Endocytosis of VEGFR-2 on endothelial progenitor cells by dopamine attenuates tumour growth and vasculogenesis

Abstract

Introduction: Endothelial progenitor cells (EPC’s) andvasculogenesis are intricately involved in the initial neovascularisation of tumours. EPC’s are characterised by the expression of VEGFR-2 and AC133. Vasculogenesis offers a novel possibility for the targeting of anti-cancer therapies. Methods: Ex-vivo expanded EPC’s were cultured from peripheral blood samples from healthy male volunteers. To endocytose VEFGR-2 on EPC’s, cells were treated with incremental doses of dopamine and VEGFR-2 expression was analysed using anti-VEGFR-2 mAb on flow cytometry. Dil-labelled ex-vivo expanded EPC’s were transplanted into tumour bearing (3LL tumour cell line) MF1-nude mice (n=12). The mice received a daily i.p. injection of PBS or Dopamine (50 mg/kg). Tumour volume was assessed on alternate days. Vasculogenesis and angiogenesis were assessed using fluorescent microscopy and CD31 staining. To determine if Dopamine has a direct effect on 3LL cells, cells were cultured with incremental doses of dopamine for 48 h and cell apoptosis (PI-staining), proliferation (BrdU-Assay) and Cytotoxicity (LDH-Assay) were analysed. Statistical analysis was performed on SigmaStat using ANOVA. Results: Ex-vivo expanded EPC cultures were 80–85% pure. Dopamine caused an endocytosis of VEGFR-2 on EPC’s with the maximal effect seen at 1μM (p < 0.05). Dopamine had no effect on 3LL cell apoptosis, proliferation or cytotoxicity in all doses performed. Dopamine, by endocytosing VEGFR-2 on EPC’s, attenuated tumour growth by decreasing both tumour volume (p < 0.05; 241.8 +/- 106.8 v’s 59.7 +/- 20.5 at day 10) and tumour weight (p < 0.05). Absolute and relative levels of vasculogenesis were decreased in the dopamine group as compared to controls (p < 0.05). Conclusion: Induction of VEGFR-2 endocytosis on EPC’s decreases tumour growth and vasculogenesis. This data suggests that VEGFR-2 is a target for attenuating tumour vasculogenesis.