Inhibition of angiogenic activity of hypoxic fibroblast cell line MRC-5 in vitro by topotecan

Tumor stroma plays an important role in cancer development. Stromal fibroblasts often represent the majority of stromal cells within various types of human carcinomas, and they are competent to promote the growth of cancer cells and to enhance tumor angiogenesis. However, the effect of known anti-cancer drugs on stromal cells has not been thoroughly investigated. Topotecan (TPT) is a semi-synthetic analogue of camptothecin, and several studies have shown that TPT inhibited angiogenesis via its direct effect on vascular endothelial cells. Here, we studied the effect of TPT on pro-angiogenesis action of hypoxic fibroblasts (MRC-5 cells). Growth inhibition was analyzed by MTT assay, while transwell assay and tube formation assay were used to evaluate the inhibition by TPT of hypoxic MRC-5 cell angiogenic activity in vitro. ELISA and Western blot analysis were used to investigate the related mechanism. Pretreatment of MRC-5 with TPT remarkably attenuated the induction of migration and tube formation of HUVECs by conditioned medium from hypoxic MRC-5 cells. In addition, topotecan decreased hypoxia-induced VEGF production by MRC-5 cells. Moreover, topotecan inhibits HIF-1 alpha and alpha-SMA protein expression in hypoxic MRC-5 cells. Our data suggest that TPT inhibits hypoxic fibroblast angiogenic activity via downregulation of HIF-1 alpha and prevention of fibroblast differentiation to myofibroblast.