Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. herb pair suppresses breast cancer liver metastasis by targeting ECM1-mediated cholesterol biosynthesis pathway.

BACKGROUND: Liver metastasis is a frequent event in breast cancer that causes low survival rate and poor prognosis. Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. (CR), a traditional Chinese herb pair, is used for the treatment of breast cancer liver metastasis or cholesterol gallstone disease in clinics. PURPOSE: This study attempted to investigate the potential therapeutic target and mechanism of CR herb pair on breast cancer liver metastasis. METHODS: The anti-metastatic and cholesterol-lowering activities of CR extract were evaluated in triple-negative breast cancer (TNBC) cell lines and an experimental liver metastasis model. The role of extracellular matrix protein 1 (ECM1) in the cholesterol biosynthesis pathway was determined by the knockdown and overexpression of ECM1 gene of TNBC cells. Changes in the gene and protein expression levels of ECM1 and the cholesterol biosynthesis pathway after CR treatment were detected in vitro and in vivo by real-time PCR and Western blot. RESULTS: The invasive and metastatic potentials and hypercholesterol levels of TNBC cells were positively associated with ECM1 expression. ECM1 knockdown reduced tumor cholesterol levels via downregulating cholesterol biosynthesis genes, including ACAT2, HMGCS1, HMGCR, MVK, and MVD, whereas ECM1 overexpression elicited the opposite effects. CR herb pair exerts the potential therapeutic effects on TNBC liver metastasis, which is partially mediated by disrupting ECM1-activated cholesterol biosynthesis process in TNBC cells. CONCLUSION: This study reveals that ECM1 is a novel target for the activation of cholesterol biosynthesis to promote TNBC liver metastasis occurrence. CR herb pair, an ECM1 inhibitor, maybe be considered to serve as an adjuvant therapeutic drug for liver metastasis in clinical practice.

Exploration of potential mechanism of Rougan formula against hepatic fibrosis by network analysis and experimental assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: Rougan Formula (RG) has long been clinically applied to treat hepatic fibrosis in patients with different chronic liver diseases. However, the core active substances and the potential pharmacological mechanisms of RG remain unclear. AIM OF THE STUDY: The purpose of this study is to explore bioactive components, key targets, and potential mechanisms of RG by performing network pharmacological analyses and experimental model validation. MATERIALS AND METHODS: All chemical components in RG extract were identified using ultraperformance liquid chromatography-quadrupole/time-of-flight tandem mass technology. The candidate components and drug targets of RG, as well as disease-related genes, were extracted from TCMSP and GeneCards databases. The potential pathways related to genes were predicted by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The core bioactive components, key targets, and signaling pathways were ultimately obtained by analyzing protein-protein interaction (PPI) and component-target-pathway (C-T-P) networks. Subsequently, the efficacy and underlying mechanisms of RG on hepatic fibrosis were experimentally validated in transforming growth factor-beta 1 (TGF-ß1)-induced hepatic stellate cell activation model and CCL4-induced hepatic fibrosis mouse model. RESULTS: A total of 52 components in RG extract were obtained, and 22 of them were selected as the core bioactive components. Five hundred and thirty-nine overlapped targets were determined by matching drug targets with disease-related targets. The results of PPI and C-T-P network analyses revealed 100 key targets and 19 signaling pathways associated with RG efficacy. In vitro and in vivo studies further verified that RG exerted a significant anti-hepatic fibrotic effect by suppressing the activation of hepatic stellate cells by downregulating the TGF-ß1/Smads signaling pathway. CONCLUSIONS: These results may provide some evidence for further clinical research and development of RG formula as an effective and safe drug for hepatic fibrosis treatment.

Osthole inhibits the migration and invasion of highly metastatic breast cancer cells by suppressing ITGα3/ITGß5 signaling.

Metastasis is the leading cause of death in breast cancer patients. Osthole, as an active compound detected in the traditional Chinese medicine Wenshen Zhuanggu Formula, has shown a promising anti-metastatic activity in human breast cancer cells, but the underlying mechanisms remain ambiguous. In this study we elucidated the anti-metastatic mechanisms of osthole in highly metastatic breast cancer cells and a zebrafish xenograft model. We showed that the expression of integrin α3 (ITGα3) and integrin ß5 (ITGß5) was upregulated in highly metastatic MDA-MB-231, MDA-MB-231BO breast cancer cell lines but was downregulated in poorly metastatic MCF-7 breast cancer cell line, which might be the key targets of osthole's anti-metastatic action. Furthermore, we showed that knockdown of ITGα3 and ITGß5 attenuated breast cancer cell migration and invasion possibly via suppression of FAK/Src/Rac1 pathway, whereas overexpression of ITGα3 and ITGß5 caused the opposite effects. Consistently, osthole significantly inhibited breast cancer metastasis by downregulating ITGα3/ITGß5 signaling in vitro and in vivo. These results provide new evidence that osthole may be developed as a candidate therapeutic drug for metastatic breast cancer.

Curcumin-loaded liposomes with the hepatic and lysosomal dual-targeted effects for therapy of hepatocellular carcinoma.

Curcumin can induce cancer cell apoptosis through lysosomal permeabilization pathway. However, the poor selectivity of curcumin restricts its use in the therapy of hepatocellular carcinoma. Because galactose group can recognize ASGPR overexpressed on hepatoma cells and morpholine group can target to the lysosome, they are integrated into a dual-targeted lipid material with low toxicity. The corresponding galactose-morpholine modified liposomes loaded with curcumin (Gal-Mor-LPs) were prepared and evaluated in comparison with conventional liposomes (LPs) and galactose modified liposomes (Gal-LPs). The in vitro and in vivo hepatic targeting capacity of liposomes followed a trend of LPs < Gal-LPs < Gal-Mor-LPs. The endocytosis of Gal-Mor-LPs was competitively inhibited by galactose, which confirmed the galactose modified liposomes entered hepatoma cells via ASGPR-mediated pathway. Gal-Mor-LPs displayed more excellent lysosomal targeting efficacy than LPs and Gal-LPs due to the attraction of acidic lysosome on basic morpholine group of Gal-Mor-LPs. The in vivo tumor inhibition effects of formulations also followed a trend of free curcumin < LPs < Gal-LPs < Gal-Mor-LPs, confirming that hepatic and lysosomal dual-targeting vehicle can improve the antitumor efficacy of curcumin. Moreover, the curcumin-loaded liposomes modified with galactose and morpholine moieties show good biocompatibility in vivo.

WSZG inhibits BMSC-induced EMT and bone metastasis in breast cancer by regulating TGF-ß1/Smads signaling.

Bone metastasis of breast cancer causes severe skeletal-related events and poor prognosis. Wensheng Zhuanggu Formula (WSZG), a traditional Chinese prescription, is used to adjunctively treat breast cancer bone metastases in clinical practice. This study was undertaken to investigate the antibone-metastatic activities and mechanisms of WSZG extract by evaluating the effect of this formula on the cross-talk between bone marrow-derived mesenchymal stem cells (BMSCs) and breast cancer cells in triggering epithelial-mesenchymal transition (EMT) in vivo and in vitro. The results demonstrated that BMSCs might enhance the invasive and metastatic potentials of breast cancer cells as a consequence of EMT induction through direct cell-to-cell contact. WSZG treatment remarkably suppressed motility, invasion, EMT-related gene, and protein markers in BMSC-conditioned breast cancer cells and ameliorated bone metastases and damages in nude mice following co-injection of BMSCs and MDA-MB-231BO breast cancer cells. Further investigation showed that the transforming growth factor-ß1 (TGF-ß1)/Smads pathway was an important mechanism enabling BMSCs to induce EMT occurrence of breast cancer cells. WSZG treatment reversed BMSC-induced EMT by downregulating TGF-ß1/Smads signaling. Thus, WSZG extracts may be regarded as a potential antibone-metastatic agent for breast cancer therapy.

Time-resolved fluorescent immunochromatographic assay-based on three antibody labels for the simultaneous detection of aflatoxin B1 and zearalenone in Chinese herbal medicines.

A time-resolved fluorescent immunochromatographic assay (TRFICA) was successfully developed for the sensitive, simultaneous, and quantitative detection of aflatoxin B1 (AFB1) and zearalenone (ZEN) in Chinese herbal medicines. Eu-nanospheres (EuNPs) with unique optical properties increased the stability and sensitivity of the immunochromatographic assay. To obtain stable quantitative results, we applied a three-label system in which monoclonal antibodies for AFB1 and ZEN were conjugated to the EuNPs as detection probes on the test line (T line), and EuNP-labelled chicken IgY conjugates acted as the reference on the control line (C line). The fluorescence intensities of the T and C lines were recorded, and the T/C ratio was employed as the quantitative signal for the elimination of strip variation and matrix effects. The parameters that affected the TRFICA were optimised. Under optimal conditions, the established TRFICA gave good linear ranges from 0.60 µg/kg to 3.92 µg/kg for AFB1 and from 0.40 µg/kg to 1.28 µg/kg for ZEN. The limits of detection for AFB1 and ZEN were as low as 0.60 and 0.40 µg/kg, respectively, in Chinese herbal medicines Semen coicis, Rhizoma dioscoreae, and Platycodon grandiflorus, respectively. The average recoveries of the spiked samples were 73%-95% for AFB1 and 75.83%-90% for ZEN, both with a relative standard deviation of < 9.08%. The results of 15 actual samples detected by the developed TRFICA showed a satisfactory correlation with those of ultra-performance liquid chromatography tandem mass spectrometry. Therefore, the TRFICA is a simple, rapid, and sensitive approach to quantitatively detect mycotoxins in Chinese herbal medicines.

Comparative pharmacokinetics of six coumarins in normal and breast cancer bone-metastatic mice after oral administration of Wenshen Zhuanggu Formula.

ETHNOPHARMACOLOGICAL RELEVANCE: Wenshen Zhuanggu Formula (WSZG) is a traditional Chinese medicine (TCM) prescription used in clinics for adjuvant treatment of breast cancer bone metastases in Longhua Hospital in China. WSZG has been reported to decrease the risk of bone metastases and alleviate the severity of bone lesions in a breast cancer xenograft model. AIM OF THE STUDY: The present study aimed at investigating the pharmacokinetic behaviors of six coumarins in normal and breast cancer bone-metastatic mice following oral administration of WSZG extract. MATERIALS AND METHODS: A bone-metastatic mouse model was established by intracardiac injection of MDA-MB-231BO breast cancer cells, and WSZG extract (1.60 g/kg) was given orally to the model and normal mice for 4 weeks. Then, the blood pharmacokinetic parameters of six bioactive components from WSZG (psoralen, isopsoralen, bergapten, xanthotoxin, osthole, and imperatorin) were analyzed by liquid chromatography tandem mass spectrometry. RESULTS: There were significant differences in pharmacokinetic behaviors between normal and pathological states. Compared with normal mice, the model mice showed significantly increased AUC0-t and AUC0-∞ of the bioactive compounds (P < 0.05) and significantly decreased total blood clearance (CLZ/F) (P < 0.05). CONCLUSIONS: The different pharmacokinetic behaviors might be partly ascribed to intestinal functional disorders and imbalance of gastrointestinal microbiota under the morbid state. The findings provide some valuable information to evaluate the clinical efficacy and safety of this TCM formula.

Effects of dietary lipids on the hepatopancreas transcriptome of Chinese mitten crab (Eriocheir sinensis).

Fish oil supplies worldwide have declined sharply over the years. To reduce the use of fish oil in aquaculture, many studies have explored the effects of fish oil substitutions on aquatic animals. To illustrate the effects of dietary lipids on Chinese mitten crab and to improve the use of vegetable oils in the diet of the crabs, 60 male juvenile Chinese mitten crabs were fed one of five diets for 116 days: fish oil (FO), soybean oil (SO), linseed oil (LO), FO + SO (1:1, FSO), and FO + LO (1:1, FLO). Changes in the crab hepatopancreas transcriptome were analyzed using RNA sequencing. There were a total 55,167 unigenes obtained from the transcriptome, of which the expression of 3030 was significantly altered in the FLO vs. FO groups, but the expression of only 412 unigenes was altered in the FSO vs. FO groups. The diets significantly altered the expression of many enzymes involved in lipid metabolism, such as pancreatic lipase, long-chain acyl-CoA synthetases, carnitine palmitoyltransferase I, acetyl-CoA carboxylase, fatty acid synthase, and fatty acyl Δ9-desaturase. The dietary lipids also affected the Toll-like receptor and Janus activated kinase-signal transducers and activators of transcription signaling pathways. Our results indicate that substituting fish oil with vegetable oils in the diet of Chinese mitten crabs might decrease the digestion and absorption of dietary lipids, fatty acids biosynthesis, and immunologic viral defense, and increase ß-oxidation by altering the expression of the relevant genes. Our results lay the foundation for further understanding of lipid nutrition in Chinese mitten crab.

Wenshen Zhuanggu formula effectively suppresses breast cancer bone metastases in a mouse Xenograft model.

Wenshen Zhuanggu formula (WSZG) is a traditional Chinese medicine used as an adjuvant for the prevention of bone metastases in breast cancer patients. In this study we investigated the efficacy of WSZG in preventing bone metastases and the potential mechanisms in a mouse xenograft model of breast cancer bone metastases. This model was established by injection of human MDA-MB-231BO-Luc breast cancer cells alone or a mixture of the cancer cells with bone marrow-derived mesenchymal stem cells (BMSCs) into left ventricle of the heart in female nude mice. Then the mice were treated with WSZG (3.25, 6.5 or 13.0 mg·kg-1·d-1, ig) for four weeks, whereas zoledronic acid (100 µg/kg per week, ig) was used as a positive control. The occurrence and development of bone metastases were monitored via bioluminescent imaging, and bone lesions were assessed using micro-CT. Intracardiac injection of the mixture of MDA-MB-231BO-Luc breast cancer cells with BMSCs significantly facilitated the bone metastatic capacity of the breast cancer cells, and aggravated bone lesions in the mouse xenograft model of breast cancer bone metastases. Administration of WSZG dose-dependently inhibited the incidence and intensity of bone metastases and protected against bone lesions by suppressing osteoclast formation and tumor cell infiltration. Furthermore, administration of WSZG caused a marked reduction in the expression of CCL5/CCR5 and IL-17B/IL-17BR in bone metastatic tissues. The results demonstrate that WSZG exerts potential therapeutic effects in a mouse xenograft model of breast cancer bone metastases, which are partially mediated by weakening the interaction between BMSCs and breast cancer cells in the tumor microenvironment.

[Attenuation and mechanism of endoplasmic reticulum stress-mediated hepatocyte apoptosis in rats with alcohol-induced liver injury by qinggan huoxue recipe and its disassembled formulas].

OBJECTIVE: To explore attenuation and mechanism of endoplasmic reticulum stress (ERS)-mediated hepatocyte apoptosis in rats with alcohol-induced liver injury by Qinggan Huoxue Recipe (QGHXR) and its disassembled formulas (Qinggan Recipe and Huoxue Recipe respectively). METHODS: A rat model of chronic alcoholic liver injury was successfully established using a compound reagent of alcohol, corn oil, and pyrazol. The modeled rats were randomly divided into the model group, the QGHXR group, the Qinggan Recipe (QGR) group, and the Huoxue Recipe group (HXR). The CCl4 control group and the normal control group were also set up. There were ten rats in each group. All rats of modeled groups were gastrogavaged with alcohol compound reagent every morning. Rats in the QGHXR group (at the daily dose of 9. 5 g/kg, QGR group (at the daily dose of 3.0 g/kg), and HXR group (at the daily dose of 6.5 g/kg) were administered with corresponding medicines by gastrogavage every afternoon. Equal volume of normal saline was given to rats of the model group by gastrogavage. CCl4 was intraperitoneally injected at the dose of 0.3 mL/kg to rats in the CCl4 control group, once per week. Normal saline was given to rats in the normal control group by gastrogavage. The treatment was lasted for two weeks. Pathological changes of the liver were observed by histopathology. Serum total homocysteine (tHCY) level was detected by ELISA. The hepatocyte apoptosis rate was detected using flow cytometry. The gene and protein expressions of eukaryotic translation initiation factor 2 alpha (elF-2alpha), phosphorylation elF-2alpha (pelF-2alpha), glucose-regulated protein 78 (GRP78), and Caspase-3 in the liver were examined using Real-time PCR and Westen blot respectively. RESULTS: Compared with the normal control group, typical pathological changes of chronic alcoholic liver injury such as steatosis, inflammation, and even fibrosis occurred in model rats. The hepatocyte apoptosis obviously increased, with the apoptosis rate reaching the five-fold of that in normal rats. Besides, early apoptosis dominated. The serum tHCY level significantly increased. The expressions of p-elF-2alpha, GRP78, and Caspase-3 protein obviously increased (P < 0.01). Expressions of GRP78 and Caspase-3 mRNA significantly increased (P < 0.05, P < 0.01). Compared with the model group, the degrees of the liver injury and the hepatocyte apoptosis in the QGHXR group, the QGR group, and the HXR group were significantly alleviated. The serum tHCY level was significantly lowered. The protein expressions of p-elF-2a, GRP78, and Caspase-3 obviously decreased (P < 0.01). mRNA expressions of GRP78 and Caspase-3 obviously decreased in the QGHXR group (P < 0.05, P < 0.01). Only GRP78 mRNA expression obviously decreased in the QGR group (P < 0.05). CONCLUSION: QGHXR and its disassembled formulas could attenuate ERS-mediated hepatocyte apoptosis in alcohol-induced liver injury rats by lowering the serum tHCY level and expressions of ERS apoptosis correlated factors.