Huaier inhibits cholangiocarcinoma cells through the twist1/FBP1/Wnt/ß-catenin axis.

BACKGROUND: Although Huaier granules can be used as prospective anti-cholangiocarcinoma drugs, the mechanism of action of Huaier granules in cholangiocarcinoma is not clear. The anti-cholangiocarcinoma effect of Huaier granules was validated in cell line research. In vitro experiments were conducted to investigate the signalling pathways affected by Huaier in CCA cells. METHODS AND RESULTS: Real-time quantitative PCR (RT‒qPCR) and Western blot analysis were performed to analyse gene expression in CCA cells. MTT assays, scratch tests, and Transwell assays were used to explore the effects on the proliferation and metastasis of CCA cells. Chromatin immunoprecipitation assays were performed to reveal the potential underlying mechanisms involved. Twist1 was upregulated in human CCA tissues. In addition, its expression levels were negatively related to FBP1 expression levels. Mechanistically, Twist1 can bind to the region of the FBP1 promoter to reduce its expression. Huaier plays an indispensable role in suppressing Twist1 expression to inhibit the Twist1/FBP1/Wnt/ß-catenin axis. Then, we verified the effect of Huaier in vitro. CONCLUSIONS: These findings suggested that Huaier granules were capable of inhibiting CCA development through regulating the Twist1/FBP1/Wnt/ß-catenin signalling axis and provided a novel orientation for the development of novel anti-CCA drugs.

Huaier suppresses cell viability, migration and invasion in human non-small cell lung cancer via lncRNA DLEU2/miR-212-5p/ELF3 axis.

Accumulating studies suggest that Huaier exerts anti-tumor effects through intricate mechanisms. Despite extensive research on its efficacy in lung cancer, further investigation is required to elucidate the molecular mechanism of Huaier. The involvement of long noncoding RNAs (lncRNAs) in the anti-lung cancer effects of Huaier remains unknown. In this study, we found Huaier suppressed cell viability, migration and invasion in non-small cell lung cancer (NSCLC) cells. LncRNA sequencing analysis revealed Deleted in lymphocytic leukemia 2 (DLEU2) to be significantly downregulated in Huaier-treated NSCLC cells. Furthermore, DLEU2 silencing was observed to suppress NSCLC progression, while DLEU2 overexpression attenuated the anti-tumor effects of Huaier in NSCLC, thereby promoting cell viability, migration and invasion of NSCLC. The ceRNA role of DLEU2 had been demonstrated in NSCLC, which directly interacted with miR-212-5p to rescue the repression of E74 Like ETS Transcription Factor 3 (ELF3) by this microRNA. Additionally, Huaier was found to regulate the expression of miR-212-5p and ELF3. Functionally, miR-212-5p inhibitor or ELF3 overexpression reversed the effects of DLEU2 silencing or Huaier treatment, resulting in increased colony formation, migration and invasion in NSCLC. Taken together, these results illuminate the mechanism underlying Huaier's anti-tumor effects via the DLEU2/miR-212-5p/ELF3 signaling pathway, which offers novel insights into the anti-tumor effects of Huaier and constitutes a promising therapeutic target for the treatment in NSCLC.

[Huaier triggering mitochondria apoptosis in colorectal cancer cells through oxidative stress].

This study investigates the specific mechanisms of Huaier-induced mitochondrial apoptosis in colorectal cancer. HCT116 and SW480 cells were subjected to Huaier treatment. Cell proliferation and migration capabilities were examined through CCK-8 and scratch experiments, respectively. Apoptotic cells were clarified with Annexin-PE staining. DCFH-DA staining, malondialdehyde(MDA), and glutathione(GSH) were used to evaluate the oxidative stress damage level of cells. MitoSOX and JC-1 probes were used to selectively target mitochondria reactive oxygen species(mtROS) and mitochondria membrane potential(MMP) for the evaluation of mitochondria damage. Western blot(WB) experiment was performed to determine apoptosis proteins and PINK1/Parkin pathway. Experiments reveal that in different concentrations of Huaier treatment, the proliferation and migration capabilities of HCT116 and SW480 cells were both restrained. Additionally, mitochondrial apoptosis was activated. Compared with the control group, excessive ROS in colorectal cancer cells was generated in the Huaier group, while MDA increased, and GSH decreased, indicating oxidative stress damage. mtROS increased, and MMP decreased in colorectal cancer cells treated with Huaier, indicating mitochondrial damage. WB result revealed that Huaier suppressed the PINK1/Parkin pathway, hindered the clearance of impaired mitochondria, and subsequently facilitated apoptosis. In conclusion, Huaier impairs colorectal cancer cells through oxidative stress and mitochondria damage. Furthermore, it suppressed the PINK1/Parkin pathway, promoting mitochondria apoptosis in colorectal cancer cells.

Metabolomic comparison between natural Huaier and artificial cultured Huaier.

Vanderbylia robiniophila (Murrill) B.K. (Huaier) is a kind of higher fungal fruiting body that is parasitic on the trunk of Sophora japonica and Robinia pseudoacacia L.. As a traditional Chinese medicine with a history of more than 1,600 years, Huaier has attracted wide attention for its excellent anticancer activity. A systematic study on the metabolome differences between natural Huaier and artificial cultured Huaier was conducted using liquid chromatography-mass spectrometry in this study. Principal component analysis and orthogonal projection on latent structure-discriminant analysis results showed that cultured Huaier evidently separated and individually separated from natural Huaier, indicating metabolome differences between natural and cultured Huaier. Hierarchical clustering analysis was further performed to cluster the differential metabolites and samples based on their metabolic similarity. The higher contents of amino acids, alkaloids and terpenoids in natural Huaier make it an excellent choice as a traditional Chinese medicine for anticancer or nutritional supplementation. The results of the Bel-7,402 and A549 cell cytotoxicity tests showed that the anticancer activity of natural Huaier was better than that of cultured Huaier. This may be due to the difference in chemical composition, which makes the anticancer activities of natural and cultured Huaier different.

Huaier Polysaccharide Attenuates Doxorubicin-Induced Acute Cardiotoxicity by Regulating Ferroptosis.

We studied the effects of Huaier polysaccharide (HP) in doxorubicin-induced myocardial injury in mice. The content of HP in Trametes robiniophila Murr medicinal fungus determined by the phenol-sulfuric acid method was 85.25%. In the in vitro model, the viability of H9c2 cells was significantly increased after HP treatment compared to the control, while doxorubicin (DOX) decreased this parameter. The inhibitory effect of DOX on cell viability was attenuated after HP treatment. In the in vivo model, the body weight of mice in DOX and DOX+HP groups was significantly decreased compared to the control group. ECG showed significantly elevated ST segment in the DOX group, while in the DOX+HP group, ECG was close to normal. The levels of cardiotoxicity markers cTnI and lactate dehydrogenase in the DOX+HP group were significantly lower than in the DOX group. In the DOX group, the myocardial tissue had obvious structural disorder and interfibrillar vacuoles. In the DOX+HP group, the cardiomyocytes were neatly arranged without interfibrillar vacuoles. The expression of the ferroptosis marker glutathione peroxidase 4 was increased in the DOX+HP group compared to the DOX group. Thus, our study reveals that HP attenuated DOX-induced myocardial injury in mice probably by regulating ferroptosis.

[Anti-tumor effect of Huaier extract supernatant on human gastric cancer HGC-27 and MGC-803 cells].

The purpose of this study was to investigate the effect of Huaier extract supernatant(HES) on the proliferation, apoptosis, autophagy, and migration of human gastric cancer HGC-27 and MGC-803 cells and its molecular mechanisms. The main components in HES were preliminarily analyzed by high-performance liquid chromatography-mass spectrometry(HPLC-MS). Methyl thiazolyl tetrazolium(MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine(EdU) staining assay were used to explore the effect of HES on the proliferation of human gastric cancer HGC-27 and MGC-803 cells. Hoechst staining and flow cytometry assay were used to determine the effect of HES on apoptosis of human gastric cancer HGC-27 and MGC-803 cells. Acridine orange staining and cell scratch assay were used to determine the effect of HES on autophagy and migration of human gastric cancer HGC-27 and MGC-803 cells, respectively. Western blot was used to investigate the regulatory effect of HES on the expression levels of proteins related to apoptosis, epithelial-mesenchymal transition(EMT), and signaling pathways in human gastric cancer HGC-27 and MGC-803 cells. The results showed that HES mainly contained some components with high polarities. HES significantly reduced the cell viability of human gastric cancer cells in a dose-and time-dependent manner. The IC_(50 )values after 48 h of HES treatment in human gastric cancer HGC-27 and MGC-803 cells were 7.56 and 10.77 g·L~(-1), respectively. Meanwhile, HES inhibited the colony-forming ability and short-term proliferation of human gastric cancer cells. The apoptosis rates of HGC-27 and MGC-803 cells treated with 8 g·L~(-1) HES for 72 h were 62.13%±8.92% and 54.50%±3.26%, respectively. HES also promoted autophagy in human gastric cancer cells and impaired their migration ability in vitro. Moreover, HES up-regulated the cleavage of the apoptosis marker poly ADP-ribose polymerase(PARP) and the protein expression level of the epithelial cell marker E-cadherin, and down-regulated the protein levels of phosphorylated-mammalian target of rapamycin(p-mTOR), phosphorylated-S6(p-S6), and phosphorylated-extracellular signal-regulated kinase(p-ERK) in human gastric cancer cells. Therefore, HES is one of the effective anti-tumor components of Huaier, which inhibits the proliferation and migration of human gastric cancer cells, and induces apoptosis and autophagy. Moreover, the mTOR signal and ERK signal may be involved in the anti-gastric cancer effect of HES. This study provides novel references for the in-depth research and clinical application of Huaier. It is also of great significance to promote the scientific development and utilization of Huaier.

Huaier shows anti-cancer activities by inhibition of cell growth, migration and energy metabolism in lung cancer through PI3K/AKT/HIF-1α pathway.

Huaier has been verified to have anti-cancer effects on many tumours. However, little information is available about the effects of Huaier on non-small cell lung cancer (NSCLC). We sought to probe the anti-cancer effects and related mechanisms of Huaier on lung cancer. A549 cells were pre-treated with 2, 4 and 8 mg/mL Huaier at different time points. Thereafter, cell viability was analysed by CCK-8 and the migration and invasion were detected by Scratch test and Transwell chamber migration assay. Moreover, ELISA, Western blot, shRNA transfection and RT-PCR were conducted to discover the related gene and protein expressions of energy metabolism and phosphatidylinositol 3-kinase (PI3K)/AKT/hypoxia-inducible factor 1α (HIF-1α) pathway. Furthermore, tumour xenografts were accomplished to inspect the anti-cancer effects of Huaier. Our consequences suggested that Huaier considerably repressed cell viability and migration in a dose-dependent way. In addition, Huaier statistically suppressed glycolysis, glucose transport and lactic acid (LA) accumulation. Besides, we detected that Huaier could inactivate the PI3K/AKT/HIF-1α pathway. The in vivo data confirmed that Huaier obviously decreased tumour volume and tumour growth, reduced the glycolysis, glucose transport and HIF-1α expression in the tumour-bearing tissues. Our results suggested Huaier revealed anti-tumour effects in both in vivo and in vitro possibly through PI3K/AKT/HIF-1α pathway.

p-MEK expression predicts prognosis of patients with adenocarcinoma of esophagogastric junction (AEG) and plays a role in anti-AEG efficacy of Huaier.

The incidence rate of adenocarcinoma of the esophagogastric junction (AEG) is increasing worldwide with poor prognosis and unclear pathogenesis. Trametes robiniophila Murr. (Huaier), a traditional Chinese medicine has been used in the clinical treatment of a variety of solid tumors, including AEG. However, its anticancer components and molecular mechanisms are still unclear. In our previous studies, we have found that Huaier n-butanol extract (HBE) shows the most potent anticancer activity among different extracts. In the present study, we aimed to investigate the clinical relevance of p-MEK expression in AEG patients and the role of the MEK/ERK signaling pathway in the anti-AEG efficacy of HBE in vitro and in vivo. We herein demonstrate that p-MEK expression in AEG tissues was significantly higher than that in paracancerous tissues and correlated with a poor prognosis in AEG patients. We further found that HBE inhibited the colony formation, migration, and invasion in AEG cell lines in a concentration-dependent manner in vitro. HBE also suppressed the growth of AEG xenograft tumors without causing any host toxicity in vivo. Mechanistically, HBE caused the inactivation of the MEK/ERK signaling pathway by dephosphorylating MEK1 at S298, ERK1 at T202, and ERK2 at T185 and modulating the expression of EMT-related proteins. In summary, our results demonstrate that the high expression of p-MEK may be an independent factor of poor prognosis in patients with AEG. The clinically used anticancer drug Huaier may exert its anti-AEG efficacy by inhibiting the MEK/ERK signaling pathway.

Anti-tumor effect of Huaier extract against neuroblastoma cells in vitro.

Huaier extract, the main active constituent proteoglycan, has anti-tumor activity in various experimental and clinical settings. However, the potential anti-neuroblastoma and associated mechanisms have not been investigated. Therefore, in this study, we aimed to elucidate the potential role of Huaier extract in 3 human neuroblastoma cell lines. Our study demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability in a dose-dependent manner. Huaier extract induced cell cycle arrest at G0/G1 phase in neuroblastoma and decreased the cell cycle related protein expression of cyclin D3. Western blotting analysis also showed that Huaier extract induced neuroblastoma cell apoptosis and autophagy. Signaling analysis indicated that Huaier extract suppressed the MEK/ERK and mTOR signaling pathways simultaneously. In conclusion, we verify that Huaier extract causes cell proliferation inhibition, apoptosis, autophagy, and cell cycle arrest in G0/G1 phase via MEK/ERK and mTOR signaling. Huaier extract may act as a complementary agent for treating neuroblastoma.

[Systematic evaluation of Huaier Granules adjuvant treatment of primary liver cancer].

To systematically evaluate the efficacy and safety of Huaier Granules in the adjuvant treatment of primary liver cancer. The databases of CNKI, Wanfang, VIP, CBMdisc, PubMed, Cochrane Library and EMbase were searched by computer to screen out the randomized controlled trial on Huaier Granules combined with Western medicine in the treatment of primary liver cancer from the establishment of the databases to January 2020. Data extraction and quality evaluation were conducted for the included literature. Meta-analysis was conducted with RevMan 5.3 software, and evidence quality evaluation was conducted for the outcomes by GRADE profiler software. A total of 24 articles were included, with a total sample size of 2 664 cases. Meta-analysis showed that as compared with Western medicine alone, Huaier Granules combined with Western medicine could improve the objective remission rate(RR=1.38, 95%CI[1.26, 1.51], P<0.000 01), disease control rate(RR=1.29, 95%CI[1.10, 1.52], P=0.002) and 6-month survival rate(RR=1.20, 95%CI[1.10, 1.32], P<0.000 1), 1-year survival rate(RR=1.39, 95%CI[1.23, 1.58], P<0.000 01), 2-year survival rate(RR=1.95, 95%CI[1.28, 2.96], P=0.002), KPS score(MD=17.15, 95%CI[6.47, 27.83], P=0.002) and the improvement rate of KPS score(RR=2.02, 95%CI[1.47, 2.77], P<0.000 1), AFP decline rate(RR=1.40, 95%CI[1.20, 1.62], P<0.000 1), CD3~+(MD=17.34, 95%CI[9.28, 25.40], P<0.000 1), CD4~+(MD=8.62, 95%CI[1.59, 15.64], P=0.02), CD8~+(MD=1.95, 95%CI[-3.93, 7.82], P=0.52), CD4~+/CD8~+(MD=0.42, 95%CI[-0.33, 1.17], P=0.27); reduce the level of AFP(MD=-71.57, 95%CI[-80.42,-62.72], P<0.000 01), recurrence rate(RR=0.76, 95%CI[0.67, 0.85], P<0.000 01), and incidence of adverse reactions(RR=0.60, 95%CI[0.41, 0.89], P=0.01) in patients with primary liver cancer. According to the GRADE system, the evidence for outcome measures was low to very low. The results show that Huaier Granules have certain efficacy and high safety in adjuvant treatment of primary liver cancer, but its effect in reducing adverse reactions and improve immunity remains to be verified. Due to the poor quality of the included studies and evidences, the conclusions still need to be further verified by multi-center, large sample, and randomized double-blind controlled studies.

An immune-stimulating proteoglycan from the medicinal mushroom Huaier up-regulates NF-κB and MAPK signaling via Toll-like receptor 4.

Trametes robiniophila Murr. (Huaier) is a mushroom with a long history of use as a medicinal ingredient in China and exhibits good clinical efficacy in cancer management. However, the antitumor components of Huaier and the underlying molecular mechanisms remain poorly understood. Here, we isolated a proteoglycan with a molecular mass of ∼5.59 × 104 Da from Huaier aqueous extract. We named this proteoglycan TPG-1, and using FTIR and additional biochemical analyses, we determined that its total carbohydrate and protein compositions are 43.9 and 41.2%, respectively. Using biochemical assays and immunoblotting, we found that exposing murine RAW264.7 macrophages to TPG-1 promotes the production of nitric oxide (NO), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) through Toll-like receptor 4 (TLR4)-dependent activation of NF-κB and mitogen-activated protein kinase (MAPK) signaling. Of note, the TPG-1 treatment significantly inhibited the tumorigenesis of human hepatoma HepG2 cells likely at least in part by increasing serum levels of TNFα and promoting leukocyte infiltration into tumors in nude mice. TPG-1 also exhibited good antitumor activity in hepatoma H22-bearing mice and had no obvious adverse effects in these mice. We conclude that TPG-1 exerts antitumor activity partially through an immune-potentiating effect due to activation of the TLR4-NF-κB/MAPK signaling cassette. Therefore, TPG-1 may be a promising candidate drug for cancer immunotherapy. This study has identified the TPG-1 proteoglycan as an antitumor agent and provided insights into TPG-1's molecular mechanism, suggesting a potential utility for applying this agent in cancer therapy.

[Effect of Huaier aqueous extract on growth and metastasis of human non-small cell lung cancer NCI-H1299 cells and its underlying mechanisms].

This study aims to investigate the effect of Huaier aqueous extract on the growth and metastasis of human non-small cell lung cancer NCI-H1299 cells and its underlying mechanisms. MTT assay was used to detect the effect of Huaier aqueous extract on the proliferation of NCI-H1299 cells. Flow cytometry was used to examine the effect of Huaier aqueous extract on the apoptosis, cell cycle, and ROS level of NCI-H1299 cells. Wound healing assay was used to evaluate the effect of Huaier aqueous extract on the migration ability of NCI-H1299 cells. Western blot was used to detect the levels of proteins involving apoptosis, epithelial-mesenchymal transition(EMT), and MAPK signaling pathway in NCI-H1299 cells exposed to Huaier aqueous extract. The results showed that Huaier aqueous extract inhibited the proliferation of NCI-H1299 cells, and induced cell-cycle arrest at the phase S. Huaier aqueous extract promoted the apoptosis of NCI-H1299 cells by down-regulating the expression of anti-apoptotic protein Bcl-2. Moreover, Huaier aqueous extract increased ROS level and induced ferroptosis in NCI-H1299 cells. EMT played a critical role in cancer metastasis. Huaier aqueous extract reduced the migration ability of NCI-H1299 cells by inhibiting EMT of NCI-H1299 cells. In addition, this study revealed that Huaier aqueous extract inhibited MAPK signaling pathway in human non-small cell lung cancer NCI-H1299 cells, which may be one of Huaier's mechanisms in inhibiting growth and metastasis of NCI-H1299 cells. This study provides a new theoretical basis for the clinical treatment of lung cancer with Huaier, and important reference significance for further studies on the anti-tumor mechanisms of Huaier.

The synergistic antitumor effect of Huaier combined with 5-Florouracil in human cholangiocarcinoma cells.

BACKGROUND: 5-Florouracil (5-FU) is a commonly used chemotherapeutic drug for cholangiocarcinoma, whereas it has unsatisfactory effect, and patients often have chemo-resistance to it. The combination of chemotherapeutic agents and traditional Chinese medicine has already exhibited a promising application in oncotherapy. Huaier extract (Huaier) has been used in clinical practice widely, exhibiting good anti-tumor effect. This paper aims to investigate the possibility of combination 5-FU and Huaier as a treatment for cholangiocarcinoma. METHODS: A series of experiments were performed on the Huh28 cells in vitro, which involved cell proliferation, colony formation, apoptosis, cell cycle, migratory and invasive tests. Besides, western blots were also performed to examine the potential mechanism of 5-FU. RESULTS: The combination effect (antagonism, synergy or additive) was assessed using Chou-Talalay method. Using the CCK-8 and Colony formation assay, the anti-proliferation effect of 5-FU combined with Huaier was observed. Apoptosis inducing and cell cycle arrest effect of the combination of two drugs were assessed by flow cytometry. To determine the combined treatment on cell immigration and invasion ability, wound healing and Transwell assay were performed. The above experiment results suggest that the combined 5-FU and Huaier, compared with treatment using either drug alone, exhibited stronger effects in anti-proliferation, cycle arrest, apoptosis-induced and anti-metastasis. Further, western blot results reveal that the inhibition of STAT3 and its target genes (e.g. Ki67, Cyclin D1, Bcl-2 and MMP-2) might be set as the potential therapeutic targets. Besides, the inhibition of combination treatment in proteins expression associated with proliferation, apoptosis, cell cycle and metastasis was consistent with that of previous phenotypic experiments. CONCLUSIONS: Huaier combined with 5-FU exhibited a synergistic anti-tumor effect in Huh28 cell. Furthermore, the mechanisms might be associated with the activation and translocation of STAT3, as well as its downstream genes.

The effects of polysaccharides from Auricularia auricula (Huaier) in adjuvant anti-gastrointestinal cancer therapy: A systematic review and network meta-analysis.

The aim of this study was to assess the comparative efficacy and safety of polysaccharides from Auricularia auricula (Huaier) for patients with gastrointestinal cancers (GICs) through a systematic review and network meta-analysis. We performed a network meta-analysis to identify evidence from clinical trials. We searched databases for publications up to February 2018. The prespecified primary efficacy outcomes were clinical therapeutic effects, which included the treatment response rate, 0.5-year overall survival rate, 1-year overall survival rate, 2-year overall survival rate, KPS improved rate and AFP decreased rate. The safety outcomes were the side effects of Huaier. The secondary efficacy outcome was immune function. Subgroup analyses and meta-regression were performed according to the various cancer types in all of the efficacy and safety outcomes. The study is registered with PROSPERO (CRD42018086481). A total of 33 trials, involving 2884 patients and 10 treatment arms, were eligible. Huaier significantly increased the treatment response rate (2.48, 1.83-3.35) and survival rate (0.5-year, 1-year and 2-year) and improved immune function without increasing the incidence of adverse effects. Significant efficacy was observed in most subgroups. Network meta-analysis revealed that Huaier was very suitable for combination therapy with TACE and 125I particle implantation. Similarly, Huaier also had a good adjuvant therapeutic effect on enhancing platinum (L-OHP and DDP) and adriamycin (ADM). Huaier offers clear advantages for patients with GICs. Moreover, patients should be encouraged to accept Huaier treatment, especially HCC patients undergoing combination therapy with TACE and 125I particle implantation.

The Anticancer Effect of Huaier Extract in Renal Cancer 786-O Cells.

BACKGROUND: Trametes robiniophila Murr (Huaier) has been used as an adjuvant therapy of tumor in traditional Chinese medicine for many years, but the underlying mechanisms are largely unknown. In the present study, we tested the inhibitory effect of Huaier extract on renal cancer 786-O cells and explored the possible mechanisms. METHODS: 786-O cells were treated by gradient concentrations of Huaier extract, cell viability, invasion, migration and apoptosis were assessed by cell counting kit 8, cell scratch, transwell, and flow cytometry assay in vitro. The changes in protein level were detected by western blot analysis. Finally, the anticancer effect of Huaier was tested in vivo by nude mouse tumorigenicity assay. RESULTS: Viability of 786-O cells was suppressed by Huaier in a time- and dose-dependent manner; cell invasion and migration were also dramatically inhibited. Flow cytometry assays showed that Huaier could induce cell apoptosis. Western blotting analysis indicated that Huaier suppressed the activation of PI3K/AKT/mTOR/p70S6K/4E-BP1 signaling pathway. We also found that Huaier could partly reverse the epithelialmesenchymal transition (EMT) process. In vivo experiment indicated that tumor growth in the xenograft mouse model was suppressed by Huaier. CONCLUSION: Huaier plays an anticancer effect partially through the suppression of the PI3K/AKT/mTOR/p70S6K/4E-BP1 pathway and by reversing the EMT process. Huaier may act as an effective agent for treating renal cell carcinoma.

[Huaier aqueous extract inhibits proliferation of human hepatoma SK-HEP-1 cells through up-regulation of autophagy].

The purpose of this study was to investigate the effect of Huaier on autophagy of human hepatoma SK-HEP-1 cells and the effect of autophagy on the proliferation of SK-HEP-1 cells. CCK-8 assay was used to evaluate the effect of Huaier on the proliferation of SK-HEP-1 cells under different concentrations and different times. Acridine orange staining was used to measure the effect of Huaier on the autolysosome formation in SK-HEP-1 cells. Immunofluorescence assay was applied to examine the effect of Huaier on the expression and distribution of autophagy marker LC3 in SK-HEP-1 cells. In addition， LC3 expression was also checked by immunoblot analysis in the presence of Huaier. At last， the effects of Huaier in combination with autophagy inhibitor bafilomycin A1 on the proliferation of SK-HEP-1 cells was detected by CCK-8 assay. The results showed that Huaier aqueous extract significantly inhibited the proliferation of human hepatoma SK-HEP-1 cells in a dose- and time-dependent manner. Huaier aqueous extract dramatically promoted the formation of autolysosome in SK-HEP-1 cells. Moreover， Huaier markedly increased the number and intensity of intracellular LC3 fluorescent puncta and up-regulated LC3-Ⅱ expression. These data indicated that Huaier evidently activated autophagy of SK-HEP-1 cells. Additionally， autophagy inhibition significantly attenuated the sensitivity of SK-HEP-1 cells to Huaier treatment. Therefore， autophagy activation is involved in the inhibitory effects of Huaier on the proliferation of human hepatoma SK-HEP-1 cells.

[Killing effect of Huaier combined with DC-CIK on nude mice bearing colon cancer HT29 stem cells in vivo].

To compare the therapeutic effects of different treatment methods on the nude mice bearing colon cancer HT29 cells. BalB/C nude mice colon cancer stem cell models were established and randomly divided into the following four groups, with 8 nude mice in each group: blank control group, DC-CIK group, Huaier group, and Huaier combined with DC-CIK group (combined treatment group). The mice in DC-CIK group and combined treatment group received 1×106 DC-CIK cells treatment by tail vein injectionafter the tumor stem cells were inoculated for 4 days,2 times a week for three weeks. The mice in Huaier group and combined treatment group received intragastric administration at the dose of 20 g/60 kg body weight, 0.2 mL/time, once a day for a total of three weeks. The mice in control group received equal volume of normal saline. Tumor size and body weight of nude mice were measured every 2 days during treatment for three weeks in each group. After the treatment, the nude mice were sacrificed to measure the tumor weight and the tumor inhibition rate was calculated. The RT-PCR method was used to detect the expression levels of the key genes in the signal pathway. After the end of the treatment, the quality of the tumor in the Huaier group, DC-CIK group and combined treatment group was significantly lower than that in the control group; the quality in combined treatment group was significantly lower than that in Huaier group and DC-CIK group.Among them, the tumor inhibition rate reached 46.77% in the combined treatment group. In respect of changes in expression levels of key genes in the signaling pathway, the mRNA expression levels of key genes PI3KR1 and Akt in PI3K/Akt pathway, key genes Wnt1 and CTTNB1 in Wnt/ß-catenin pathway, and key genes Notch1, Notch2, Notch3 in Notch pathway in the combined treatment group were lower than those in DC-CIK group and Huaier group. The Huaier combined with DC-CIK group showed best therapeutic effect among different treatment methods for HT29 stemcell colon tumors in nude mice, providing a new idea for clinical treatment of colon cancer.

Effect of Huaier On the Proliferation of Mesangial Cells in Anti-Thy-1 Nephritis.

BACKGROUND/AIMS: To determine whether an aqueous extract of Trametes robiniophila Murr. (Huaier) suppresses anti-Thy-1 mesangial proliferative glomerulonephritis (MsPGN) in vivo and platelet-derived growth factor (PDGF)-BB-induced mesangial cell proliferation in vitro. METHODS: Male Wistar rats were randomly categorized into 5 groups: Sham, Thy-1, and 3 Huaier-treated groups (low, medium, and high dose). Two weeks after treatment, urinary proteins were quantified and renal pathological changes were examined. MAX interactor 1 (Mxi-1) and proliferating cell nuclear antigen (PCNA) expression levels in isolated glomeruli, rat mesangial cell viability, cell-cycle distribution, and cell-cycle pathways were assessed. RESULTS: Huaier diminished the proliferative damages and urinary protein secretion in Thy-1 rats. PCNA was downregulated, whereas Mxi-1 was upregulated in the isolated glomeruli of Huaier-treated groups compared with the Thy-1 group. Huaier inhibited PDGF-BB- stimulated proliferation of rat mesangial cells in a time- and dose-dependent manner (50% inhibitory concentration = 6.19 mg/mL) and induced G2 cell-cycle arrest. Cell-cycle pathway proteins were downregulated, whereas Mxi-1 was upregulated in Huaier-treated mesangial cells compared with PDGF-BB-stimulated cells. CONCLUSION: Huaier reduces urinary protein excretion and relieves hyperplasia in mesangial cells in anti-Thy-1 MsPGN as well as inhibits PDGF-BB-stimulated proliferation and DNA synthesis of rat mesangial cells in vitro, suggesting its novel therapeutic potential in MsPGN.

Huaier Extract Inhibits Breast Cancer Progression Through a LncRNA-H19/MiR-675-5p Pathway.

BACKGROUND/AIMS: Increasing evidence indicates that Huaier extract has promising therapeutic effects against cancer. However, the mechanisms that underlie its anti-tumor effects remain unclear. In recent years, various studies have shown that long noncoding RNAs (lncRNAs) play a critical role in the regulation of cancer development and progression. Here, we explored the role of lncRNAs in Huaier-induced tumor suppression. METHODS: Microarray profiling was performed to identify the candidate lncRNAs affected by Huaier extract. Quantitative realtime PCR (qPCR) was used to evaluate the transfection efficiency and the influence of Huaier extract on H19 expression. The effect of Huaier extract on the cell viability was examined by MTT. Moreover, the rates of apoptotic cells were detected using flow-cytometric analysis. Western blot analysis was applied to show the protein levels of CBL. RESULTS: Microarray data derived from Huaier-treated breast cancer cells identified H19 as a potential target. Huaier extract reduced the expression of H19. The over-expression of H19 inhibited the cytotoxic effects of Huaier extract; in contrast, reduced H19 expression enhanced the function of Huaier extract. MiR-675-5p was identified as a mature product of H19. Moreover, Huaier extract reduced the miR-675-5p expression. Upregulating miR-675-5p reversed the inhibitory effects of Huaier extract, whereas downregulating miR-675-5p sensitized breast cancer cells to the effect of Huaier extract. In addition, Huaier extract increased the expression of CBL protein, a direct target of miR-675-5p. CONCLUSION: Collectively, the data demonstrate that Huaier extract reduces viability and induces apoptosis in breast cancer cells via H19-miR-675-5p-CBL axis regulation.

[Study on effect and mechanism of Huaier aqueous extract on growth and invasion of human prostate cancer PC3 cells].

The purpose of this study was to investigate the effect and mechanism of Huaier aqueous extracton growth and invasion of human prostate cancer PC3 cells. CCK-8 assay was used to evaluate the inhibitory effect of Huaier aqueous extract on proliferation of PC3 cells. The effects of Huaier aqueous extract on cell cycle and apoptosis of PC3 cells were analyzed by flow cytometry. Moreover, wound healing assay and Transwell assay were performed to determine the effect of Huaier aqueous extract on invasion and migration abilities of PC3 cells. PC3 cells treated with Huaier aqueous extract were subjected to western blotting for protein levels of EMT markers and phosphorylation levels of key proteins in MAPK pathway. Results revealed that Huaier aqueous extract significantly inhibited the proliferation of PC3 cells in a dose-dependent and time-dependent manner. Huaier aqueous extract dramatically increased the apoptosis rate and induced S-phase arrest in PC3 cells.Furthermore, Huaier suppressed invasion and migration abilities of PC3 cells, and facilitated MET process of PC3 cells via down-regulation of N-cadherin and TCF8/ZEB1 and up-regulation of E-cadherin. In addition, Huaier reduced the phosphorylation of JNK and ERK. Therefore, the regulatory effects of Huaier on EMT and MAPK pathway may be responsible for the suppressive effect of Huaier on growth and invasion of PC3 cells.