Developing an Efficient Processing System Treatment for the High Concentration of Eucalyptus Chemical Mechanical Pulp Wastewater.

The current wastewater treatment method shows low efficiency in treating wastewater with high concentrations of chemical mechanical pulp (CMP). Therefore, a chlorine dioxide Pretreatment Anaerobic Treatment (DPAT) was developed and applied to treat the CMP wastewater to obtain higher efficiency, obtaining the following results: The biodegradability of CMP wastewater improved after chlorine dioxide pretreatment. The COD of wastewater treated with chlorine dioxide was reduced from 5634 mg/L to 660 mg/L. The removal rate for chemical oxygen demand (COD) was 88.29%, 29.13% higher than the common anaerobic treatment. The reasons for the high efficiency of the DPAT treatment were that chlorine dioxide pretreatment removed the toxic substances in the original wastewater and thereby promoted the proliferation and growth of the anaerobe. The results show that pretreatment with chlorine dioxide can effectively enhance the biodegradability of high-concentration CMP wastewater. Therefore, DPAT treatment of high-concentration CMP wastewater is beneficial to environmental protection.

Natural ingredients from Chinese materia medica for pulmonary hypertension.

Pulmonary hypertension (PH) is a severe pathophysiological condition characterized by pulmonary artery remodeling and continuous increases in pulmonary artery pressure, which may eventually develop to right heart failure and death. Although newly discovered and incredible treatment strategies in recent years have improved the prognosis of PH, limited types of effective and economical drugs for PH still makes it as a life-threatening disease. Some drugs from Chinese materia medica (CMM) have been traditionally applied in the treatment of lung diseases. Accumulating evidence suggests active pharmaceutical ingredients (APIs) derived from those medicines brings promising future for the prevention and treatment of PH. In this review, we summarized the pharmacological effects of APIs derived from CMM which are potent in treating PH, so as to provide new thoughts for initial drug discovery and identification of potential therapeutic strategies in alternative medicine for PH.

Trametes robiniophila Murr in the treatment of breast cancer.

Breast cancer is the leading cause of cancer death among women across the world. Trametes robiniophila Murr (Huaier), a traditional herbal medicine, has been used in China to protect human health for about 1600 years. Recent years, Huaier had been proven to be effective for multiple types of malignancies. This systematic review focused on breast cancer treatment, summarizing the curative function of Huaier aqueous extract and polysaccharides in preclinical researches. Huaier could markedly inhibit breast cancer progression with low toxicity, enhance immune response and increase the sensitivity to radiation and chemotherapy. The therapeutic effect of Huaier granule in clinical studies was also included. This review amalgamated the current studies and highlighted the promising role of Huaier and its polysaccharides as complementary alternative medicine in breast cancer treatment.

Huaier Suppresses Breast Cancer Progression via linc00339/miR-4656/CSNK2B Signaling Pathway.

Huaier, as known as Trametes robiniophila Murr, is a traditional Chinese medicine. Various studies have demonstrated that Huaier could inhibit cancer progression and improve the prognosis of patients. In the present study, we comprehensively screened the expression profiles of lncRNAs, miRNAs, and mRNAs in Huaier-treated breast cancer cells. Using bioinformatic analysis, hub genes were identified and functionally annotated. Weighted gene coexpression network analysis was applied to construct the molecular network influenced by Huaier. Linc00339 was then found to play a critical role in Huaier-mediated cancer suppression. To validate the effects of linc00339 and identify the downstream targets, we performed in vitro and in vivo experiments. Finally, we identified that Huaier could inhibit the proliferation of breast cancer cells through modulating linc00339/miR-4656/CSNK2B signaling pathway.

CCL20 triggered by chemotherapy hinders the therapeutic efficacy of breast cancer.

Chemotherapeutic resistance in triple-negative breast cancer (TNBC) has brought great challenges to the improvement of patient survival. The mechanisms of taxane chemoresistance in TNBC have not been well investigated. Our results illustrated C-C motif chemokine ligand 20 (CCL20) was significantly elevated during taxane-containing chemotherapy in breast cancer patients with nonpathologic complete response. Furthermore, CCL20 promoted the self-renewal and maintenance of breast cancer stem cells (BCSCs) or breast cancer stem-like cells through protein kinase Cζ (PKCζ) or p38 mitogen-activated protein kinase (MAPK)-mediated activation of p65 nuclear factor kappa B (NF-κB) pathway, significantly increasing the frequency and taxane resistance of BCSCs. Moreover, CCL20-promoted NF-κB activation increased ATP-binding cassette subfamily B member 1 (ABCB1)/multidrug resistance 1 (MDR1) expression, leading to the extracellular efflux of taxane. These results suggested that chemotherapy-induced CCL20 mediated chemoresistance via up-regulating ABCB1. In addition, NF-κB activation increased CCL20 expression, forming a positive feedback loop between NF-κB and CCL20 pathways, which provides sustained impetus for chemoresistance in breast cancer cells. Our results suggest that CCL20 can be a novel predictive marker for taxane response, and the blockade of CCL20 or its downstream pathway might reverse the taxane resistance in breast cancer patients.

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.

Huaier aqueous extract protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting NLRP3 inflammasome activation.

The use of Trametes robiniophila Murr. (Huaier) as a complementary therapy for cancer has recently become increasingly common in China. However, whether Huaier can regulate host immune responses, especially innate immunity, remains largely unknown. The NLRP3 inflammasome is a multimeric complex consisting of NLRP3, ASC and caspase-1. NLRP3 inflammasomes respond to a variety of endogenous (damage-associated molecular patterns) and exogenous (pathogen-associated molecular patterns) stimuli, and play crucial roles in host defense against pathogens and multiple diseases such as ulcerative colitis (UC). In this study, we investigated the anti-inflammatory effect of Huaier in dextran sulfate sodium (DSS)-induced murine colitis and revealed the underlying mechanisms by targeting NLRP3 inflammasomes. In C57BL/6 mice, oral administration of Huaier attenuated DSS-induced colon shortening and colonic pathological damage. Furthermore, we analyzed the effect of Huaier on NLRP3 inflammasome activation in macrophages. Huaier inhibited NLRP3 inflammasome activation-induced IL-1ß secretion and caspase-1 cleavage. Moreover, Huaier decreased NLRP3 protein expression via promoting NLRP3 degradation through the autophagy lysosome pathway. Therefore, our findings demonstrate a novel function for Huaier in the regulation of NLRP3 inflammasome activation and suggest a potential role for Huaier in NLRP3 inflammasome-associated diseases.

Radiosensitization effect of Huaier on breast cancer cells.

Radiotherapy is a critical treatment strategy for breast cancer. However, its wide application is sometimes restricted by radioresistance and radiotoxicity. Trametes robiniophila Murr. (Huaier), an officinal fungus used as a traditional Chinese medicine (TCM), is reported to have multi-biological functions during cancer treatment. Yet, its radiosensitization effects have not been evaluated to date. In the present study, using HTA 2.0 transcriptome microarray assay, Huaier was found to downregulate genes related to the cell cycle, cell division, cell cycle phases and DNA repair. This investigation utilized a colony formation assay to confirm the ability of Huaier to sensitize breast cancer cells to radiotherapy. Flow cytometry, immunofluorescence staining and western blotting were used to illustrate the sensitization mechanism. Our findings suggest that Huaier causes G0/G1 arrest through downregulation of cell cycle-regulating proteins in MCF-7 and MDA-MB-468 cells, prolongs the persistence of γ-H2Ax foci after radiotherapy and interferes with the homologous recombination (HR) pathway of DNA repair by downregulating RAD51. These results suggest that Huaier has the ability to sensitize breast cancer cells to radiotherapy through regulation of the cell cycle and DNA repair pathway. Thus, Huaier may be a promising radiosensitizer for the treatment of breast cancer.

Huaier extract synergizes with tamoxifen to induce autophagy and apoptosis in ER-positive breast cancer cells.

Tamoxifen (TAM) is the most widely used endocrine therapy for estrogen receptor (ER)-positive breast cancer patients, but side effects and the gradual development of insensitivity limit its application. We investigated whether Huaier extract, a traditional Chinese medicine, in combination with TAM would improve treatment efficacy in ER-positive breast cancers. MTT, colony formation, and invasion and migration assays revealed that the combined treatment had stronger anticancer effects than either treatment alone. Huaier extract enhanced TAM-induced autophagy, apoptosis, and G0/G1 cell cycle arrest, as measured by acidic vesicular organelle (AVO) staining, TUNEL, flow cytometry, and western blot. Additionally, combined treatment inhibited tumorigenesis and metastasis by suppressing the AKT/mTOR signaling pathway. Huaier extract also enhanced the inhibitory effects of TAM on tumor growth in vivo in a xenograft mouse model. These results show that Huaier extract synergizes with TAM to induce autophagy and apoptosis in ER-positive breast cancer cells by suppressing the AKT/mTOR pathway.

Huaier aqueous extract inhibits cervical cancer cell proliferation via JNK/p38 pathway.

Although the anticancer effects of Huaier extract have been widely investigated, including anti-proliferate, anti-angiogenic and anti-metastatic activities, the mechanisms are not well understood. This study aimed to elucidate the inhibitory effect of Huaier extract on tumor growth in cervical cancer cells and its molecular mechanisms. Cell viability and motility were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony assays, migration, and invasive assays, respectively. The distribution of the cell cycle was analyzed by flow cytometry. Huaier inhibited cell viability of SiHa and C33A cells in a time- and dose-dependent manner; cell migration and invasiveness were also suppressed; Huaier was able to cause G2/M cell cycle arrest in C33A cells. The western blot results confirmed Huaier dose-dependently increased expression of phosphorylated c-Jun N-terminal kinase (JNK), p-38 and downregulated the expression of phosphorylated extracellular signal-regulated kinase (ERK) in a time- and dose-dependently manner. In vivo experiments showed that Huaier significantly suppressed the tumor volume of SiHa cell xenografts. These data suggest that Huaier may inhibit tumor proliferation in cervical cancer via the JNK/p38 signaling pathway.

Huaier Extract Induces Autophagic Cell Death by Inhibiting the mTOR/S6K Pathway in Breast Cancer Cells.

Huaier extract is attracting increased attention due to its biological activities, including antitumor, anti-parasite and immunomodulatory effects. Here, we investigated the role of autophagy in Huaier-induced cytotoxicity in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cells. Huaier treatment inhibited cell viability in all three cell lines and induced various large membranous vacuoles in the cytoplasm. In addition, electron microscopy, MDC staining, accumulated expression of autophagy markers and flow cytometry revealed that Huaier extract triggered autophagy. Inhibition of autophagy attenuated Huaier-induced cell death. Furthermore, Huaier extract inhibited the mammalian target of the rapamycin (mTOR)/S6K pathway in breast cancer cells. After implanting MDA-MB-231 cells subcutaneously into the right flank of BALB/c nu/nu mice, Huaier extract induced autophagy and effectively inhibited xenograft tumor growth. This study is the first to show that Huaier-induced cytotoxicity is partially mediated through autophagic cell death in breast cancer cells through suppression of the mTOR/S6K pathway.

The anticancer effect of Huaier (Review).

Trametes robiniophila Murr. (Huaier) is a sandy beige mushroom found on the trunks of trees and has been widely used in traditional Chinese medicine (TCM) for ~1,600 years. The anticancer effects of Huaier have attracted increasing worldwide interest in recent years. Accumulating evidence suggests that the anticancer mechanism of Huaier may be associated with various biological activities, such as inhibition of cell proliferation, anti-metastasis, interference with tumor angiogenesis and tumor-specific immunomodulatory effect. Animal and experimental studies suggest that Huaier is a promising anticancer agent. Further clinical research is warranted to illustrate the untapped chemopreventive and therapeutic potential of Huaier either alone or in conjunction with existing therapies.

Identification of multi-target effects of Huaier aqueous extract via microarray profiling in triple-negative breast cancer cells.

Breast cancer is one of the most common malignant tumors in the world. Long-term maintenance treatment is important for breast cancer. However, effective maintenance treatment is lacking for triple-negative breast cancer (TNBC). Traditional Chinese medicine (TCM) has shown its potential anticancer roles as an effective maintenance treatment for TNBC. However its mechanisms remained unclear. In this study, we detected the differentially expressed genes (DEGs) after treatment with Huaier aqueous extract by using microarray profiling in MDA-MB-231 cells. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene-gene interaction network were conducted to confirm the altered biological functions induced by Huaier extract. Screening of DEGs gave 387 genes (226 upregulated and 161 downregulated) in MDA-MB-231 cells which were regulated significantly by Huaier extract. GO and KEGG pathway analysis suggested that a number of functions were affected by Huaier, including proliferation, apoptosis, migration, and angiogenesis. Gene-gene interaction network showed the detailed molecular signal-net. Based on microarray data, we studied several functions of Huaier extract and in return verified the results of microarray profiling. This study had important guidance roles and indicated new research directions.

Huaier aqueous extract inhibits stem-like characteristics of MCF7 breast cancer cells via inactivation of hedgehog pathway.

The theory of targeting cancer stem-like cells (CSCs) provides novel strategy for cancer treatment. In the present study, we examined the inhibitory effect of Huaier aqueous extract on eradicating breast cancer stem cells and explored the underlying mechanisms. Our data demonstrated that various concentrations of Huaier extract significantly decreased the viabilities, numbers, and sizes of mammospheres. After incubation with Huaier extract for 24 h, the clonogenicity of MCF7 cell line was obviously impaired, along with less holoclones. In addition, Huaier extract reduced the number of cells expressing CD44+/CD24- and decreased the level of stem cell markers (OCT-4, NESTIN, and NANOG). The hedgehog (Hh), notch, and Wnt/ß-catenin pathways were essential stem cell signaling pathways involved in regulating CSC renewal and maintenance. We reported that the inhibitory effect of Huaier extract was partly depended on the inactivation of Hh pathway. These findings provided experimental evidence that Huaier extract was a promising therapeutic drug for eliminating the breast cancer stem cells.

Nitidine chloride induces apoptosis, cell cycle arrest, and synergistic cytotoxicity with doxorubicin in breast cancer cells.

Medicinal plant extracts have been widely used for cancer treatment. Nitidine chloride (NC) is a natural bioactive alkaloid that has recently been reported to have diverse anticancer properties. We aimed to investigate the cytotoxic effects of NC and the effectiveness of combinatorial treatment including NC and doxorubicin in breast cancer cells. Using MTT and flowcytometry assays, we found that NC induced cell growth inhibition and G2/M cell cycle arrest in a time- and dose-dependent manner both in MCF-7 and MDA-MB-231 breast cancer cell lines. Cancer cell growth inhibition was associated with increased levels of the p53 and p21 proteins. Apoptosis induction by NC treatment was confirmed by JC-1 mitochondrial membrane potential, annexin V-positive cell, and TUNEL staining. Using western blot analysis, we found that NC upregulated the pro-apoptotic proteins Bax, cleaved caspase-9 and -3 and cleaved PARP and that it downregulated the anti-apoptotic proteins Bcl-2 and PARP. By using the PI3K/Akt inhibitor LY294002, we further demonstrated that NC-induced apoptosis might be Akt-specific or dependent. In addition, NC exhibited a synergistic effect with doxorubicin on the growth inhibition of the human breast cancer cell lines MCF-7 and MDA-MB-231. Our study demonstrated the anticancer effect of NC on breast cancer and highlighted the potential clinical application of NC.

Huaier aqueous extract suppresses human breast cancer cell proliferation through inhibition of estrogen receptor α signaling.

Estrogen receptor α (ERα) has been reported to play a critical role in promoting the growth of breast tumor cells. In the present study, we explored the effect of Huaier extract on estrogen receptor α signaling in breast cancer cell lines. Our data demonstrated that Huaier extract effectively inhibited the proliferation of the MCF-7, T47D and ZR-75-1 human breast cancer cell lines. For the mechanism analysis, we demonstrated that Huaier extract significantly reduced the mRNA and protein levels of ERα in all three ERα-positive cell lines. The downregulation of ERα protein levels was correlated with activation of the proteasomes. We demonstrated that Huaier extract markedly decreased the expression of both ERα and its downstream genes, inhibited the estrogen-stimulated proliferation and reversed the estrogen-induced activation of the nuclear factor κB (NFκB) pathway. Our study provides evidence that Huaier extract is a novel estrogen receptor modulator and is a promising drug for the prevention and treatment of ERα-positive human breast cancers.

Anti-angiogenic and antitumor activities of Huaier aqueous extract.

Traditional Chinese medicine, a rich source of potent cancer chemopreventive agents, is attracting increasing attention worldwide. Recently, the anticancer activity of Trametes robiniophila Μurr. (Huaier) has been widely investigated. However, the mechanisms are not yet fully understood. This study aimed to elucidate the inhibitory effect of Huaier extract on angiogenesis and tumor growth. Incubation with Huaier extract inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) and mouse mammary tumor cells (4T1). In addition, treatment with Huaier extract decreased the motility and tube formation of HUVECs in a dose-dependent manner in vitro. As determined by western blot analysis, Huaier extract dose-dependently decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK), transcription factor p65, c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription 3 (STAT3) and the expression of vascular endothelial growth factor (VEGF). In ex vivo experiments, new vessel growth was suppressed as shown by chick embryo chorioallantoic membrane (CAM) and rat aortic ring assays in the presence of Huaier extract. To further evaluate the inhibitory effect, 4T1 cells were injected subcutaneously into BALB/c mice. The administration of Huaier extract suppressed tumor volume, decreased microvessel density and induced apoptosis. These data suggest that Huaier extract may serve as a potent anti-angiogenic and antitumor agent.

Radiosensitization effects of berberine on human breast cancer cells.

Berberine, an isoquinoline derivative alkaloid, has recently been shown to have antitumor activity. The present study aimed to investigate the effects of the concomitant administration of berberine and radiation on breast cancer. The effects of berberine on the radiosensitivity of MCF-7 and MDA-MB-468 cells were evaluated by using cell clonogenic assays. Cells pre-treated with berberine or dimethyl sulfoxide (DMSO) for 24 h were irradiated using a Faxitron Cabinet X-ray System to deliver the indicated doses (0, 1, 2, 3 and 4 Gy). Changes in cell cycle distribution were determined by flow cytometry. γ-H2AX foci were detected by immunofluorescence staining. The levels of Ku70, Ku86 and RAD51 proteins were evaluated by western blot analysis. We observed that berberine increased the MCF-7 and MDA-MB-468 cell radiosensitivity with cell clonogenic assays. the radiation-induced G2/M cell cycle delay was reduced in the MCF-7 cells pre-teated with berberine. Berberine pre-treatment prolonged the persistence of DNA double-strand breaks in the MCF-7 cell line. In comparison with the control cells, the protein levels of RAD51 were decreased in the MCF-7 and MDA-MB-468 cells treated with berberine, and in the cells pre-treated with 15 µM berberine for 24 h, the level of RAD51 protein decreased significantly at the indicated time-points (0, 2, 6 and 24 h) following X-ray exposure. In conclusion, berberine sensitizes human breast cancer cells to ionizing radiation by inducing cell cycle arrest and the downregulation of the homologous recombination repair protein, RAD51. Berberine may be a promising radiosensitizer for the treatment of breast cancer.

Huaier aqueous extract inhibits proliferation of breast cancer cells by inducing apoptosis.

Aqueous extract of Trametes robiniophila murr (Huaier) has been commonly used in China for cancer complementary therapy in recent years; however, the mechanisms of its anticancer effects are largely unknown. In the present study, we aim to investigate its inhibitory effect on both MCF-7 and MDA-MB-231 cells, and explore the possible mechanisms of its anticancer effect. Cell viability and motility were measured by MTT and invasive assays, migration and scratch assays in vitro, respectively. The distribution of cell cycle, PI-Annexin-V staining and Rhodamine 123 assay were analyzed by flow cytometry, and western blot were used to test the apoptotic pathways. We found that Huaier extract could strongly inhibit cell viability of MCF-7 and MDA-MB-231 cells in a time- and dose-dependent manner; however, MDA-MB-231 cells showed more susceptibility to the treatment. Furthermore, cell invasiveness and migration were also suppressed with exposure to Huaier extract. We also indicated that Huaier could induce G0/G1 cell-cycle arrest, p53 accumulation and activation selectively in MCF-7 cells. Inspiringly, the PI-Annexin-V staining assay and western blot analysis confirmed cell apoptosis executed by caspase-3. Decreased mitochondrial membrane potential by Rhodamine 123 assay and down-regulation of Bcl-2 and up-regulation of BCL2-associated X protein (BAX) indicated that Huaier induced apoptosis through the mitochondrial pathway. Caspase activation during Huaier-induced apoptosis was confirmed by pan-caspase inhibitor, Z-VAD-fmk. As expected, the inhibitor decreased Huaier-induced apoptosis in both cell lines. Based on our findings, Huaier can induce cell apoptosis in both ER-positive and ER-negative breast cancer cell lines and is an effective complementary agent for breast cancer treatment.

Prognostic value of Bcl-2 in invasive breast cancer receiving chemotherapy and endocrine therapy.

Apoptosis induced by anticancer agents is a mechanism of treatment activity, overexpression of antiapoptotic genes could produce drug resistant tumors. We have demonstrated that the susceptibility of Bcl-2-negative tumors to a series of anticancer drugs was significantly higher than that of Bcl-2-positive tumors. The purpose of this study was to examine if negative Bcl-2 expression has treatment benefit in breast cancer patients received chemotherapy and endocrine treatment. A cohort of 147 Japanese women with invasive ductal carcinoma was studied. All the patients received postoperative adjuvant therapy consisting of combination chemotherapy with cyclophosphamide, epirubicin, and fluorouracil, and tamoxifen therapy. The expression of Bcl-2, estrogen receptor (ER) and MIB-1 was evaluated in breast cancer surgical specimens. Bcl-2 immunostaining was inversely correlated with increasing histologic grade (p=0.0095) and MIB-1 (p=0.0128). Furthermore, a positive correlation between Bcl-2 and ER was observed (p<0.0001). Unexpectedly, survival curves determined by the Kaplan-Meier method and univariate analysis demonstrated that Bcl-2-positivity was associated with favorable prognosis (p=0.0430). Cox proportional hazard model demonstrated that positive Bcl-2 expression still has favorable survival (p=0.0242) after consideration of other prognostic factors. Our results imply that Bcl-2 is a significant favorable prognostic factor for breast cancer with chemotherapy and endocrine therapy.