Norcantharidin: A potential antiangiogenic agent for gallbladder cancers in vitro and in vivo

Abstract

Abstract. Our objective was to explore the antiangiogenic activity of norcantharidin (NCTD) as an angiogenic inhibitor for gallbladder cancers. In vitro and in vivo experiments to determine the effects of NCTD on HUVECs, chicken CAM capillaries and gallbladder cancer xenograft angiogenesis in nude mice were respectively done. The MTT method was used to assay the cytotoxicity of NCTD on HUVECs. Immunofluorescence was used to evaluate HUVEC apoptosis. The scraping line method, matrigel invasion assay and tube formation assay were used to detect the migration, invasion and tube formation of HUVECs. A digital camera was used to observe chicken CAM capillaries. Experiments with NCTD in a xenograft model were used to observe the effect of NCTD on xenograft growth and survival of mice with xenografts. CD 34 immunohistochemistry, flow cytometry and micro-MRA were used, respectively, to determine MVD, cell apoptosis and hemodynamic analysis of the xenografts. Immunohistochemistry and RT-PCR were used, respectively, to detect the expression of VEGF, Ang-2, TSP, TIMP-2 proteins/mRNAs of the xenografts. The xenograft MVD associated with tumor volume, the PCNA/apoptosis ratio and related-protein expression was evaluated simultaneously. We found that NCTD effectively inhibited the proliferation, migration, invasion and capillary-like tube formation of HUVECs in vitro; it reduced angiogenesis and directly destroyed the formed CAM capillaries in vivo. In the experiments in mice, NCTD not only inhibited significantly xenograft proliferation and growth, prolonged survival time of mice with xenografts, decreased the xenograft MVD and vascular perfusion, but also, similarly to ES, decreased significantly the expression of VEGF or Ang-2 protein/mRNA, increased the expression of TSP or TIMP-2 protein/mRNA. Moreover, the xenograft MVD was positively related with tumor volume, PCNA/apoptosis ratio, and VEGF or Ang-2 expression, respectively (all P<0.05), but negatively correlated with TSP or TIMP-2 expression (both P<0.05). These data showed that NCTD could serve as a potential antiangiogenic agent for gallbladder cancers.

Inhibition of Tumor Vasculogenic Mimicry and Prolongation of Host Survival in Highly Aggressive Gallbladder Cancers by Norcantharidin via Blocking the Ephrin Type a Receptor 2/Focal Adhesion Kinase/Paxillin Signaling Pathway

“…Lately, there is a growing interest that some anticancer researches for NCTD have begun to focus on antiangiogenesis and anti-VM. We firstly reported the in vitro and in vivo antiangiogenic activity of NCTD as a potential angiogenic inhibitor for gallbladder cancers [41], [61]. Chen et al also reported the in vitro and in vivo antiangiogenic effect of NCTD on CT26 cells by down-regulation of MAPK expression of phosphorylated (p)-JNK and p-ERK, and considered NCTD is a synthetic, small-molecule compound possessing antiangiogenic activity [62].…”

“…Tumor inhibitory rate  =  (volume in control group - volume in experimental group)/volume in control group×100%. The dose of NCTN in the in vivo experiment is 28 mg/kg, i.e., a safe dose of NCTD 1/5 LD 50 [41]. Survival time of each group was also evaluated.…”

“…It has been reported that NCTD inhibits the growth of a variety of human tumor cells, including HepG2, K562, HL60, A375-S2, HT29 and GBC-SD cells, and is clinically used to treat human cancers, e.g., hepatic, gastric, colorectal and ovarian cancers [33]–[38]. We have reported that NCTD has multiple antitumor activities against gallbladder cancers in vitro and in vivo [39]–[41]. However the exact mechanism responsible for the NCTD antitumor is not thoroughly elucidated.…”

Repression of YAP by NCTD disrupts NSCLC progression

“…NCTD has been found to induce both in vitro and in vivo inhibitions in growth of a variety of human tumor cells via modulating functions of cell cycle kinases leading to tumor cell cycle arrests. NCTD also can activate the mitochondrial pathway and ROS accumulation to induce tumor cell apoptosis [38–39] and significantly reduce the tumor angiogenesis [40–42] However, the underlying mechanisms by which NCTD inhibits lung cancer progression and metastasis are still largely undefined.…”

Intraarterial administration of norcantharidin attenuates ischemic stroke damage in rodents when given at the time of reperfusion: novel uses of endovascular capabilities

“…3,41 Studies have demonstrated potent antimetastatic and antiangiogenic properties of NCTD in addition to its potential to generate tolerogenic skin grafts. 15,17,20,41,42 It is eliminated in 6 hours from all tissues of rats and mice except the liver and kidneys. 8 The dose used in this work has been shown to block both MMP-9 as well as hypervascularization and the associated MAPK/vascular endothelial growth factor (VEGF) pathways in vitro and in vivo.…”