Huaier Aqueous Extract Inhibits Ovarian Cancer Cell Motility via the AKT/GSK3β/β-Catenin Pathway

Abstract

Traditional Chinese medicine has gained popularity due to its ability to kill tumor cells. Recently, the apoptotic and anti-angiogenic effects of Trametes robiniophila murr (Huaier) have been investigated. The aim of this study was to investigate its effect on cell mobility and tumor growth in ovarian cancer. Cell viability and motility were measured using SRB, scratch and migration assays. Cell apoptosis was analysed by annexin V/PI staining. Using a reverse-phase protein array (RPPA) assay, we analyzed the levels of 153 proteins and/or phosphorylations in Huaier-treated and untreated cells. Huaier inhibited cell viability and induced both early and late apoptosis in SKOV3, SKOV3.ip1 and Hey cells in a time- and dose-dependent manner. Cell invasiveness and migration were also suppressed significantly. The RPPA results showed significant differences (of at least 30%; P <0.05) in the levels of 7 molecules in SKOV3 cells and 10 in SKOV3.ip1 cells between the untreated and treated cells. Most of the molecules identified play roles in cell proliferation, apoptosis or cell adhesion/invasion. Western blot analysis further validated that Huaier treatment resulted in decreased AKT phosphorylation, enhanced expression of total GSK3β, inhibition of the phosphorylation of GSK3β on S9, reduction of both cytoplasmic β-catenin expression and nuclear β-catenin translocation, and transcriptional repression of several Wnt/β-catenin target genes (DIXDC1, LRP6, WNT5A, and cyclin D1). After knocking down GSK3β, β-catenin expression could not be inhibited by Huaier. Finally, Huaier inhibited the growth of ovarian tumor xenografts in vivo. These studies indicate that Huaier inhibits tumor cell mobility in ovarian cancer via the AKT/GSK3β/β-catenin signaling pathway.

The anticancer effect of Huaier (Review)

“…To detect whether Huaier inhibits proliferation though the pAKT/mTOR/S6 pathway, the protein levels of pAKT, AKT, pS6 (S235-236) and pS6 (S240-244) were evaluated by western blot analysis. The results showed that phosphorylation of S6 at S235-236 and S240-244 was significantly downregulated in three ovarian epithelial cancer cell lines (SKOV3, SKOV3.ip1 and HEY) after 72 h of Huaier treatment, in accordance with the decreased pAKT expression in SKOV3 and SKOV3.ip1 cells, indicating that the pAKT/ mTOR/S6 kinase pathway is involved in the anti-proliferative effects of Huaier (17).…”

“…The same trend was also found in Huaier polysaccharide (HP)-treated hepatocarcinoma cells (MHCC97-H) (21), Huaier extract-treated melanoma cells (A875) (20), ovarian epithelial cancer cells (SKOV3, SKOV3. ip1 and HEY) (17) and breast cancer cells (MCF-7 and MDA-MB-231) (19). The underlying mechanism associated with the proliferation inhibitory effect of Huaier was further revealed in which several well-studied proteins and classic pathways were involved (Fig.…”

“…The classic wnt/β-catenin signaling pathway is essential to human beings since aberrant wnt/β-catenin signaling leads to a series of human diseases including various types of cancers (29). It was demonstrated that Huaier inhibits the proliferation of cancer cells via the wnt/β-catenin signaling pathway (17). The detection of decreased wnt target gene expression after Huaier treatment provides evidence in support of wnt/β-catenin pathway inhibition.…”

“…In previous studies, the MTT, CCK-8 cell viability or sulforhodamine B (SRB) assay has been used to measure the cell viability after Huaier treatment (16,17,19,20). The results showed that w-NTRP (a neutral water-soluble polysaccharide isolated from the fruit bodies of Huaier) dose-dependently inhibited the proliferation of three human cholangiocarcinoma cell lines (QBC939, Sk-ChA-1 and MZ-ChA-1) in vitro (16).…”

“…However, the anticancer effects of Huaier which have attracted increasing worldwide interest have only been researched in recent years (14)(15)(16). Accumulating evidence suggests that the anticancer mechanisms of Huaier effects may be associated with various biological activities, such as inhibition of cell proliferation (15), anti-metastasis (17), interference with tumor angiogenesis (14) and tumor-specific immunomodulatory effect (16,18). Huaier was found to be a putative anticancer agent with broad therapeutic value following elaborate molecular studies conducted in the recent past.…”