Thymidine phosphorylase (TP) has emerged as a promising target for antiangiogenesis treatment of
cancer. Angiogenesis, the formation of blood vessels, is essential for
tumors to grow in order to be supplied with nutrients and
oxygen. The association of TP with angiogenesis was demonstrated in several clinical studies in various tissue types. It has been postulated that the angiogenic effect of TP is related to its enzymatic activity, which catalyzes the breakdown of
thymidine to
thymine and deoxyribose-1-phosphate (dR-1-P). The latter, in its parent form or in its
sugar form,
deoxyribose, may play a role in angiogenesis. It may interfere in cellular energy metabolism or be substrate in a chemical reaction generating
reactive oxygen species. L-
deoxyribose and a specific TP inhibitor, TPI, can reverse these effects, supporting the role of the enzymatic reaction and that of the
sugar. Although TP is usually high in the
tumor, we also observed a high expression in
tumor-associated stromal cells and macrophages. In order to elucidate the mechanism of TP induced angiogenesis we have investigated the association of TP with angiogenesis, the effect of
thymidine and its metabolites on angiogenic parameters (e.g. invasion), the modulation by TPI, the formation and retention of the
sugar metabolites of
thymidine, and the potential signalling pathways involved in the angiogenic process. We used cell lines without/low TP expression (Colo320 and RT112) and TP transfected variants (Colo320TP1 and RT112/TP). Intrinsic TP expression in
cancer cells did not stimulate these cells to invade more. On the other hand, Colo320 and Colo320TP1 cells could attract endothelial cells to a high extent, but Colo320TP1 did not attract them to a higher extent. RT112/TP cells attracted more endothelial cells than RT112 (2 fold). The difference between the RT112's and Colo320's may be related to different formation of
sugars. Exposure of
tumor cells to
thymidine resulted in a rapid formation of dR-1-P, which was rapidly degraded to
deoxyribose and further metabolized to other
sugar derivatives. Of the possible
sugars that can be produced by the conversion of TdR, dR-5-P seems to accumulate the most. dR accumulated 3 fold higher extent in RT112/TP than in Colo320/TP1 cells. dR could be converted to
advanced glycation endproducts (AGE), however this was to a lower extent than
ribose.
Thymidine also induced several signalling pathways in the cells, involved in migration and invasion, such as the
Focal adhesion kinase (FAK), which subsequently stimulated p70/S6 phosphorylation. The latter is a downstream
kinase of
rapamycin and its phosphorylation is inhibited by
rapamycin, an mTOR inhibitor. The association between
rapamycin and TP was shown by the protection by
thymidine of
rapamycin induced cytotoxicity, while TPI inhibited the effect of
thymidine addition. These studies clearly show a mechanistic link between TP, signalling pathways, and cell migration.