马钱子碱抑制乳腺癌细胞体外血管生成拟态的形成及其可能机制研究

Brucine inhibits vasculogenic mimicry of breast cancer cell lines in vitro and the related mechanism

背景与目的:乳腺癌等多种高度侵袭性的肿瘤中存在血管生成拟态(vasculogenic mimicry,VM)现象,可能导致传统的抗血管治疗效果不理想。马钱子碱是马钱子的主要生物碱单体,具有潜在的抗血管生成作用。本研究旨在探讨马钱子碱对人乳腺癌细胞系MDA-MB-231体外形成血管生成拟态的影响及其可能的作用机制。方法:采用MTT法测定马钱子碱对人乳腺癌细胞增殖的抑制作用;应用AnnexinⅤ-FITC/碘化丙啶(propidium iodide,PI)双染流式细胞术检测细胞凋亡情况,以Hoechst 33342/PI(HO/PI)染色进行细胞死亡检测。在Matrigel基质胶上三维培养人乳腺癌细胞,研究在体外是否可以形成血管生成拟态,并观察马钱子碱对这一过程的影响。采用蛋白质印迹法(Western blot)检测血管生成拟态相关标志分子表达的变化,如血管内皮生长因子A(vascular endothelial growth factor-A,VEGF-A)、血管内皮钙黏蛋白(vascular endothelial cadherin,VE-cadherin)、EphA2和基质金属蛋白酶(matrix metalloprotein,MMP)。结果:马钱子碱可有效地抑制乳腺癌细胞增殖,且随药物浓度增加,细胞凋亡与坏死比例增大,细胞死亡率升高;马钱子碱可抑制乳腺癌细胞血管生成拟态的形成,呈剂量依赖性;马钱子碱处理后血管生成拟态标志蛋白(VEGF/VE-cadherin/EphA2/MMP-9/MMP-2)的表达水平明显下调。结论:马钱子碱可以抑制乳腺癌细胞系MDA-MB-231增殖,诱导其凋亡,并抑制其体外血管生成拟态的形成,其机制可能与VEGF/VE-cadherin/EphA2/MMP-9/MMP-2的表达下调有关。

Background and purpose： The existence of vasculogenic mimicry（VM） in a large variety of highly invasive tumors, such as breast cancer, may lead to less effective traditional anti-angiotherapy. Brucine, a natural plant alkaloid, isolated from the seeds of medicinal herb, Strychnos nux-vomica Linn, has the potential antiangiogenic effect. This study aimed to explore the effect of brucine on vasculogenic mimicry formation of breast cancer cell lines MDA-MB-231 in vitro and the potential mechanisms. Methods： The inhibitory effect of brucine on growth of MDAMB-231 cells was measured by MTT method. Annexin Ⅴ-FITC/PI binding assay was used to measure the apoptosis rate of MDA-MB-231 cell after treatment with different concentrations of brucine. Hoechst 33342/propidium iodide（HO/PI） dual staining was used to assay cell death detection. A three-dimensional system of breast cancer cell lines MDAMB-231 on Matrigel collagen matrix was used to investigate whether breast cancer cells can develop vasculogenic mimicry, and to determine the effects of brucine on this process. Furthermore, the protein level of the related marker molecules（VEGF, VE-cadherin, EphA2, MMP-2 and MMP-9） of VM were determined by Western blot. Results： Brucine displayed significant anti-proliferative effect on MDA-MB-231 cells in a dose-and time-dependent manner. Brucine could significantly induced MDA-MB-231 cells apoptosis and necrosis, increased the death rate. Brucine inhibited the development of vasculogenic mimicry by the MDA-MB-231 cells. Brucine also decreased VEGF, VEcadherin, EphA2, MMP-2 and MMP-9 in a dose-dependent manner. Conclusion： Brucine could inhibit the proliferation of MDA-MB-231 cell and induce apoptosis. It inhibits the development of vasculogenic mimicry by regulating the expression of VEGF, VE-cadherin, EphA2, MMP-2 and MMP-9.