel drugs with low toxicity for the prevention and treatment of breast cancer. From this perspective, the study of natural products extracted from traditional Chinese Medicine (TCM) remains one of the most challenging fields in cancer research; in China, over the past few decades, compounds used in TCM have been mined for the discovery of potential anticancer agents.7–9
Trametes robiniophila Murr, commonly called “Huaier” in Chinese, was recorded for the first time in Zhou Hou Fang 1500 years ago. It is a type of officinal fungus and has been used as a TCM to treat a wide variety of diseases.10 In recent decades, Huaier extract has been reported to be effective in the treatment of cancers, including hepatocellular carcinoma and breast cancer,11,12 and displays various biological activities, such as apoptosis, anti-angiogenesis, drug resistance reversal, anti-metastasis, and system immune activation.
Radix Isatidis (Isatis Tinctoria), named “Banlangen” in traditional Chinese medicine, is used to reduce inflammatory factors in the blood and to relieve convulsions. Many chemical compounds have been discovered in Radix Isatidis, including tryptanthrin B, indirubin, and organic acids. The functions of Radix Isatidis are widely reported to include antiviral, fever detoxification, and anti-inflammatory activities.13 Indirubin and tryptanthrin B have been proven to be the main active components from Radix Isatidis from the perspective of their anticancer effects. Indirubin, an anticancer drug with an established clinical effect, has a clear effect on the treatment of chronic granulocytic leukemia.14 Tryptanthrin B possessed antitumor activity to BEL-7402 human hepatocellular carcinoma cell and the A2780 human ovarian cancer cell, in vitro.15
Bi-directional solid fermentation, a new Chinese herbal fermentation technology with low toxicity, was able to enhance the properties of medicines and improve absorption.16–18 The bi-directional solid fermentation product extract of Trametes Robiniophila Murr (Huaier) with Radix Isatidis (TIF) was developed based on the new bi-directional fermentation technology of modern Chinese medicine, established by Professor Yi Z in the 1980s.19 Huaier was developed as a medicinal fungi, and the associated Huaiqi fungal substance has achieved a good clinical tests.20 Radix Isatidis is rich in nutrients and bioactive composition, and has a wide range of clinical applications, including a significant anticancer effect. In this study, we acquired TIF and aimed to evaluate its effect on breast cancer cells and explore the underlying mechanisms.
The breast cancer cell lines SK-BR-3 and MDA-MB-231 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Radix Isatidis (Isatis Tinctoria) was purchased from Tongrentang Chinese Medicine -Since 1669 (Beijing, China). Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Gibco-BRL (Rockville, IN, USA) and fetal bovine serum (FBS) and 0.25% trypsin–EDTA were supplied by Hyclone (Beijing, China). DMSO and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were supplied by Amresco (Beijing, China). A BCA protein assay kit, Annexin V-FITC Apoptosis Detection Kit, and Cell Cycle and Apoptosis Analysis Kit were purchased from Beyotime Biotechnology (Shanghai, China). Penicillin-Streptomycin Liquid was supplied by Solarbio Bioscience & Technology Co. (Shanghai, China). The RT-PCR Kit, 6 × Loading Buffer, 100 bp DNA Ladder, RNAiso Plus, and Regular Agarose were purchased from TaKaRa Bio Group (Daliang, China). BD Matrigel Basement Membrane Matrix was purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA) and Crystal Violet Staining Solution was supplied by Sigma–Aldrich (St Louis, MO, USA). Antibodies against p53, MMP-9, β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
2.2. Preparation of TIF, Radix Isatidis, and Huaier extract
Samples of T. robiniophila Murr were obtained from The Northeast Food Medicine Institute of Fungi (Heilongjiang, China). The cultures were maintained on nutrient agar (NA) slants [20.0% (w/v) potato, 2.0% (w/v) glucose, and 2.0% (w/v) agar at 4 °C]. The activated cultures were then inoculated into fluid medium [2.5% (w/v) glucose, 0.05% (w/v) yeast powder, 2.5% (w/v) bran, 0.1% (w/v) KH2PO4, 0.05% (w/v) MgSO4·7H2O] and incubated at 28 °C for 7 days. Then, bidirectional solid fermentation was used to acquire TIF. The cultures were used to inoculate Radix Isatidis prior to fermentation. For each sample, Radix Isatidis (10 g) was placed in a flask and then steamed at 121 °C for 30 min; subsequently, the samples were inoculated with fungus inoculum (10 mL) and incubated at 28 °C with a relative humidity of 80% for 40 days. A new type of mushroom was acquired, which was inoculated on the culture medium composed of Radix Isatidis. This was powdered and the obtained powder (50 g) was extracted with 70% ethanol (500 mL) by refluxing for 2 × 1 h periods. After merging and filtering, the supernatant was collected, concentrated under vacuum, and dried in a drying oven at 60 °C for 48 h. This obtained dry extract powder was TIF. The same method was used to prepare Radix Isatidis and ordinary Huaier, which was inoculated on agar medium. Three different extracts (0.5 g) were dissolved in complete medium (50 mL), sterilized by filtration through a 0.22 μm filter, and diluted to produce a 10 mg/mL stock solution suitable for long term storage at 4 °C.
2.3. Cell culture
Cells (SK-BR-3 and MDA-MB-231) were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in 5% CO2 at 37 °C.
2.4. MTT assay
The cell viability was determined by using an MTT assay, as previously described.21 The cells in suspension were digested using trypsin, adjusted to a concentration of 2.5 × 104 cells/mL, and pated in 96-well plates (200 μL/well). After 24 h, complete medium was replaced by serum-free medium or serum-free medium containing TIF, Radix Isatidis, and Huaier extracts, for 24, 48, or 72 h, respectively. In the preliminary experiments, we found that the SK-BR-3 cells were more sensitive to 6 mg/mL extracts; meanwhile, the MDA-MB-231 cells were more sensitive to 4 mg/mL extracts. Therefore, we treated cells with these concentrations in all subsequent experiments. After incubation at 37 °C for the indicated period, the medium was removed from each well and the cells were washed with PBS. MTT solution (20 μL; 5 mg/mL in PBS) was added to each well of the plate and the plates were incubated at 37 °C in a 5% CO2 incubator for 4 h. Finally, DMSO (100 μL) was added to dissolve purple formazan crystals. The color intensity at 570 nm was measured by using an ELISA reader.
2.5. Cell adhesion assay
The SK-BR-3 and MDA-MB-231 cells were digested by trypsin and the final cell concentration was adjusted to 2 × 105/mL. The cell suspension (2 mL) was added to 6-well plates. After 24 h, the complete medium was replaced with serum-free medium and serum-free medium containing the three different extracts. The cells were incubated for another 48 h at 37 °C in an atmosphere of 5% CO2, the cell concentration was then adjusted to 1 × 106/mL, and 200 μL of the cell suspension was placed in 24-well plates. After incubation for 4 h, the culture medium was discarded, and the wells were washed with the PBS three times, to remove non-adherent cells. The cells were fixed with methanol for 20 min, stained with crystal violet for 20 min, and then washed twice with fresh water. The remaining adherent cells were observed and photographed by using an inverted microscope.
2.6. In vitro scratch assay
To evaluate the effect of the test compounds on cell mobility, a scratch assay was performed on SK-BR-3 and MDA-MB-231 cells. As the cells were attached to the wall, they were first detached by trypsin and adjusted to a concentration of 2 × 105 cells/mL. The cell suspension (2 mL) was added to the 6-well plates. When the cells reached a sub-confluent state, the complete medium was replaced with serum-free medium. After starvation for 12 h starvation, a straight cell-free wound was created with a 10 μL pipette tip and washed twice with PBS. The cells were maintained in serum-free medium or serum-free medium containing the three different extracts at 4 mg/mL. The scratch width was measured at 0, 24, and 48 h, and the migration distances were quantitatively analyzed.
2.7. Cell invasion assay
SK-BR-3 and MDA-MB-231 cells were maintained in serum-free medium for 12 h to avoid the interference of serum. Transwell chambers were placed in 24-well plates to produce the Transwell system. Matrigel (BD Biosciences, San Jose, CA, USA) was dissolved into serum-free medium in a 1:5 ratio, and then 60 μL of solution was gently added to the upper chamber. Next, the Transwell system was incubated at 37 °C for 4 h. The cells were collected and re-suspended in serum-free medium or serum-free medium containing the three different extracts. Briefly, 1 × 105 cells suspended in 200 μL serum-free medium (or serum-free medium containing the extracts) were added to the upper chamber, and 750 μL complete medium (containing 10% FBS) was added to the lower chamber. After incubation for 24 h, the cells on the upper surface of the membrane were wiped off with cotton swabs, and the cells on the lower surface of the membrane were fixed in methanol for 20 min, were stained with crystal violet for 20 min, and washed twice with PBS. The cells that had successfully invaded the lower layer were observed and photographed in five random fields by using an inverted microscope.
2.8. Cell cycle analysis
Cell cycle analysis was performed by the standard method22 with some modifications. The cells were digested by trypsin, resuspended, and washed twice with PBS buffer. The cell concentration was adjusted to 5 × 105/mL and 1 mL of the suspension was seeded into a 25 cm2 culture flask. After incubation overnight, the cells became attached to the side wall, and the complete medium was replaced with serum-free medium or serum-free medium containing the three different extracts. The cells were incubated for a further 48 h at 37 °C in an atmosphere of 5% CO2, digested by trypsin, and washed with PBS. The cells were the transferred into a 1.5 mL EP tube and washed once more with PBS; subsequently, the supernatant was discarded, the cells were washed with cold PBS, and fixed overnight with 70% cold ethanol containing 3% FBS at 4 °C. The next day, the fixed cells were centrifuged at 1000 g for 5 min and washed twice with PBS. The cells were then resuspended with 200 μL RNase A (1 mg/mL) at 37 °C for 10 min, which was followed by the addition of 300 μL propidium iodine (PI, 100 μL/mL) and incubation in the dark, at 18–21 °C (room temperature) to stain the DNA of cells. The DNA content of the cells was analyzed by using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and the data were analyzed by ModFitLT V2.0 software (Becton Dickinson).
2.9. Detection of apoptosis in the cells by PI-Annexin-V staining
The cell concentration was adjusted to 5 × 105/mL and 1 mL of the suspension was seeded in 25 cm2 culture flasks. After 24 h, complete medium was replaced with serum-free medium or serum-free medium containing the three different extracts and the cells were incubated for 48 h, respectively. The Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) was used in accordance with the manufacturer's instructions. Briefly, the cells were digested by trypsin, washed with PBS, transferred to 1.5 mL EP tube, washed once more with PBS, and the supernatant was discarded. Subsequently, 2.5 μL Annexin V-FITC and 5 μL propidium iodide (PI) were added to the cells and for 15 min at room temperature in the dark. Finally, 250 μL of the binding buffer was added and the cells were analyzed by using a flow cytometer (BD Biosciences).
2.10. Detection of gene expression changes by semi-quantitative RT-PCR
The cell concentration was adjusted to 5 × 105/mL and 1 mL of the suspension was seeded in 25 cm2 culture flasks. After 24 h, the complete medium was replaced with serum-free medium or serum-free medium containing the three different extracts, which were incubated for 48 h. Total RNA was isolated with TRIZOL reagent in accordance with the manufacturer's protocol. The purity of RNA was assessed spectrophotometrically by measurement of the absorbance at 260 and 280 nm. The RNA sample was diluted to 500 ng/μL and stored at −80 °C until required for further experiments.
cDNA was synthesized from 1 μL of total RNA by a PrimerScript RT Reagent kit (TaKaRa). PCR was performed by using this cDNA as a template. The PCR reaction conditions were 94 °C pre-denaturation for 2 min; 94 °C denaturation for 30 s; annealing for 30 s; 72 °C extension for 1 min, and a final extension step of 5 min at 72 °C. The sequences of the primer sets and the PCR conditions for each gene are in Table 1.
The PCR products were separated by 1% agarose gel electrophoresis and detected under ultraviolet illumination. Images of the gel were computed by using Lane 1D gel analysis software, which generated densitometry data for the band intensities in different sets of experiments.
2.11. Detection of protein expression changes by western blotting
SK-BR-3 and MDA-MB-231 cells were treated with the three different extracts for 48 h. The cells administered each treatment were collected separately and lysed in lysis buffer in the presence of protease inhibitors. Protein samples (80 μg) were separated by 12% SDS-PAGE and electroblotted to a PVDF membrane by using a semi-dry blotting apparatus (Bio-Rad, Hercules, CA, USA). After blocking with 5% nonfat milk, the membranes were incubated overnight at 4 °C with the primary antibodies and then labeled with the secondary antibody. The protein bands were visualized by using the Pro-lighting HRP agent. The protein β-actin was used as an endogenous control; the control sample comprised cells cultured in the complete medium in the absence of any extract.
2.12. Statistical analysis
The software SAS V9.1 (SAS Institute Inc., Cary, NC, USA) was used for statistical analysis. Significant differences were determined by using one-way ANOVA with post-hoc Dunnett's test for the comparison of at least three treatment groups and Student's t-test for comparisons of two groups. A value of P < 0.05 was accepted as significant. The data were expressed as the mean ± standard deviation and experiments were repeated three times.
3.1. TIF inhibits cell viability of both SK-BR-3 and MDA-MB-231 cells
To investigate the in vitro antitumor activity of TIF on breast cancer SK-BR-3 and MDA-MB-231 cells, we performed an MTT assay to determine the percentage of viability of cancer cells after treatment with the three different extracts for the indicated period. Compared with untreated cells, the three different extracts at 4 mg/mL inhibited the viability of SK-BR-3 and MDA-MB-231 in a time-dependent manner at 24, 48, and 72 h (Fig. 1). The viability of the MDA-MB-231 cells treated with TIF was significantly lower than that of the other groups (p < 0.05). In addition, TIF inhibited the viability of SK-BR-3 cells (11.98% ± 5.91%), but the inhibition was less than that of Huaier (7.87% ± 3.34%).
3.2. TIF inhibits adhesion of both SK-BR-3 and MDA-MB-231 cells
The adhesion of tumor cells to the extra-cellular matrix and basement membranes were considered to be the initial step in the invasion process of metastatic tumor cells. The results shown in Fig. 2 revealed that, compared with untreated cells, the adherent ability of SK-BR-3 and MDA-MB-231 cells was significantly decreased after treatment with the three different drugs for 48 h (p < 0.01). The adherence ability of both cell lines treated with TIF was significantly lower than that of the other groups.
3.3. Cell motility decreases after exposure to TIF
The invasion, migration, and scratch assays were performed to determine whether TIF affected the motility of SK-BR-3 and MDA-MB-231 cells in vitro.
An in vitro scratch assay was used to assess how cell migration was affected by administration of the extracts. As indicated in Fig. 3, the wound healing capacity was significantly inhibited in SK-BR-3 and MDA-MB-231 cells treated with TIF for 48 h compared with untreated cells (29.82% ± 5.72% and 10.43% ± 9.09% versus 52.29% ± 8.76% and 58.10% ± 7.33%, p < 0.05). In addition, the wound closure rate of TIF-treated SK-BR-3 and MDA-MB-231 cells was slower than the other two drugs groups (p < 0.05). Thus, TIF significantly inhibited the migration of SK-BR-3 and MDA-MB-231 cells.
Next, we investigated the inhibitory effect of TIF on cell invasion by using a Transwell system coated with Matrigel. The SK-BR-3 and MDA-MB-231 cells were treated with serum-free medium or serum-free medium containing the three different extracts for 48 h. As shown in Fig. 4, the treatment with TIF dramatically decreased the number of SK-BR-3 and MDA-MB-231 cells invading through the Matrigel-coated membrane compared with other groups (p < 0.05). Therefore, the invasive capacity of SK-BR-3 and MDA-MB-231 cells was also inhibited by TIF.
3.4. TIF treatment induces cell-cycle arrest
In addition, flow cytometry was used to determine the ability of the test compounds to induce cell cycle arrest or affect the cell cycle distribution of SK-BR-3 and MDA-MB-231 cells. Before processing and analysis, the SK-BR-3 and MDA-MB-231 cells were treated with serum-free medium or serum-free medium containing the three different extracts for 48 h, respectively. SK-BR-3 cells treated with TIF extract exhibited increased arrest in the G2 phase. Compared with the control cells, the percentage of cells in the G2 phase was increased to 11.80% ± 3.58% (p < 0.05); The percentage of cells in the G0/G1 phase was decreased and the percentage of cells in the S phase was increased. Similarly, MDA-MB-231 cells treated with TIF extract also exhibited increased arrest in the G2 phase compared with the control cells (38.62% ± 2.68%, p < 0.01) and the percentages of cells in the G0/G1 and S phases were significantly decreased (p < 0.05). These results demonstrated that the TIF extract can suspend the proliferation of SK-BR-3 and MDA-MB-231 cells via cell cycle arrest in the G2 phase, but that the extract's ability to arrest SK-BR-3 cells is weaker than that to arrest MDA-MB-231 cells (Fig. 5). The result revealed that TIF mainly induced G2/M arrest, and the sub-G1 population did not appear in the cell cycle distribution curve. In the next experiment, we detected the apoptosis of the cells by PI-Annexin-V staining. In general, these results revealed that TIF inhibited the proliferation of the breast cancer cells SK-BR-3 and MDA-MB-231 via cell-cycle arrest in the G2 phase.
3.5. Cell apoptosis analysis by flow cytometry
Detection of intact cells, early apoptotic cells, and late apoptotic cells or dead cells was conducted by using PI-annexin-V double staining. Before processing and analysis, the SK-BR-3 and MDA-MB-231 cells were treated with serum-free medium or serum-free medium containing three different extracts for 48 h, respectively. Compared with the control cells, the ratio of late apoptotic/dead cells and the ratio of early apoptotic cells were increased in both SK-BR-3 and MDA-MB-231 cells after the three different treatments (Fig. 6). Furthermore, the apoptosis rate of TIF-treated MDA-MB-231 cells was significantly higher than that of other groups (p < 0.01). As expected, treatment with TIF induced apoptosis in both SK-BR-3 and MDA-MB-231 cells.
3.6. Effects of TIF on apoptosis and migration-related gene and protein expression
SK-BR-3 and MDA-MB-231 cells were treated with serum-free medium or serum-free medium containing the three different extracts for 48 h. The total RNA was extracted from SK-BR-3 and MDA-MB-231 cells and the effect of TIF on the expression of apoptosis- and migration-related genes was detected by sqRT-PCR. The results are shown in Fig. 7; compared with the control group, the expression level of p53 was significantly upregulated in SK-BR-3 cells treated with Huaier and TIF extracts (p < 0.05); the expression of Caspase-3 was also upregulated, but the result was not significant. However, the expression of p53 and Caspase-3 was significantly upregulated in MDA-MB-231 cells after treatment with the Huaier and TIF extracts (p < 0.05). Compared with other groups, TIF not only significantly downregulated the MMP-9 and Snail genes in SK-BR-3 cells (p < 0.05), but also significantly downregulated the MMP-2 and MMP-9 genes in MDA-MB-231 cells (p < 0.05).
Next, we detected the expression of proteins related to apoptosis and metastasis by using western blotting (Fig. 8). In SK-BR-3 cells treated with TIF, the protein expression of p53 was increased, but it was not stronger than treatment with two other extracts; in addition, the expression of MMP-9 was significantly decreased and weaker than other two groups. In MDA-MB-231 cells, TIF significantly increased the expression of p53 and significantly decreased the expression of MMP-9 compared with the other groups.
The p53 signaling pathway has been shown to mediate a variety of intrinsic and extrinsic stress responses, including apoptosis, in various cells.23 P53 tumor suppressor genes can influence cell cycle progression, DNA damage repair, genomic stability, and the apoptotic pathway through the promotion of apoptosis.24 Mutations of the p53 gene have been discovered in many human tumors. Caspase-3 is considered one of the key apoptosis executors, because most of the factors which trigger apoptosis do so through the caspase-3-mediated signaling pathway.25 In this study, the MTT assay showed that TIF significantly inhibited the proliferation of breast cancer cells. Then, cell-cycle analysis revealed that TIF could arrest the cell cycle at the G2/M phase in SK-BR-3 and MDA-MB-231 cells, and the ability to arrest SK-BR-3 cells was weaker than that to arrest MDA-MB-231 cells. Because TIF mainly induced G2/M arrest, the lack of the sub-G1 population in the cell cycle distribution curve does not indicate that no apoptosis occurred after TIF treatment.26,27 Hence, we next detected the apoptosis of the cells by PI-Annexin-V staining; we found that treatment after TIF indeed induced apoptosis in breast cancer cells. In addition, the expression level of the p53 gene was upregulated in SK-BR-3 and MDA-MB-231 cells after treatment with TIF. Western blotting indicted that TIF also upregulated p53 protein expression in both cell lines; it was thought that TIF may induce the apoptosis of breast cancer cells through the upregulation of p53 and Caspase-3 expression.
During metastasis, cancer cells must cross several ECM barriers, crossing the epithelial basement membrane and invading the surrounding stroma, entering blood vessels or lymphatics, extravasate, and establishing new proliferating colonies. Studies have shown that matrix metalloproteinases (MMPs) have long been associated with cancer-cell invasion and metastasis, and that MMP-9 and MMP-2 were most closely related to tumor invasion and metastasis.28 MMP-2 and MMP-9 were thought to be associated with breast cancer, particularly in relation to the invasion and metastasis.29 Snail is a member of the Snail superfamily of zinc finger transcription factors. It has many functions and plays a pivotal role in the acquisition of migratory and invasive properties, and triggers the EMT through the repression of E-cadherin expression. The transcription of Snail is regulated by NF-κB and it is a transcription repressor of the metastatic suppressor gene product E-cadherin and RKIP. Snail silencing reverses the EMT phenotype, whereas its overexpression induces EMT.30 In the present study, the invasion, migration, and scratch assays showed that TIF significantly inhibited cell wound healing, decreased the number of cells passing through the matrix membrane, and inhibited the adherent ability of breast cancer cells. In addition, we also found that the gene expression of MMP-9 and Snail was downregulated in SK-BR-3. Similarly, the gene expression of MMP-9 and MMP-2 was downregulated in MDA-MB-231 cells after treatment with TIF. In addition, the protein expression of MMP-9, a key protein in the implementation of the metastasis process, was decreased in both cell lines. Therefore, TIF may inhibit the migration and invasion of breast cancer cells through the downregulation of the expression of the MMP-2, MMP-9, and Snail genes.
In conclusion, TIF affected the inhibition of the proliferation and migration of the breast cancer cell lines SK-BR-3 and MDA-MB-231 in vitro, and the observed anticancer effect was stronger than that of the Huaier extraction in MDA-MB-231 cells. In order to identify additional mechanisms that may be responsible for the action of TIF on breast cancer, further studies are required.
We gratefully acknowledge the support of the Program for New Century Excellent Talents in University of China (NCET-12–0803).
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