in this case PXD101, with 5 FU should lead to synergism in their anti proliferative eVects. The HCT116 colorectal cancer cell line was employed, which is commonly used Oridonin as a model for colorectal tumours, to examine this in some detail. Both PXD101 and 5 FU were found to be potent inhibitors of HCT116 cellular proliferation in vitro, in both WST1 and clonogenic assays, and when combined their eVects on proliferation were synergistic. It is plausible that the down regulation of TS by PXD101 is required for optimal synergy to occur since pre incubation with PXD101 for 24 h, followed by 48 h with 5 FU, was more eVective than the 48 h co incubation . In addition to the aVect of PXD101 on thymidylate synthase expression, other HDACi have been shown to eVect additional molecular pathways involved in colon cancer carcinogensis and growth.
These include down regulation of Cyclin B1 in a p21WAF1 and transcriptional dependent manner , suppression of Cox2 activation and repression of Src family kinase members . PXD101 may also regulate each of these pathways via its action as an HDACi. It is therefore conceivable that other molecular mechanisms of HDAC inhibition may be implicated Rocuronium molecular weight in the synergy of anti proliferative actions of PXD101 and 5 FU. In addition to the synergistic eVects on proliferation, co incubation of PXD101 and 5 FU enhanced apoptosis compared with single compound treatment. The PARP cleavage experiment, however, showed no increased cleavage over single compound alone when the PXD101 pre incubation followed by 5 FU schedule was used.
This is in line with the fact that PARP cleavage is an early stage event in apoptosis . Owing to this, it Bcr-Abl hemmer is likely to be diYcult to distinguish any diVerences in cleavage between single compound and combinations at such an extended period post compound treatment outset. Daptomycin ic50 To our knowledge, no in vivo data using HDACi/ 5 FU combinations have been previously described. In line with our in vitro data, results presented here show that both enhanced survival and tumour reduction in vivo over and above single compound treatment can be achieved using PXD101/5 FU combination. It should be noted however, that there was evidence of increased toxicity when doses of 30 mg/kg 5 FU alone in combination with PXD101 in vivo, in a preliminary toxicology study using B6D2F1 mice .
Due to these observations, a sub therapeutic concentration of 5 FU was tested and tolerated in the nude mouse HCT116 xenograft when combined with PXD101. Even at this lower concentration, signiWcant beneWcial responses were nausea observed when compared to each monotherapy. These data indicate that PXD101 signiWcantly enhances the anti tumour eYcacy of 5 FU in vivo. After the success of the HCT116 study, a second xenograft model using HT29 cells was then established to examine this combination using an elevated dose of 5 FU in an attempt to enhance the eVect of the combination. Two toxic deaths were observed, however, when a dose of 100 mg/kg PXD101 was combined with 30 mg/kg 5 FU. After reduction of PXD101 to 60 mg/kg no toxic deaths occurred, although weight loss was observed which could be attributed to the elevated dose of 5 FU since the mice recovered after cessation of the 5 FU treatment. This is in agreement with the preliminary toxicology study.