Antiangiogenic potential of camptothecin and topotecan

Purpose: To determine the inhibitory nature of sublethal doses of camptothe cin (CPT) and topotecan (TPT) treatments on normal human endothelial cells in vitro, as well as the in vivo antiangiogenic activity as compared to ano ther antiangiogenic compound, TNP-470 and to a nonspecific cytotoxic agent, cisplatin. Methods: Growth inhibition was determined by the crystal Violet assay to measure relative cell numbers. H-3-thymidine uptake was used to d etermine the inhibitory effect of CPT and TPT on DNA synthesis in vitro. Ce ll viability was determined using trypan blue exclusion assays. Cell cycle response to CPT was determined by flow cytometric analysis of propidium iod ide-stained nuclei. In vivo inhibition of angiogenesis was determined by th e disc angiogenesis system (DAS), where surgical sponge discs were placed s ubcutaneously in the rat dorsum and the ability of systemic treatment with liposomal CPT (LCPT), TPT, TNP-470 or cisplatin to inhibit Vascular growth into the discs was evaluated. Quantitation of vascular growth was determine d using toluidine blue staining of sectioned discs followed by digital imag e analysis. Results: Treatment with 50 nM CPT or TPT inhibited human umbili cal venular endothelial cell (HUVEC) growth as shown by crystal violet stai ning, but was not cytotoxic to the cells. This was evidenced by the fact th at cell numbers did not increase or decrease with treatment, but remained s tatic while cells were Viable for over 96 h posttreatment. 3H-thymidine upt ake in HUVEC was inhibited as early as 5 min, reached a maximum inhibition at 24 h and lasted over 96 h posttreatment. Cell cycle analysis of CPT-trea ted HUVEC showed arrest in S-phase at 12 h with a concurrent decrease in po pulation of cells in GI. Accumulation of cells at the G(2)/M-phase was disc ernible at 24 h along with the S-phase inhibition. Treatment df rats with 1 mg/kg LCPT or TPT every other day for 14 days resulted in approximately 30 % inhibition of vascular growth into the discs. This inhibition was similar to the inhibition seen with TNP-470, an established and potent angiogenic inhibitor. In contrast, cisplatin was not as effective in inhibiting vascul ar growth into the discs. Conclusions: In this work we showed that CPT and TPT inhibit human endothelial cell growth in vitro in a non-cytotoxic manne r and that this inhibition lasts more than 96 h after drug removal. We also showed that LCPT and TPT, unlike a nonspecific cytotoxic agent, cisplatin, are as effective as TNP-470 in inhibiting angiogenic growth in the in vivo disc angiogenesis model. From this observation we propose that in addition to their proven tumoricidal activities, camptothecins may have an indirect in vivo antitumor effect mediated through the inhibition of angiogenesis.