Blockage of 2-Deoxy-d-Ribose-Induced Angiogenesis with Rapamycin Counteracts a Thymidine Phosphorylase-Based Escape Mechanism Available for Colon Cancer under 5-Fluorouracil Therapy

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Abstract
Purpose: Colorectal neoplasms remain a leading cause of cancer-related deaths. A recognized weakness of conventional 5-fluorouracil (5-FU) therapy relates to expression of the intracellular enzyme, thymidine phosphorylase (TP). Although TP promotes 5-FU cytotoxicity, TP-derived 2-deoxy-d-ribose (dRib) counterproductively stimulates tumor angiogenesis. Here, the newly discovered antiangiogenic drug rapamycin was combined with 5-FU to counteract the potential escape mechanism of dRib-induced angiogenesis. Experimental Design: Orthotopic tumor growth was assessed in rapamycin and 5-FU-treated BALB/c mice with TP-expressing CT-26 colon adenocarcinoma cells. To examine liver metastasis, green-fluorescent protein-transfected CT-26 cells were visualized by fluorescence microscopy after intraportal injection. Cell counting and Ki67 staining were used to determine in vitro and in vivo cell expansion, respectively. In vitro angiogenic effects of dRib were assessed with endothelial cell migration and aortic ring assays. Western blotting detected dRib effects on p70/S6 kinase activation. Results: Rapamycin treatment of mice bearing orthotopic tumors inhibited tumor growth more than did 5-FU, and mice treated with both drugs typically developed no tumors. In the liver metastasis assay, combination therapy blocked metastatic expansion of solitary tumor cells. Interestingly, complex drug activities were suggested by tumor-cell proliferation being more sensitive to 5-FU than to rapamycin in vitro, but more sensitive to rapamycin in vivo. With regard to angiogenesis, dRib-induced endothelial cell migration and aortic ring formation were completely abrogated by rapamycin, correlating with blockage of dRib-induced p70/S6 kinase activation in endothelial cells. Conclusions: Inhibition of dRib-induced angiogenesis with rapamycin counteracts a potential TP-based escape mechanism for colorectal cancer under 5-FU therapy, introducing a novel, clinically feasible, combination treatment option for this common neoplasm.