The hollow fibre assay as a model for in vivo pharmacodynamics of fluoropyrimidines in colon cancer cells

Abstract

The Hollow Fibre Assay (HFA) is usually applied as an early in vivo model for anti-cancer drug screening, but is potentially an excellent model for short-term in vivo pharmacodynamic studies. We used the model to study the in vivo role of thymidine phosphorylase/ platelet-derived endothelial cell growth factor (TP/PD-ECGF) in the cytotoxicity and pharmacodynamics of TAS-102 in colon cancer cells. TAS-102 is a new oral drug formulation, which is composed of trifluorothymidine (TFT) and thymidine phosphorylase inhibitor (TPI), which prevents TFT degradation. We compared the activity with Xeloda (capecitabine), which is activated by TP into 5FU. Hollow fibres filled with human Colo320 or Colo320TP1 colorectal cancer cells with deficient or high TP expression, respectively, were implanted subcutaneously (s.c.) at both flanks of BALB/c mice. The mice were treated orally over 5 days with TAS-102, TFT alone, 5 0 DFUR7TPI or capecitabine at their maximum tolerated dose (MTD). The cells were retrieved from the fibres and assayed for growth (MTT assay), cell cycle distribution (flow cytometry) and apoptosis induction (FragEL method). TAS-102 induced considerable growth inhibition (50%, Po0.01) to both cell lines, which was completely abolished in the absence of TPI. Capecitabine and its metabolite 5 0 DFUR reduced proliferation of Colo320TP1 cells in the fibres significantly (down to 25 -40%), but much less in Colo320 cells, whereas addition of TPI reduced the effect of 5 0 DFUR, although not completely. These differences in cytotoxic effects were reflected in the pharmacodynamic evaluation. TAS-102 induced a G2M-phase arrest (from 25 to 40%) and apoptosis (48-fold), which was more pronounced in Colo320 than in Colo320TP1. Again, omission of TPI neutralised the effect of TAS-102. Similarly, 5 0 DFUR and capecitabine induced a significant G2M-phase arrest (up to 45%) in the Colo320TP1 cell line, but less pronounced in the parental Colo320. Addition of TPI to 5 0 DFUR reduced this effect to control levels. Also induction of apoptosis was reduced in the presence of TPI. The data demonstrated that the HFA is excellently suited for studying short-term pharmacodynamic effects of fluoropyrimidines in vivo. TAS-102 is only effective in inducing cytotoxicity when systemic TPI is present, but acts against both low and high TP expressing colon cancer cells, while 5 0 DFUR needs cellular TP to exert significant activity.

Differential activation of cell death and autophagy results in an increased cytotoxic potential for trifluorothymidine compared to 5‐fluorouracil in colon cancer cells

“…15 Although these phenomena are known to be induced by microtubule inhibitors, such as taxanes, they are less expected to be associated with the activity of a nucleoside analog that targets the S-phase of the cell cycle, e.g., by TS inhibition and DNA damage induction. 24 The difference between TFT and 5-FU induced cell death was also found in in vivo experiments, in which TFT (as TAS-102) induced more tumor growth inhibition and caused more cell death than the 5-FU prodrug 5 0 -DFUR 37 . In other preclinical in vivo studies, TAS-102 also showed a better antitumor effect than various other 5-FU formulations, all at their MTD.…”

“…Cells were retrieved and growth inhibition was determined by the MTT-assay and cell death by the TUNEL assay as described previously. 37 …”

“…37 The protocol was approved by the local Animal Ethical Committee in accordance with the UKCCR guidelines. In brief, Colo320 cells (7.5Â10 6 cells/ml) were transplanted into a 2 cm hollow fiber in BALB/c mice.…”

Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

“…Flow cytometry was used to determine cell cycle distribution as well as the amount of apoptotic cells within the cell populations exposed to the drugs alone or in combination as described previously (Temmink et al, 2007). Cells at a density of 50 Â 10 3 cells per well were plated into flat bottom 6-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and allowed to attach for 24 h prior to drug treatments at IC 50 concentrations.…”

Use of the in Vivo Hollow Fiber Assay in Natural Products Anticancer Drug Discovery

“…Along these lines, Hall and colleagues have used the hollow fiber assay to study the effects of cell cycle inhibitors on Rb expression and phosphorylation and PCNA expression 37. Temmink and colleagues used the model to study the in vivo role of thymidine phosphorylase/platelet-derived endothelial cell growth factor in the cytotoxicity and pharmacodynamics in colon cancer cells of a formulation of trifluorothymidine and a thymidine phosphorylase inhibitor 50. Sader et al have adapted the assay to study the molecular events involved as a human prostate cancer cell line (LNCaP) progresses to hormone independence 51.…”