The Mechanisms Of Huaier Inhibiting Breast Cancer Progression

BACKGROUND:Breast cancer is the most common cancer in women. Worldwide, About 1.4 million new cases of breast cancer are expected to occur among women each year. And over 500 thousand women die of breast cancer. In China, the incidence and mortality of breast cancer were increasing every year. Therefore, exploring the molecular mechanisms of breast cancer and finding new treatments are important tasks of translational medicine.In China, Huaier has been used as an important medical fungi for 1,600 years. In recent years, the antitumor effects of Huaier have got more and more attentions. As reported, Huaier exerted important tumor prevention effects in hepatocellular carcinoma, cholangiocarcinoma, ovarian, melanoma, colorectal, gastric cancer, cervical cancer, pulmonary cancer and fibrosarcoma. Our previous study demonstrated that Huaier could induce cell apoptosis, arrest cell cycle and inhibit cell migration and invasion. However, the underlying mechanisms are not fully understood.In this study, we explored the effects of Huaier on autophagy, proliferation, ERa signaling, cancer stem cell and angiogenesis. We used molecular biology and clinical data to study the inhibitory effects of Huaier. This study provided pharmacological evidences for clinical use of Huaier.METHODS AND RESULTS:In this study, we intended to understand the inhibitory effects of Huaier, including the following five parts:Part I Huaier induces autophagic cell death in breast cancer cellsMETHODS:We used MTT to examine the effect of Huaier on cell proliferation. AO/EB staining and the TUNEL assay were used to detect other modes of cell death induced by Huaier. Transmission electron microscopy, MDC staining, immunofluorescence, western blot and flow cytometry were applied to detect the Huaier-induced autophagy. To further investigate whether the induction of autophagy contributed to Huaier-induced cell death, we used the autophagy inhibitors 3-MA, CQ and siRNA against LC3B. Using western blot, we examined the autophagic pathways. Then breast cancer cells were implanted subcutaneously into the right flank of BALB/c nu/nu mice, we measured the expression of associated proteins using immunohistochemical staining and western blot.RESULTS:Huaier significantly decreased cell viability in all three cancer cell lines. Data from AO/EB staining and the TUNEL assay demonstrated that Huaier could induce necrosis and apoptosis. Using transmission electron microscopy and MDC staining, Huaier could increase the number of autophagosomes in the cytoplasm of cells. The increased expression of LC3B was indicated by using immunofluorescence and western blot. Increased formation of AVOs was confirmed by flow cytometry. Inhibition of autophagy recovered the Huaier-induced cell cytotoxicity. Western blot suggested that Huaier could activate AMPK and inhibit mTOR/S6K pathway. In vivo data demonstrated that Huaier treatment inhibited xenograft tumor growth, increased the expression of LC3B, Atg7, Beclin-1, and reduced the expression of p62/SQSTM1.Part Ⅱ Huaier inhibits the proliferation of breast cancer through miR-99a-HOXAl pathwayMETHODS:We first examined the effect of Huaier on the expression of HOXA1. To better understand the functional role of HOXA1, we performed loss-of-function studies using small interfering RNA against HOXA1. qPCR was used to detect the transfection efficiency. MTT and transwell were applied to explore the functions of HOXA1. Then, we observed the effect of HOXA1 on the anti-cancer effects of Huaier.To explore the mechanisms of Huaier inhibiting HOXA1, the miRNA expression profile was used. qPCR was applied to detect the effect of Huaier on miR-99a expression. MTT and transwell were applied to explore the functions of miR-99a. Luciferase activity assays were used to confirm the relationship between miR-99a and HOXA1.To determine whether miR-99a-induced inhibition of cell proliferation, migration and invasion could be reversed by restoration of HOXA1 expression, we transfected a vector expressing HOXA1 without its 3’UTR into MCF7-miR-99a or MCF7-pMSCV cells. MTT and transwell were used to examine the proliferation, migration and invasion.RESULTS:Using qPCR, we demonstrated that Huaier could inhibit the expression of H0XA1. Knockdown of HOXA1 inhibited the proliferation, migration and invasion of cells. Overexpression of H0XA1 partially abrogated Huaier-mediated tumor suppression.Data from miRNA microarray showed that Huaier could inhibit the expression of miR-99a. Relative to 31 samples of normal breast tissues, miR-99a expression was significantly down-regulated in tissues from 110 cases of primary breast cancers (P= 0.0031). Additionally, in 84%(26 of 31) of breast cancers, miR-99a expression was reduced relative to the corresponding non-tumorous breast tissues from the same patients. miR-99a level was obviously associated with overall survival (P= 0.040) and easier metastasis. Overexpression of miR-99a inhibited breast cancer proliferation, migration and invasion. Knockdown miR-99a got the reverse effects. Using bioinformatics analysis, HOXA1 was predicted to be the target of miR-99a. Western blot confirmed that overexpression of miR-99a resulted in reduced HOXA1 expression. luciferase activity assay demonstrated that HOXA1 was the direct target of miR-99a. Overexpression of HOXA1 could reverse the inhibitory effects of miR-99a.Part Ⅲ Huaier suppresses estrogen receptor a signaling of breast cancer cellsMETHODS:We first examined the effect of Huaier on the growth of ERa-positive human breast cancer cell lines using MTT. qPCR and western blot were applied to detect the mRNA and protein levels of ERa. To explore the molecular mechanisms of Huaier-induced ERa degradation, MG132 was used. Then we used qPCR to the transcription levels of estrogen responsive genes after Huaier treatment. MTT and western blot assays were used to detect the effect of Huaier on E2-induced proliferation and NF-κB activation.RESULTS:Huaier significantly inhibited the proliferation of these three cell lines in a dose- and time- dependent manner (P<0.05). Meanwhile, Huaier could decrease the mRNA and protein levels of ERa dose-and time-dependently. Addition of MG132 before Huaier treatment markedly rescued the downregulation of ERa (P<0.05). Significant reduction of mRNA levels of Cathepsin D, pS2 and PR could be observed dose- and time-dependently (P<0.05). Huaier significantly inhibited E2-induced cell proliferation, which was associated with the inactivation of NFkB pathway.Part IV Huaier inhibits stem-like characteristics of breast cancer cellsMETHODS:Mammosphere viability was first assessed by MTT assay. the number and size of mammospheres were also observed. Clonogenic assay was used to detect the colony formation ability. We examined the effect of Huaier on CD44+/CD24-breast cancer cells using flow cytometry. qPCR was used to analyze the expression of several stem cell markers. To further investigate the mechanisms underlying the inhibitory effect of Huaier, we explored several pathways involved in CSC development.RESULTS:Data from MTT indicated that Huaier could inhibit the viability of MCF7 mammospheres. Huaier significantly inhibited the number and size of primary spheres. In Clonogenic assay, Huaier reduced colony formation ability and declined the percentage of holoclones. As shown in flow cytometry, Huaier significantly suppressed the number of cells expressing CD44+/CD2-. The percentage of CD44+/CD24- cells in adherent parental cells was obviously declined by 50.23 (2 mg/ml),61.36 (4 mg/ml), and 62.73%(8 mg/ml), as compared with vehicle control. The mRNA levels of OCT-4, NESTIN, and NANOG were significantly reduced after treatment with Huaier in a time-and dose-dependent manner. Huaier obviously inhibited the activation of Hh pathway detected by western blot assay.Part V Huaier inhibits angiogenesis of breast cancerMETHODS:We examined cell viability of HUVEC by using MTT assay. To further explore the underlying mechanism of the anti-proliferative effect of Huaier, we applied flow cytometry. scratch assay and transwell assay were used to examine the influence of Huaier on cell motility and invasion. Tube formation assay, CAM assay and aortic ring assay were used to detect the effect of Huaier on angiogenesis. We used western blot analysis to investigate the associated endothelial signaling pathways. Mouse breast cancer cells 4T1 were implanted into BALB/c mice to the anti-angiogenic and antitumor effects of Huaier in vivo.RESULTS:Data from MTT demonstrated that Huaier suppressed the proliferation of the HUVECs. Using flow cytometry, Huaier could induce apoptosis and arrest cell cycle at G0/G1 phase. After treatment with Huaier, the migration and invasion of the HUVECs was dose- and time-dependently inhibited. Data from tube formation assay, CAM assay and aortic ring assay indicated that Huaier significantly inhibited angiogenesis. The anti-angiogenic effect of Huaier was associated with reduced expression of VEGF and inactivation of ERK, JNK, STAT3 and NFkB pathways. Meanwhile, Huaier significantly inhibited the growth of xenograft in vivo and decreased MVD.CONCLUSIONS1. Huaier induces autophagic cell death by inhibiting the mTOR/S6K pathway.2. Huaier inhibits the proliferation of breast cancer through miR-99a-HOXA1 pathway.3. Huaier suppresses estrogen receptorasignaling in breast cancer cells.4. Huaier inhibits stem-like characteristics of breast cancer cells via inactivation of hedgehog pathway5. Huaier inhibits angiogenesis of breast cancer.SIGNIFICANCESThis study identified the anti-tumor activity of Huaier and its molecular mechanisms (autophagy, proliferation, ERa signaling, cancer stem cell and angiogenesis). These results indicated the important roles of Huaier in breast cancer treatment and provided the rationale for its clinical use.