Abstract #5503: Differential activation of cathepsin B and autophagy results in an increased sensitivity to trifluorothymidine compared to 5-fluorouracil in colorectal cancer cells
Authors: Irene Bijnsdorp, Godefridus Peters, Masakazu Fukushima, Frank Kruyt
Journal: Cancer Research (2009)

Trifluorothymidine (TFT) is part of the oral formulation TAS-102. Although 5-fluorouracil (5-FU) and TFT have the same targets (e.g. inhibition of thymidylate synthase and incorporation into the DNA), they only show moderate cross-resistance. TFT is more active than 5-FU in various colorectal cancer cells, especially regarding the long-term survival. However, this could not be related to the activation of different apoptotic, caspase dependent pathways. In order to explain these differences in cytotoxicity, we analyzed whether caspase independent cell death mediated by cathepsin B played a role and whether autophagy was induced. Autophagy can be a survival mechanism after treatment with anticancer drugs. Therefore, cleavage of cathepsin B, LC3BI/II (autophagy marker) and caspase 3 were determined by Western blotting. The induction of apoptosis/necrosis was determined by annexinV/PI staining with FACS analysis. All experiments were performed in the absence/presence of the specific cathepsin B inhibitor CA074-me and at equitoxic drug concentrations (IC 80 ) as determined by the sulphorhodamine B assay. In all cell lines, TFT had a slightly higher induction, which was about 15-20%. In WiDR (mt p53) and Lovo92 (wt p53), cathepsin B was cleaved to a higher extent after TFT, compared to 5-FU. In WiDR cells TFT and 5-FU induced both apoptotic (12 vs 10%) and necrotic cell (8 vs 7%) death, respectively. In Lovo92 cells, apoptosis was induced by TFT (13%) and 5-FU (10%), and necrosis was not induced. In Colo320, only a necrotic type of cell death (10 vs 6%) was induced, respectively. To determine whether cathepsin B was involved in the induction of necrosis, the inhibitor CA074-me was added. In WiDR cells, CA074-me reduced the TFT and 5-FU induced apoptosis by 63 and 32 % and necrosis by 68 and 54 %, respectively. In Lovo92 the apoptotic reduction was 77 and 100% after TFT and 5-FU, respectively. Colo320 (mt p53 pathway) hardly cleaved cathepsin B and the induced necrosis could not be inhibited by CA074-me. Since cathepsin B also reduced apoptosis in WiDR and Lovo92, we determined the effect of cathepsin B inhibition on caspase 3 cleavage. Interestingly, in Lovo92 and WiDR cells, cathepsin B inhibition also inhibited the cleavage of caspase 3. This indicates a role of cathepsin B in cross-activation of the apoptotic pathway. In all tested cell lines, 5-FU strongly induced autophagy as indicated by increased expression and cleavage of LC3B. This was not seen after TFT exposure. Inhibition of cathepsin B by CA074-me increased LC3B expression and cleavage in TFT-treated cells, suggesting a role for cathepsin B in suppressing autophagy. In conclusion, we found that 5-FU in contrast to TFT induces autophagy that is known to counteract cell death, thus providing an explanation for the stronger anti-tumor activity of TFT in clonogenic assays.   Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5503.