Metabolomics and molecular docking-directed antiarthritic study of the ethyl acetate extract from Celastrus orbiculatus Thunb.

ETHNOPHARMACOLOGICAL RELEVANCE: Celastrus orbiculatus Thunb., an important folk medicine, has long been used for the treatment of rheumatoid arthritis and its ethyl acetate extract (COE) has been reported to possess anticancer, antiinflammation and antiarthritic effects. However, the therapeutic effect and mechanism of COE treatment in rheumatoid arthritis has been rarely studied especially from the perspective of metabolomics. AIM OF STUDY: To reveal the therapeutic effects of COE on adjuvant-induced arthritis (AIA) rats through histopathological analysis, non-targeted metabolomics, and molecular docking study. MATERIALS AND METHODS: Forty-three Wistar rats were randomly divided into normal group, AIA model group, methotrexate group, and COE groups (80 mg/kg, 160 mg/kg and 320 mg/kg of ethyl acetate extract). Paw swelling and arthritis score were monitored through the experiment. Serum levels of tumor necrosis factor α (TNF-α) and nitric oxide were determined and histopathological evaluation was performed. Furthermore, Ultra-high performance liquid chromatography-linear trap quadrupole-Orbitrap-based metabolomics was employed to characterize metabolic changes of AIA rats after COE treatment and molecular docking was performed to predict the potential phytochemicals of COE against TNF-α. RESULTS: COE at three dosages could significantly relieve paw swelling and reduce arthritis scores of AIA rat. Histopathological analysis revealed remarkable decrease in synovial inflammation and bone erosion after COE treatment, especially at middle and high dosage. Additionally, COE down-regulated serum levels of TNF-α and nitric oxide. Serum metabolomics showed that 22 potential biomarkers for the COE treatment of AIA rats were identified, which were closely related to fatty acid metabolism, glycerophospholipid catabolism, and tryptophan metabolism. The molecular docking models predicted that olean-type triterpenes in COE may contribute most to therapeutic effects of rheumatoid arthritis through targeting TNF-α. CONCLUSIONS: COE could significantly relieve the arthritic symptoms in AIA rats and the ultra-high performance liquid chromatography-mass spectrometry based metabolomics proved to be an efficient method to characterize subtle metabolic changes of AIA rats after COE treatment.

A Study on the Effect and Mechanism of Xiaoaiping (XAP) Injection and S-1 Combination Therapy in Inhibiting the Invasion and Metastasis of Human GC Cells.

BACKGROUND: This study aimed to determine the effect and mechanism of Xiaoaiping (XAP) injection combined with S-1 in inhibiting the invasion and metastasis of human GC cells. METHODS: BGC-823 and MGC-803 cells were incubated in vitro, and the effects of treatment on the cytotoxicity and proliferation of BGC-823 and MGC-803 cells were evaluated by MTT assay. Cell adhesion tests and Transwell assays were used to detect the effects of Xiaoaiping injection combined with S-1 on the metastatic ability of BGC-823 and MGC-803 cells. The expression of VEGF, Metalloproteinases (MMPs) and proteins related to the Epithelial-Mesenchymal Transition (EMT) were detected by Western blotting. Meanwhile, a tumour model was established in nude mice, and the effect of XAP combined with S-1 on BGC-823 cells in vivo was studied. RESULTS: Compared with the single drug group, the combination of XAP with S-1 increased the inhibition rate (P<0.05). The adhesion test showed that the combination group significantly inhibited the adhesion of BGC-823 and MGC-803 cells (P<0.05). The combination of XAP with S-1 reduced the migration and invasion potential of human GC BGC-823 and MGC-803 cells. Western blotting showed that the expression of VEGF, MMP-9, Ncadherin and vimentin was decreased and E-cadherin expression was increased in the combination group compared with these expression values in either the XAP or S-1 alone group (P<0.05). In vivo, we found that XAP combined with S-1 had a significant inhibitory effect on the growth of tumours compared with XAP or S-1 alone. Immunohistochemistry showed that XAP combined with S-1 was able to enhance the levels of E-cadherin and downregulate N-cadherin and vimentin. CONCLUSION: The combination of XAP with S-1 can enhance the inhibitory effect of a single drug on proliferation, invasion and metastasis. The mechanism may be related to the decrease in the expression of VEGF and MMP-9 proteins and the effect on EMT.

28-Hydroxy-3-oxoolean-12-en-29-oic Acid, a Triterpene Acid from Celastrus Orbiculatus Extract, Inhibits the Migration and Invasion of Human Gastric Cancer Cells In Vitro.

Gastric cancer is the fifth most common tumor and has the third-highest mortality rate among various malignant tumors, and the survival rate of patients is low. Celastrus orbiculatus extract has been shown to inhibit the activity of a variety of tumors. This study explored the inhibitory effect of the oleanane-type triterpenoid acid 28-hydroxy-3-oxoolean-12-en-29-oic acid molecule from Celastrus orbiculatus extract on gastric cancer cell invasion and metastasis and determined its mechanism. 28-Hydroxy-3-oxoolean-12-en-29-oic acid was first diluted to various concentrations and then used to treat SGC-7901 and BGC-823 cells. Cell proliferation was assessed by an MTT (thiazole blue) assay. Transwell and wound healing assays were used to assess cell invasion and migration. High-content imaging technology was used to further observe the effects of the drug on cell invasion and migration. Western blotting was used to assess the effects on the expression of matrix metalloproteinases (MMPs) and the effects on epithelial-mesenchymal transition (EMT)-related proteins and phosphorylation-related proteins. We found that 28-Hydroxy-3-oxoolean-12-en-29-oic acid inhibited the migration and invasion of SGC-7901 and BGC-823 gastric cancer cells in a dose-dependent manner. Consequently, 28-hydroxy-3-oxoolean-12-en-29-oic acid decreased the expression of EMT-related proteins and MMPs in gastric cancer cells and reduced protein phosphorylation, inhibiting the migration and invasion of gastric cancer cells.

Celastrus Orbiculatus Extracts Inhibit the Metastasis through Attenuating PI3K/Akt/mTOR Signaling Pathway in Human Gastric Cancer.

BACKGROUND: Rapamycin receptor inhibitors have been applied in the clinic and achieved satisfactory therapeutic effect recently. The mechanisms did not clearly show how the Celastrus Orbiculatus Extracts (COE) inhibited the expression of the mammalian Target of Rapamycin (mTOR) in human gastric cancer cells. The aim of this study was to investigate whether the COE inhibited the metastasis through the mTOR signaling pathway in human gastric cancer MGC-803 cells. METHODS: The abnormal expression level of mTOR protein was detected by immunohistochemistry in human gastric cancer tissue. The MGC-803/mTOR- cells were constructed by knockdown of mTOR using lentivirus infection technique. The human gastric cancer MGC-803/mTOR- cells were treated with different concentrations (20, 40, 80 µg/ml) of COE for 24 hours. The ability of cell metastasis was analyzed by the cell invasion and migration assay. The expression levels of PI3K/Akt/mTOR signaling pathway were detected by Western Blotting. RESULTS: COE inhibited the proliferation, invasion and migration of MGC-803/mTOR- cells in a concentrationdependent manner. The expression of E-cadherin protein increased, and the expression of N-cadherin and Vimentin decreased simultaneously in the MGC-803/mTOR- cells. 4EBP1, p-4EBP1, P70S6k, p-P70S6k, mTOR, p-mTOR, PI3K and Akt proteins in MGC-803/mTOR- cells were reduced in a dose-dependent manner. CONCLUSION: COE could not only inhibit cell growth, invasion and migration, but also inhibit the epithelialmesenchymal transition of gastric cancer cells. The molecular mechanism of COE inhibited the metastasis which may be related to the PI3K/Akt/mTOR signal pathway. This study provides ideas for the development of new anti-gastric cancer drugs.

Huachansu Capsule inhibits the proliferation of human gastric cancer cells via Akt/mTOR pathway.

BACKGROUND: For decades, the traditional Chinese medicine preparation, Huachansu Capsule (HCS), has been applied to a variety of solid tumors and leukemias with significant curative effects. More importantly, HCS has few side effects on cardiovascular and gastrointestinal functions in patients. However, the potential mechanism of the anti-tumor activity of HCS has not been fully revealed. The current study investigated the in vivo and in vitro effects of HCS on the proliferation and apoptosis of human gastric cancer (GC) cells and explored the underlying mechanism. MATERIALS AND METHODS: HCS was first diluted to varying concentrations followed by the treatment to MGC-803 and BGC-823 GC cells. Cell proliferation was evaluated by Cell Counting Kit-8 assay. Cell invasion and migration were assessed using Transwell membrane chambers. Apoptosis and cell cycle arrest in GC cells induced by HCS were detected by flow cytometry. Western blotting assays were used to measure the influence of HCS on apoptosis-related proteins, including B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and Cleaved-Caspase-3. Additionally, mammalian target of rapamycin (mTOR) signaling pathway-related proteins such as phosphorylated (p)-Akt, p-mTOR and p-4E-BP1 were detected. Transmission electron microscopy was used to observe the microstructure of apoptotic cells. An animal imaging technique was used to analyze the influence of HCS on the growth of GC cells in vivo and immunohistochemistry assays were performed to investigate the signal transduction pathways influenced by HCS. RESULTS: HCS significantly inhibited the proliferation, invasion and migration of MGC-803 and BGC-823 GC cells. It also induced cell cycle arrest at the G2/M phase and increased the cell apoptotic rate. Additionally, the HCS treatment downregulated the protein levels of Bcl-2, but upregulated the protein expression of Bax and cleaved-caspase 3. Furthermore, HCS downregulated the levels of p-Akt, p-mTOR and p-4E-BP1, suggesting that HCS inhibited tumor growth of GC via suppressing the Akt/mTOR pathway. CONCLUSION: This study indicated that HCS has significant anti-proliferative and apoptotic effects both in vitro and in vivo, and that HCS can inhibit tumor growth of GC via suppressing the Akt/mTOR pathway and induce apoptosis through the intrinsic pathway. Our study provides a scientific basis for the clinical application of HCS.

Celastrus orbiculatus Extracts Inhibit Human Hepatocellular Carcinoma Growth by Targeting mTOR Signaling Pathways.

OBJECTIVE: To characterize the molecular mechanism underlying the antineoplastic activity of Celastrus orbiculatus Thunb. extracts (COE). METHODS: The human hepatocellular carcinoma HepG2 cells with mammalian target of rapamycin (mTOR) knockdown expressed (HepG2/mTOR) were constructed using molecular biological technology. In vitro, the HepG2/mTOR- cells were treated with COE at various concentrations (10, 20, 40, 80, 160 and 320 µ g/mL). Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. According to the half-maximal inhibitory concentration (IC50) value (140 mg/L), the concentrations of COE in the subsequent experiment was set to alleviate cytotoxicity. The HepG2/mTOR- cells were divided into 5 groups: negative control (untreated), COE treatment groups (40, 80, 120 mg/L COE) and positive control group (cisplatin, DDP, 2 mg/L), respectively. Wild-type HepG2 cells were used as a blank control. The effects of COE on the cell apoptosis were analyzed by flow cytometry and transmission electronic microscopy (TEM), respectively. The protein expression levels of mTOR signal pathways were determined by Western blotting. In vivo, HepG2/mTOR- cells (2 × 106 cell/mice) were subcutaneously injected into the right flank of nude mice. Thirty-six female nude mice were randomly assigned to 6 groups according to body weight (6 mice per group) as follows: solvent vehicle control, Banmao Capsule treated group (BM, 195 mg/kg), Tegafur, Gimeracil and Oteracil Potassium Capsules (10 mg/kg) treated group, and different dosages of COE (10, 20, 40 mg/kg) groups. Tumor growth was monitored and immunohistochemical staining was used to examine the expression of apoptosis-related proteins in tumor tissues. RESULTS: COE inhibited the proliferation significantly in a concentration-dependent manner in HepG2/mTOR- cells (P<0.01). COE significantly induced the apoptosis of HepG2/mTOR- cells (P<0.01), and the apoptotic bodies can be observed under TEM. COE significantly inhibits the proteins expression of mTOR-related signal pathways. In vivo, COE significantly inhibited tumor growth in nude mice (P<0.01). Moreover, the results showed that COE down-regulated the expression of Bcl-2 and Bcl-xL, and up-regulated the levels of Bax and caspase-3 protein (P<0.01). CONCLUSION: COE was a potential chemotherapeutic drug in HCC treatments via targeting mTOR signal pathway.

Extracts of Celastrus Orbiculatus Inhibit Cancer Metastasis by Down-regulating Epithelial-Mesenchymal Transition in Hypoxia-Induced Human Hepatocellular Carcinoma Cells.

OBJECTIVE: To evaluate the effects of Celastrus Orbiculatus extracts (COE) on metastasis in hypoxia-induced hepatocellular carcinoma cells (HepG2) and to explore the underlying molecular mechanisms. METHODS: The effect of COE (160, 200 and 240 µ g/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch-wound and transwell assays, respectively. CoCl2 was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immunofluorescence analysis, respectively. RESULTS: COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner (P<0.01). Furthermore, the expression of epithelial-mesenchymal transition (EMT) related markers were also remarkably suppressed in a dose-dependent manner (P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1 α (Hif-1 α) and Twist1 were suppressed by COE. Additionally, the Hif-1 α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced HepG2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect (P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced HepG2 cells. CONCLUSIONS: COE significantly inhibited the tumor metastasis and EMT by suppressing Hif-1 α/Twist1 signaling pathway in hypoxia-induced HepG2 cell. Thus, COE might have potential effect to inhibit the progression of HepG2 in the context of tumor hypoxia.

Celastrus orbiculatus extracts induce apoptosis in mTOR-overexpressed human hepatocellular carcinoma HepG2 cells.

BACKGROUND: Celastrus orbiculatus (Celastraceae) are used as traditional Chinese medicine to treat inflammation and cancer. This study aims to evaluate the effect of Celastrus orbiculatus extract (COE) on the apoptosis in human hepatic carcinoma HepG2 cells with mTOR overexpression. METHODS: The stable expression of mTOR in HepG2 cells (HepG2/mTOR+) were established by lipofectin transfection of GV238-mTOR recombinant plasmids and further antibiotic selection. Human hepatic carcinoma HepG2/mTOR+ cells were treated with different concentrations (20, 40, 80, 160, and 320 µg/mL) of COE for 24 h. The cell proliferation upon COE treatment was detected by MTT. Apoptosis was measured by Flow Cytometry. The activity of mTOR signaling pathway was detected by Western Blotting. RESULTS: COE significantly inhibited the proliferation of HepG2/mTOR+ cells. The expression levels of Bax and Caspase-3 protein were increased in the HepG2/mTOR+ cells in a dose-dependent manner. The proteins expression of Bcl2, Bcl-2 L12, mTOR, phospho-mTOR, 4EBP1, phospho-4EBP1, P70S6k, and phospho-P70S6k in HepG2/mTOR+ cells were reduced in dose-dependent manners. Furthermore, COE and mTOR inhibitor rapamycin (RAPA) synergistically induced apoptosis in HepG2/mTOR+ cells by regulating apoptosis-related proteins and inhibiting mTOR signaling pathways. CONCLUSION: COE could inhibit the proliferation of HepG2/mTOR+ cells, and induce the cell apoptosis. The mechanisms may be related to the regulation of the expression of Bcl-2, Bcl-2 L12, and mTOR signaling pathways. These data suggest that COE may be a potential treatment for human hepatocellular carcinoma.

Polypharmacological Profiles Underlying the Antitumor Property of Salvia miltiorrhiza Root (Danshen) Interfering with NOX-Dependent Neutrophil Extracellular Traps.

Danshen, the dried root of Salvia miltiorrhiza, one of the most investigated medicinal plants with well-defined phytochemical constituents, has shown prominent clinical outcomes for antioxidant, anti-inflammatory, and anticoagulant activities to attain vascular protection and additional benefits for cancer therapy. More recently, activation of neutrophil and excessive formation of neutrophil extracellular traps (NETs) have been observed in pathological conditions of metastatic cancers; thus, we hypothesized that suppression of NETs could account for an essential cellular event underlying Danshen-mediated reduction of the incidence of metastasis. Using an experimental pulmonary metastases model of red fluorescent protein- (RFP-) labeled gastric cancer cells in combination with macroscopic ex vivo live-imaging system, our data indicated that Danshen impaired the fluorescent intensity and quantity of metastatic nodules. Moreover, Danshen could prevent neutrophil trafficking to the metastatic sites with decreased plasma levels of neutrophil elastase (NE) and procoagulant potential featured by fibrinogen. We further established phorbol 12-myristate 13-acetate- (PMA-) induced NET formation of human neutrophils and screened representative active compounds derived from the hydrophilic and hydrophobic fractions of Danshen using qualitative and quantitative methods. As a result, we found that salvianolic acid B (Sal B) and 15,16-dihydrotanshinone I (DHT I) exhibited superior inhibitory activities on NET formation and significantly attenuated the levels of citrullinated histone H3 (citH3), a biomarker for NET formation. Multitarget biochemical assays demonstrated that Sal B and DHT I distinctly modulated the enzymatic cascade involved in NET formation. Sal B and DHT I could disrupt NET formation at the earlier stage by blocking the activities of myeloperoxidase (MPO) and NADPH oxidase (NOX), respectively. Lastly, combining treatment of Sal B and DHT I under subED50 doses displayed remarkable synergism effect on NET inhibition. Altogether, these data provide insight into how promiscuous compounds from herbal medicine can be effectively targeted NETs towards hematogenous metastasis of certain tumors.

Jinlong Capsule (JLC) inhibits proliferation and induces apoptosis in human gastric cancer cells in vivo and in vitro.

BACKGROUND: As a representative traditional Chinese medicine made by modern pharmaceutical technology, Jinlong Capsule (JLC) has been used for several decades to treat liver cancer with significantly improved clinical outcomes as adjuvant therapy. JLC consists of three medicinal animals including freshly prepared Bungarus, Agkistrodon and Gecko. The active components were extracted by the process of modern cryogenic and biochemical separation from raw animals. However, the specific molecular mechanisms underlying the antitumor activities of JLC were not fully investigated. In the current study, experiments were carried out to examine the effect of JLC on anti-proliferative, pro-apoptotic activities of human gastric cancer (GC) cell lines in vivo and in vitro. METHODS: MTT assay was used to observe the viability of MGC-803 and BGC-823 cells treated with JLC. Apoptosis and cell cycle distribution of MGC-803 and BGC-823 cells induced by JLC were analyzed by flow cytometry. Western blot assay was used to detect the effect of JLC on apoptosis-related proteins, including Bax, Bcl-2, survivin and caspase-3. Transmission electron microscopy (TEM) was used to evaluate the microstructure of apoptotic GC cells. Tumor growth in vivo was monitored using live-imaging system. Immunohistochemical staining (IHC) was used to examine the expression of apoptosis-related proteins in tumor tissues. RESULTS: Our data indicated that JLC inhibited proliferation and induced apoptosis of MGC-803 and BGC-823 cells in a concentration-dependent manner. JLC significantly inhibited tumor growth in nude mice. Both in vivo and in vitro studies showed that JLC could downregulate the expression of Bcl-2 and survivin, whereas upregulate the levels of bax and caspase-3. JLC had significant antitumor effects in human GC through cell cycle arresting. Besides, JLC altered the microstructure of GC cells. CONCLUSION: These findings demonstrate that JLC can be considered as a promising candidate in GC treatment.

Traditional Chinese Medicine Integrated with Chemotherapy for Stage II-IIIA Patients with Non-Small-Cell Lung Cancer after Radical Surgery: A Retrospective Clinical Analysis with Small Sample Size.

OBJECTIVE: This study was designed to evaluate the clinical efficacy of combined traditional Chinese medicine (TCM) and conventional chemotherapy versus conventional chemotherapy in patients with stage II-IIIA non-small-cell lung cancer (NSCLC) after radical surgery. METHODS: A retrospective cohort study was conducted in patients with stage II-IIIA NSCLC from Subei People's Hospital and Yangzhou Traditional Chinese Medicine Hospital in Yangzhou City of Jiangsu Province from 2012 to 2016. Patients were divided into two groups: the TCM user group (patients receiving treatment with integrated TCM and conventional chemotherapy) and the non-TCM user group (patients receiving conventional chemotherapy only). The two groups were compared for their median disease-free survival (DFS) and median overall survival (OS). RESULTS: A total of 67 patients with stage II-IIIA NSCLC were enrolled between January 2012 and December 2016. The median DFS for the non-TCM user group was 601 days (95% confidence interval [CI], 375.7-826.3). The median DFS for TCM user group could not be calculated. However, log-rank analysis showed that the median survival time in the TCM user group was significantly longer than that of the non-TCM user group (P < 0.05). In addition, several significant risk factors were detected for predicting disease prognosis in patients with NSCLC, such as age, ECOG, lymphatic metastasis, and body mass index (BMI). For patients harboring these independent risk factors, the DFS of TCM user group was much longer than that of non-TCM user group (P < 0.05). CONCLUSION: Adjuvant therapy with TCM may reduce the rate of tumor recurrence and metastasis and prolong DFS of patients with stage II-IIIA NSCLC.

Zusanli (ST36) Acupoint Injection with Neostigmine for Paralytic Postoperative Ileus following Radical Gastrectomy for Gastric Cancer: a Randomized Clinical Trial.

Background: The Zusanli (ST36) acupoint has been associated with treatment of various gastrointestinal conditions. There have been no studies of acupuncture therapy for paralytic postoperative ileus (PPOI). Materials and methods: Patients with PPOI following gastrectomy for gastric cancer were randomized to receive ST36 acupoint injection with neostigmine, gluteal intramuscular injection with 1.0 mg neostigmine, ST36 acupuncture alone, or standard therapy. The main outcome was the effectiveness rate for recovery of peristalsis. Secondary outcomes were time to bowel sound recovery, time to first flatus, and time to first defecation. Tertiary outcomes were drug-related adverse events, including abdominal pain, diarrhea, nausea, vomiting, tearing, delirium, seizure, and anxiety. Results: ST36 acupoint injection with neostigmine and gluteal intramuscular injection of neostigmine gave a higher rate of peristalsis recovery, and the ST36 acupoint injection group showed significantly higher total effectiveness rate than that of the intramuscular injection group. These interventions gave significantly shorter times to bowel sound recovery, shorter times to first flatus and first defecation compared with ST36 acupuncture and standard post-operative therapy (P < 0.01). ST36 acupoint injection group gave shorter time to bowel sound recovery, shorter time to first flatus and first defecation than those of the intramuscular injection group (P < 0.01). Drug-related adverse events in the intramuscular injection group were more serious than in the ST36 acupoint injection group (P < 0.05). Conclusion: ST36 acupoint injection with neostigmine is safe and effective for treatment of PPOI.

Efficacy of extracts of Celastrus orbiculatus in suppressing migration and invasion by inhibiting the EZH2/ROCK1 signaling pathway in human nasopharyngeal carcinoma.

Celastrus orbiculatus extract (COE) has been used in folk medicine in China for the treatment of a number of diseases. In the laboratory, COE exhibits a variety of anticancer functions, including inhibition of metastasis. However, the underlying molecular anti-metastatic mechanism in nasopharyngeal carcinoma (NPC) cells remains unclear. The aim of the present study was to determine whether the anti-metastatic effect of COE was involved in inhibiting migration and invasion of human NPC cells. In vitro, cell viability and apoptosis of 5-8F cells were analyzed using an MTS assay and flow cytometry, respectively. Invasion and migration of 5-8F cells were analyzed using a Transwell assay. Protein and mRNA expression levels of 5-8F cells were analyzed by western blot analysis and the reverse transcription-quantitative polymerase chain reaction, respectively. COE significantly decreased cell viability in 5-8F cells and inhibited enhancer of zeste homolog 2 (EZH2) and Rho-associated coiled coil-containing protein kinase 1 (ROCK1) expression at the mRNA and protein levels. Furthermore, COE decreased the migration and invasion of 5-8F cells in a dose-dependent manner. The results of the present study suggested that COE prevents migration and invasion by suppressing the EZH2/ROCK1 signaling pathway in NPC cells. On the basis of the results of the present study, COE may be a novel anticancer agent for the treatment of metastasis in NPC.

Celastrus orbiculatus extract suppresses the epithelial-mesenchymal transition by mediating cytoskeleton rearrangement via inhibition of the Cofilin 1 signaling pathway in human gastric cancer.

Celastrus orbiculatus is a traditional medicinal plant used in the anti-inflammatory and analgesic treatment of various diseases. A previous study demonstrated that ethyl acetate extract of C. orbiculatus (COE) exhibited significant antitumor effects. However, studies concerning the effects and mechanism of COE in terms of suppressing the epithelial-mesenchymal transition (EMT) in human gastric adenocarcinoma cells have not been performed at present. The present study hypothesized that COE may inhibit EMT in gastric adenocarcinoma cells by regulating cell cytoskeleton rearrangement. The effect of COE on the viability of AGS cells was detected by MTT assay. An EMT model was induced by transforming growth factor-ß1. Cell cytoskeleton staining, laser scanning confocal microscopy and electronic microscopy were used to detect the changes in cell morphology and microstructure of gastric adenocarcinoma cells prior and subsequent to COE treatment. Invasion and migration assays were used to observe the effect of COE on the metastatic ability of AGS cells in vitro. The effect of COE on the expression of Cofilin 1 and EMT biomarkers, including Epithelial-cadherin, Neural-cadherin, Vimentin and matrix metalloproteinases, was examined by western blotting in AGS cells. The correlation between Cofilin 1 and EMT was investigated with immunofluorescence and cytoskeleton staining methods. The results demonstrated that COE may significantly inhibit the process of EMT in AGS cells, and that this was concentration-dependent. In addition, COE significantly downregulated the level of Cofilin 1 in a concentration-dependent manner. In conclusion, these results suggested that Cofilin 1 was directly involved in the process of EMT in AGS cells, and that it served an important function. COE may significantly inhibit EMT in AGS cells, potentially by inhibiting the activation of the Cofilin 1 signaling pathway. The present study may provide a basis for the development of novel anticancer drugs and the development of novel therapeutic strategies, targeting Cofilin 1 protein.

Anticancer efficacy of the ethyl acetate extract from the traditional Chinese medicine herb Celastrus orbiculatus against human gastric cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine (TCM) herb Celastrus orbiculatus is an important folk medicinal plant in China that has been used as an anti-inflammatory, antitumor, and analgesic in various diseases. The ethyl acetate extract of C. orbiculatus (C. orbiculatus extract, COE) was reported to show significant antitumor effects. However, no study in China or abroad has reported the effect and mechanism of COE in triggering apoptosis of gastric cancer (GC) cells. AIM OF STUDY: To further uncover the molecular mechanism underlying COE's apoptotic and anti-proliferative effects and lay a foundation for the development of novel, effective antitumor TCM agents. MATERIALS AND METHODS: The effect of COE on AGS and BGC-823 GC cell viability was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis of AGS and BGC-823 cells induced by COE was analyzed using flow cytometry and a mitochondrial membrane potential assay kit (JC-1). The proliferating GC cells were identified and examined using a 5-bromo-2'-deoxyuridine (BrdU) staining kit and flow cytometric analysis. A western blot assay was used to detect the effect of COE on apoptosis-related proteins, B-cell lymphoma-2 (Bcl-2), Bcl-extra-large (Bcl-xL), Bcl-2-like protein 12 (Bcl-L12), Bcl-2-associated X protein (Bax), and caspase as well as proliferation-related proteins, phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR)/p70s6k. Transmission electron microscopy (TEM) and an animal imaging technique were used to evaluate the microstructure of apoptotic GC cells and the effect of COE on tumor cell growth in vivo, respectively. RESULTS: The results indicate that COE significantly inhibited proliferation and induced apoptosis of GC AGS and BGC-823 cell lines both in vivo and in vitro. COE significantly decreased the cell mitochondrial membrane potential. Moreover, COE downregulated the levels of Bcl-2, Bcl-xL, and PI3K/Akt/mTOR/p70s6k while those of Bax and caspase were upregulated. More interestingly, COE altered the microstructure of the mitochondria. CONCLUSION: All these data collectively indicate that COE not only has significant antiproliferative effects but also has both in vivo and in vitro apoptotic effects. In addition, COE altered the structure and function of the mitochondria, which is another potential pathway for the antitumor activity of COE. These findings may provide a basis for the development of new anticancer TCM candidates.

Anti-metastatic Efficacy of Traditional Chinese Medicine (TCM) Ginsenoside Conjugated to a VEFGR-3 Antibody on Human Gastric Cancer in an Orthotopic Mouse Model.

Background/Aim: The aim of the present study was to investigate the efficacy of a traditional Chinese medicine (TCM), VEGFR-3 antibody-conjugated ginsenoside Rg 3 nanoemulsion (VRIN), targeting lymphangiogenesis, on the inhibition of tumor growth and metastasis in an orthotopic mouse model of human gastric cancer. Materials and Methods: An orthotopic nude-mouse model of gastric cancer was established with the red fluorescent protein (RFP)-expressing human gastric cancer cell line NUGC-4-RFP. The tumor-bearing mice were treated with vehicle (0.2 ml normal saline every other day, iv), 5-FU (20 mg/kg once a week, i.p.) and VRIN (1 mg/kg every other day, i.v.). Real-time fluorescence imaging was performed to assess tumor inhibition in each group. Metastasis was evaluated by open fluorescence imaging at autopsy. The expression of lymphangiogenesis-related factors VEGF-C, VEDF-D and VEGFR-3 in the tumors were analyzed by immunohistochemistry and real-time RCP. Results: VRIN and 5-FU significantly inhibited primary tumor growth as compared to vehicle control (p<0.05). However, significant inhibition of lymph-node metastasis was only found in the VRIN-treated group (p<0.05). The expression of VEGF-C, VEGF-D and VEGFR-3 in the tumor was suppressed by VRIN treatment (p<0.05). Expression of VEGF-D and VEGFR-3 in the 5-FU-treated group was not significantly increased (p>0.05). No obvious toxicity was found in VRIN- and 5-FU-treated groups. Conclusion: Lymphangiogenesis-targeted ginsenoside Rg 3 immune-nanoemulsion inhibited tumor growth and reduced lymphatic metastasis by suppressing expression of VEGF-C, VEGF-D and VEGFR-3 in an orthotopic mouse model of human gastric cancer. Our study demonstrates the potential of TCM as an effective targeted treatment for metastatic gastric cancer.

Celastrus orbiculatus extract triggers apoptosis and autophagy via PI3K/Akt/mTOR inhibition in human colorectal cancer cells.

Celastrus orbiculatus is used as a folk medicine in China for the treatment of numerous diseases. The ethyl acetate extract of Celastrus orbiculatus (COE) also displays a wide range of anti-cancer activities in the laboratory. However, the effectiveness of COE-induced autophagy and its mechanism of action in colorectal cancer cells have not been investigated thus far. The present study analyzed the effect of COE on HT-29 cell viability, apoptosis and autophagy using MTT assay, flow cytometry, transmission electron microscopy and western blotting. Additionally, the autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin were used to further explore the effects of COE-induced autophagy in HT-29 cells. The present study also examined whether the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) signaling pathway was involved in the regulation of COE-induced autophagy. The results revealed that COE inhibited HT-29 cell proliferation and decreased cell survival in a time- and dose-dependent manner, and that COE possessed the ability to induce both apoptosis and autophagy in HT-29 cells. Furthermore, autophagy and apoptosis induced by COE synergized to inhibit colorectal cancer growth. In addition, COE treatment decreased the phosphorylation of Akt and its downstream effectors mTOR and p70S6K. Taken together, these results demonstrate that both autophagy and apoptosis were activated during COE treatment of HT-29 cells, and that COE-induced autophagy decreases the viability of HT-29 cells via a mechanism that may depend on the PI3K/Akt/mTOR/p70S6K signaling pathway. Furthermore, compounds that induce autophagy administered in combination with COE may be an attractive strategy for enhancing the anti-tumor potency of COE in colorectal cancer.

Celastrus orbiculatus extract inhibits the migration and invasion of human glioblastoma cells in vitro.

BACKGROUND: Gliomas are highly aggressive tumors of the nervous system, and current treatments fail to improve patient survival. To identify substances that can be used as treatments for gliomas, we examined the effect of Celastrus orbiculatus extract (COE) on the invasion and migration of human glioblastoma U87 and U251 cells in vitro. METHODS: The effects of COE on cell viability and adhesion were tested using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and cell adhesion assay, respectively. The effects of COE on cell migration and invasion were assessed by a wound-healing assay and transwell migration and invasion assays. The effects of COE on the expression of epithelial-mesenchymal transition (EMT)-related proteins and matrix metalloproteinases (MMPs) were evaluated using western blot and gelatin zymography, respectively. Finally, the effect of COE on actin assembly was observed using phalloidin-tetramethylrhodamine isothiocyanate labeling and confocal laser scanning microscopy. RESULTS: We found that COE inhibited the adhesion, migration, and invasion of U87 and U251 cells in a dose-dependent manner. COE reduced N-cadherin and vimentin expression, increased E-cadherin expression, and reduced MMP-2 and MMP-9 expression in U87 and U251 cells. Furthermore, COE inhibited actin assembly in U87 and U251 cells. CONCLUSIONS: COE attenuates EMT, MMP expression, and actin assembly in human glioblastoma cells, thereby inhibiting their adhesion, migration, and invasion in vitro.

Antimetastatic effects of Celastrus orbiculatus on human gastric adenocarcinoma by inhibiting epithelial-mesenchymal transition and NF-κB/snail signaling pathway.

AIM OF THE STUDY: Celastrus orbiculatus has been used as a folk medicine in China for the treatment of many diseases. In the laboratory, the ethyl acetate extract of Celastrus orbiculatus (COE) displays a wide range of anticancer functions. However, the inhibition of the metastasis mechanism of COE in gastric cancer cells has not been investigated so far. The present study was undertaken to determine if the antimetastatic effects of COE were involved in inhibition of the epithelial-mesenchymal transition (EMT) of human gastric adenocarcinoma SGC-7901 cells. METHODS: The adhesion, invasion, and migration of SGC-7901 cells were determined by COE treatment in vitro, using Matrigel-coated plate, transwell membrane chamber, and wound healing models, respectively. In vivo, the growth-inhibiting and antimetastatic effects of COE on the nude mice model of gastric cancer were tested and the mechanisms were explored. The expression of EMT markers and nuclear factor κB (NF-κB)/Snail signaling pathway were evaluated by using western blotting and immunohistochemistry. RESULTS: Treatment with COE dose-dependently inhibited the proliferation, adhesion, invasion, and migration of SGC-7901 cells in vitro, which was realized by enhancing the expression of E-cadherin and reducing N-cadherin and vimentin expression. Moreover, COE suppressed the activation of NF-κB/Snail signaling pathway induced by tumor necrosis factor-α. In addition, COE effectively suppressed tumor growth and metastasis in the nude mice model due to reduced expression of N-cadherin, vimentin, NF-κB p65, and Snail and increased expression of E-cadherin in the tumor tissues. CONCLUSION: Our findings provided new evidence that COE is an effective inhibitor of metastatic potential of SGC-7901 cells through suppression of EMT and NF-κB/Snail signal pathway. Based on these findings, COE may be considered a novel anticancer agent for the treatment of metastasis in gastric cancer.

Research on the efficacy of Celastrus Orbiculatus in suppressing TGF-ß1-induced epithelial-mesenchymal transition by inhibiting HSP27 and TNF-α-induced NF-κ B/Snail signaling pathway in human gastric adenocarcinoma.

BACKGROUND: Celastrus orbiculatus has been used as a folk medicine in China for the treatment of many diseases. In the laboratory, the ethyl acetate extract of Celastrus orbiculatus (COE) displays a wide range of anticancer functions. However, the inhibition of the metastasis mechanism of COE in gastric cancer cells has not been investigated so far. METHODS: The present study was undertaken to determine if the anti-metastasis effect of COE was involved in inhibiting of epithelial-mesenchymal transition (EMT) of human gastric adenocarcinoma SGC-7901 cells. In vitro, a well-established experimental EMT model involving transforming growth factor ß1 (TGF-ß1) was applied. Viability, invasion and migration, protein and mRNA expression of tumor cells were analyzed by MTT assay, transwell assay, western blot and real-time PCR, respectively. The molecular targets of COE in SGC-7901 cells were investigated by two-dimensional gel electrophoresis (2-DE) and MALDI-TOF-TOF mass spectrometer. Overexpression of heat shock protein 27 (HSP27) was performed by transfected with the recombinant retroviral expression plasmid. In vivo, the anti-metastasis mechanisms of COE in the peritoneal gastric cancer xenograft model was explored and the effect was tested. RESULTS: The non-cytostatic concentrations of COE effectively inhibited TGF-ß1 induced EMT process in SGC-7901 cells, which is characterized by prevented morphological changes, increased E-cadherin expression and decreased Vimentin, N-cadherin expression. Moreover, COE inhibited invasion and migration induced by TGF-ß1. Using a comparative proteomics approach, four proteins were identified as differently expressed, with HSP27 protein being one of the most significantly down-regulated proteins induced by COE. Moreover, the activation of nuclear factor κB (NF-κB)/Snail signaling pathway induced by tumor necrosis factor-α (TNF-α) was also attenuated under the pretreatment of COE. Interestingly, overexpression of HSP27 significantly decreases the inhibitory effect of COE on EMT and the NF-κB/Snail pathway. Furthermore, COE significantly reduced the number of peritoneal metastatic nodules in the peritoneal gastric cancer xenograft model. CONCLUSIONS: Taken together, these results suggest that COE inhibits the EMT by suppressing the expression of HSP27, correlating with inhibition of NF-κB/Snail signal pathways in SGC-7901 cells. Based on these results, COE may be considered a novel anti-cancer agent for the treatment of metastasis in gastric cancer.