Astragaloside IV suppresses transforming growth factor-β1-induced epithelial–mesenchymal transition through inhibition of Wnt/β-catenin pathway in glioma U251 cells

ABSTRACT

Astragaloside IV (AS#IV) has previously demonstrated antitumoractivity. We investigated the effect and mechanisms of AS#IV in relation to epithelial–mesenchymal transition (EMT), viainterference with the Wnt/β-catenin signaling pathway in gliomaU251 cells. Induction of glioma U251 cells by transforming growthfactor (TGF)#β1 activated EMT, including switching E#cadherin toN-cadherin and altering the expression of Wnt/β-catenin signalingpathway components such as vimentin, β-catenin, and cyclin-D1.AS-IV inhibited the viability, invasion, and migration of TGF-β1-induced glioma U251 cells. AS-IV also interfered with the TGF#β1-induced Wnt/β-catenin signaling pathway in glioma U251 cells.These findings indicate that AS#IV prohibits TGF#β1-induced EMTby disrupting the Wnt/β-catenin pathway in glioma U251 cells. AS#IV may thus be a potential candidate agent for treating glioma andother central nervous system tumors.

Graphical abstract

Astragaloside IV interfered with the TGF-β1-induced Wnt/β-catenin signaling pathway in glioma cells.

KEYWORDS:

• Astragaloside
• central nervous system tumors
• epithelial–mesenchymal transition
• glioma U251 cells
• transforming growth factor-β1

Glioma often occurs in the brain and spinal cord and accounts for nearly 70% of all brain tumors [1,2]. Gliomas generally begin in glial cells, which surround the nerve cells and support their function [1]. Glioma cells have prominent characteristics, including high malignancy and strong proliferation, migration, and invasion [3]. Glioma is among the most deadly forms of cancer, causing approximately 13,000 deaths annually in the United States [2]. Despite extensive research and the use of various treatment modalities including radiotherapy, chemotherapy, and surgery, the prognosis for glioma patients has not improved significantly [1,4,5]. There is thus a need to understand the molecular mechanisms involved in the pathogenesis of glioma. Epithelial–mesenchymal transition (EMT), characterized by the expression of certain stem cell proteins, is an important inducer of the cancer stem cell phenotype [6]. Similar to other cancer types, glioma cells express high levels of these stem cell markers, associated with invasive features and resistance to chemotherapy and radiotherapy, demonstrated both in vitro and in the clinic [7–10].

EMT is a multistage reprogramming phenomenon in which epithelial cells are converted into cells with mesenchymal properties [11]. The most notable features of EMT include a loss of cellular polarity and adhesion, and increased invasion and migration capacities. Several studies have demonstrated the vital role of EMT in tumor progression and metastasis [11] and the crucial role of transforming growth factor (TGF)‐β1 in mediating EMT [12]. Downregulation of TGF‐β1 signaling is thus considered to prevent EMT in various kinds of tumor cells.

The Wnt/β-catenin signaling pathway is highly conserved and occurs in most eukaryotic cells [13] where it is indispensable for cell growth and development, metabolism, and for sustaining stem cell niches [14]. Several studies have demonstrated abnormal activation of the Wnt/β-catenin signaling pathway [15] in various cancers, including prostate cancer [16], colorectal cancer [17], and glioma [18]. This pathway has therefore gained attention for its pathogenic role in tumor development [19]. Astragaloside IV (AS-IV) is a lanolin alcohol-shaped tetracyclic triterpenoid saponin and one of the main active constituents of Astragalus membranaceus Bunge [20,21]. AS-IV has shown a wide range of pharmacological roles in numerous disorders such as cardiovascular and gastrointestinal disorders, and cancer [21]. However, the effects of AS-IV on EMT in glioma cells has not been reported.

In this study, we investigated the role of AS-IV on the TGF-β1-induced EMT-related Wnt/β-catenin signaling pathway in glioma U251 cells, to provide possible clues for the future treatment of glioma.

Materials and methods

Cell culture and treatment

The human glioma cell line U251 was purchased from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium (Gibco, Carlsbad, CA, USA). The U251 cells with overexpressed β-catenin were purchased from Beijing Kawin Technology Co., Ltd.

Approximately 1.5 × 10⁴ U251 cells per well were cultured in two batches for 48 h: one batch was cultured without TGF‐β1, and the other was cultured with TGF‐β1 (10 ng/mL) with or without 20, 40, or 80 μg/mL AS‐IV. Using the same approaches, β-catenin was also overexpressed in some experiments.

Western blot assay

To obtain cell lysates, U251 cells were washed with ice-cold phosphate-buffered saline and lysed in 1 × sodium dodecyl sulfate buffer. Western blotting was carried out using the following specific primary antibodies: anti-E-cadherin, anti‐N‐cadherin, anti‐vimentin, anti-β-catenin, and anti‐β‐actin (all 1:1000; all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), as described previously [22].

Invasion and migration assays

We carried out cell invasion and migration assays, as described previously with some minor changes . In wound healing test, cells were inoculated into 24 well culture plate with a certain density. After 24 hs of growth, the fusion degree of monolayer cells should reach 70–80%. Do not change the media. Use a new 1 mL gun head to gently scratch between monolayer culture cells, and the scratches will cross through the hole. In this way, the gap distance is equal to the outer diameter of the end of the gun head. Gap distance can be adjusted by different gun heads. The scratches are in a straight line in the same direction. Make another scratch perpendicular to the direction of the first scratch, and the scratch of each hole is cross type. After scratches, the plate holes were cleaned twice with medium to remove the exfoliated cells. Fresh medium was added to each well. (the medium contains certain components. Cells grow for 48 hs (or as time requires). The cells were washed twice with 1 x PBS and fixed with 3.7% paraformaldehyde for 30 min. 1% crystal violet (dissolved in 2% ethanol) was dyed for 30 min.

In transwell experiment, transwell chambers with 8‐μm pore size inserts (Corning, Cambridge, MA, USA) were used for both assays. For the invasion assay, the upper chamber was supplemented with Matrigel, and 1.5 × 10⁴ cells per well were seeded followed by the addition of AS‐IV 20, 40, and 80 μg/mL. The medium in the lower chamber was supplemented with 10% fetal bovine serum as a chemoattractant. The cells were incubated for 48 h and noninvasive cells were removed using a wet cotton swab. The invaded cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet solution. The cells at the bottom were counted under a microscope (Nikon, Tokyo, Japan) at a magnification of 200 × . The same procedure was applied for migration assays, without the addition of Matrigel.

Flow cytometry for apoptosis assays

U251 cells in the logarithmic growth phase were treated with TGF-β1 and 20, 40, 80 μg/mL AS-IV. And overexpressed β-catenin U251 cells were treated with TGF-β1 and AS-IV. Cells in both the control group and the experimental groups were collected 24 h after the treatment. U251 cells were prepared and evaluated according to the instructions with the Annexin FITC/PE apoptosis detection kit. Cell apoptosis assays were performed using flow cytometry (BD Biosciences, San Jose, CA, USA). The cells were washed with phosphate-buffered saline (PBS), followed by trypsinization. After detachment, the cells were collected, washed twice and resuspended in 500 µL of PBS. We used a flow cytometer at excitation/emission wavelengths of 488/525 nm to measure the fluorescence.

Detection of cell proliferation activity and cytotoxicity

U251 cells or overexpressed β-catenin U251 cells were seeded into each well of the 96 well plate . The plate were pre cultured in the incubator for 24 hs (37 °C, 5% CO2). Add 10 μL of TGF-β1 or AS-IV of different concentrations to the culture plate. Incubate the plates in the incubator for an appropriate period of time 48 hs. Add 10 μL CCK8 solution to each well (be careful not to generate bubbles in the hole, they will affect the reading of OD value). Incubate the plates in the incubator for 1–4 hs. Then measured the absorbance at 450 nm by enzyme scale.

Statistical analysis

Each experiment was performed at least 3 times, and all data were presented as mean ± standard deviation. A p-value <0.05 was considered statistically significant. For comparisons between three or more groups, one-way analysis of variance was used, followed by Tukey’s multiple comparison tests. Statistical analysis was performed using the GraphPad Prism software, version 5.0 (GraphPad Software, Inc., San Diego, CA, USA).

Results

Activation of Wnt/β-catenin pathway in U251 cells

We investigated TGF-β1-induced activation of the Wnt/β-catenin pathway during EMT in U251 cells. Western blotting demonstrated weaker E-cadherin and stronger vimentin, N-cadherin, Β-catenin, and cyclin-D1 expression in TGF-β1-induced, compared with control glioma U251 cells (Figure 1(a)). This suggested that TGF-β1 played an essential role in the switch from E-cadherin to N-cadherin and in up-regulating the remaining EMT factors in the Wnt/β-catenin pathway.

Figure 1. The effects of TGF-β1 on Wnt/β-catenin pathway expression and related proteins of EMT in U251 cells.

TGF-β1 promotes U251 cell migration and invasion

We evaluated the effects of TGF-β1 on U251 cell migration and invasion. We subjected U251 cells to migration assay after treatment with TGF-β1 for 48 h. Migration was significantly enhanced in U251 cells treated with TGF-β1, compared with the control group (p < 0.05) (Figure 1(b)). TGF-β1 treatment for 48 h also significantly increased the invasion ability of U251 cells compared with control cells, as demonstrated by Transwell assay (p < 0.05) (Figure 1(c)).

AS-IV inhibits TGF-β1-induced EMT

We hypothesized that AS‐IV might suppress the TGF-β1-induced Wnt/β-catenin pathway during EMT. We therefore investigated the protein expression levels of Wnt/β-catenin pathway-related factors by western blot (Figure 2(a)), respectively. Protein expression levels of E‐cadherin were decreased by TGF‐β1. AS‐IV 20, 40, and 80 μg/mL progressively reversed this suppression and enhanced the protein expression levels of N‐cadherin, vimentin, β-catenin, and cyclin-D1. Thus, AS-IV reversed the induction of EMT in U251 cells by TGF‐β1, suggesting efficient suppression of the Wnt/β-catenin pathway during EMT by AS‐IV.

Figure 2. The effects of AS-IV on TGF treated U251 cells.

AS-IV inhibits TGF-β1-induced U251 cell migration and invasion

EMT is thought to be a critical initial step in the invasion and migration of tumor cells [23]. TGF-β1 treatment enhanced these two parameters in U251 cells. We conducted migration (Figure 2(b)) and Transwell assays (Figure 2(c)) to verify if AS-IV could prevent the invasion and migration of TGF-β1-stimulated U251 cells. AS-IV reversed the invasion and migration of TGF-β1-induced U251 cells in concentration-dependent manners. These findings suggested that AS-IV hindered the TGF-β1-induced invasion and migration of U251 cells.

Overexpressed β-catenin reverses AS-IV effect

AS-IV has been reported to downregulate β-catenin [24]. We therefore determined if AS-IV could counteract the overexpression of β-catenin. AS‐IV suppressed TGF‐β1‐induced EMT by interfering with the Wnt/β-catenin pathway. We investigated expression levels of Wnt/β-catenin pathway by western blot to imitate EMT progression in the TGF‐β1‐AS-IV induced with overexpressing β-catenin U251 cells (Figure 3(a)). E‐cadherin expression level in TGF-β1-induced U251 cells was progressively increased by AS-IV at 20, 40, or 80 μg/mL in a concentration-dependent manner, indicating switching of N-cadherin to E‐cadherin. This induction was reversed by overexpression of β-catenin. Similarly, protein expression levels of vimentin, β-catenin, and cyclin-D1 in TGF‐β1-induced U251 cells were gradually decreased by AS-IV at 20, 40, and 80 μg/mL, and this status was reversed by overexpression of β-catenin. These results indicated that β-catenin counteracted the effect of AS‐IV; thus, reinstating the TGF‐β1‐induced Wnt/β-catenin pathway and EMT in U251 cells. AS-IV treatment of TGF-β1-stimulated U251 cells impaired cell invasion and migration. According to the above test results, AS-IV inhibited Wnt signal pathway of U251 cells most obviously at 80 μg/mL, so in β- Catenin overexpression experiment, we used the concentration of AS-IV 80 μg/mL to test, so as to facilitate the observation of obvious results. We therefore determined if β-catenin could counteract this effect of AS-IV in U251 cells by migration (Figure 1(b)) and Transwell assays (Figure 3(c)), respectively. β-catenin restored the invasion and migration abilities of TGF-β1-treated U251 cells, thus neutralizing the effect of AS-IV. These findings suggested that overexpression of β-catenin, which is downregulated by AS-IV, could counteract the effects of AS-IV and restore the role of TGF‐β1 by activating the Wnt/β-catenin pathway for EMT in glioma cells.

Figure 3. The effect of AS-IV on TGF-β1 treated overexpressed β-catenin U251 cells.

AS-IV promots TGF-β1-induced apoptosis of U251 cells

Apoptosis assays were conducted via flow cytometry. The results showed that TGF can reduce apoptosis (Figure 1(d)) AS-IV can promot apoptosis and inhibit the effect of TGF on cells, and with the increase of as-iv concentration, the effect of promoting apoptosis is more obvious (Figure 2(d)). AS-IV has a very significant apoptosis promoting effect on cells, and it also has a significant apoptosis promoting effect on overexpress β-catenin cells affected by TGF at the same time (Figure 3(d)).

AS-IV inhibits cell proliferation and Wnt signaling pathway

Compared with the normal U251 cells, the OD value of U251 cells with TGF increased significantly (P < 0.05), indicating that TGF can promote the proliferation of U251 cells. (Figure 1(e)) AS-IV can significantly reduce the OD value of U251 cells treated by TGF, and the OD value gradually decreases with the increase of as-iv concentration (P < 0.05). (Figure 2(e)) For U251 cells with overexpressed β-catenin and affected by TGF, the addition of AS-IV can still reduce the OD value, but the degree of reduction is less than that of normal U251 cells (P < 0.05). (Figure 3(e))

Discussion

This study showed that AS-IV could inhibit EMT-related Wnt/β-catenin signaling in glioma cells. TGF-β1 altered the protein expression levels of Wnt/β-catenin signaling pathway/EMT markers, including E-cadherin, vimentin, N-cadherin, Β-catenin, and cyclin-D1 in glioma cells. These effects were accompanied by changes in the migration and invasion capabilities of glioma cells, determined by migration and Transwell assays. AS-IV treatment reversed the effects of TGF-β1, while overexpression of β-catenin, which is downregulated by AS-IV, counteracted the effect of AS-IV, further validating the suppression of TGF-β1-induced EMT in glioma cells. At the same time, AS-IV promotes the apoptosis of glioma cells with Wnt signaling pathway activated, and the effect of AS-IV counteracts the cell proliferation induced by TGF-β and overexpression β-catenin. The results of CCK8 experiments of U251 cells showed that AS-IV could inhibit the proliferation of U251 cells induced by TGF and the inhibition was enhanced with the increase of AS-IV concentration. For the CCK8 experiments of U251 cells with overexpressed β-catenin, it is suggested that AS-IV can inhibit the proliferation of U251 cells, and AS-IV can inhibit the Wnt signaling pathway to promote the proliferation of U251 cells.

Three Wnt signaling pathways have been identified [25]: (1) a JNtC-mediated planar cell polarity pathway required for embryo development; (2) a Ca²⁺-mediated Wnt/Ca²⁺ pathway involving calmodulin, the transcription factor NF-AT, and Ca²⁺-dependent kinases, which antagonizes the Wnt/β-catenin pathway; and (3) a Wnt/β-catenin pathway, which is the most extensively studied and highly conserved pathway [26].

The Wnt/β-catenin pathway is unusually activated in various tumors [27]. Paul et al. investigated the molecular mechanism of the Wnt/β-catenin pathway in tumor development [28] and concluded that Wnt forms a trimer complex on the cell membrane by binding to frizzled protein and low-density lipoprotein receptor-related protein. This trimer complex then enters the cytoplasm, resulting in the phosphorylation and activation of the cytoplasmic protein disheveled (Dsh). Activated Dsh hinders glycogen synthase kinase, which in turn prevents degradation of phosphorylated β-catenin, resulting in its accumulation in the cytoplasm. This β-catenin translocates from the cytoplasm into the nucleus and forms a complex with the TCF transcription factor, resulting in abnormal transcription of downstream target genes such as c-myc, VEGF, cyclin-D1, and matrix metalloproteinase-7. These genes/proteins are mostly responsible for cell growth, apoptosis, angiogenesis, and tumor invasion and migration, thus endorsing tumor development and cell cycle progression. Disruption of any constituent of this pathway results in signaling abnormalities and may ultimately cause malignant cellular alterations and diseases [29] .

EMT is a highly conserved and fundamental phenomenon, critical for tumorigenesis [30]. Abnormal EMT activation increases mesenchymal features [5] and is thus a major factor promoting cancer cell initiation, invasion, metastasis, and chemoresistance [5]. During EMT, the cellular adhesion molecule E-cadherin is repressed, allowing tumor cells to spread throughout the body [5]. Some EMT-suppressive drugs have recently been described, with possible antitumor roles [31]. AS‐IV is an active, traditional Chinese drug [21] with antitumor properties [20,21]. AS‐IV inhibited breast cancer [32] and lung cancer cells [33] by interfering with Vav3‐mediated Rac1/MAPK signaling and the PKC‐α‐ERK1/2‐nuclear factor (NF)‐κB pathways, respectively.

Zhu and Wen recently validated the antitumor potential of AS‐IV by inhibiting the PI3 K/Akt/NF‐κB pathway in gastric cancer cells. They demonstrated that AS‐IV (20, 40, and 80 μg/mL) significantly reduced the sustainability of gastric cancer cell lines [4]. AS‐IV has also been shown to suppress EMT in hepatoma cells, thus, inhibiting metastasis [34]. However, the ability of AS-IV to suppress EMT in cancer cells by interfering with the Wnt/β-catenin pathway has not been investigated. In the present study, we therefore examined the ability of AS-IV to disrupt EMT in glioma cells by interfering with the Wnt/β-catenin pathway. TGF‐β1 causes abnormal translocation of β‐catenin into the nucleus, where it triggers certain genes required for EMT activation [35]. We therefore induced EMT in glioma cells using TGF‐β1. During EMT, E‐cadherin switched to N‐cadherin, which has a vital role in enhancing cell motility and migration [12,36]. And the results in this study strongly suggest that TGF‐β1‐induced EMT in glioma cells was inhibited by AS‐IV. The discovery of novel EMT regulators may support the development of new treatments for glioma. A recent study showed that AS-IV inhibited the TGF‐β1‐induced PI3 K/Akt/NF‐κB pathway [4]. The present study also demonstrated that AS‐IV stopped the TGF‐β1‐induced activation of the Wnt/β-catenin pathway for EMT in glioma cells.

In conclusion, we evaluated the effects and mechanisms of AS‐IV on TGF‐β1‐induced EMT in glioma cells. AS‐IV appears to inhibit TGF‐β1‐induced EMT by interfering with the Wnt/β-catenin pathway in glioma cells. These observation suggest that AS‐IV may have great therapeutic potential for preventing and curing gliomas and other central nervous system tumors.

Authors contributions

Research idea and project design: H JM, Z LJ. Data collection and dataset setup:S XH, YZ. Data analysis: S XH, WS. Manuscript editing: H JM, Z LJ. All authors contributed to the development of the manuscript, and approved the final version.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

No funding was received to perform this study.