5093

Gene expression is controlled in part, by deacetylation of specific lysine residues in histone tails and this process controls repression of many genes involved in cell cycle progression. Histone deacetylase inhibitors (HDACi) are currently being developed as anti-tumour agents and have been shown to inhibit effectively the growth of hyper-proliferating cancer cells. It has also been demonstrated that combinations of HDACi with well-established chemotherapeutics can synergise with their anti-tumour effects. We have investigated in detail the effects of PXD101 (HDACi in phase I clinical trials) in combination with the thymidylate synthesis inhibitor 5-fluorouracil (5-FU), on tumour cell proliferation and apoptosis both in vitro and in vivo. The human HCT116 cell line (an established model of colon cancer) was studied using WST-1 proliferation assays. Synergistic inhibition of proliferation was obtained with combination index (CI) values of 0.7 or below over a wide range of concentrations, as calculated by CalcuSyn™. Several different incubation schedules were tested and the greatest degree of synergy was obtained when cells were incubated in PXD101 for 24 hours, followed by a further 48 hours in 5-FU alone. Furthermore, synergism was confirmed in both HCT116 and P388 mouse leukemia cells in clonogenic assays when PXD101 and 5-FU were combined. 5-FU combined with PXD101 also increased DNA fragmentation (demonstrated using FACS TUNEL assay) and increased PARP cleavage in HCT116 cells, compared to each compound alone. Incubation with PXD101 alone for 24 hours also down-regulated thymidylate synthase (TS) expression in HCT116 cells. High level TS expression in colon cancer is a marker of poor prognosis. Quantitative RT-PCR has been used to explore the effects of PXD101 on other enzymes involved in the 5-FU metabolic/mechanism of action pathways. In vivo studies using the mouse P388 i.p. tumour model showed an increase in median survival of 3 days (log rank analysis p < 0.002) compared to each drug alone, when PXD101 and 5-FU were combined. HCT116 xenograft models also showed a beneficial outcome when PXD101 and 5-FU were combined. Tumour volume was significantly reduced in HCT116 xenografts with combinations compared to each drug alone.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]