Dual functions of a monoclonal antibody against cell surface F1F0 ATP synthase on both HUVEC and tumor cells1

Abstract

To generate a monoclonal antibody (McAb) against cell surface F1F0 ATP synthase (ATPase) and observe its antitumoral activity on both human umbilical vein endothelial cells (HUVEC) and tumor cells. Hybridoma cells secreting McAb against ATPase were produced by polyethylene glycol-mediated fusions and screened by ELISA. The specificity of McAb was demonstrated by immunofluorescence and confocal imaging, as well as flow cytometry analysis. After the blockade of surface ATPase with McAb on HUVEC and human breast adenocarcinoma MDA-MB-231 cells, an ATP determination kit and CellTiter96 AQueous Assay (MTS) assay were used to detect the effect of the antibody on extracellular ATP modification and cell proliferation. A cellular cytotoxicity assay in combination with doxorubicin, and a cell migration assay on MDA-MB-231 cells were used to determine the antitumoral activity. Finally, a HUVEC tube formation assay was used to detect the antiangiogenic effect of McAb178-5G10. A monoclonal antibody (McAb178-5G10) against the beta-subunit of ATPase was generated, and its reactivity toward HUVEC and tumor cells was studied. We demonstrate that McAb178-5G10 binds to ATPase at the cell surface, where it is able to inhibit ATP synthesis. This antibody also prevents the proliferation of HUVEC and MDA-MB-231 cells. Furthermore, McAb178-5G10 enhances the tumoricidal effects of doxorubicin (P<0.05), inhibits the migration of MDA-MB- 231 in transwell assays (P<0.01), and disrupts HUVEC tube formation on Matrigel (P<0.01). McAb178-5G10 binds preferentially to cell surface ATPase, blocks ATP synthesis, and exhibits both antiangiogenic and antitumorigenic effects.