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1.
Gut ; 67(3): 497-507, 2018 03.
Article in English | MEDLINE | ID: mdl-28077438

ABSTRACT

OBJECTIVE: Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. DESIGN: Gemcitabine metabolites were analysed in LSL-KrasG12D/+ ; LSL-Trp53R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. RESULTS: Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. CONCLUSIONS: Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine.


Subject(s)
Antimetabolites, Antineoplastic/pharmacokinetics , Carcinoma, Pancreatic Ductal/metabolism , Deoxycytidine/analogs & derivatives , Fibroblasts/metabolism , Liver Neoplasms/metabolism , Pancreatic Neoplasms/metabolism , 5'-Nucleotidase/metabolism , Actins/metabolism , Animals , Antimetabolites, Antineoplastic/therapeutic use , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/secondary , Cell Line, Tumor , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/metabolism , Deoxycytidine/pharmacokinetics , Deoxycytidine/therapeutic use , Floxuridine/analogs & derivatives , Floxuridine/metabolism , Humans , Liver/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/secondary , Mice , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Primary Cell Culture , Tumor Microenvironment , Gemcitabine
2.
Br J Cancer ; 111(2): 318-25, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24874484

ABSTRACT

BACKGROUND: The modest benefits of gemcitabine (dFdC) therapy in patients with pancreatic ductal adenocarcinoma (PDAC) are well documented, with drug delivery and metabolic lability cited as important contributing factors. We have used a mouse model of PDAC: KRAS(G12D); p53(R172H); pdx-Cre (KPC) that recapitulates the human disease to study dFdC intra-tumoural metabolism. METHODS: LC-MS/MS and NMR were used to measure drug and physiological analytes. Cytotoxicity was assessed by the Sulphorhodamine B assay. RESULTS: In KPC tumour tissue, we identified a new, Kennedy pathway-linked dFdC metabolite (gemcitabine diphosphate choline (GdPC)) present at equimolar amounts to its precursor, the accepted active metabolite gemcitabine triphosphate (dFdCTP). Utilising additional subcutaneous PDAC tumour models, we demonstrated an inverse correlation between GdPC/dFdCTP ratios and cytidine triphosphate (CTP). In tumour homogenates in vitro, CTP inhibited GdPC formation from dFdCTP, indicating competition between CTP and dFdCTP for CTP:phosphocholine cytidylyltransferase (CCT). As the structure of GdPC precludes entry into cells, potential cytotoxicity was assessed by stimulating CCT activity using linoleate in KPC cells in vitro, leading to increased GdPC concentration and synergistic growth inhibition after dFdC addition. CONCLUSIONS: GdPC is an important element of the intra-tumoural dFdC metabolic pathway in vivo.


Subject(s)
Carcinoma, Pancreatic Ductal/drug therapy , Deoxycytidine/analogs & derivatives , Pancreatic Neoplasms/drug therapy , Animals , Antimetabolites, Antineoplastic/pharmacokinetics , Antimetabolites, Antineoplastic/pharmacology , Carcinoma, Pancreatic Ductal/metabolism , Choline/metabolism , Deoxycytidine/metabolism , Deoxycytidine/pharmacokinetics , Deoxycytidine/pharmacology , Diphosphates/metabolism , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Pancreatic Neoplasms/metabolism , Tumor Cells, Cultured , Gemcitabine
3.
Br J Cancer ; 107(10): 1692-701, 2012 Nov 06.
Article in English | MEDLINE | ID: mdl-23037716

ABSTRACT

BACKGROUND: Amplification of aurora kinase A (AK-A) overrides the mitotic spindle assembly checkpoint, inducing resistance to taxanes. RNA interference targeting AK-A in human pancreatic cancer cell lines enhanced taxane chemosensitivity. In this study, a novel AK-A inhibitor, CYC3, was investigated in pancreatic cancer cell lines, in combination with paclitaxel. METHODS: Western blot, flow cytometry and immunostaining were used to investigate the specificity of CYC3. Sulforhodamine B staining, time-lapse microscopy and colony-formation assays were employed to evaluate the cytotoxic effect of CYC3 and paclitaxel. Human colony-forming unit of granulocyte and macrophage (CFU-GM) cells were used to compare the effect in tumour and normal tissue. RESULTS: CYC3 was shown to be a specific AK-A inhibitor. Three nanomolar paclitaxel (growth inhibition 50% (GI(50)) 3 nM in PANC-1, 5.1 nM in MIA PaCa-2) in combination with 1 µM CYC3 (GI(50) 1.1 µM in MIA PaCa2 and 2 µM in PANC-1) was synergistic in inhibiting pancreatic cell growth and causing mitotic arrest, achieving similar effects to 10-fold higher concentrations of paclitaxel (30 nM). In CFU-GM cells, the effect of the combination was simply additive, displaying significantly less myelotoxicity compared with high concentrations of paclitaxel (30 nM; 60-70% vs 100% inhibition). CONCLUSION: The combination of lower doses of paclitaxel and CYC3 merits further investigation with the potential for an improved therapeutic index in vivo.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bone Marrow Cells/drug effects , Paclitaxel/pharmacology , Pancreatic Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Aurora Kinase A , Aurora Kinases , Bone Marrow Cells/enzymology , Bone Marrow Cells/pathology , Cell Line, Tumor , Drug Synergism , Granulocyte-Macrophage Progenitor Cells/drug effects , Granulocyte-Macrophage Progenitor Cells/metabolism , Humans , Paclitaxel/administration & dosage , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/pathology , Protein Kinase Inhibitors/administration & dosage , Protein Serine-Threonine Kinases/metabolism , Stem Cells/drug effects , Stem Cells/enzymology , Stem Cells/pathology
4.
Drug Metab Dispos ; 29(1): 30-5, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11124226

ABSTRACT

In this study we have evaluated the application and reliability of using fluorescence (FLUO)-based high throughput screening assays with recombinant CYPs (rCYP). This was accomplished by screening 29 clinically important antiparasitic drugs for inhibition of the five major drug-metabolizing CYPs (-1A2, -2C9, -2C19, -2D6, and -3A4). Data from FLUO/rCYP assays were compared with that obtained by conventional HPLC assays using human liver microsomes (HLM) and rCYPs. The K(i) values showed good correlations: FLUO/rCYP and HPLC/rCYP (r(2) = 0.81), HPLC/rCYP and HPLC/HLM (r(2) = 0.82), and FLUO/rCYP and HPLC/HLM (r(2) = 0.72). Niclosamide had substrate-dependent contrasting effects on CYP2C9 activity with an apparent activation (400%) of 7-methoxy-4-trifluoromethylcoumarin demethylase activity and potent inhibition (K(i) = 6.00 microM) of diclofenac 4-hydroxylase activity. Potent inhibitors of CYP1A2 were artemisinin, dihydroartemisinin, thiabendazole, primaquine, and niclosamide (K(i) = 0.43, 3.67, 1.54, 0.22, and 2.70 microM, respectively). Proguanil, cycloguanil, amodiaquine, and desethylamodiaquine inhibited CYP2D6 (K(i) = 6.76, 5.97, 2.1, and 4.13 microM, respectively). Considering the C(max) of these drugs, artemisinin, thiabendazole, primaquine, amodiaquine, and desethylamodiaquine may cause clinically important interactions because they are predicted to inhibit 67 to 99% of the activities of the CYPs they interact with. In addition, our results suggest CYP1A2 inhibition as the mechanism behind the observed thiabendazole/theophylline and primaquine/antipyrine interactions in vivo.


Subject(s)
Antiprotozoal Agents/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Antiprotozoal Agents/pharmacokinetics , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme System/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacokinetics , Humans , In Vitro Techniques , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Spectrometry, Fluorescence
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