Wogonoside inhibits invasion and migration through suppressing TRAF2/4 expression in breast cancer

Wogonoside (>98% purity, Langze Pharmaceutical Co., Ltd., Nanjing, China) was dissolved in dimethylsulfoxide (DMSO) as a stock solution, stored at −20 °C, and diluted with medium before each experiment [17, 18]. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was purchased from Sigma Chemical Co. (St. Louis, MO, USA). A nuclear/cytosol fractionation kit (KeyGEN, Nanjing, China) was used according to the manufacturer’s directions. Human recombinant TNF-α and TGF-β1 were from PeproTech Inc. (PeproTech, IL, USA). Primary antibodies against CD44v6 were form Abcam plc. (Abcam, Cambridge, UK), antibodies against E-cadherin, vimentin, p-IκBα and β-Tubulin were from Cell Signaling Technology (CST, MA, USA), antibodies against MMP-9, p-IKKα, IKKα, GAPDH, Lamin A, TNF-α, TRAF2 and TRAF4 were from Bioworld (Bioworld, MN, USA), antibodies against MMP-2, IκBα, NF-κB p65 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies against Twist1 were form Signalway antibody (SAB, MD, USA). IRDye^®800-conjugated secondary antibodies were from Rockland Inc. (PA, USA).

Four-week-old female BALB/c nude mice (Slaccas Shanghai Laboratory Animal Co., Ltd., Shanghai, China) were used for the orthotopic model of MDA-MB-231 cells. The animals were maintained in a pathogen-free environment (23 ± 2 °C, 55 ± 5% humidity) on a 12 h light/12 h dark cycle with food and water supplied ad libitum throughout the experimental period. Animal study and euthanasia was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the China Pharmaceutical University.

Human breast cancer MDA-MB-231 cells, MCF7 cells, MDA-MB-435 cells, BT-474 cells were originally obtained from the Cell Bank of the Shanghai Institute of Cell Biology. The MDA-MB-231 cells, MDA-MB-435 cells and BT-474 cells were cultured in DMEM medium (Gibco, Grand Island, NY) and MCF7 cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) both containing 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin, in a stable environment with 5% CO₂ at 37 °C.

Cells were plated at a density of 5 × 10³ cells/well in 96-well plates. After 24 h culture, the cells were exposed to different concentrations of wogonoside for 48 h in a 5% CO₂ incubator at 37 °C. Then, MTT was added to the medium and the cells were incubated at 37 °C for 4 h. The supernatant was removed and dimethyl sulfoxide was used to dissolve the precipitate. The absorbance was measured spectrophotometrically at 570 nm.

The 96-well plates were coated with matrigel (Corning, NY, USA) overnight at 4 °C and blocked with 1% BSA for 4 h at 37 °C. After cells were treated with different concentrations of wogonoside for 48 h, cells were collected in serum-free medium at 5 × 10⁵ cells/ml. Aliquots (100 μl) of the cell suspensions were seeded into the wells and incubated for 1 h at 37 °C. After that, unattached cells were washed thrice with PBS and the attached cells were determined by MTT assay.

Cells were seeded in a six-well plate and allowed to attach overnight, with growth to 80% confluence. The cell monolayers were then wounded with white pipette tips and washed twice with phosphate-buffered saline. The cells were then incubated with wogonoside in medium supplemented with 1% serum for 48 h. The number of migrated cells was determined under an inverted microscopy.

The transwell chambers (12 mm in diameter, 8 μm pore-size, Millipore, Billerica, MA) were loaded with 0.1 ml of matrigel (Corning, NY, USA) in a 24 well plate at 37 °C for 1 h. After cells were pretreated of wogonoside for 48 h, cells were collected in serum-free medium at a final concentration of 2 × 10⁵ cells/ml. 400 μl cell suspensions were then placed in the upper transwell chamber, and 600 μl medium containing 10% fetal bovine serum was added to the lower compartment. Followed by incubation for 24 h, cells on the upper surface were removed, and invasive cells on the lower surface were fixed with 100% methanol and stained with hematoxylin and eosin. Then quantified by manual counting and three randomly chosen fields were analyzed for each group.

Cells were harvested after pretreatment of wogonoside for 48 h. Western blot was performed as previously described [20]. The membrane was blocked with 5% BSA in PBS at 37 °C for 1 h and incubated overnight at 4 °C with the indicated antibodies, and then with IRDye^®800-conjugated secondary antibody for 1 h at 37 °C. The samples were visualized with the Odyssey Infrared Imaging System (LI-COR Inc., Lincoln, NE, USA).

Cells were pre-treated with wogonoside for 48 h. The mRNA levels of E-cadherin, vimentin, MMP-9 and Twist1 were then determined with a method described previously [20]. The primer sets used for the PCR amplifications were as follows: Twist1 (forward, 5′- GGAGTCCGCAGTCTTACGAG-3′, reverse, 5′- TCTGGAGGACCTGGTAGAGG-3′), E-cadherin (forward, 5′-CCACCAAAGTCACGCTGAAT-3′, reverse, 5′-GGAGTTGGGAAATGTGAGC-3′), MMP-9 (forward: 5′-GCAGAGGAATACCTGTACCGC-3′, reverse, 5′-AGGTTTGGAATCTGCCCAGGT-3′), vimentin (forward, 5′-ATGAAGGTGCTGCAAAAC-3′, reverse, 5′-GTGACTGCACCTGTCTCCGGTA-3′), human GAPDH (forward, 5′-TGGGTGTGAACCATGAGAAG-3′, reverse, 5′-GCTAAGCAGTTGGTGGTGC-3′).

Cells were seeded in six-well plates at 70% confluency. The transient transfection assay was performed by using Lipofectamine^®2000 Transfection Reagent (Thermo, MA, USA) according to the manufacturer’s protocol. Briefly, 8 μl Transfection Reagent and the Twist plasmids (1 μg, Addgene), TRAF2 plasmids (1 μg, Addgene), TRAF4 plasmids (1 μg, Addgene) were respectively diluted in 250 μl medium gently. Then mix the above two diluted medium gently and incubate for 20 min at room temperature. Finally, the complexes were added to each well containing cells and media. The plate was rocked back and forth and incubated at 37 °C in a CO₂ incubator for 12 h.

The concentration of cytokines in supernatant was detected by ELISA kit (Boster Biotechnology, Wuhan, China). Briefly, the supernatant diluted with sample diluent buffer was added to the microwell (100 μl/well) and the plates were then stored in incubator at 37 °C for 90 min. The supernatant was discarded and antibody diluted with antibody diluent buffer was added to the microwell (100 μl/well) and stored in incubator at 37 °C for 1 h. Then plates were washed with PBS for 3 times and Avidin-Biotin-Peroxidase-Complex (ABC) diluted with ABC diluent buffer was added to the microwell. After incubated at 37 °C for 30 min, ABC was discarded and plates were washed with PBS for 5 times. Add the TMB color developing agent to the microwell (90 μl/well) at 37 °C for 20 min and then add the TMB stop solution (100 μl/well). The absorbance was measured at 450 nm.

Orthotopic injections were performed following the previous study with minor modifications [21]. MDA-MB-231 cells (1 × 10⁵/25 μl) were mixed with 25 μl matrigel on ice, and then the cell suspension was quickly injected into the fourth Mammary Fat Pad (MFP). Animal was observed for 30 min until fully recovery. Seventy-seven days later, the mice were randomly divided into three groups (8 mice/group): the negative group (intraperitoneal injection of 0.9% normal saline); the wogonoside-treated group (gavage of 80 mg/kg wogonoside at a frequency of once every other day); and the gemcitabine-positive group (intraperitoneal injection of 80 mg/kg gemcitabine at a frequency of one time every 2 day). Ninety-eight days later, the nude mice were killed and the tumor xenografts were segregated and measured. Additionally, to the blank control group (16 mice, without any drug administration), 8 mice were killed when they lived to 63-day and 98-day respectively, then the tumor xenografts were segregated and measured. Visceral tissue resected from control and test mice were fixed in formalin and tested with H&E staining. Tumor volume (TV) was calculated using the following formula:

D and d are the longest and the shortest diameters, respectively. At the same time the animals were weighed twice per week and monitored for mortality throughout the experimental period.

Relative tumor volume (RTV) was calculated according to the equation:

V ₀ is the tumor volume at day 0 and V _t is the tumor volume at day t. And the evaluation index for inhibition was of relative tumor growth ratio

T _RTV and C _RTV represented RTV of treated and control groups, respectively.

This study was approved in SPF Animal Laboratory of China Pharmaceutical University. In all experiments, the ethics guidelines for investigations in conscious animals were followed, with approval from the local Ethics Committee for Animal Research.

The expression of Twist1, E-cadherin, vimentin, MMP-9, TNF-α, TRAF2, and TRAF4 in nude mice model was assessed to the method described previously [22], using a goat-anti-rabbit antibody and an Ultra-Sensitive TMSAP kit. All reagents used in the experiments were supplied by Maixin-Bio Co., Fuzhou, China.

The data were obtained from at least three independent experiments and all data in different experimental groups were expressed as the mean ± SD. We compared TNF-α-treated group or TNF-α + TGF-β1-treated group to control group in vitro, and the saline-treated group to control group in vivo. Differences between groups were tested with One-Way ANOVA analysis of variance and Dunnett’s post hoc test. The changes in tumor weight and tumor volume over time were tested using a random effects mixed model. Metastasis incidence rates were evaluated using percentages of animals withmetastases, and tested using Fisher’s exact test. The significance of differences is indicated at *p < 0.05 and **p < 0.01.

The anti-metastatic effect of wogonoside was assessed with orthotopic model of MDA-MB-231 cells in vivo (Fig. 1a). As it is shown in Fig. 1b, during the 21-day treatment, tumor volume was reduced by wogonoside or gemcitabine (80 mg/kg), which showed inhibitory effects on tumor growth of MDA-MB-231 cells. The tumor weight was also decreased compared with the control group (Fig. 1c). The inhibitory rate of wogonoside was about 46%, while that of gemcitabine was approximately 71%. However, as shown in Table 1, eight (100%), three (37.5%) and four (50%) mice were found of metastasis after pathological examination in the control, gemcitabine-treated and wogonoside-treated group respectively. The probability of metastasis was down-regulated by wogonoside (50%), while that of gemcitabine was 62.5%. In the orthotopic model of MDA-MB-231 cells, cancer cells could metastasize to several organs, including brain, lung, liver and bone. Afterwards, we analyzed the incidence of metastasis in each organ according to the pathological section. The results showed that wogonoside could suppress the formation of metastases in brain, lung, liver and bone (Fig. 1d). The instance of organ metastasis was then added up to indicate the risk of metastases in each organ. For example, breast-to-lung cancer metastases were found in 8 mice (100%) in the control group, and lung metastases were found in 4 animals (50%) in the wogonoside-treated group [23] (Table 2). In the primary tumor, we tested the expression of metastasis-associated proteins with immunohistochemistry and western blot assay. It demonstrated that wogonoside could increase the expression of E-cadherin and decrease the expression of MMP-9, vimentin and Twist1 (Fig. 1e and f).

Mice tumors resected at different time periods were shown in Fig. 2a. By Elisa assay and immunohistochemical analysis, we found that the content of TNF-α in 98-day primary tumors was increased, compared to 63-day primary tumors (Fig. 2b and c). Meanwhile, the expressions of TNF-α, TRAF2 and TRAF4 were also enhanced in 98-day primary tumors (Fig. 2d). Coincidentally, as shown in Fig. 2e and f, the immunohistochemistry assay and western blot assay verified that wogonoside could reduce the protein expression of TNF-α, TRAF2, and TRAF4. Therefore, we wondered that whether wogonoside inhibited metastasis through suppressingTRAF2/4 expression.

As shown in Fig. 3a, the result of MTT assay revealed that the treatment of TNF-α (20 ng/ml) and wogonoside (0, 50, 100 and 150 μM) caused no significant cytotoxicity in MDA-MB-231, MDA-MB-435, and BT-474 cells. These concentrations were then applied to all subsequent experiments. Cancer cell adhesion to basement membranes is important for tumor invasion since it is a key step in proteinase-dependent cell locomotion. The results of cell attachment assay showed that the adhesive capabilities of TNF-α-induced MDA-MB-231, MDA-MB-435, and BT-474 cells were decreased after treatment of wogonoside (Fig. 3b).

We next examined the effect of wogonoside on the TNF-α-induced migration of these three cells. As shown in Fig. 3c and d, migrated cells were quantified by scratch width. The inhibitory rate of wogonoside (50, 100 and 150 μM) was about 1%, 43% and 63% in MDA-MB-231 cells, 12%, 49% and 69% in MDA-MB-435 cells, 1%, 20% and 34% in BT-474 cells, respectively.

Then, we investigated the effects of wogonoside on the TNF-α-induced invasion of MDA-MB-231, MDA-MB-435, and BT-474 cells in vitro. We found that cells in the control group were able to invade freely through the matrigel, whereas this ability was inhibited by wogonoside. As shown in Fig. 3e and f, wogonoside could inhibit the invasion of MDA-MB-231, MDA-MB-435, and BT-474 cells in a concentration-dependent manner, and the inhibition rate at 150 μM was about 57% in MDA-MB-231 cells, 60% in MDA-MB-435 cells, and 24% in BT-474 cells.

In the process of cancer metastasis, MMP-9, CD44v6 and vimentin are responsible for cell migration, invasion and cell-matrix adhesion. Therefore, the effects of wogonoside on the expression of MMP-9, MMP-2, CD44v6, vimentin and Twist1 in MDA-MB-231 and MDA-MB-435 cells were detected by western blot analysis. As shown in Fig. 4a, we observed that wogonoside reduced the expression of MMP-9, MMP-2, CD44v6 and vimentin in TNF-α-induced MDA-MB-231 and MDA-MB-435 cells. TNF-α could induce Twist1 expression and overexpression of Twist1 could promote further metastasis, which increased the expression of MMP-9, MMP-2, CD44v6 and vimentin in cancer cells. Therefore, we detected Twist1 expression. As shown in Fig. 4b and c, wogonoside decreased the protein and mRNA expression of Twist1, which suggested that wogonoside could inhibit Twist1 expression at the transcriptional level. The inhibition rate of Twist1 mRNA level was about 9%, 37% and 65% in TNF-α-induced MDA-MB-231 cells and 7%, 21% and 45% in TNF-α-induced MDA-MB-435 cells.

Previous study has demonstrated that NF-κB pathway is involved with the regulation of Twist1 expression at the level of transcription. Therefore, we examined the key kinases in NF-κB signaling and the nuclear translocation of p65. The results showed that wogonoside inhibited the phosphorylation of IκBα and IKKα in TNF-α-induced MDA-MB-231 and MDA-MB-435 cells (Fig. 5a). And the nuclear translocation of NF-κB p65 was also inhibited by wogonoside in TNF-α-induced MDA-MB-231 and MDA-MB-435 cells (Fig. 5b).

Since TRAF2 complex is the upstream of NF-κB signaling, which could be responsible for the phosphorylation of IKK, we detected the inhibitory effect of wogonoside on TRAF2 expression. The results showed that wogonoside could inhibit TRAF2 expression in TNF-α-induced MDA-MB-231 and MDA-MB-435 cells (Fig. 5c). In addition, according to the experiment of orthotopic model, wogonoside could decrease TRAF2 and TRAF4 in vivo. Therefore, we further investigated whether TRAF4 expression was also decreased in these two cells. We found that wogonoside mainly inhibited the nuclear protein level of TRAF4 instead of the cytoplasmic protein level (Fig. 5d).

Since the treatment of TNF-α alone in MCF7 cells could not induce EMT efficiently, we added TGF-β1 (5 ng/ml) to promote the EMT process. An MTT assay showed that TNF-α (20 ng/ml) and TGF-β1 (5 ng/ml) or the combination of wogonoside, TNF-α and TGF-β1 had no cytotoxic effect (Fig. 6a). These concentrations were used in the following experiments. As shown in Fig. 6b, we found that MCF7 cells transformed from an epithelial morphology to an elongated fibroblast-like cell morphology under the treatment of TNF-α and TGF-β1. However, wogonoside (150 μM) could inhibit TNF-α + TGF-β1-induced morphological changes in MCF7 cells. The results of cell attachment assay showed that the adhesive capabilities of TNF-α + TGF-β1-induced MCF7 cells were decreased after treatment of wogonoside (Fig. 6c). Afterwards, we investigated the anti-invasive and anti-migratory effects of wogonoside on TNF-α + TGF-β1-triggered EMT with matrigel invasion and wound healing assays. Wogonoside inhibited the migration of TNF-α + TGF-β1-stimulated MCF7 cells across the wounded space in a concentration-dependent manner (Fig. 6d and e). And treatment with wogonoside could also reduce the invasiveness of TNF-α + TGF-β1-induced MCF7 cells through matrigel (Fig. 6f and g).

As TNF-α and TGF-β1 triggered EMT process in MCF7 cells, we investigated the effect of wogonoside on EMT biomarkers. Quantization of western blot assay showed that wogonoside up-regulated the protein and mRNA expression of E-cadherin while it down-regulated the protein and mRNA expression of MMP-9 and vimentin in a concentration-dependent manner (Fig. 6h and i).

As shown in Fig. 7a, the expression of TRAF2, TRAF4, and Twist1 in MDA-MB-231, MCF7, MDA-MB-435, and BT-474 cells were detected by western blot analysis. There were different protein levels of Twist1 in four cells: MDA-MB-231 and MDA-MB-435 with high expression (100%), MCF7 with medium expression (75%), and BT-474 with low expression (55%).

Since MCF7 cells exhibited lower expression of Twist1, TRAF2 and TRAF4, we transfected the overexpressed plasmids of TRAF2, TRAF4 and Twist into MCF7 cells. The results showed that transfection of TRAF2 and TRAF4 plasmids could induce Twist1 expression while transfection of Twist plasmids could induce TRAF2/4 expression. On the other hand, the overexpressed proteins of TRAF2, TRAF4 and Twist1 were all decreased by wogonoside (Fig. 7b).

Indeed, the stimulation of TNF-α and TGF-β1 could also enhance the expression of TRAF2, TRAF4 and Twist1 in MCF7 cells. Hence, we investigated whether the mechanism of wogonoside against EMT was involved with TRAF2/4 expression. The results showed that wogonoside inhibited the total protein of TRAF2 and the nuclear protein level of TRAF4 in TNF-α + TGF-β1-induced MCF7 cells (Fig. 7c and d). Correspondingly, wogonoside could inhibit the phosphorylation of IκBα and IKKα in TNF-α + TGF-β1-induced MCF7 cells (Fig. 7e). Meanwhile, as shown in Fig. 7f, nuclear translocation of NF-κB p65 was also inhibited by wogonoside.