Effects of Transferrin Receptor Blockade on Cancer Cell Proliferation and Hypoxia-Inducible Factor Function and Their Differential Regulation by Ascorbate

Dylan T. Jones, Ian S. Trowbridge, Adrian L. Harris
2006 Cancer Research  
Cellular iron is needed for cell survival and hydroxylation of hypoxia-inducible factor-1A (HIF-A) by prolyl hydroxylases (PHD). One mechanism of iron uptake is mediated by the cell surface transferrin receptor (TfR). Because iron is required for cell growth and suppression of HIF-A levels, we tested the effects of the two anti-TfR monoclonal antibodies (mAb) E2.3 and A27.15 on growth of breast cancer cells and induction of HIF-A and hypoxia-regulated genes. Treatment with both mAbs together
more » ... th mAbs together synergistically inhibited cell proliferation in a doseresponsive manner by up to 80% following 8 days of exposure, up-regulated HIF-1A and HIF transcription targets, downregulated TfR expression, and down-regulated cellular labile iron pool by 60%. Because combined treatment with anti-TfR mAbs resulted in the up-regulation of the hypoxia pathway, which may increase tumor angiogenesis, we analyzed the effects of ascorbate on cell viability and HIF-1A levels in cells treated with both anti-TfR mAbs together, as ascorbate has been shown to be required by PHD enzymes for full catalytic activity. Ascorbate at physiologic concentrations (25 Mmol/L) suppressed HIF-1A protein levels and HIF transcriptional targets in anti-TfR mAb-treated cells but did not suppress the antiproliferative effect of the mAbs. These results indicate that the addition of ascorbate increased the activity of the PHD enzymes in down-regulating HIF but not the proliferation of iron-starved anti-TfR mAb-treated cells. The use of anti-TfR mAbs and ascorbate in inhibiting both cell proliferation and HIF-1A and angiogenesis under normoxic conditions may be of therapeutic use. (Cancer Res 2006; 66(5): 2749-56) Materials and Methods Cell culture. Breast carcinoma MDA-468, MDA-231, and MCF-7 cell lines were obtained from the Cancer Research UK Cell Service and maintained in DMEM supplemented with 10% fetal bovine serum, 2 Amol/L L-glutamine, 50 IU/mL penicillin, and 50 Ag/mL streptomycin sulfate. L-Ascorbic acid sodium salt was obtained from Sigma (Poole, United Kingdom). Anti-TfR mAbs A27.15 and E2.3 were provided as a kind gift by The Salk Institute,
doi:10.1158/0008-5472.can-05-3857 pmid:16510596 fatcat:qzaqheqwybbifhl6atf3tjye7y