Marimpietri Danilo, Nico Beatrice, Vacca Angelo, Mangieri Domenica, Catarsi Paolo, Ponzoni Mirco, Ribatti Domenico
Differentiation Therapy Unit Laboratory of Oncology, G Gaslini Childrens' Hospital, Genoa 16148, and Department of Human Anatomy and Histology, University of Bari Medical School, Italy.
Oncogene. 2005 Oct 13;24(45):6785-95. doi: 10.1038/sj.onc.1208829.
The aim of this study was to evaluate the synergistic antiangiogenic effect of low dose of vinblastine (VBL) and rapamycin (RAP) in neuroblastoma (NB). Both in vitro (endothelial cells proliferation assay; TUNEL assay; phosphatidylserine exposure and cell cycle analysis) and in vivo (chick embryo chorioallantoic membrane, CAM) assays were used. Each compound alone was able to induce a significant dose- and time-response inhibition of in vitro endothelial cells (EC) growth. Interaction index evaluation indicates that a synergistic effect was found when both drugs were combined at very low doses. Comparable effects were obtained when EC were preincubated with conditioned medium (CM) derived from the human NB cell line HTLA-230. Morphological changes were induced by each drug, and their combination resulted in a clear and stronger effect. Apoptosis was demonstrated by the TUNEL assay and confirmed by Annexin V-FITC staining of EC treated with VBL, showing an increase in the percentage of cells with a G2-M and sub-G1 DNA content, whereas in those treated with RAP a block in the G1 cell fraction and inhibition of progression to the S phase were observed. Here too, the combination resulted in a synergistic cell cycle arrest and induction of apoptosis. Similar results were obtained in vivo with the CAM assay. The angiogenic responses induced by HTLA-230-derived CM, NB tumor xenografts, and human NB biopsy specimens were inhibited by each drug and more significantly by their combination. The observation that these well-known drugs display synergistic effects as antiangiogenics when administered frequently at very low dose may be of significance in the designing of new ways of treating NB.
本研究的目的是评估低剂量长春碱（VBL）和雷帕霉素（RAP）在神经母细胞瘤（NB）中的协同抗血管生成作用。采用了体外实验（内皮细胞增殖试验；TUNEL试验；磷脂酰丝氨酸暴露和细胞周期分析）和体内实验（鸡胚绒毛尿囊膜，CAM）。每种化合物单独使用时都能在体外显著抑制内皮细胞（EC）生长，呈现剂量和时间依赖性。相互作用指数评估表明，两种药物以极低剂量联合使用时具有协同作用。当EC与源自人NB细胞系HTLA - 230的条件培养基（CM）预孵育时，也获得了类似的效果。每种药物都能诱导形态学变化，它们的联合使用产生了明显更强的效果。TUNEL试验证实了细胞凋亡，并用VBL处理的EC的膜联蛋白V - FITC染色进一步确认，结果显示G2 - M期和亚G1期DNA含量的细胞百分比增加，而用RAP处理的细胞中观察到G1期细胞分数阻滞和向S期进展的抑制。同样，联合使用导致细胞周期协同阻滞和凋亡诱导。在体内CAM试验中也获得了类似结果。HTLA - 230衍生的CM、NB肿瘤异种移植物和人NB活检标本诱导的血管生成反应均被每种药物抑制，联合使用时抑制作用更显著。当以极低剂量频繁给药时，这些知名药物作为抗血管生成剂显示出协同作用，这一观察结果可能对设计治疗NB的新方法具有重要意义。
