Cancer is a major cause of morbidity and mortality worldwide and remains a formidable disease. According to the National Cancer Institute (NCI), it was estimated that there would be 1,665,540 new cases of all cancer types and 585,720 people would succumb to cancer in 2014. Conventional treatment for cancer patients include surgery, radiotherapy and chemotherapy. Over the last few decades, adjuvant chemotherapy following surgery has been proven to decrease recurrence and improve patient survival, yet may subsequently promote other malignancies such as ovarian (1), bowel (2), gastro-oesophageal (3) and pancreatic (4) cancers. Recently, several advanced alternative treatments including gene and targeted therapy are attracting worldwide attention (5,6). However, these treatments are not effective, and resistance against chemotherapy, toxicity, side-effects and the unaffordable cost for most patients require the search for comparatively natural products or non-toxic drugs (7). Therefore, it is of urgent need in the biomedical science to develop novel anticancer agents with fewer and less debilitating side-effects and reduced drug resistance to satisfy the unfulfilled therapeutic demand of cancer patients.

Agents derived from natural sources exert a multitude of effects on chronic inflammation-driven diseases including cancer (8). Recently, much attention has been focused on medicinal plants or herbs as potential sources of new therapeutic anticancer drugs due to their lack of toxic effects, relatively lower cost and enhanced effectiveness (9–11). It was reported that these agents increase chemotherapeutic efficacy, reduce toxicity, and prolong the survival time, improve the immune functions and the quality of life of cancer patients (12).

Trametes robiniophila Murr. (Huaier) is a sandy beige mushroom found on the trunks of trees and was first recorded by Shi-Zhen Li, a famous Chinese practitioner in the Ming Dynasty. Huaier has been widely used in traditional Chinese medicine (TCM) for ~1,600 years (13). However, the anticancer effects of Huaier which have attracted increasing worldwide interest have only been researched in recent years (14–16). 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. Irrespective of its effectiveness as a complementary therapy for cancer, to date, there is no systematic review available concerning the anticancer effects of Huaier and the underlying mechanisms. Understanding the mechanisms involved in the anticancer action of Huaier should provide useful information for its possible application in clinical cancer therapy and also in cancer prevention. This review briefly summarizes the literature published to date reporting the anticancer effect of Huaier and its underlying mechanisms.

Cell proliferation is an indispensible process in the occurrence and development of cancer. 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). 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. 1).

The p53 tumor-suppressor gene can influence cell cycle progression, DNA damage repair, genomic stability and thus inhibit the proliferation of cancer cells (22). To determine whether the proliferation inhibitory effect of Huaier is due to p53-induced cell-cycle arrest, cell cycle distribution of Huaier-treated MCF-7, MDA-MB-231 and A875 cells was analyzed by flow cytometry (19,20). G0/G1 arrest was found in the MCF-7 and MDA-MB-231 cells, suggesting that Huaier inhibits cell proliferation via cell-cycle arrest at the G0/G1 phase (19). The time- and dose-dependent increase in the fraction of cells in the G2/M phase and the decrease in PCNA protein (a molecular marker for proliferation) expression in the A875 cells treated with Huaier extract confirmed that Huaier extract inhibited melanoma A875 cell proliferation via cell-cycle arrest at the G2/M phase (20). The enhanced p53 expression detected in the MCF-7, MDA-MB-231 and A875 cells led to the conclusion that Huaier inhibits cell proliferation via p53-induced cell-cycle arrest (19,20).

As one of the major causes of cancer treatment failure and ultimately mortality in cancer patients (32), metastasis is an extremely complicated multi-step process involving the separation of cancer cells from their primary site by penetrating the stromal tissue, circulation through the blood vessels or the lymph nodes, adhesion to the basement membrane and invasion of the target organ for distinct metastasis (33), thereby causing poor prognosis and survival of patients. Moreover, over 90% of cancer-related deaths are due to metastatic disease (34). Recent studies have shown that Huaier extract suppresses metastasis in ovarian cancer SKOV3, SKOV3.ip1 and HEY cell lines and in the human breast cancer MCF-7 and MDA-MB-231 cell lines, as well as in hepatocarcinoma MHCC97-H cells (17,19,21), indicating that Huaier may serve as a potent anti-metastatic agent for cancer therapy. Migration and scratch assays in vitro were performed to determine the cell migratory ability. In comparison with untreated cells, significant inhibition of migrational movement was observed in migration assays in MDA-MB-231 and MCF-7 cells treated with 4 mg/ml Huaier (19). In scratch assays, the migration index (corresponding to wound-healing capacity) was significantly inhibited in Huaier-treated SKOV3, SKOV3. ip1 and HEY cells compared with untreated cells, respectively (17). HP exerted the same effect in the scratch assays. The cell migration of human hepatocellular carcinoma cell line MHCC97-H was controlled in a time-dependent manner by HP, being inhibited by up to 28.57, 47.81 and 57.19% at 25, 50 and 100 µg/ml, respectively (21). Invasion assays were conducted to compare the invasive potential of Huaier extract-treated cells including MDA-MB-231, SKOV3, SKOV3. ip1, HEY and HP-treated MHCC97-H cells with untreated cells. The results revealed that following Huaier treatment the numbers of invading cells through the Matrigel-coated membrane were significantly decreased compared with the untreated groups (17,19,21). In cell adhesion assays performed on MHCC97-H cells, the numbers of adhesive cells on Matrigel-coated plates were stained with hematoxylin and eosin (H&E) reagent and counted under an inverted microscope. HP treatment significantly inhibited cell adhesion to the Matrigel-coated substrate in a dose-dependent manner; the reduction was 28.55, 47.48 and 56.47% with HP at 25, 50 and 100 µg/ml, respectively (21). The underlying mechanisms of these effects were revealed in several studies suggesting that the GSK3β/β-catenin and AEG-1 pathways were involved (17,19,21) (Fig. 2). These previous results suggest that Huaier may serve as a potent anti-metastatic agent of enormous clinical value in cancer therapy.

Angiogenesis is a complex process which is defined as the formation of new blood vessels from pre-existing ones. It involves a multi-step process which includes degradation of the extra cellular matrix, migration, proliferation, sprouting, elongation and tube formation of endothelial cells (40,41). Angiogenesis plays an essential role in tumor growth, metastasis and recurrence (42–45). Without vascularization, solid tumors grow only to 1–2 mm (46). Thus, preventing angiogenesis is a potential strategy for cancer therapy and many anti-angiogenic substances have entered the clinic and have been used as therapeutic options for various types of cancer (47).

Wang et al (14) reported that exposure to Huaier aqueous extract led to cell-cycle arrest, decreased mobility and reduced angiogenesis ability of human umbilical vein endothelial cells (HUVECs), indicating that Huaier may inhibit tumor-induced angiogenesis. The study showed that Huaier increased the proportion of HUVECs in the G0/G1 phase in a dose-dependent manner (from 36.79±2.25% in the control group to 62.41±9.77% in the 8 mg/ml Huaier group) and promoted the accumulation of p21, a well-studied cyclin-dependent kinase inhibitor, thus resulting in cell-cycle arrest. The motility of HUVECs was then examined using modified scratch and cell migration assays, both of which showed that the motility of the HUVECs was dose- and time-dependently inhibited (P<0.01). To further evaluate the effect of Huaier on angiogenesis, tube formation, chick embryo chorioallantoic membrane (CAM) and aortic ring assays were subsequently performed in vitro and ex vivo with direct Huaier treatment. The assays revealed that Huaier extract caused a marked decrease in the angiogenesis ability of HUVECs (14). The potential signaling pathways underlying the potent anti-angiogenic activity of Huaier extract were also uncovered. Vascular endothelial growth factor (VEGF), one of the most potent pro-angiogenic factors both physiological and pathological, is a highly specific mitogen for vascular endothelial cells and a vascular permeability enhancer which is essential for endothelial cell proliferation, migration and anti-apoptosis (48,49). Studies have demonstrated that the overexpression of VEGF in cancer patients is associated with poor prognosis and decreased survival (50,51). Huaier extract inhibited the expression of VEGF and the activation of ERK in a dose-dependent manner and eventually exhibited anti-angiogenic activity. Huaier extract also suppressd the phosphorylation of JNK, STAT3 and p65 (major components in the NF-κB complex), all of which are important pathways that regulate cell migration.

The anti-angiogenic activity in vivo was evaluated in BALB/c mice (14). Tumor tissues from the mice injected with 4T1 cells were stained with H&E, CD34 and TUNEL. Significantly reduced blood vessels and enhanced apoptosis were observed in mice administered a 100-µl solution containing 50 mg Huaier extract by gavage daily. The same effect was also found in tumor-bearing New Zealand rabbits (52). Lower expression of VEGF and P53 and significantly decreased microvessel density (MVD) were observed in the rabbits following treatment with Huaier. These results suggest that Huaier extract may serve as a potent anti-angiogenic and anticancer agent with broad therapeutic value.

Apoptosis is a genetically encoded program of cell suicide, characterized by the morphological features observed upon cell death which include nuclear condensation, nuclear and cellular fragmentation, membrane blebbing and phagocytosis of the dying cell in the absence of inflammation (53). It is a process that plays an indispensable role in the control of the growth and development of organisms, and serves as a natural barrier to cancer development (54). Blockage of apoptosis may determine the sensitivity of cancer cells to a wide range of diverse chemotherapy agents and explain the frequently observed phenomenon of multidrug resistance. Thus, apoptosis induction is considered to be a crucial strategy for cancer prevention and treatment since successful eradication of cancer cells through apoptosis is one of the ultimate aims of chemotherapy (55). Morphological changes have been observed in cancer cells exposed to Huaier extract. The majority of Huaier-treated melanoma A875 and breast cancer MCF-7 cells became enlarged, irregular-shaped and showed vacuolated changes in the cytoplasm (19,20). These morphological changes demonstrated cell damage following treatment with Huaier extract. Experiments in vitro showed that Huaier inhibited the proliferation of melanoma (A875), lung adenocarcinoma (A549), breast cancer (MCF-7 and MDA-MB-231), hepatocellular carcinoma (Hep-G2) and ovarian cancer (SKOV3, SKOV3.ip1 and HEY) cells by inducing apoptosis (15,17,19,20,52). These previous studies have confirmed that Huaier induces the apoptosis of cancer cells, suggesting that Huaier is an effective anticancer agent for the treatment of cancer patients.

Mitochondria are the targets of several molecular pro-apoptotic signal transduction pathways (56). Intrinsic control of apoptosis requires activation of cytosolic caspases by mitochondrial cytochrome c release and involves regulation of mitochondrial outer membrane permeabilization by Bcl-2 family proteins (57). Mitochondrial-mediated apoptosis has been studied in breast cancer MCF-7, melanoma A875, HCC Hep-G2 and ovarian epithelial cancer SKOV3.ip1 cell lines (17,19,20,52). Following PI/Annexin V double staining performed in MCF-7, A875 and SKOV3.ip1 cells treated with Huaier extract, apoptosis was induced as confirmed by increased late apoptosis or cell death rate (UR) and the early apoptosis rate (LR). Decreased MMP also confirmed Huaier-induced apoptosis in MCF-7 cells (19). Subsequent western blot analysis suggested that this effect was at least partly induced via the mitochondrial pathway. After treatment with Huaier aqueous extract for increasing time periods (48 and 72 h) and concentrations (4 and 8 mg/ml), the expression of p53, phosphorylated-p53 (p-p53), bcl-2 and BAX were tested with western blot analysis. The expression of p53 and p-p53 were upregulated, indicating the accumulation and activation of p53 in response to Huaier treatment in the breast cancer cell line MCF-7. Meanwhile, treatment with Huaier extract suppressed Bcl-2 expression and upregulated BAX expression in a time- and dose-dependent manner, suggesting mitochondrial-mediated apoptosis (19). The same trend was also found in melanoma A875 and ovarian cancer SKOV3.ip1 cells (17,20). In addition, caspase-3 is the most studied caspase concerning apoptosis induced by natural products (58). It has been reported in previous studies that the activation of caspase-3 was significantly increased in A875, MCF-7 and MDA-MB-231 cells following Huaier treatment, confirmed by increased cleaved caspase-3 expression and decreased pro-caspase-3 expression (19,20). As detected by immunohistochemistry, Huaier-treated tumor-bearing New Zealand rabbits had lower expression of P53 and Bcl-2 and higher expression of Bax with the differences being significant when compared with the control group (52). The results revealed that Huaier extract promoted cell apoptosis through the mitochondrial pathway, thus inhibiting tumor occurrence and progression.

MicroRNAs may act as oncogenes or tumor suppressors involved in the tumorigenesis and aggression of human cancers (59). It was observed in a recent study that Huaier treatment (2–8 mg/ml for 24–72 h) induced cell apoptosis of pulmonary adenocarcinoma A549 cells via upregulation of miR-26b-5p (15), an miRNA known to be significantly downregulated in various types of cancer and to induce apoptosis in cancer cells (60,61). Flow cytometric analysis showed that cells treated with 4 mg/ml Huaier and an miR-26b-5p inhibitor exhibited a 50% decrease in the rate of apoptosis as compared with the group treated with Huaier and the inhibitor control, indicating that Huaier induced apoptosis via miR-26b-5p. EZH2, a target gene of miR-26b-5p, is involved in the apoptosis of cancer cells and was found to be downregulated in A549 cells exposed to Huaier. EZH2 is the histone H3 lysine 27 methyltransferase of polycomb-repressive complex 2 and is overexpressed in multiple cancer types (62). Several EZH2-related proteins were detected by western blot analysis in both A549 and non-small cell lung cancer (NSCLC) H1299 cell lines and decreased expression of both β-catenin and bcl-2 was found in consensus with the previous finding that EZH2 can ultimately enhance Bcl-2 expression via the activation of Wnt/β-catenin signaling (63). Thus, Huaier may induce apoptosis in lung cancer cells via the miR-26b-5p-EZH2-mediated Wnt/β-catenin pathway (Fig. 3).

Cancer stem cells (CSCs) represent a small subset of cancer-initiating cells endowed with self-renewal and multi-lineage differentiation capacity that promote tumor growth and recurrence (64–66). Clinically, CSCs resist conventional cancer therapies including chemotherapy and radiation therapy and are involved in relapse which is one of the most important features leading to the poor clinical outcome of cancer patients (67–69). Therefore, major clinical challenges towards the complete eradication of cancers are likely to target CSCs (70). In the past few years, the potential role of naturally occurring agents as potent antitumor agents functioning by targeting CSCs has been highlighted (71).

Huaier extract inhibited the number and the size of spheroids formed in both colorectal cancer cells and mammospheres of breast cancer MCF7 cells at a significantly lower concentration than those exerting anti-proliferative impact on bulk cancer cells, indicating that CSCs were preferentially targeted by Huaier extract (72,73). Considering that CD44, CD24 and ALDH are cell surface markers for breast cancer MCF7 and colorectal cancer (CRC) cells, respectively, a decrease in the cell population of CD446⁺/CD24⁻ breast cancer cells and ALDH-positive CRC cells suggested an inhibitory effect of Huaier on CSCs.

The underlying mechanism of the inhibitory effect of Huaier on CSCs was subsequently studied, among which downregulation of the Wnt/β-catenin pathway and inhibition of the hedgehog (Hh) pathway were highlighted (Fig. 4). The Wnt/β-catenin pathway is one of the critical pathways demonstrated to mediate the self-renewal of CSCs (74). The activation of Wnt target genes depends on mediation by β-catenin, which enters the nucleus to activate the TCF/LEF transcription factor (74). Treatment of the CRC cells with Huaier led to dose-dependent downregulation of the levels of total β-catenin protein, decreased activation of TCF/LEF in the nucleus and reduced expression of cyclin D1, one of the Wnt/β-catenin downstream genes (72). Therefore, the downregulation of the Wnt/β-catenin self-renewal pathway may be a potential target of the Huaier extract. The inhibitory effect of Huaier extract on CSCs was also found to be partly dependent on the inactivation of the Hh pathway which is associated with both normal mammary gland development and breast cancer progression (73,75). After treatment with Huaier extract, the expression of Gli1 was obviously declined, showing that Huaier extract was highly effective to eradicate breast (CSCs) through regulating Hh signaling (73). These results identified Huaier as an effective agent of great clinical value to eradicate CSCs and to improve current cancer treatment.

Tumor growth and the immune system are intertwined in a complex competition where tilting the subtle balance between tumor-specific immunity and tolerance can ultimately decide the fate of the host (76). Recently, experimental and clinical testing of novel types of immunotherapeutic agents have been rapidly developing due to the advances in tumor immunology and a better understanding of the mechanisms regulating the immune response (77). The water extract from Huaier consists mainly of polysaccharide protein which has been proved to be of great clinical value as the main active ingredient in the anticancer effects and immunity-enhancing actions of Huaier (Fig. 5) (16). Lymphocytes are the key effector cells of the mammalian immune system, whose proliferation is considered an indicator of immunopotentiation. W-NTRP (a neutral water-soluble polysaccharide isolated from the fruit bodies of Huaier) activated different subpopulations of lymphocytes and was suggested to be a potent immunomodulating and immunoenhancing agent (16). TP-1 (a Huaier polysaccharide) administered at two doses (50 or 100 mg/kg) significantly prompted the T- or B-lymphocyte proliferation induced by ConA or LPS, respectively, when compared to those in H22-bearing mice (Kunming mice weekly transplanted with murine H22 ascitic hepatoma cells into the peritoneal cavities). Li et al (78) also noted increased CD4⁺ T cells and decreased CD8⁺ T cells induced by TP-1 in tumor-bearing mice (78). Macrophages and NK cells are capable of inducing the death of tumor cells and play essential roles in host anticancer immune response (79). Macrophages are an essential component of the host defense against tumor growth by releasing various cell factors, such as TNF-α, NO, reactive oxygen intermediates (ROI) and other substances to kill tumor cells (80). It was reported that W-NTRP significantly enhanced macrophage phagocytosis, increasing NO production and iNOS activity (16). An increased quantity of NK cells and enhanced NK cell activity were detected after exposure to Huaier polysaccharide (18,78). Huaier polysaccharide (TP-1) also modulated the cytokine release, as confirmed by the increased IFN-γ and IL-2 expression and inhibition of IL-10 expression. Taken together, these results suggest that the polysaccharide isolated from Huaier has an immunoregulatory effect and potent antitumor activities with great clinical value.

Huaier, as a type of officinal fungi, has been used in China for nearly 1,600 years, and has been reported to exert potent anticancer activities. This review aimed at obtaining a clear picture regarding the anticancer effects of Huaier and the underlying mechanisms. The anticancer potential of Huaier, including proliferation inhibitory, anti-metastasis, anti-angiogenesis, apoptosis induction, tumor-specific immunomodulatory and cancer stem cell inhibitory activities, have been validated in in vitro studies and in various animal models, indicating its potential therapeutic value against various types of cancers. Another encouraging finding was that Huaier may serve as an efficient anticancer agent with little toxicity. Preliminary toxicological evaluation demonstrated that TP-1 (a Huaier polysaccharide) had no obvious systemic toxicity on the kidney and liver of tumor-bearing mice at the therapeutic dose (25 and 50 mg/kg). This was confirmed by the results that the tumor-caused changes in hepatic function markers (ALT and AST) and renal function parameters (BUN, UA, and CRE) (78) were ameliorated, or even restored to the normal level after TP-1 treatment. Xenograft experiments in mice were also performed. The weight of tumors isolated from the Huaier-treated groups (at concentration of 2.5 g/kg/day) was significantly decreased compared to the control group with no significant difference in body weight, which indicated no obvious toxicity to mice at the curative dose (17). Several molecular targets and classic pathways of Huaier which play an important role in the development of cancer have also been identified. These results highlight the possible application of Huaier in cancer chemoprevention and lay a solid foundation for its clinical use in humans, opening a more effective anticancer treatment option even though most of the research is still in an experimental stage. We hope and believe that Huaier may provide a benefit in the treatment of malignant tumors as a prospective anticancer drug candidate.

Although the findings documented in this review are quite encouraging for the use of Huaier as a novel anticancer agent, several limitations exist in the current scenario. Most of the studies were conducted using Huaier aqueous extract, and the observed biological effects could be due to the combined effects of various components. Thus, further research on the specific effective anticancer components of Huaier is warranted. Although animal and experimental studies have been successfully carried out showing Huaier’s efficacy as an anticancer agent, no information on the absorption, distribution, metabolism, and excretion of Huaier in humans, or clinical trial was found in the search of the available literature. Therefore, clinical studies should be carried out to illustrate the untapped chemopreventive and therapeutic potential of Huaier either alone or in conjunction with existing therapies.