Antiangiogenic potential of camptothecin and topotecan.

Abstract

To determine the inhibitory nature of sublethal doses of camptothecin (CPT) and topotecan (TPT) treatments on normal human endothelial cells in vitro, as well as the in vivo antiangiogenic activity as compared to another antiangiogenic compound, TNP-470 and to a nonspecific cytotoxic agent, cisplatin. Growth inhibition was determined by the crystal violet assay to measure relative cell numbers. (3)H-thymidine uptake was used to determine the inhibitory effect of CPT and TPT on DNA synthesis in vitro. Cell viability was determined using trypan blue exclusion assays. Cell cycle response to CPT was determined by flow cytometric analysis of propidium iodide-stained nuclei. In vivo inhibition of angiogenesis was determined by the disc angiogenesis system (DAS), where surgical sponge discs were placed subcutaneously 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 determined using toluidine blue staining of sectioned discs followed by digital image analysis. Treatment with 50 nM CPT or TPT inhibited human umbilical venular endothelial cell (HUVEC) growth as shown by crystal violet staining, but was not cytotoxic to the cells. This was evidenced by the fact that cell numbers did not increase or decrease with treatment, but remained static while cells were viable for over 96 h posttreatment. (3)H-thymidine uptake 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-treated HUVEC showed arrest in S-phase at 12 h with a concurrent decrease in population of cells in G(1). Accumulation of cells at the G(2)/M-phase was discernible at 24 h along with the S-phase inhibition. Treatment of 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 vascular growth into the discs. In this work we showed that CPT and TPT inhibit human endothelial cell growth in vitro in a non-cytotoxic manner 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.