ICAM1 promotes bone metastasis via integrin-mediated TGF-ß/EMT signaling in triple-negative breast cancer.

Bone-related events caused by breast cancer bone metastasis substantially compromise the survival and quality of life of patients. Because triple-negative breast cancer (TNBC) lacks hormone receptors and Her2-targeted therapeutic options, progress in the treatment of TNBC bone metastasis has been very slow. Intercellular adhesion molecule 1 (ICAM1) is highly expressed in various cancers and plays an important role in tumorigenesis and metastasis. However, the effect and mechanism of ICAM1 in TNBC bone metastasis are still unknown. We found that ICAM1 was highly expressed in TNBC and correlated with prognosis in TNBC patients. Cell lines with high expression of ICAM1 exhibited enhanced bone metastasis in tumor-bearing mice, and silencing ICAM1 expression significantly inhibited bone metastasis in mice. ICAM1 interacted with integrins to activate the epithelial-to-mesenchymal transition program through TGF-ß/SMAD signaling, ultimately enhancing cell invasiveness. Therefore, the findings of the present study provide a strong rationale for the application of ICAM1-targeted therapy in TNBC patients with bone metastasis.

GeneChip expression profiling identified OLFML2A as a potential therapeutic target in TNBC cells.

Background: An elevated level of olfactomedin-like-2A (OLFML2A) is unfavorable for female breast cancer patients. Patients with a high mRNA level of OLFML2A receive a poor prognosis. Therefore, we speculate that inhibiting the expression of this gene may be beneficial to breast cancer patients. We previously found that silencing the OLFML2A gene by using mRNA interference significantly inhibited proliferation and migration in triple-negative breast cancer (TNBC) cells. Methods: Cell activity and proliferation were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Celigo analyses. Cell migration and invasion were determined by wound-healing and transwell invasion assays. The mechanism of the inhibition of a small hairpin RNA that targets OLFML2A (shOLFML2A) was determined by using a GeneChip array, real-time quantitative PCR (RT-qPCR), and western blot analysis. Results: Gene silencing by shOLFML2A induces apoptosis by promoting S phase arrest in TNBC cells. In addition, shOLFML2A decreased the progression of epithelial-mesenchymal transition (EMT). Additionally, microarray analysis showed that shOLFML2A significantly upregulated 428 genes and downregulated 712 genes. These significantly changed genes regulated DNA synthesis, chromosome alignment, microtubules and the cytoskeleton, cell movement, the cell cycle, cell necrosis, and apoptosis because they promoted G2/M DNA damage checkpoint regulation and p53 signaling, and because they inhibited integrin, hepatocyte growth factor (HGF), nerve growth Factor (NGF), and other tumor-promoting signaling pathways. Conclusions: shOLFML2A reduces cell proliferation, migration, and invasion and promotes cell apoptosis. Therefore, the results of the present study suggest that OLFML2A is a potential therapeutic target for TNBC.

Simultaneous Quantification of Four Compounds in Rat Plasma by HPLC-MS/MS and Its Application to Pharmacokinetic Study after Oral Administration of Pomegranate Flowers.

Pomegranate flowers (PFs) were reported to possess various biological activities such as antidiabetic, anti-inflammatory and hepatoprotective activities, and using to treat diabetes. Although chemical constituents and pharmacological activities of PFs have been studied, unfortunately, there was no report on the pharmacokinetic profile of PFs in vivo. In this study, a selective high-performance liquid chromatography triple quadrupole tandem mass spectrometry (HPLC-QQQ-MS/MS) method was developed and validated for simultaneous quantification of four compounds (corilagin, ellagic acid, gallic acid and brevifolincarboxylic acid) in rat plasma after oral administration of PFs. The good linearity concentration ranges for the four analytes were from 2.5 to 3000 ng/mL with coefficient value R2 > 0.99 in calibration curves. The intra- and inter-day accuracy of the four analytes was in the range of 85.33-102.50%, with relative standard deviation (RSD) of <14.81%. The stability results showed that accuracy of the four analytes was in the range of 81.88-104.74%, with RSD of <14.86%. The validation method was successfully applied to pharmacokinetic profiles of the four analytes in rats after oral administration of PFs extract. This pharmacokinetic study can provide better understanding to clarify in vivo mechanisms of PFs and may facilitate its further development as therapeutic agent.

Amorphophalli Rhizoma inhibits breast cancer growth, proliferation, migration, and invasion via the PI3K/AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Amorphophalli Rhizoma (APR) is widely used as an adjuvant treatment for advanced and metastatic triple-negative breast cancer (TNBC), but its effects, potential active ingredients, and mechanism of action on estrogen receptor-positive (ER+) and human epidermal growth factor receptor-positive (HER2+) breast cancer cells were not reported. AIM OF THE STUDY: The present study investigated the effects and mechanism of APR on ER+ and HER2+ breast cancer cells. MATERIALS AND METHODS: Rotary evaporation was used to prepare different extracts of APR. Cell activity was assessed using the cell counting kit-8 (CCK-8) method. Wound healing assays were used to assess cell migration, and a cell invasion assay was performed using a Transwell chamber with Matrigel matrix. A xenograft model was used to analyze the inhibitory effects of APR on tumor growth. Bioinformatics analyses were used to explore the potential mechanism of APR in breast cancer. RT-qPCR and Western blotting were performed to reveal the molecular mechanism. RESULTS: The ethyl acetate extract of APR showed the strongest tumor inhibitory effect on ER+ and HER2+ breast cancer cells compared to petroleum ether or N-butanol extracts. APR inhibited ER+ and HER2+ breast cancer cell growth, proliferation, migration, and invasion via the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. CONCLUSIONS: APR had a significant inhibitory effect on ER+ and HER2+ breast cancer cells via the PI3K/AKT signaling pathway. Therefore, APR may be useful for preventing ER+ and HER2+ breast tumor growth, proliferation, migration, and invasion.

Huangjinsan ameliorates adenine-induced chronic kidney disease by regulating metabolic profiling.

Chronic kidney disease is an increasingly serious public health problem worldwide. Our recent studies have shown that Huangjinsan has a renal protective effect on chronic kidney disease, but the specific mechanism by which this effect occurs is not clear. To study the therapeutic effect of Huangjinsan on chronic kidney disease and to explore its possible mechanism of action through nontargeted metabolomics methods, a chronic kidney disease rat model was induced by adenine, and the Huangjinsan extract was given by oral gavage. Body weight, the kidney index, pathological sections, and a series of biochemical indicators were measured. High-performance liquid chromatography quadrupole time-of-flight mass spectrometry was used to analyze the changes in the plasma metabolome. Huangjinsan significantly reduced indicators of kidney damage, including total protein, albumin, the total protein to creatinine ratio, and the albumin to creatinine ratio in urine, as well as IL-2, MCP-1α, and blood urea levels in plasma. Based on nontargeted metabolomics, 13 metabolites related to chronic kidney disease were discovered. These metabolites are closely related to glycerophospholipid metabolism, arginine and proline metabolism, and sphingolipid metabolism. We found that Huangjinsan can restore the renal function of adenine-induced chronic kidney disease by regulating the metabolic profile.

Baicalin Inhibits Influenza A Virus Infection via Promotion of M1 Macrophage Polarization.

BACKGROUND AND AIMS: The natural compound baicalin (BA) possesses potent antiviral properties against the influenza virus. However, the underlying molecular mechanisms of this antiviral activity and whether macrophages are involved remain unclear. In this study, we, therefore, investigated the effect of BA on macrophages. METHODS: We studied macrophage recruitment, functional phenotypes (M1/M2), and the cellular metabolism via flow cytometry, qRT-PCR, immunofluorescence, a cell culture transwell system, and GC-MS-based metabolomics both in vivo in H1N1 A virus-infected mice and in vitro. RESULTS: BA treatment drastically reduced macrophage recruitment (CD11b+, F4/80+) by approximately 90% while maintaining the proportion of M1-polarized macrophages in the bronchoalveolar lavage fluid of infected mice. This BA-stimulated macrophage M1 phenotype shift was further verified in vitro in ANA-1 and primary peritoneal macrophages by measuring macrophage M1 polarization signals (CD86, iNOS, TNF-α, iNOS/Arg-1 ratio, and IL-1ß cleavage). Meanwhile, we observed an activation of the IFN pathway (upregulation of IFN-ß and IRF-3), an inhibition of influenza virus replication (as measured by the M gene), and distinct cellular metabolic responses in BA-treated cells. CONCLUSION: BA triggered macrophage M1 polarization, IFN activation, and other cellular reactions, which are beneficial for inhibition of H1N1 A virus infection.

Determination of the antidiabetic chemical basis of Phellodendri Chinensis Cortex by integrating hepatic disposition in vivo and hepatic gluconeogenese inhibition in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Phellodendri Chinensis Cortex (PCC) has been an herb clinically used to treat diabetes, but the chemical basis of its antidiabetic effects has remained unclear. AIM OF THIS STUDY: Based on the efficacy of herbal medicine resulting from the cooperative response of the effective compounds in the target organs with sufficient exposure, the in vivo hepatic disposition and in vitro hepatic gluconeogenesis inhibition were integrated to elucidate the chemical basis for the antidiabetic effect of orally administered PCC from a target organ, liver, perspective. MATERIALS AND METHODS: With a developed and validated HPLC-MS/MS method, three alkaloids and five metabolites were determined in the portal vein plasma, liver, and systemic plasma of rats orally administered PCC. The inhibition of hepatic gluconeogenesis by the eight compounds was evaluated in primary hepatocytes. RESULTS: The in vivo results showed that magnoflorine was present at the highest concentration among the target constituents in the plasma, where berberine showed a low concentration. In contrast, berberine showed the highest concentration in the liver, and its five metabolites exhibited substantial hepatic accumulation. This discrepancy was strongly associated with the hepatic disposition of the compounds. The hepatic disposition prevented the transfer of 96.1% of the phellodendrine, 71.1% of the berberine and 47.5% of the magnoflorine from the portal vein plasma to the systemic plasma, which corresponded to their hepatic distribution and hepatic metabolism. In vitro, berberine, M1, M4 and M5 significantly and dose-dependently inhibited hepatic glucose production. By integrating the hepatic exposure and inhibitory activity data, we estimated that berberine contributed the most (74%) to the total glucose production inhibition of the orally administered PCC decoction, followed by M4 (14%), M1 (11%) and M5 (1%). CONCLUSION: This study was the first to comprehensively describe the pharmacokinetic profiles and hepatic disposition of alkaloids in PCC, and concluded that berberine and its metabolites contributed the most to the total hepatic gluconeogenesis inhibition by orally administered PCC. These results reveal the chemical basis for the antidiabetic effect of orally administered PCC decoction, providing scientific evidence to support the clinical usage of PCC in diabetes treatment.

Synthesis and Cytotoxic Activity of Novel C-23-Modified Asiatic Acid Derivatives.

We selectively oxidized the C-23 hydroxyl group in an asiatic acid (AA) derivative and then, for the first time with AA, modification of the C-23 carboxyl group was conducted to synthesize a series of new AA derivatives. The evaluation of their cytotoxic activities against two human cancer cell lines (SKOV-3 and HCT116) using the MTT assay in vitro revealed a distinctive structure activity relationship (SAR) associated with the intramolecular hydrogen bonding of the amide moiety at C-23. According to the established SAR, the cytotoxic activities of four promising compounds were then evaluated against MCF-7, A549, A2780, HepG2 and HL-60 cancer cell lines. Compound 10 had the best cytotoxic activity among all tested derivatives in the HL-60 cell line, giving IC50 = 0.47 µM, while showing no cytotoxic effect against human normal cells (HUVEC).

Correction to: Identifying the active components of Baihe-Zhimu decoction that ameliorate depressive disease by an effective integrated strategy: a systemic pharmacokinetics study combined with classical depression model tests.

[This corrects the article DOI: 10.1186/s13020-019-0254-9.].

Identifying the active components of Baihe-Zhimu decoction that ameliorate depressive disease by an effective integrated strategy: a systemic pharmacokinetics study combined with classical depression model tests.

BACKGROUND: Modern pharmacological studies have demonstrated that Baihe-Zhimu decoction (BZD) has antidepressant effects. However, the complex composition and lack of clear evaluation standards for BZD make it less likely to be understood and accepted than evidence-based active natural compounds. METHODS: In this study, an effective method for the identification of antidepressant components was demonstrated and applied to BZD. The first step was to evaluate the efficacy of BZD by the forced swimming test (FST) and the tail suspension test (TST), followed by successive quantitative analyses of the absorbed constituents at different stages, such as before hepatic disposition, liver distribution, after hepatic disposition and brain distribution after the oral administration of BZD. Finally, the compounds detected in the brain were confirmed by activity testing. RESULTS: Our investigation observed that timosaponin BII and timosaponin BIII were accurately determined in the brain after oral administration of BZD, and they were further confirmed to reduce the immobility time in the FST and TST. As described above, timosaponin BII and timosaponin BIII were used to scientifically and reasonably explain the effective chemical basis of the effect of BZD on depression. CONCLUSIONS: This research affords an effective method to discover lead molecules for antidepressants from traditional Chinese medicine.

A comprehensive study of pomegranate flowers polyphenols and metabolites in rat biological samples by high-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Pomegranate flowers is an ancient medicine that has commonly been used to treat various diseases such as diabetes. However, no reports are available on the metabolic profile of pomegranate flowers in vivo. In the present study, with the aid of HPLC-Q-TOF-MS2, 67 compounds were identified in pomegranate flowers extract, including 18 ellagitannins, 14 gallic acid and galloyl derivatives, five anthocyanins and 18 flavonoids. Seven compounds were firstly identified. In vivo, 22 absorbed compounds and 35 metabolites were identified in rat biosamples (urine, feces, plasma and tissues) after orally administered with pomegranate flowers extract. This result showed that not all compounds abundant in pomegranate flowers extract could be absorbed well in plasma and tissues. This finding also suggested a potential correlation between study on metabolic profile of these compounds in vivo and study on strategy of screening bioactivity of the isolates with in vitro cell systems evaluation. Notably, mono-glucuronide conjugated metabolite of ellagitannin compound (corilagin) was firstly identified. In addition, this is first report to identify phase II conjugate metabolites of ellagitannins in vivo after oral administration of ellagitannins-rich extracts (or foods). Thus, characterizing its multiple constitution, absorption and metabolic fate of these compounds in vivo is helpful to better analyze the active components in pomegranate flowers.

Determining the protective effects of Yin-Chen-Hao Tang against acute liver injury induced by carbon tetrachloride using 16S rRNA gene sequencing and LC/MS-based metabolomics.

Yin-Chen-Hao Tang (YCHT), consisting of Artemisia annua L., Gardenia jasminoides Ellis, and Rheum Palmatum L., has been used to relieve liver diseases in China for thousands of years. Several protective mechanisms of YCHT on liver injury have been investigated based on metabolomics, but the effects of YCHT on the alterations in the gut microbiota are still unclear. In this study, an integrated approach based on 16S rRNA gene sequencing combined with high-performance liquid chromatography-mass spectrometry (HPLC-MS) metabolic profiling was performed to assess the effects of YCHT on liver injury induced by carbon tetrachloride (CCl4) through the regulation of the relative abundances of gut microbiota and their relationships with biomarker candidates. A total of twelve significantly altered bacterial genera and nine metabolites were identified, which returned to normal levels after YCHT treatment. The relative abundances of the identified microbiota, including significantly elevated amounts of p_Firmicutes, c_Clostridia, o_Clostridiales, f_Ruminococcaceae, g_[Eubacterium]_coprostanoligenes_group, s_uncultured_bacterium_f_Lachnospiraceae and remarkedly increased amounts of p_Bacteroidetes, c_Bacteroidia, o_Bacteroidales, f_Bacteroidaceae, g_Bacteroides and s_uncultured_bacterium_g_Bacteroides, were found in model rats compared with controls. Potential biomarkers, including lower levels of LysoPC (16:1(9Z)/0:0), LysoPC (20:3(5Z,8Z,11Z)), LysoPC (17:0), LysoPC (20:1(11Z)) and 3-hydroxybutyric acid and higher amounts of ornithine, L-kynurenine, hippuric acid and taurocholic acid are involved in several custom metabolic pathways, such as arginine and proline metabolism, tryptophan metabolism, glycerophospholipid metabolism and primary bile acid biosynthesis. Interestingly, there was a strong correlation between the perturbed gut microbiota in genera c_Clostridia and o_Clostridiales and the altered plasma metabolite 3-hydroxybutyric acid. This finding means that the hepatoprotective effects of YCHT may be due to the regulation of the production of the functional metabolite 3-hydroxybutyric acid through changes in the proportions of c_Clostridia and o_Clostridiales. These results showed that the hepatoprotective effects of YCHT not only focused on custom metabolic pathways but also depended on the changes in the gut microbiota in liver injury. These findings suggest that the 16S rRNA gene sequencing and LC-MS based metabolomics approach can be applied to comprehensively evaluate the effects of traditional Chinese medicines (TCMs).

Metabonomics Study on the Hepatoprotective Effect of Panax notoginseng Leaf Saponins Using UPLC/Q-TOF-MS Analysis.

Alcohol liver disease is a major public health problem associated with lifestyle. Our recent study demonstrated that the roots of Panax notoginseng saponins (PNS) exert hepatoprotective effects against alcohol consumption. Considering that the leaves of Panax notoginseng saponins (LPNS) have similar chemical ingredients with PNS, increased attention should be given to the hepatoprotective effects of LPNS. In this study, a metabonomic approach based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) was developed to evaluate the hepatoprotective effect of LPNS on alcoholic fatty liver and elucidate the interaction mechanisms. Results showed that the ethanol-induced metabolic perturbations were restored after treatment with LPNS. Furthermore, 12 potential biomarkers (11 upregulated and 1 downregulated) were identified by V-plot and orthogonal partial least square discriminant analysis. Changes in the levels of these metabolites indicated that glycerophospholipid and fatty acid metabolism were disturbed in alcoholic fatty liver mouse. Our findings demonstrated that the UHPLC-QTOF/MS-based metabonomic method may provide a useful means for exploring biomarkers involved in alcoholic fatty liver and elucidating the therapeutic effects of LPNS. This work also showed that the metabonomic approach is a powerful and promising tool for the evaluation of the efficacy of traditional Chinese medicine and elucidation of related mechanisms.

Exploration of the hepatoprotective chemical base of an orally administered herbal formulation (YCHT) in normal and CCl4-intoxicated liver injury rats. Part 1: Metabolic profiles from the liver-centric perspective.

ETHNOPHARMACOLOGICAL RELEVANCE: Yin-Chen-Hao Tang (YCHT), derived from "Treatise on Febrile Diseases" in ancient China, has been a very popular hepatoprotective three-herb formula in China and Japan, although its chemical base remains unclear. AIM OF THIS STUDY: As the first step in revealing the hepatoprotective chemical base of YCHT, we aimed to clarify the absorbed ingredients and associated metabolic pathways for orally dosed YCHT in both normal and liver injury rats from a liver-centric perspective. MATERIALS AND METHODS: With the aid of 10 reference compounds, the absorbed ingredients and generated metabolites were systematically characterized by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF) in the portal vein plasma (the plasma before hepatic disposition) - liver - systemic plasma (the plasma after hepatic disposition), following oral administration of YCHT in normal and CCl4-induced liver injury rats. RESULTS: A total of 38 compounds with six chemical structures, consisting of 10 prototypes and 28 metabolites generated through 9 biotransformations, were absolutely or tentatively identified, and 25 compounds were first reported on YCHT treatments. Among them, 8 compounds were absolutely confirmed by comparing with standard substances, and some had published hepatoprotective activities. Compared with the 35, 15, and 29 compounds identified in the portal vein plasma, liver, and systemic plasma of normal rats, respectively, the corresponding numbers of characterized compounds were 37, 13 and 29 in the liver injury rats. CONCLUSIONS: Sulfation and glucuronidation were the predominant biotransformations, and intestinal metabolism, prior to hepatic metabolism, occurred for most compounds. CCl4-induced liver injury caused only slight changes in the metabolic profiles of rats orally administered YCHT. These results provided the precondition for further quantitative analysis and pharmacodynamic screening of compounds in YCHT.

Evaluation of the chemical consistency of Yin-Chen-Hao-Tang prepared by combined and separated decoction methods using high-performance liquid chromatography and quadrupole time-of-flight mass spectrometry coupled with multivariate statistical analysis.

In this study, Yin-Chen-Hao-Tang prepared by two decoction methods, namely, combined decoction (modern decoction method) and separated decoction (traditional decoction method), was analyzed by high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry. The acquired datasets containing sample codes, tR -m/z pairs and ion intensities were processed with multivariate statistical analyses, such as principal component analysis and an orthogonal partial least squared discriminant analysis model, to globally compare the chemical differences between the different decoction samples. Then, the chemical differences between the combined and separated decoctions were screened out by S-plots generated from the orthogonal partial least squared discriminant analysis model and compared with chemical information from an established in-house library. The six components that contributed the most to the chemical differences were identified as chlorogenic acid, caffeic acid, geniposide, genipin, scopoletin, and 3,5-dicaffeoylquinic acid. The concentrations of genipin and caffeic acid from the separated decoction were higher than those from the combined decoction, indicating that the separated decoction may present a stronger hepatoprotective effect. However, the results still require further investigation through pharmacological and clinical studies. Our findings not only establish a strategy to evaluate chemical consistency of Yin-Chen-Hao-Tang but also provide the scientific basis for using traditional separated decoction method.

Enhanced Anti-diabetic Effect of Berberine Combined With Timosaponin B2 in Goto-Kakizaki Rats, Associated With Increased Variety and Exposure of Effective Substances Through Intestinal Absorption.

Objective: Inspired by the traditionally clinical application of herb pair Zhimu-Huangbo to treat diabetes, a combination of plant ingredients, timosaponin B2 (TB-2) and berberine (BBR), was evaluated for their anti-diabetic efficacy and cooperative mechanisms. Methods: The efficacy and pharmacokinetics of orally administered TB-2 (33.3 mg/kg/day), BBR (66.7 mg/kg/day), and TB-2+BBR (100 mg/kg/day) were evaluated in spontaneously non-obese diabetic Goto-Kakizaki (GK) rats, and metformin (200 mg/kg/day) was used as a positive control. The comparative exposure of the parent drugs, timosaponin A3 (TB-2 metabolite), and M1-M5 (BBR metabolites) was quantified in the portal vein plasma (before hepatic disposition), liver, and systemic plasma (after hepatic disposition) of normal rats on single and combination treatments. Cooperative mechanism of TB-2 and BBR on intestinal absorption and hepatic metabolism was investigated in Caco-2 cells and primary hepatocytes, respectively. Results: After a 6-week experiment, non-fasting and fasting blood glucose levels and oral glucose tolerance test results showed that TB-2+BBR treatments (100 mg/kg/day) displayed significantly anti-diabetic efficacy in GK rats, comparable to that on metformin treatments. However, no significant improvement was observed on TB-2 or BBR treatments alone. Compared to single treatments, combination treatments led to the increased circulating levels of BBR by 107% in GK rats. In normal rats, the hepatic exposure of BBR, timosaponin A3, and M1-M5 was several hundred folds higher than their circulating levels. Co-administration also improved the levels in the plasma and liver by 41-114% for BBR, 141-230% for TB-2, and 12-282% for M1-M5. In vitro, the interaction between TB-2 and BBR was mediated by intestinal absorption, rather than hepatic metabolism. Conclusion: Combining TB-2 and BBR enhanced the anti-diabetic efficacy by increasing the in vivo variety of effective substances, including the parent compounds and active metabolites, and improving the levels of those substances through intestinal absorption. This study is a new attempt to assess the effects of combined plant ingredients on diabetes by scientifically utilizing clinical experience of an herb pair.

Genomic and GeneChip expression profiling reveals the inhibitory effects of Amorphophalli Rhizoma in TNBC cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Amorphophalli Rhizoma has been widely used as an adjuvant treatment for advanced or metastatic breast cancer, pancreatic cancer, hepatoma, and malignant lymphoma, but its molecular mechanism of action for treatment of metastatic triple-negative breast cancer (TNBC) is generally poorly understood. AIM OF THE STUDY: To investigate genomic changes related to the inhibitory effect of Amorphophalli Rhizoma and to elucidate the molecular mechanism of this inhibition in MDA-MB-231 TNBC cells. MATERIALS AND METHODS: Gene chip analysis was employed to explore genomic changes caused by Amorphophalli Rhizoma in TNBC cells. Potential classical signaling pathways, upstream regulators, functions, regulatory effects and gene interaction networks were analyzed by Ingenuity Pathway Analysis (IPA). Real-time quantitative PCR (RT-qPCR) and RNA interference (RNAi) assays were used to clarify the roles of potential target genes. RESULTS: In total, 536 significantly upregulated and 648 significantly downregulated genes were identified between the group treated with Amorphophalli Rhizoma extract and that treated with vehicle. Many of these differentially expressed genes (DEGs) in TNBC cells are involved in DNA replication, recombination and repair, the cell cycle, and cellular assembly and organization. Attenuation of KNL1, OLFML2A, RTKN2 and SGO1 gene expression by Amorphophalli Rhizoma significantly induced cell cycle arrest and suppressed cell proliferation and migration. CONCLUSIONS: The inhibitory effects of Amorphophalli Rhizoma in TNBC cells likely occur through regulation of the spindle checkpoint, chromosomal and centrosomal instability, and cell membrane stability.

Exploration of the hepatoprotective chemical base of an orally administered herbal formulation (YCHT) in normal and CCl4-intoxicated liver injury rats. Part 2: Hepatic disposition in vivo and hepatoprotective activity in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Yin-Chen-Hao Tang (YCHT) has been a very popular, hepatoprotective three-herb formula with an unclear chemical base. AIM OF THIS STUDY: To reveal the hepatoprotective chemical base of oral-dosed YCHT, we bridged the hepatic disposition of six compounds in vivo and their hepatoprotection in vitro. MATERIALS AND METHODS: In vivo, following the oral administration of YCHT in normal and CCl4-induced liver injury rats, the determinations of chlorogenic acid, 4-hydroxyacetophenone, geniposide, genipin, rhein and emodin were conducted in the portal vein plasma, the liver, and the systemic plasma. In vitro, the hepatoprotective activities of these compounds were determined in the CCl4-induced HepG2 cells. RESULTS: Consistent with the highest content in YCHT, geniposide had the highest exposure in vivo. Inconsistent with the negligible content, rhein, 4-hydroxyacetophenone, emodin and genipin showed substantial hepatic accumulations. In contrast, chlorogenic acid, an ingredient that has a high content in YCHT, elicited no hepatic exposure. In normal rats, the hepatic disposition prevented the compounds entering into the systemic plasma from the portal vein plasma by 44.9-100%, except for rhein. CCl4-induced liver injury caused a decreased hepatic exposure of 4-hydroxyacetophenone, rhein and emodin by 50%. In vitro, all six compounds exerted the hepatoprotection by increasing cell viability, decreasing hepatic marker enzymes and inhibiting lipid peroxidation at varying levels. CONCLUSION: Geniposide, rhein, emodin, 4-hydroxyacetophenone and genipin directly resisted liver injury in oral-dosed YCHT, while chlorogenic acid likely played an indirect role. This study proved that YCHT exerted hepatoprotection through multiple components and multiple actions. However, close attention should be paid to the possible side effects and oral dosage of YCHT in clinics.

Wenshen Zhuanggu formula mitigates breast cancer bone metastasis through the signaling crosstalk among the Jagged1/Notch, TGF-ß and IL-6 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Advanced breast cancer frequently metastasizes to the bone, resulting in patient morbidity and mortality. The interaction of tumor cells with osteoclasts and osteoblasts seriously affects the occurrence and development of bone metastasis in breast cancer. The signaling crosstalk among the Jagged1/Notch, TGF-ß and IL-6 signaling pathways plays a significant role in the context of bone metastasis. AIM OF THE STUDY: Although Wenshen Zhuanggu (WSZG) formula efficiently decreased the risk of bone metastases in tumor-bearing mice, it remains unclear how WSZG formula regulates the interaction of cancer cells with osteoclasts and osteoblasts in bone metastasis of breast cancer. MATERIALS AND METHODS: In this study, we investigated the role of WSZG formula in the progress of bone metastasis in breast cancer and focused on the cell-cell interactions of tumor cells with osteoclasts and osteoblasts. Western blotting and quantitative real-time PCR were utilized to evaluate the inhibitory activities of WSZG formula on Jagged1 expression both in vivo and in vitro. Osteoblast co-culture and osteoclastogenesis co-culture were applied to analyze the effects of WSZG formula on the interaction of tumor cells with osteoclasts and osteoblasts. A breast cancer xenograft model was also used to test the inhibitory effects of WSZG formula on bone metastasis in breast cancer. RESULTS: WSZG formula decreased Jagged1 expression in osteolytic lesions in the breast cancer xenograft model. Additionally, WSZG formula decreased Jagged1 expression in tumor cell culture alone or co-culture with pre-osteoclasts and osteoblasts. In addition, WSZG formula decreased Jagged1 expression in Jagged1-overexpressing tumor cells. CONCLUSION: The results of this study suggest that WSZG formula mitigates breast cancer bone metastasis through the Jagged1/Notch signaling pathway mediated by TGF-ß and IL-6.

Metabolism, pharmacokinetics, and hepatic disposition of xanthones and saponins on Zhimu treatments for exploratively interpreting the discrepancy between the herbal safety and timosaponin A3-induced hepatotoxicity.

Timosaponin A3, a saponin in Zhimu, elicited hepatotoxicity via oxidative stress. However, the clinical medication of Zhimu has been historically regarded as safe, probably associated with the antioxidants it contains. However, the related information on the in vivo levels of timosaponin A3 and antioxidants remained unclear on Zhimu treatments. Therefore, a combination of the in vitro metabolism, including microbiota-mediated and liver-mediated metabolism, and in vivo pharmacokinetics and hepatic disposition, was conducted for three xanthones (neomangiferin, mangiferin, and norathyriol) and three saponins (timosaponin B2, timosaponin B3, and timosaponin A3) on Zhimu treatments. Consequently, following oral administration of Zhimu decoction to rats, those saponins and xanthones were all observed in the plasma with severe liver first-pass effect, where mangiferin was of the maximum exposure. Despite the ignorable content in the herb, timosaponin A3 elicited sizable hepatic exposure as the microbiota-mediated metabolite of saponins in Zhimu. The similar phenomenon also occurred to norathyriol, the microbiota-mediated metabolite of xanthones. However, the major prototypes in Zhimu were of limited hepatic exposure. We deduced the hepatic collection of norathyriol, maximum circulating levels of mangiferin, and timosaponin B2 and mangiferin interaction may directly or indirectly contribute to the whole anti-oxidation of Zhimu, and then resisted the timosaponin A3-induced hepatotoxicity. Thus, our study exploratively interpreted the discrepancy between herbal safety and timosaponin A3-induced hepatotoxicity. However, given the considerable levels and slow eliminated rate of timosaponin A3 in the liver, more attention should be paid to the safety on the continuous clinical medication of Zhimu in the future.