Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Shanghai, PR China.
Int J Oncol. 2012 May;40(5):1501-14. doi: 10.3892/ijo.2011.1314. Epub 2011 Dec 21.
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. CD₃₄ 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.
我们的目的是探索去甲斑蝥素（NCTD）作为血管生成抑制剂对胆囊癌的抗血管生成活性。分别进行了体外和体内实验，以确定 NCTD 对 HUVECs、鸡胚绒毛尿囊膜（CAM）毛细血管和裸鼠胆囊癌异种移植血管生成的影响。MTT 法检测 NCTD 对 HUVECs 的细胞毒性。免疫荧光法评估 HUVEC 细胞凋亡。划痕线法、基质胶侵袭实验和管形成实验分别检测 HUVEC 迁移、侵袭和管形成。数字相机观察鸡胚 CAM 毛细血管。NCTD 在异种移植模型中的实验用于观察 NCTD 对异种移植生长和荷瘤小鼠生存的影响。CD₃₄免疫组化、流式细胞术和微磁共振血管造影术（micro-MRA）分别用于确定 MVD、细胞凋亡和异种瘤的血流动力学分析。免疫组化和 RT-PCR 分别用于检测异种瘤中 VEGF、Ang-2、TSP、TIMP-2 蛋白/mRNA 的表达。同时评估异种瘤 MVD 与肿瘤体积、PCNA/凋亡比值和相关蛋白表达的相关性。我们发现，NCTD 能有效抑制 HUVECs 的体外增殖、迁移、侵袭和毛细血管样管形成；它减少了血管生成，并直接破坏了体内形成的 CAM 毛细血管。在小鼠实验中，NCTD 不仅显著抑制了异种移植的增殖和生长，延长了荷瘤小鼠的生存时间，降低了异种瘤的 MVD 和血管灌注，而且与 ES 类似，显著降低了 VEGF 或 Ang-2 蛋白/mRNA 的表达，增加了 TSP 或 TIMP-2 蛋白/mRNA 的表达。此外，异种瘤 MVD 与肿瘤体积、PCNA/凋亡比值和 VEGF 或 Ang-2 表达呈正相关（均 P<0.05），与 TSP 或 TIMP-2 表达呈负相关（均 P<0.05）。这些数据表明，NCTD 可作为胆囊癌潜在的抗血管生成药物。
