Huaier Extract Inhibits Proliferation and Promotes Apoptosis via the MAPK Pathway in Human Osteosarcoma Cells

Background: Osteosarcoma is a common pediatric bone malignancy. Huaier, a traditional Chinese medicine, attracts increasing attention for its antitumor effect. The aim of this study is to investigate the inhibitory effect and molecular mechanisms of Huaier in osteosarcoma cells. Methods: Bioinformatics was performed to determine the biological processes and pathways connected to Huaier. The CCK-8 method and ow cytometry were used to detect the cell viability, cell cycle distribution and apoptosis of osteosarcoma cells (MG-63 and MNNG/HOS). Western blot was applied to assess the expression of proteins involved in apoptosis, cell cycle and the MAPK pathway. Results: Huaier could inhibit osteosarcoma cells proliferation by arresting cells in the G0/G1 phase. The extract also suppressed invasion and migration, while promoting the apoptosis of osteosarcoma cells in a time- and dose-dependent manner. Under Huaier stimulation, the expression of p-ERK/ERK, Cyclin D1 and Bcl-2 decreased, while the expression of p-JNK/JNK, p-P38/P38, P21, P27, Bax and Caspase3 increased in osteosarcoma cells. Conclusions: Our ndings demonstrated for the rst time that Huaier extract could inhibit proliferation and promote apoptosis in osteosarcoma cells via the MAPK pathway in vitro, suggesting that Huaier may be developed as a chemopreventive medicine for the treatment of osteosarcoma. mg/mL and 7.3% at 4 mg/mL. MNNG/HOS cells in G2/M phases cell cell-cycle-associated carried by Western blotting. treatment the of Cyclin D1 and increased the of P21 and P27 (Figs. P-P38/P38 results P-ERK. JNK JNK1, JNK2, JNK3, of which JNK1 and JNK2 are widely present in various cells (Bode and Dong 2007). P38 is generally divided into four subtypes, of which is P38α (Ono Han Bcl2 downstream protein of JNK1 and JNK2, P38α

In this study, we observed the effects of Huaier extract on two widely used OS cell lines, MG-63 and MNNG/HOS, and demonstrated for the rst time that Huaier extract could inhibit proliferation and promote apoptosis in OS cells via the MAPK pathway in vitro.

Cell culture and reagents
Human broblast cell line MRC-5 and human MG-63 and MNNG/HOS osteosarcoma cells, were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM) that supplemented 1% penicillin streptomycin and 15% fetal bovine serum (Gibco, Grand Island, NY, USA) and at a temperature of 37 °C.
Colony formation assay MNNG/HOS cells were plated at a density of 700 cells per well in 6-well plates and MG-63 cells were plated at a density of 1000 cells per well in triplicate. 24 h later, the cells were treated with various concentrations of Huaier (0, 2 and 4 mg/mL) and then incubated at 37 °C in a 5% CO 2 incubator for 10 days. Washing with PBS was followed, then xing with methanol and staining with 1% crystal violet. Finally, we counted the colonies which contained more than 50 cells.

Cell viability assay
Cell Counting Kit-8 assay was performed to evaluate the cell viability activity according to the manufacturer's instructions.
MNNG/HOS, MG-63 and MRC-5 cells were harvested into 96 -well plates at a density of 5 × 10^3 cells per well with 200 ul culture medium. After 24 hours of incubation, cells were treated with various concentrations of Huaier for indicated times. Then the cell viability was measured using cell counting kit -8(CCK8, solarbio, Beijing, China). Finally, the absorbance of plate was read using the automated microplate spectrophotometer (Bio-Rad Laboratories, USA) at 450 nm. The data was repeated three times.

Invasion assay
Transwell invasion assay was performed to detect the effect of Huaier on the invasion ability of MNNG/HOS and MG-63 cells. We put 8 uM pore-sized Transwell inserts which were covered by matrigel into 24-well plate full of 700 ul of medium including 20% FBS.
We added complete medium without FBS to the upper layer of the well plate. Cells (2 × 10^4) were inoculated with or without Huaier for 24 h in an incubator that was maintained at 37 °C and 5% CO2. After 24 h, we washed the cells twice with PBS, carefully detached the cells from the upper layer plate with a cotton swab and then we xed the cells with 100% methanol for 30 min. Finally cells were stained with 1% crystal violet for 15 min and were counted by using a light microscope (Olympus BX53, Tokyo, Japan).

Scratch assay
The migration ability of the cells was tested using a scratch test. The MNNG/HOS and MG-63 cells were seeded into 6-well plates at 5 × 10^5 per well. After 12 h, the cells were cultured in complete medium to a con uent state. Next, the cells were scraped vertically with a 20 µ L pipette tip. Then, the cells were washed three times with PBS. Finally, we treated cells with various concentrations of Huaier for 24 h in Serum-free medium. After 24 h, the width of the scratches was observed under a microscope (Olympus BX53, Tokyo, Japan) and statistical analysis was performed.
Cell cycle analysis MG-63 and MNNG/HOS cells were seeded into 6-well plate at a density of 2 × 10^5/well and treated with different concentrations of Huaier (0, 2, 4 mg/mL) for 24 h. Osteosarcoma cells were harvested by EDTA-free trypsinization and then washed with pre-chilled PBS. Then, we placed it overnight at 7 °C in 75% alcohol. The next day, we stained the cells with propidium iodide (PI) and RNase A according to the instructions of the KGI Biocycle Kit (Nanjing, China). Since the stain is a photosensitizer, we stain in the dark. After incubation for 30 min, we analyzed the stained cells by BD Accuri C6 Plus (Becton -Dickinson, San Jose, CA) software. All experiments were repeated three times.
Apoptosis ow-cytometry assay Cell apoptosis was detected by Annexin-V-FITC and PI staining. Osteosarcoma cells were plated at 6-well plate and treated with various concentration (0, 2, 4 mg/mL) of Huaier for 24 h. Next, we collected cells with EDTA-free trypsin followed by Annexin V FITC Apoptosis Assay Kit (Hanbio, Shanghai, China) instructions for operation. For the reason phosphatidylserine can bind to Annexin V-FITC, we determined the proportion of cells that were early apoptotic; PI was a DNA-binding dye which has excitation and emission wavelengths of 617 nm and 536 nm, respectively. It can produce red uorescence but no membrane permeability, not through the living cell membrane. So, we use it to determine the proportion of cells with late apoptosis/death. Annexin V-FITC and PI are photosensitizers and we use them in the dark. We immediately analyzed stained cells using BD Accuri C6 Plus (Becton -Dickinson, San Jose, CA) software.

Western blot analysis
We scraped the cells into EP tubes with cell scraping and lysed the supernatant proteins with RIPA lysis buffer (Betotium Institute of Biotechnology, Beijing, China). All operations were performed on ice. We measured the protein concentration of the sample using the BCA assay kit (Beyotime Biotechnology Institute). The remaining protein was boiled at 100 ° C for 5 min, and stored at minus 20 degrees. We separated the total protein by 10-15% SDS-PAGE gel electrophoresis then transferred it to the polyvinylidene uoride (PVDF) membrane by electrolysis on ice. After blocking the PVDF membrane with 5% BSA in TBST for 45 minutes, the primary antibody of the target protein was incubated for 2 h, and then the membrane was washed three times with TBST for ten minutes each time. Next, goat anti-rabbit secondary antibody cultured with horseradish peroxidase were incubated for 1 h. The membrane was still washed three times with TBST for ten minutes each time. Finally, the immune response zone was detected using an ECL detection system (SmartChemi 420, Beijing, China). Every experiment was performed three times.

Drug-target search and functional enrichment analysis
The BATMAN-TCM is a traditional Chinese medicine database which contains detailed information on various drugs and their related information. In this study, the main target genes of Huaier were found using Chinese medicine database. Next, we use BATMAN-TCM to perform pathway enrichment analysis and gene ontology. BATMAN-TCM provides researchers with a comprehensive and systematic functional annotation tool to discover the biological implications behind the list of submitted genes.
Gene ontology analysis includes molecular functions (MF), cellular components (CC), and biological processes (BP). P < 0.05 was considered statistically signi cant. The terms of GO results were also ranked by P value. We used the same manner to carry out the KEGG pathway.

Statistical analysis
Statistical analysis was performed using Graghpad prism 7 software and Student's t-test was carried out to analyze statistical differences. P < 0.05 was considered as a statistically signi cant difference. The result is shown as the mean ± SD. All experimental data was repeated at least three times.

Drug-target search, enrichment analysis and Gene Ontology
Huaier was input into the Chinese medicine database BATMAN. Then, the main corresponding target genes of Huaier were detected (Table 1). Subsequently, we performed enrichment analysis on pathways and GO functions. To assess the functional characteristics of the Huaier-mediated genome, we carried out GO annotations on all genes. The top 12 items of GO and KEGG enrichment analysis were then selected based on the P-value ranking (Figs. 1a, b). Among the top 12 KEGG terms and 12 GO terms, we subsequently selected some terms of interest (Table 2). Functional analysis showed that the genes associated with Huaier are mainly related to cell growth. In addition, the KEGG results showed that the MAPK signaling pathway is involved in the antitumor effect mediated by Huaier.   (Figs. 4a, b). Treatment with Huaier for 24 h resulted in nearly 40% apoptosis and death in MG-63 cells, while the apoptosis and death rate of the control cells was only 18% (Fig. 4c). Similar effects were observed for the MNNG/HOS cells (Fig. 4d). To further understand the mechanisms of cell apoptosis, we examined the changes of apoptosis-associated regulatory proteins using Western blotting.

Huaier may suppress OS cells via the MAPK signaling pathway
To determine whether the MAPK signaling pathway is involved in the OS cancer cell proliferation and apoptosis induced by Huaier, the expression levels of ERK, JNK and P38-MAPK were tested by Western blot. We treated MG-63 and MNNG/HOS cells with Huaier for 24 hours. The total ERK content was basically unchanged while the P-ERK/ERK content was signi cantly reduced. Furthermore, both P-JNK/JNK and P-P38/P38 levels increased, which was consistent in both cell lines (Figs. 6a-c). However, the underlying mechanism should be explored further.

Discussion
Multiple studies have shown that Huaier suppresses tumor development by inducing cell apoptosis. Consequently, ow cytometry was used to detect the apoptotic rate of Huaier-treated OS cells. As expected, the rate of apoptosis and death of OS cells increased signi cantly after exposure to Huaier. Apoptosis is a crucial biological process in the regulation of cancer development and treatment response. There are two distinct apoptotic pathways, namely, the extrinsic membrane death-receptor pathway and the intrinsic mitochondrial pathway. Many chemopreventive medicines promote cancer cell apoptosis through the mitochondrial pathway (Liu et al. 2012). Bcl-2 and Bax are two key proteins involved in the intrinsic mitochondrial pathway (Li et al. 2009). Bax can induce mitochondria to release cytochrome C, which enhances cell apoptosis. In contrast, the function of Bcl-2 is to suppress apoptosis by blocking the release of cytochrome C (Walensky 2006). Cytochrome C activates the down-regulation of the caspase cascade, while Caspase3 ends this process (Kroemer et al. 2007). By assessing the expression of apoptosis-related proteins, we showed that Bcl-2 decreased while Cleaved-Caspase3 and Bax increased signi cantly under treatment with Huaier. These nding suggest that Huaier promoted apoptosis in OS cells by triggering the mitochondrial-signaling pathway.
To detect the migration and invasion ability of OS cells under the Huaier treatment, we performed the wound healing test and Transwell assay. Obtained results showed that Huaier can inhibit the migration and invasion of OS cells. The low ve-year survival rate of OS is closely related to its strong metastatic ability. Therefore, Huaier could be a potential drug for the treatment of OS metastasis. Based on the abovementioned results, Huaier could inhibit the proliferation and promote the apoptosis of OS cells. To explore the underlying mechanisms, we used Western blotting to detect the expression of major proteins in the MAPK signaling pathway which was predicted to be involved in the antitumor effect of Huaier by bioinformatics analysis. Results showed that P-

Conclusions
In this study, we showed that Huaier is a potential drug for the treatment of OS. Huaier inhibited the proliferation, suppressed the invasion and migration, and promoted the apoptosis of OS cells in a time-and dose-dependent manner. Furthermore, the MAPK pathway was shown to be involved in the anticancer effect of Huaier (Fig. 7). Further studies should assess the effects of Huaier on OS in vivo, possibly using a xenograft mouse model of OS. Authors' contributions XG performed the experiment, and was a major contributor in writing the manuscript, YZ and PY analyzed the data, RT, DL, YK, HL and ML critically revised the manuscript. All authors read and approved the nal manuscript.   The MAPK pathway is involved in the anticancer effect of Huaier