Inhibition Of Hepatocellular Carcinoma Angiogenesis And Enhancement On Function Of Dendritic Cells By Extracts From Celastrus Orbiculatus

Tumor angiogenesis, the development of new blood vessels from the existing vasculature, is considered to be a key step in tumor growth, invasion, and metastasis, and it is required for the proper nourishment and removal of metabolic wastes from tumor sites. Cell proliferation and migration initiate angiogenesis in response to chemotactic agents. One such agent is the vascular endothelial growth factor (VEGF), which is expressed and generated by most cancer cell types and is a potent proangiogenic factor that functions in tumor vascular development.Recently VEGF was identified as one of the factors responsible for defective dendritic cells (DCs) maturation. DCs are the most potent and professional antigen presenting cells that determine either T helper lymphocytes typel (Thl) or Th2polarization of naive T cells, and they have been a promising tool for cancer immunotherapy. The immature state of DCs is known to be appropriate for antigen processing, and in turn, they must be matured to fully activated DCs, which express high levels of cell surface MHC-antigen complex and costimulatory molecules, for sufficiently productive immunological response.The Celastraceae plant Celastrus orbiculatus has been used for thousands of years in China as a remedy against arthritis and other inflammatory diseases. In the laboratory, C. orbiculatus ethyl acetate extract (COE) displayed a variety of biological effects toward tumor cells including antiproliferation, antiangiogenesis, and the induction of apoptosis; these effects contribute to its potent antitumor activity. However, the mechanisms underlying the antitumor activity of COE remain largely unknown, and the possibility of utilizing COE clinically requires further investigation.In this study, we have shown for the first time that COE suppresses VEGF expression in mouse hepatoma (Hepal-6) cells at both the mRNA and protein levels. Simultaneously, it has been observed that COE inhibited the formation of the capillary-like network in human umbilical vein endothelial cells (HUVECs).Further more, we investigated whether COE would promote maturation and function of DCs. To answer this question, we studied the effects of COE on DCs development, maturation, and cytokines production. It was found that COE not only had dramatic effects on DCs development and maturation, but also significantly affected DCs function of immunoregulation.In in vivo studies, COE not only attenuates the growth of malignant hepatocellular carcinomas but also stimulate maturation of DCs, associated with strongly enhanced CD8+CTL responses. Meanwhile, COE strongly suppress VEGF protein expression in tumor sections.In conclusion, the C. orbiculatus extract targets both tumor cells and proliferating endothelial cells and inhibits tumor angiogenesis by modulating the VEGF signaling pathway. In addition, COE has potent anticancer effects and enhances DCs function by inducing DCs migration and their capacity for stimulation of lymphocytes. This work may lead to new therapeutic options and improved understanding of the interaction of phytochemicals in hepatic cancer cells.The findings will be described in three parts as follows.Part I:Extracts from Celastrus Orbiculatus Inhibit Hepatocellular Carcinoma Angiogenesis in vitroObjective:To discuss the antitumor mechanism preliminarily by observing effects of Celastrus orbiculatus ethyl acetate extracts (COE) on vascular endothelial growth factor (VEGF) mRNA and protein expression in hepatoma (Hepal-6) cells of mice.Methods:The effects of COE at various concentrations (10,20,40,80, or160μg/mL) on the variation of cell number were determined by the3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-diphenytetrazoliumromide (MTT) assay. Apoptotic cells were detected by the Annexin V assay. VEGF production in the conditioned medium was analyzed with the mouse VEGF-A ELISA kit. The mRNA and protein expressions of VEGF were detected by reverse transcription-PCR and Western Blotting, respectively. To investigate the effects of COE on angiogenesis, we examined the impact of COE on HUVECs. HUVECs adhered to the extracellular matrix (Matrigel) within18h, displayed high motility and cell-cell communication, and formed an anastomosing network of capillary-like tubes.Results:We found that10to160μg/mL COE inhibited the proliferation and growth of Hepal-6cells. The rates of inhibition were7.51±0.73%,23.39±1.02%,36.64±2.13%,41.16±3.15%, and52.47±3.23%for10,20,40,80, and160μg/mL of COE, respectively. Cells in early apoptosis are Alexa Fluor488-Annexin V positive and PI negative, and cells in late apoptosis are both Alexa Fluor488-Annexin V and PI positive. When Hepal-6cells were treated with COE at doses of10to160μg/mL for12h, the numbers of early apoptotic cells (Annexin V+PI-) were12.0±2.3%,13.9±1.5%,15.3±2.8%,17.2±3.4%, and21.7±5.6%, respectively. COE significantly inhibited VEGF expression at both mRNA and protein levels in a dose-dependent manner. COE (10,20,40,80or160μg/mL) significantly inhibited tube formation on Matrigel in a dose-dependent manner and in contrast to the negative control.Conclusion:COE targets both tumor cells and proliferating endothelial cells and inhibits tumor angiogenesis by modulating the VEGF signaling pathway, therefore suggest that VEGF could be chosen as an therapeutic target for COE in the context of cancer chemoprevention and anticancer therapy.Part Ⅱ:Extracts from Celastrus Orbiculatus Enhance Maturation and Function of Dendritic Cells in vitroObjective:To examine the immunoregulation effects of Celastrus orbiculatus ethyl acetate extracts (COE), a traditional Chinese medicine, on maturation and function of dendritic cells in vitro.Methods:After treated with COE in different nontoxic concentration (10,20,40,80or160μg/mL) for5days, the surface immunological molecules and cytokine secretion of mice bone marrow-derived DCs in response to COE were analyzed by flow cytometric analysis (FCM) and enzyme linked immunosorbent assay (ELISA), respectively. To examine whether the addition of COE enhanced the function of DCs, we evaluated the allostimulatory capacity of the DCs.Results:COE stimulated IL-2and IFN-y 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 receptor7(CCR7) expression. COE increased DCs produce IFN-y and IL-2in a dose-dependent manner when the concentration of COE less than40μg/mL, decreased DCs produce IL-10and IL-4also in a dose-dependent manner. After addition different nontoxic concentration (10,20,40,80and160μg/mL) of COE, IL-10protein production of DCs were301.0±15.9pg/mL,233.2±24.7pg/mL (P<0.05),185.7±11.3pg/mL (P<0.05),157.6±21.8pg/mL (P<0.01), and145.5±12.2pg/mL (P<0.01) compared with349.7±17.3pg/mL of the solvent vehicle control group. After addition different nontoxic concentration (10,20,40,80or160μg/mL) of COE, IL-4protein production of DCs were445.6±17.8pg/mL,424.5±15.6pg/mL (P<0.05),324.6±25.2pg/mL (P<0.05),179.8±17.0pg/mL (P<0.01), and53.2±16.3pg/mL (P <0.01) compared with558.9±11.7pg/mL of the solvent vehicle control group.COE strongly increased allogeneic T-cell proliferation of DCs in a dose-dependent manner. After addition different nontoxic concentration (10,20,40,80or160μg/mL) of COE, proliferation of T lymphocytes were45.0±1.3%(P>0.05),58.3±6.2%(P<0.01),63.6±6.1%(P<0.01),78.1±7.3%(P<0.01), and105.0±5.7%(P<0.01) compared with control group (42.4±5.1%).Therefore, COE treatment not only enhanced the phenotypic maturation of DCs, but also reinforced their function.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. Part III:Inhibition of Hepatocellular Carcinoma Angiogenesis and Enhancement on Function of Dendritic Cells by Extracts from Celastrus Orbiculatus in VivoObjective:The goal of this study was to characterize the antiangiogenic and immunoregulatory activity of Celastrus orbiculatus ethyl acetate extracts (COE). Methods:Hepal-6cells (1×106) suspended in0.1mL of serum-free RPMI1640were inoculated s.c. in the left flank of each mouse. Mice were randomly assigned to six groups (ten mice per group) according to body weight as follows:untreated control, solvent vehicle control (DMSO), cisplatin-treated group, and different dosages COE-treated groups. On day14after inoculation, the animals received200μL of a vehicle [1%DMSO and99%PBS] or COE at different dosages (10,20or40mg/kg/d) by gavage. In the cisplatin-treated group, mice were injected intraperitoneally with1mg/kg/d of cisplatin. Mice received28doses and24h after the last dose, they were sacrificed and the tumors were removed and weighed. The histologic TUNEL staining and VEGF expression of the Hepal-6tumor tissues were observed. The relative proportions of mature DCs and CD8+T cells were measured in mononuclear cells that had been isolated from spleen by FACS.Results:Endpoint tumor mass showed dose-dependent growth suppression by COE, wherein the mean tumor masses of10,20, and40mg/kg/d groups were0.83±0.10g,0.69±0.10g (P<0.05), and0.51±0.15g(P<0.01) compared with1.08±0.10g of the solvent vehicle control group. The mean apoptotic cells of the10,20and40mg/kg/d COE and cisplatin groups were8.52±0.43,26.46±3.76,50.41±6.79, and54.70±5.31, respectively. The mean apoptotic cells of the solvent vehicle control (DMSO) group was6.36±0.69. VEGF protein production in the serum of the10,20and40mg/kg/d COE and cisplatin groups were245.12±22.99pg/mL (P<0.05),72.08±16.97pg/mL (P<0.05),45.24±21.36pg/mL (P<0.01), and70.22±29.59pg/mL (P<0.01) compared with332.02±57.64pg/mL of the solvent vehicle control group. COE suppressed VEGF mRNA expression in a dose-dependent manner in Hepal-6cells. Western blotting assays indicated that incubation with COE at a concentration of10μg/mL, COE began to inhibit VEGF protein expression in a dose-dependent manner. The mean MVD of the10,20and40mg/kg/d COE cisplatin groups were14.33±1.69,11.18±1.35,6.76±1.48and6.85±1.14, respectively. The mean MVD of the solvent vehicle control (DMSO) group was16.43±2.53. COE can not only suppress growth of malignant hepatocellular carcinomas but also stimulate maturation of DCs, associated with strongly enhanced CD8+CTL responses.Conclusion:COE has satisfactory anti-tumor activity, and the effects are associated with induction tumor cell apoptosis and inhibition of angiogenesis. This study provides evidence that COE can inhibit VEGF expression in Hepal-6cells that lead to inhibition of tumor angiogenesis, and indicate that the stimulation of maturation and function of DCs by COE contributes to its immunoregulatory effects. These results support the role of COE as a therapeutic strategy to target both the hepatocellular carcinoma angiogenesis inhibitors and immunoregulatory effects.