Improved antiangiogenic and antitumour activity of the combination of the natural flavonoid fisetin and cyclophosphamide in Lewis lung carcinoma-bearing mice

Abstract

The natural flavonoid fisetin was recently identified as a lead compound that stabilizes endothelial cell microtubules. In this study, we investigated the antiproliferative and antiangiogenic properties of fisetin in vitro and in vivo. Fisetin cytotoxicity was evaluated using Lewis lung carcinoma cells (LLC), endothelial cells and NIH 3T3 cells. Endothelial cell (EC) migration and capillary-like structure formation were evaluated using EAhy 926 cells. In vivo tumour growth inhibition studies were performed using LLC-bearing mice treated with fisetin and/or cyclophosphamide (CPA). The fisetin IC50 was 59 μM for LLC and 77 μM for EC cells, compared to 210 μM for normal NIH 3T3 cells (24 h). Fisetin inhibited EC migration and capillary-like structure formation at non-cytotoxic concentrations (22–44 μM). In mice, fisetin inhibited angiogenesis assessed using the Matrigel plug assay. In LLC-bearing mice, fisetin produced a 67% tumour growth inhibition (223 mg/kg, intraperitoneal), similar to the 66% produced by low-dose CPA (30 mg/kg, subcutaneous). When fisetin and CPA were combined, however, a marked improvement in antitumour activity was observed (92% tumour growth inhibition), with low systemic toxicity. Tumour histology showed decreased microvessel density with either fisetin or CPA alone, and a dramatic decrease after the fisetin/CPA combination. We have shown that fisetin not only displays in vitro and in vivo antiangiogenic properties, but also can markedly improve the in vivo antitumour effect of CPA. We propose that this drug combination associating a non-toxic dietary flavonoid with a cytotoxic agent could advantageously be used in the treatment of solid tumours.