Huaier extract, a natural, traditional Chinese medicine, has got growing attention in recent years for its anti-tumor effect. Previous studies on Huaier extract showed that a variety of cancer cells are suppressed by Huaier via inhibition of proliferation and promotion of apoptosis. In addition, multiple signaling pathways and biological processes were found to be involved in the anticancer effect of Huaier, such as JAK2/STAT3 (Yang et al. 2016), autophagy (Wang et al. 2015), Wnt signaling (Zhang et al. 2013), and the PIK3/AKT signaling pathway (Xie et al. 2015), among others. However, the effect of Huaier on OS and the associated mechanisms are yet to be elucidated. In this study, we observed that Huaier could inhibit OS cell proliferation by blocking G0/G1 into S phase. It could also suppress invasion and migration and promote apoptosis of OS cells in a time- and dose-dependent manner. The MAPK pathway, among the most frequently mutated signaling pathway in human cancers, has been recognized as a promising target of cancer therapy (Bao et al. 2016). Furthermore, we found the MAPK pathway is involved in the anti-OS effect of Huaier.
We analyzed the target genes of Huaier extract using bioinformatics. The results showed that Huaier is closely related to cell growth. Moreover, the MAPK pathway plays an important role in the biological effects of Huaier. Therefore, we used the CCK-8 and clone formation assays to show that Huaier can reduce the viability and inhibit the proliferation of OS cells. Previous studies have shown that numerous anticancer medicines can arrest the cell cycle in the G0/G1, S, and G2/M phases (Lee et al. 2007). Flow cytometry was performed to detect the cell cycle of MG-63 and MNNG/HOS cells under treatment with Huaier. In contrast to results obtained by other study (Matsumoto et al. 2008; Yan et al. 2015), Huaier increased the percentage of OS cells in the G0/G1 phase, while decreasing the cell percentage in the G2/M phase. Similar medicine-induced of G0/G1 arrest has also been detected in other cell type, such as human breast cancer cells (Zhou et al. 2018), and rheumatoid arthritis fibroblast-like synoviocytes (Zhang et al. 2018a). Cyclin D1 is a cell-cycle-associated protein which is involved in the transition from the G1 phase to the S phase (Wang et al. 2018). Overexpression of Cyclin D1 is linked to the progression and development of cancer (Cai et al. 2012). Multiple studies have shown that the expression of Cyclin D1 is decreased when the cell cycle is arrested (Zhang et al. 2018b). P21 and P27 are Cyclin-dependent kinase inhibitors, which always increase when cancer cell proliferation is induced (Zhou et al. 2008). To further explore the mechanism of Huaier-induced cell cycle arrest, Western blotting was performed to examine the expression levels of cell-cycle-associated proteins. Under treatment with Huaier, the expression of P21 and P27 increased, while Cyclin D1 expression decreased. This indicates that Huaier suppressed the proliferation of OS cells by regulating the expression of cell-cycle-associated proteins. However, the underlying mechanism should be explored further.
Multiple studies have shown that Huaier suppresses tumor development by inducing cell apoptosis. Consequently, flow 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 significantly 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 significantly under treatment with Huaier. These finding 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 five-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-ERK/ERK decreased, while P-JNK/JNK and P-P38/P38 increased. Numerous studies have shown that ERK is involved in cell proliferation, migration and invasion (Chen et al. 2018; Liu et al. 2018; Reddy et al. 2003). These results illustrated that Huaier can inhibit the proliferation and motility of OS cells by downregulating P-ERK. The JNK protein kinases include JNK1, JNK2, and 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 the most widely studied is P38α (Ono and Han 2000). Bcl2 is a downstream protein of JNK1 and JNK2, while P38α can affect Bax expression (Weston and Davis 2007). Therefore, JNK and P38 are closely related to the mitochondrial apoptosis pathway. P38α can also down-regulate the content of Cyclin family proteins, thereby negatively regulating the cell cycle (Thornton and Rincon 2009). Consequently, it may be considered that Huaier can affect proliferation and apoptosis in OS cells by influencing MAPK pathway.