Apoptosis of Hela cell induced by Celastrus orbiculatus Thunb extract and primary mechanisms

Objective： The apoptosis of Hela cells induced by ethyl acetate extract and n-butanol extract of Celastrus orbiculatus Thunb was studied in order to assess its antitumor effect. Methods： Hela cells were cultured in vitro and treated by a series of concentrations of ethyl acetate extract and n-butanol extract of Celastrus orbiculatus Thunb. Cell proliferation was detected based on MTT assay. Quantity of apoptosis were observed and analyzed by flow cytometry with Annexin V and propidium iodide double staining. P53 gene expression was detected by flow cytometry. Results： The proliferation of Hela cells was obviously inhibited by 15, 30, 60 and 120 IJg/mL extract of Celastrus orbiculatus Thunb and apoptosis of Hela was induced by dosed dependent manner. P53 gene showed increasing tendency when treated by 60-480 IJg/mL extract of Celas- trus orbiculatus Thunb. Conclusion： The ethyl acetate extract and n-butanol extract of Celastrus orbiculatus Thunb could induce apoptosis of Hela gastric cancer cells by dose dependent manner, which maybe one of the important mechanisms of Celastrus orbiculatus Thunb＇s anticancer effects. P53 protein expression in Hela was up-regulated by Celastrus orbiculatus Thunb, which maybe one of the molecular mechanisms involved in the anticancer and proapoptotic effect of Celastrus or- biculatus Thunb.

Study on Extraction Techniques of Celastrol from Celastrus orbiculatus Thunb.

[Objectives]This study was conducted to investigate the extraction technology of celastrol from Celastrus orbiculatus Thunb.[Methods]Solvent ultrasonic extraction was selected,and with the content of celastrol as the evaluation index,the effects of different solvents,extraction time,temperatures and material-to-liquid ratios on the extraction rate of celastrol were investigated by single factor and orthogonal experiments.[Results]The optimal extraction conditions were as follows:a solvent ratio of petroleum ether to ethyl acetate at 1∶1,a ratio of solvent to material at 10∶1(v/w),extraction time of 30 min,and an extraction temperature at 30℃.[Conclusions]This method has high extraction rate,and is simple and feasible.

Celastrus orbiculatus Extract Inhibits Immune Inflammatory Thrombotic State of B-Lymphoma

Objective:To investigate the inhibitory effect of Celastrus orbiculatus extracts(COE)on the proliferation of lymphoma cells and the immune regulation ability on inflammation and thrombophilia in vivo.Methods:The 38B9 lymphoma cells were treated with COE(160μg/mL)and CTX(25μmol/L).The apoptosis rate and cell cycle of each group were detected by flow cytometry.The secretion of inflammatory factors,including interleukin(IL)-6,IL-10,and tumor necrosis factorα(TNF-α),in cell supernatant was detected by enzyme-linked immunosorbent assay(ELISA).In vivo,BALB/c mice were subcutaneously injected with 38B9 lymphoma cells to establish lymphoma model.COE(3 mg·kg^(-1)·d^(-1))and CTX(40 mg·kg^(-1)·d^(-1))were administered to the model mice,respectively.The expression of plasma inflammatory factors(IL-6,IL-10 and TNF-α)and thrombus indexes,including D-dimer(D-D),von Willebrand factor(vWF)and tissue factor(TF),were detected by ELISA before tumor bearing(1 d),after tumor formation(14 d)and after intervention(21 d).PicoGreen dsDNA was used to detect the level of serum neutrophil extracellular traps(NETs).Flow cytometry was used to detect the expression of platelet activation marker calcium-dependent lectin-like receptor 2(CLEC-2).The tumor growth and survival of mice were recorded.Results:The 38B9 lymphoma cells were apoptotic after the intervention of COE and CTX.The ratio of Gz-M phase cells decreased in COE intervented cells compared with the control cells(P<0.05),and S phase cells decreased in CTX intervented cells(P<0.05).Also,the secretion level of IL-6 was significantly reduced after COE or CTX intervention(P<0.05),and IL-10 was significantly increased(P<0.05).Furthermore,the tumor mass was reduced,and the median survival time was longer in COE and CTX intervented tumor-bearing mice than in non-intervented mice.The significantly lower levels of TNF-α,IL-6,NETs,TF,DD and CLEC-2,as well as higher IL-10 were observed in COE and CTX treatment mice in comparision with the control mice(P<0.05).Conclusion:COE has a mild and stable anti-tumor effect,which can reduce the secretion of inflammatory factors by lymphoma cells and regulate thrombophilic state caused by tumor inflammatory microenvironment.

Celastrus Orbiculatus Extract Inhibits Tumor Angiogenesis by Targeting Vascular Endothelial Growth Factor Signaling Pathway and Shows Potent Antitumor Activity in Hepatocarcinomas in Vitro and in Vivo

Objective： Ce/astrus orbicu/atus Thunb. has been used for thousands of years in China as a remedy against cancer and inflammatory diseases. This study aims to investigate whether C. orbiculatus extract （COE） could inhibit angiogenesis, which is the pivotal step in tumor growth, invasiveness, and metastasis. Methods： In this study, the extract from the stem of C. orbiculatus was used. Mouse hepatic carcinoma cells （Hepat-6） were treated with COE in different nontoxic concentrations （10, 20, 40, 80, and 160 μg/mL）. The mRNA and protein expression levels of vascular endothelial growth factor （VEGF） were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively; the active fractions were further tested on C57BL/6 mice and human umbilical vein endothelial cells （HUVEC） for any antiangiogenic effects. Results： COE significantly inhibited proliferation and induced apoptosis in Hepal-6 cells and inhibited VEGF expression at both mRNA and protein levels. Furthermore, this agent inhibited the formation of the capillary-like structure in primary cultured HUVEC in a dose-dependent manner. In vivo, COE significantly reduced the volume and weight of solid tumors with low adverse effects and decreased tumor angiogenesis. Conclusions： In summary, COE could be used to treat hepatic carcinoma. The mechanisms of the antitumor activity of COE may be due to its effects against tumor angiogenesis by targeting the VEGF protein.

Inhibition of Celastrus orbiculatus Extracts on VEGF Expression in Hepatoma Cells of Mice

Objective To discuss the antitumor mechanism preliminarily by observing effects of Celastrus orbiculatus extracts(COE) on vascular endothelial growth factor(VEGF) mRNA and protein expression in hepatoma(Hepa1-6) cells of mice.Methods Hepa1-6 cells were treated with COE at different nontoxic concentration(0,10,20,40,80,and 160 μg/mL) for 16 h.The mRNA and protein expressions of VEGF were detected by reverse transcription-PCR and Western Blotting,respectively.Results COE significantly inhibited VEGF expression at both mRNA and protein levels in a dose-dependent manner.Conclusion COE can inhibit VEGF expression in Hepa1-6 cells,therefore suggest that VEGF could be chosen as an therapeutic target for COE in the context of cancer chemoprevention and anticancer therapy.

Enhancement of Extracts from Celastrus orbiculatus on Maturation and Function of Dendritic Cells in vitro and in vivo

Objective To examine the immunoregulation of Celastrus orbiculatus extracts (COE), a traditional Chinese medicine, on maturation and function of dendritic cells (DCs) in vitro and in vivo. Methods In vitro, after treated with COE in different nontoxic concentrations (0, 10, 20, 40, 80, and 160 μg/mL) for 5 d, the surface immunological molecules and cytokine secretion of mice bone marrow-derived DCs in response to COE were analyzed by flow cytometric analysis (FACS) and enzyme linked immunosorbent assay (ELISA), respectively. In vivo, mouse hepatoma cells (Hepa1-6, 1 × 106) were injected sc and were treated with different dosages of COE (10, 20 or 40 mg/kg/d). Effects on tumor growth were determined by tumor volume and histology analysis after 28 d administration of COE. The relative proportions of mature DCs and CD8+ T cells were measured in mononuclear cells that had been isolated from spleen by FACS. Results COE stimulated IL-2 and IFN-γ secretion of DCs, simultaneously enhanced the maturation of DCs by enhancing immunological molecule (CD40, CD80, CD86, H-2Kb, and I-Ab) expression in a dose-dependent manner. Furthermore, the chemotactic responses of DCs were significantly higher in COE-treated than untreated DCs, in association with higher chemokine receptor 7 expression. Furthermore, COE increased DCs produce IFN-γ and IL-2 in a dose-dependent manner when the concentration of COE less than 40 μg/mL, decreased DCs produce IL-10 and IL-4 also in a dose-dependent manner. In in vivo studies, COE can not only suppress growth of malignant hepatocellular carcinomas but also stimulate maturation of DCs, associated with strongly enhanced CD8+ CTL responses. Conclusion These data provide new insight into the mechanism of action of COE and indicate that the stimulation of maturation and function of DCs by COE contributes to its immunoregulatory effects.