Evaluating Gemcitabine Metabolism in Pancreatic Cancer Cells

The K8484 cell line was maintained in DMEM (D6429, Sigma Aldrich Co Ltd.) containing 5% FBS (10270-098, Life Technologies Ltd., Paisley, UK). MIA PaCa-2 cells were obtained from the European Collection of Cell Cultures (Public Health England, Salisbury, UK, authenticated by STR genotyping) and maintained in DMEM containing 10% FBS. Gemcitabine (3259, Tocris Bioscience) was dissolved in DMSO; linoleate (L8134, Sigma Aldrich Co Ltd.) and 6-diazo-5-oxo-L-norleucine (DON, D2141 Sigma Aldrich Co Ltd.) were dissolved in sterile water. Experiments assessing the effect of DON on dFdC metabolism were carried out in media lacking glutamine (D5671, Sigma Aldrich Co Ltd.). Growth-inhibition assays were carried out using the sulphorhodamine assay, and subsequent synergy calculations were done as previously described (Lin et al, 2012 (link)).

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Bapiro T.E., Frese K.K., Courtin A., Bramhall J.L., Madhu B., Cook N., Neesse A., Griffiths J.R., Tuveson D.A., Jodrell D.I, & Richards F.M. (2014). Gemcitabine diphosphate choline is a major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo. British Journal of Cancer, 111(2), 318-325.

Publication 2014

D6429 Gemcitabine linoleate 6-diazo-5-oxo-L-norleucine ( D2141 D5671

Assay Cell cultures Cell line Cells Dmso European Gemcitabine Glutamine Inhibition Linoleate Metabolism Sterile

Corresponding Organization :

Other organizations : Cancer Research UK, University of Cambridge, Cold Spring Harbor Laboratory

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Variable analysis

independent variables

Gemcitabine (3259, Tocris Bioscience) dissolved in DMSO
Linoleate (L8134, Sigma Aldrich Co Ltd.) dissolved in sterile water
6-diazo-5-oxo-L-norleucine (DON, D2141 Sigma Aldrich Co Ltd.) dissolved in sterile water

dependent variables

Growth-inhibition measured using the sulphorhodamine assay
Synergy calculations

control variables

K8484 cell line maintained in DMEM (D6429, Sigma Aldrich Co Ltd.) containing 5% FBS (10270-098, Life Technologies Ltd., Paisley, UK)
MIA PaCa-2 cells maintained in DMEM containing 10% FBS
Experiments assessing the effect of DON on dFdC metabolism carried out in media lacking glutamine (D5671, Sigma Aldrich Co Ltd.)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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