Abstract 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 β1 (TGF-β1) 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-β1 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-β1. 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.

Migration and invasion of fibroblast-like synoviocytes (FLSs) are critical in the pathogenesis of rheumatoid arthritis (RA). Hypoxic conditions are present in RA joints, and hypoxia has been extensively studied in angiogenesis and inflammation. However, its effect on the migration and invasion of RA-FLSs remains unknown. In this study, we observed that RA-FLSs exposed to hypoxic conditions experienced epithelial–mesenchymal transition (EMT), with increased cell migration and invasion. We demonstrated that hypoxia-induced EMT was accompanied by increased hypoxia-inducible factor (HIF)-1α expression and activation of Akt. After knockdown or inhibition of HIF-1α in hypoxia by small interfering RNA or genistein (Gen) treatment, the EMT transformation and invasion ability of FLSs were regained. HIF-1α could be blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1α activation was regulated by the PI3K/Akt pathway. Administration of LY294002 (20 mg/kg, intra-peritoneally) twice weekly and Gen (25 mg/kg, by gavage) daily for 3 weeks from day 20 after primary immunization in a collagen-induced arthritis rat model, markedly alleviated the clinical signs, radiology progression, synovial hyperplasia, and inflammatory cells infiltration of joints. Thus, results of this study suggest that activation of the PI3K/Akt/HIF-1α pathway plays a pivotal role in mediating hypoxia-induced EMT transformation and invasion of RA-FLSs under hypoxia.

Recently, Celastrus orbiculatus ethyl acetate extracts (COE) have been investigated for their anticancer effects on digestive tract tumors. However, the therapeutic effects of COE on esophageal squamous carcinoma cells (ESCC) have not been investigated. In the present study, the effects of COE on the cell cycle and apoptosis of ESCCs were assessed in vitro, and it was revealed that COE treatment triggered G0/G1 cell cycle arrest, and induced DNA damage and apoptosis in a dose-dependent manner in ESCC. Activation of the phosphatidylinositol 3-kinase/protein kinase-B/mechanistic target of rapamycin (mTOR) pathway was also suppressed by COE. Additionally, the combined treatment with COE and rapamycin (an mTOR inhibitor) acted synergistically in ECA-109 cells compared with the treatment with COE or rapamycin alone. These findings extend the understanding of the action of COE and suggest that COE has potential as a treatment option for ESCC as a single treatment or in combination.

目的 :探讨南蛇藤总萜(COE)是否通过诱导自噬来抑制结直肠癌细胞增殖.方法 :用不同浓度的COE处理体外培养的大肠癌HT-29细胞,噻唑蓝(MTT)法检查细胞存活率,透射电镜(TEM)观察细胞形态,免疫印迹法(Western blot)检测COE对细胞自噬相关蛋白表达的影响,并采用自噬抑制剂3-甲基腺嘌呤(3-MA)联合COE干预HT-29细胞,用吖啶橙染色细胞、MTT法检测3-MA对COE抑制HT-29细胞增殖的影响.结果 :COE抑制HT-29细胞的增殖,作用24h的IC50为156μg/mL;随着COE浓度的升高,TEM示细胞中自噬体明显增多,和COE治疗组比较,COE联合3-MA组的细胞生存率升高,而自噬泡(AVO)的数量显著下降,微管相关蛋白-Ⅱ(LC3-Ⅱ)表达也降低.结论 :COE可能通过上调Bcl-2同源结构域蛋白抗体-1(Beclin-1)和LC3-Ⅱ的表达来增加HT-29细胞的自噬活性,从而抑制HT-29细胞的增殖作用.

Ethnopharmacological relevance: Vasculogenic mimicry (VM) has been suggested to be present in various malignant tumors and associated with tumor nutrition supply and metastasis, leading to poor prognosis of patients. Notch1 has been demonstrated to contribute to VM formation in hepathocellular carcinoma (HCC). Celastrus orbiculatus extract (COE), a mixture of 11 terpenoids isolated from the Chinese Herb Celastrus orbiculatus Vine, has been suggested to be effective in cancer treatment. Aim of the study: In the current study, experiments were carried out to examine the effect of COE on VM formation and HCC tumor growth both in vitro and in vivo. Materials and methods: CCK-8 assay and Nikon live-work station were used to observe the viability of malignant cells treated with COE. Cell invasion was examined using Transwell. Matrigel was used to establish a 3-D culture condition for VM formation. Changes of mRNA and protein expression were examined by RT-PCR and Western Blot respectively. Tumor growth in vivo was monitored using in vivo fluorescence imaging device. PAS-CD34 dual staining and electron microscopy were used to observe VM formation. Immunohistochemical staining (IHC) was used to examine Notch1 and Heel expression in tumor tissues. Results: Results showed that COE can inhibit HCC cells proliferation and invasion in a concentration dependent manner. VM formation induced by TGF-beta 1 was blocked by COE. In mouse xenograft model, COE inhibited tumor growth and VM formation. Both in vitro and in vivo studies showed that COE can downregulate expression of Notch1 and Hes1. Conclusion: The current results indicate that COE can inhibit VM formation and HCC tumor growth by downregulating Notch1 signaling. This study demonstrates that COE is superior to other anti-angiogenesis agents and can be considered as a promising candidate in HCC treatment.

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 344,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT) 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/p7Os6k 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.

AIM:To investigate the effect of mechanical stress on periostin and semaphorin-3A expression in a murine model of postmenopausal osteoporosis and in osteoblast-like MC3T3-E1 cells.MATERIALS AND METHODS:Female mice were divided into three groups and treated with a sham operation, ovariectomy (OVX) or OVX plus treadmill training (OVX+Run). After 10 weeks, tibias were used for histological analysis. MC3T3-E1 cells were burdened by mechanical stress using a centrifuge or were treated with periostin, and the production of biologically-active semaphorin-3A was examined in vitro.RESULTS:In OVX+Run group tibias, the number of tartrate-resistant acid phosphatase-positive osteoclasts was lower than in the OVX group, and the expression of periostin and semaphorin-3A was higher. In MC3T3-E1 cells, centrifugal stress significantly increased periostin and semaphorin-3A mRNA expression. Treatment with periostin increased the semaphorin-3A level.CONCLUSION:We speculate that mechanical load may increase periostin production in osteoblasts, and periostin may inhibit osteoclast differentiation by its effects on semaphorin-3A. Our results support the concept of a positive correlation between exercise and inhibition of osteoclasts in post-menopausal osteoporosis.

Hypervascularity is one of the main characteristics of hepatocellular carcinoma (HCC). However, the mechanisms of angiogenesis in HCC remain controversial. In this study, we investigate the role of Notch1 in angiogenesis of HCC. We found that Notch1 expression was correlated with formation of vasculogenic mimicry (VM) and expression of biomarkers of epithelial-to-mesenchymal transition (EMT) in the tumor specimens. Two HCC cell lines, HepG2 and MHCC97-H, with low and high Notch1 expression, respectively, were used to study the mechanism of VM formation both in vitro and in vivo. It was found that MHCC97-H cells, but not HepG2 cells form VM when they grow on matrigel in vitro. HepG2 cells gained the power of forming VM when they were overexpressed with Notch1, while knockdown Notch1 expression in MHCC97-H cells led to the loss of VM forming ability of the cells. Similar results were found in in vivo study. High expression of Notch1 in HepG2 promoted xenograft growth in nude mice, with abundant VM formation in the tumor samples. Moreover, we observed Notch1 was associated with the EMT and malignant behavior of hepatocellular carcinoma by analyzing clinical specimens, models for in vitro and in vivo experiments. HepG2 presented EMT phenomenon when induced by TGF-β1, accompanied by Notch1 activation while MHCC97-H with knockdown of Notch1 lost the responsiveness to TGF-β1 induction. Our results suggest that Notch1 promotes HCC progression through activating EMT pathway and forming VM. Our results will guide targeting Notch1 in new drug development.

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.

Constitutive photomorphogenesis 9 signalosome (CSN) consists of a total of eight subunits (CSN1-CSN8) in mammalian cells. CSN6 may promote carcinogenesis by positively regulating v-myc avian myelocytomatosis viral oncogene homolog (Myc) and MDM2 proto-oncogene stability, and is regarded as a potential target for cancer therapy. Quercetin has a substantial anticancer effect on various human cancer cells. The present study investigated the effects of quercetin on HT-29 human colorectal cancer cell viability, apoptosis and cell cycle arrest using an MTT assay, flow cytometry, transmission electron microscopy and western blotting. It was determined that quercetin inhibited HT-29 cell viability in a dose-dependent manner. Cell shrinkage, chromatin condensation and nuclear collapse were observed in the 50, 100 and 200 mu M quercetin groups. The exposure of HT-29 cells to quercetin led to significant cell cycle arrest in the S-phase. Western blot analysis revealed that quercetin reduced the protein expression levels of phosphorylated-Akt and increased CSN6 protein degradation; therefore, affecting the expression levels of Myc, p53, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein. The overexpression of CSN6 reduced the effect of quercetin treatment on HT-29 cells, suggesting that quercetin-induced apoptosis may involve the Akt-CSN6-Myc signaling axis in HT-29 cells.

Portal vein invasion (PVI) is common in hepatocellular carcinoma (HCC) and largely contributes to tumor recurrence after radical tumor resection or liver transplantation. Vasculogenic mimicry (VM) was an independent vascular system lined with tumor cells and associated with poor prognosis of HCC. The present study was conducted to evaluate the relationship between VM and portal vein invasion. A total of 44 HCC cases receiving anatomic liver resection were included in the study and were divided into groups with and without PVI. The prevalence of VM in each group was examined by CD34-PAS dual staining. The regulatory molecules of VM formation such as Notch1, Vimentin and matrix metalloproteinases (MMPs) were investigated by immunohistochemical staining. Analysis was performed to explore the association of PVI, VM and the VM regulatory molecules. PVI was found in 40.91% (18/44) cases and VM was found in 38.64% (17/44) cases in total samples. The incidence of VM was 72.22% (13/18) in PVI group while it was 15.38% (4/26) in non-PVI group (P<0.001), VM formation was positively correlated with PVI (r=0.574, P<0.001). The VM forming regulatory molecules such as Notch1, Vimentin, MMP-2 and MMP-9 were found to be correlated with PVI in HCC patients. Taken together, our results suggested that VM formation, alone with its regulatory molecules, is the promoting factor of PVI in hepatocellular carcinoma.

Objective To evaluate leptin and bone mineral density (BMD) in a 6-month anti-TNF-α treatment ankylosing spondylitis (AS) patients, and to determine their relationship with clinical features and disease activity. Methods Thirty cases of male patients with AS of Yangzhou medical college from January 2013 to December 2015 were included in the study as experiment group. Thirty age-and weight-matched men served as controls. Clinical and biochemical parameters were assessed and serum levels of leptin were measured with radioimmunoassay, and BMD of lumbar spine and proximal femur were measured prior to and after the 6-month treatment with infliximab. Results At baseline, AS patients exhibited significantly higher leptin than controls (P<0.05), there was a significant negative correlation of bone density T scores with leptin and disease activity parameters (P<0.05). That relationship was reflected more reliably at proximal femur sites than at the lumbar spine. There were also significant differences in leptin levels, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), Bath ankylosing spondylitis disease activity index (BASDAI) and spinal pain between three groups of patients according to WHO classification of osteoporosis (normal, osteopenic and osteoporotic) (P<0.05). After the 6-month treatment with infliximab, serum levels of leptin, ESR, CRP, BASDAI, Bath ankylosing spondylitis functional index (BASFI) and spinal pain decreased while the bone density T scores increased significantly (P<0.01). Conclusions Our results indicate that an association of high levels of leptin and disease activity with low BMD in patients with AS, anti-TNF-α treatment could decrease serum levels of leptin and improve the osteoporosis of AS, suggesting that the change of leptin level may play an important role in the pathogenesis of osteoporosis in ankylosing spondylitis. Key words: Spondylitis, ankylosing; Leptin; Osteoporosis

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.

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.

Dear Editor,Rabies is a generally fatal disease caused by the rabies virus(RABV),and is transmitted mainly by Carnivora and Chiroptera(Fooks A R,et al.,2014;Tao X,et al.,2013).In China,stray dogs and some wild animals(e.g.,Chinese ferret badgers,foxes,and raccoon dogs)are the principal reservoirs for RABV(Hu R L,et al.,2009).Historically,rabies in wild foxes and raccoon dogs(Nyctereutes procyonoides)was recorded in the early

BACKGROUND:Invasion of fibroblast-like synoviocytes (FLSs) is critical in the pathogenesis of rheumatoid arthritis (RA). The metalloproteinases (MMPs) and activator of nuclear factor-kappa B (NF-κB) pathway play a critical role in RA-FLS invasion induced by interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). The present study aimed to explore the anti-invasive activity and mechanism of Celastrus orbiculatus extract (COE) on IL-1β and TNF-α combination-stimulated human RA-FLSs.METHODS:We investigated the effect of COE on IL-1β and TNF-α combination-induced FLS invasion as well as MMP expression and explored upstream signal transduction.RESULTS:COE suppressed IL-1β and TNF-α combination-stimulated FLSs invasion by inhibiting MMP-9 expression and activity. Furthermore, our results revealed that COE inhibited the transcriptional activity of MMP-9 by suppression of the binding activity of NF-κB in the MMP-9 promoter, and inhibited IκBα phosphorylation and nuclear translocation of NF-κB.CONCLUSIONS:COE inhibits IL-1β and TNF-α combination-induced FLSs invasion by suppressing NF-κB-mediated MMP-9 expression.