Evodiamine Induces Apoptosis, G2/M Cell Cycle Arrest, and Inhibition of Cell Migration and Invasion in Human Osteosarcoma Cells via Raf/MEK/ERK Signalling Pathway.

BACKGROUND Osteosarcoma is a prevalent type of bone tumor mainly reported in children and adolescents. The treatments for osteosarcoma are limited and are associated with serious adverse effects. In this study we evaluated the anticancer activity of Evodiamine, a plant-derived natural product, against a panel of osteosarcoma cells and explored the underlying mechanisms. MATERIAL AND METHODS The viability of osteosarcoma cell lines was investigated by MTT assay. Apoptosis was detected by DAPI and annexin V/PI staining and cell cycle analysis was performed by flow cytometry. The expression of the proteins was examined by Western blotting. RESULTS The results of the present study indicated that Evodiamine inhibited the proliferation of U2OS osteosarcoma cells with an IC50 of 6 µM. Further investigations indicated the antiproliferative effects of Evodiamine are due to induction of apoptosis and G2/M cell cycle arrest. The results of Western blotting revealed that the expression of several apoptosis (Cytochrome c, Bax, Bid, Caspase 3, 9, 8, and PARP) and cell cycle-related proteins (cyclin B1, Cdc25c, and Cdc2) was significantly altered. Evodiamine also suppressed the migration and invasion of U2OS osteosarcoma cells. Moreover, Evodiamine downregulated the expression of important regulatory proteins such as p-MEK and p-ERK, leading to the inhibition of Raf/MEK/ERK signalling pathways. CONCLUSIONS We found that Evodiamine exerts anticancer effects on osteosarcoma cells and has potential in the treatment of osteosarcoma.

Background

Osteosarcoma is one of the most prevalent types of bone tumors, mainly detected in adolescents and children. It has been reported that around 70% of osteosarcoma patients are ages 10–25 years [1]. The development of chemoresistance among osteosarcoma patients forms a bottleneck in the treatment of osteosarcoma [2]. Although the treatment strategies for osteosarcoma have improved, exploration of novel and efficient molecules is required to combat the development of drug resistance and to prevent osteosarcoma metastasis [3]. Consequently, new molecules are being screened for their anticancer activity against osteosarcoma. In the present study, we evaluated the anticancer activity of Evodiamine against the U2OS osteosarcoma cell line and in normal bone cells. Chemically, Evodiamine is an alkaloid that is generally isolated from Evodia rutaecarpa [4]. Many biological activities have been attributed to this naturally occurring molecule. These bioactivities include, but are not limited to, anti-inflammatory and anticancer activities [5, 6]. Several studies on Evodiamine have reported its anticancer activity. For instance, Evodiamine has been reported to inhibit the metastasis of lung and colon cancer cells [7]. Evodiamine has also been reported to exert antiproliferative effects on drug-resistant breast cancer cells [8]. The present study is the first to report the anticancer activity of Evodiamine against osteosarcoma cells, showing that Evodiamine exerts dose-dependent anticancer effects on U2OS osteosarcoma cells with no or minor effects on the growth of normal bone cells. Investigation of the underlying mechanisms revealed that Evodiamine triggers apoptosis in osteosarcoma U2OS cells, which was also associated with altered expression of apoptosis-related proteins. Furthermore, Evodiamine can prompt G2/M cell cycle arrest and inhibit the migration and invasion of U2OS cells. The Ras/MEK/ERK signalling pathway has been reported to be activated in several types of cancer cells [9]. In this study we observed that Evodiamine inhibited this pathway, indicating that Evodiamine may be an important lead molecule for the treatment of osteosarcoma.

Material and Methods

Cell lines and culture conditions

All chemicals and reagents were obtained from Santa Cruz biotechnology unless indicated otherwise. Evodiamine (98% purity by HPLC) was obtained from Sigma-Aldrich. Osteosarcoma U2OS cell line and normal bone cells were procured from the American Type Culture Collection. The cell lines were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin), and 2 mM glutamine. The cells were cultured in an incubator (Thermo Scientific) at 37°C with 98% humidity and 5% CO2.

Cell viability assay

Osteosarcoma cell viability was determined by MTT assay. In brief, the cultured U2OS osteosarcoma cells were seeded at the density of 1.5×104 in 96-well microtiter plates and cultured for 24 h at 37°C. This was followed by the addition of MTT solution in all the wells and then the absorbance at 570 nm was assessed using an ELISA plate reader.

Apoptosis assay

The osteosarcoma U2OS cells were seeded in 6-well plates (2×105 cells per well) and cultured at 37°C for 24 h. The cells were then stained with DAPI to detect the apoptosis by fluorescence microscopy, as previously reported [10]. To determine the percentage of apoptotic cells, an FITC-Annexin V/PI Apoptosis detection kit was used according to the instructions of the manufacturer.

Cell cycle analysis

The distribution of the U2OS cells in various phases of the cell cycle was assessed by flow cytometry. Briefly, 0, 3, 6, and 12 μM of Evodiamine-treated U2OS cells were harvested after 24 h of culturing at 37°C and subjected to washing with PBS. The U2OS cells were then fixed with ethanol (70%) for about 1 h and then again subjected to washing with PBS. The cells were finally resuspended in solution of PI (50 μl/ml) and RNase1 (250 μg/ml). This was followed by incubation for 30 min at room temperature and a final investigation under a fluorescence-activated cell-sorting cater-plus cytometer using 10 000 cells/group. The estimation of the percentage of cells in each phase of the cell cycle done using WinMDI software.

Cell migration and invasion assay

The cell migration of the osteosarcoma U2OS cells was determined by wound-healing assay. After culturing for 24 h at 37°C, the media and the cells were subjected to PBS washing. Thereafter, a wound was scratched using a sterile pipette tip and photographed. The cells were then grown for another 24 h and a photograph was taken again. Cell invasion of the Evodiamine-treated cells was performed by Transwell assay. Briefly, the U2OS cells were cultured at 2×105 cells/mL density followed by the addition of 200 ml of these cultured cell suspensions to the upper chamber and whole medium was added into the bottom wells. The cells were cultured for 24 h at 37°C. The U2OS cells that migrated through the chambers were subjected to fixation with methyl alcohol followed by crystal violet staining. Finally, an inverted microscope was used to count cells (200×, 10 different fields).

Western blotting

To analyze expression of proteins (Cytochrome c, Bax, Bid, Caspase 3, 9, 8, PARP, cyclin B1, Cdc25c Cdc2, MEK, and ERK), the osteosarcoma cells were lysed and the proteins were extracted. The proteins in each sample were then quantified by BCA assay and Western blotting was carried out, as described previously [11].

Statistical analysis

The experiments were carried out in triplicate and the values represent means of the 3 replicates ± SD. The t test was used for statistical analysis using GraphPad Prism 7 software. The values were considered significant at p<0.05.

Results

Evodiamine inhibits the growth of osteosarcoma cells via induction of apoptosis

The antiproliferative activity of Evodiamine was evaluated against U2OS osteosarcoma cell line and normal hFOB 1.19 cells. The results revealed that Evodiamine significantly (p<0.05) inhibited the proliferation of U2OS osteosarcoma cell line with IC50 of 6 μM. However, Evodiamine exhibited mild antiproliferative effects on the normal hFOB 1.19 cells with an IC50 of 105 μM (Figure 1A). The morphological analysis of the Evodiamine-treated cells showed blebbing and shrinkage, which are characteristic of apoptosis (Figure 1B). Consistent with the above results, DAPI and annexin V/PI staining showed that Evodiamine triggered apoptosis in U2OS cells and the percentage of the apoptotic cells was significantly enhanced (p<0.05) with increasing concentrations of Evodiamine (Figure 2A, 2B). Apoptosis is associated with altered expression of several proteins; therefore, we examined the expression of cytochrome c, Bax, Bid, Caspase 3, 9, and 8, and PARP. The results revealed that Evodiamine treatment upregulated the expression levels of cytosolic cytochrome c, mitochondrial Bax, cleaved caspase 3 and 9, and PARP (Figure 3). Taken together, these results clearly show that the antiproliferative effects of Evodiamine are at least in part due to induction of apoptosis.

Figure 1

Evodiamine exerts antiproliferative effects on U2OS osteosarcoma cells. (A) Antiproliferative effected determined by MTT assay. (B) Morphological analysis of Evodiamine-treated U2OS cells. The experiments were carried out in triplicate and expressed as mean ± SD (* p<0.05).

Figure 2

Induction of apoptosis in U2OS osteosarcoma cells as indicated by (A) DAPI staining and (B) annexin V/PI staining. The experiments were carried out in triplicate.

Figure 3

Effect of different concentrations of Evodiamine on the protein expression of apoptosis-related proteins as determined by Western blotting. The experiments were carried out in triplicate.

Evodiamine triggers G2/M cell cycle arrest in osteosarcoma cells

We also investigated whether Evodiamine triggers G2/M cell cycle arrest in U2OS osteosarcoma cells. The results showed that Evodiamine significantly (p<0.05) increases the percentage of cells in G2 phase of the cell cycle in a concentration-dependent manner (Figure 4). Moreover, protein expression analysis of cell cycle regulatory proteins revealed that Evodiamine caused significant downregulation of cyclin B1, Cdc25c, and Cdc2, as well as their phosphorylated counterparts (Figure 5).

Figure 4

Evodiamine triggers cell cycle arrest in U2OS cells. Flow cytometry analysis showing distribution of the Evodiamine-treated cells in different phases of cell cycle. The experiments were carried out in triplicate.

Figure 5

Western blots showing the effect of Evodiamine on the expression of cell cycle-related proteins. The experiments were carried out in triplicate.

Evodiamine inhibits cell migration and invasion of osteosarcoma cells

Next, the effect of Evodiamine on the migration and invasion of the U2OS cancer cells was investigated by wound-healing and Transwell assays. We found that at IC50, Evodiamine significantly (p<0.05) inhibited the migration and invasion of U2OS osteosarcoma cells (Figure 6A, 6B). Suppression of cell migration was also associated with inhibition of expression of metalloproteinases (MMP) 2 and 9 (Figure 6C).

Figure 6

Inhibition of (A) cell migration (B) cell invasion of U2OS cells treated with Evodiamine at IC50 concentration. (C) Western blots showing the effect of Evodiamine on the expression of MMP-2 and MMP-9. The experiments were carried out in triplicate.

Evodiamine inhibits the Raf/MEK/ERK signalling pathway of osteosarcoma cells

The effect of Evodiamine on the Raf/MEK/ERK signalling cascade was also examined. We found that Evodiamine significantly (p<0.05) suppressed the expression of p-MEK and p-ERK in a dose-dependent manner, while no effect was found on MEK and ERK protein expression (Figure 7).

Figure 7

Concentration-dependent effect of Evodiamine on the Ras/MEK/ERK signalling pathway as determined by Western blotting analysis. The experiments were carried out in triplicate.

Discussion

Plants are natural chemical factories producing a wide diversity of chemical scaffolds. The metabolites are produced by plants as a defense against the biotic and abiotic stresses [12]. Since plants are often exposed to extreme environmental conditions, they have evolved to produce different types of secondary metabolites. These metabolites have been shown to be beneficial in the treatment of human diseases [13]. Evodiamine is an important plant-derived alkaloid that has been reported to inhibit the growth of cancer cells [14,15]. However, the anticancer activity of Evodiamine has not been evaluated against osteosarcoma. Given this background, we examined the antiproliferative effects of osteosarcoma against a panel of osteosarcoma cell lines. We found that Evodiamine inhibited the proliferation of the U2OS osteosarcoma cells with an IC50 of 6 μM. Evodiamine was also previously reported to inhibit the growth of colon cancer, breast cancer, and hepatocellular carcinoma cells [6,16]. The morphological examination of Evodiamine-treated cells showed shrinkage and blebbing, which are characteristic of apoptosis, and this motivated us to investigate whether Evodiamine inhibits cell proliferation by triggering apoptosis. Apoptosis is an important and highly conserved type of cell death that is considered imperative for normal developmental processes, host defense, and inhibition of oncogenesis [17]. Deregulated apoptosis is a characteristic of cancer cells, and molecules that stimulate apoptosis in cancer cells may prove beneficial in the development anticancer chemotherapy [18]. Interestingly, in the present study, Evodiamine did induce apoptosis in U2OS osteosarcoma cells. It is well established that upregulation of Bax and downregulation of Bcl-2 makes the outer membrane of the mitochondria more porous and thereby facilitates the release of some mitochondrial apoptotic proteins, which ultimately cause the activation of caspases [19]. In the present study, we observed that the expression of cytc c, Bax, cleaved caspase 3 and 9, and cleaved PARP was significantly increased, indicative of intrinsic apoptosis. However, no effect was observed on the expression of caspase 8, thereby ruling out the possibility of the intrinsic apoptosis. Analysis of the Evodiamine-treated U2OS cells at different phases of the cell cycle indicated that Evodiamine prompts G2/M cell cycle arrest. Cell cycle in eukaryotes is generally driven by the stimulation of cyclin-dependent kinases and their cofactor, cyclin [20]. In the present study, we observed that Evodiamine caused a significant decrease in the expression of cyclin B1, Cdc25c, and Cdc2, favoring cell cycle arrest at G2/M phase. Metastasis of osteosarcoma is very challenging to manage; therefore, it is important to discover molecules that can inhibit the metastasis of osteosarcoma cells [21]. In the present study, we observed that Evodiamine inhibited the migration and invasion of U2OS osteosarcoma cells. Dysregulation of the RAS/extracellular signal-regulated kinase (ERK) pathway is common in cancer cells; therefore, targeting this pathway may provide a novel alternative to conventional therapy [9,22]. We examined the effect of Evodiamine on the RAF/MEK/ERK pathway and found that Evodiamine prevented the phosphorylation of MEK and ERK, suggesting that Evodiamine is a potential candidate to target this pathway.

Conclusions

We found that Evodiamine may be an important molecule for the treatment of osteosarcoma. Evodiamine inhibits the proliferation of osteosarcoma cells by induction of apoptosis and cell cycle arrest and warrants further in vivo studies.