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BACKGROUND: Liver biotransformation is the major route for drug metabolism in humans, often catalysed by cytochrome P450 (CYP) enzymes. This first-pass effect can lead to hepatotoxicity and influences the bioavailability of drugs. OBJECTIVE: We aimed to establish in vitro culture systems simulating the liver first-pass to study effects of the proteasome inhibitor MG-132 simultaneously on hepatocytes and cancer cells. METHODS: The first-pass effect was simulated by conditioned medium transfer (CMT) from pre-treated HepG2 CYP3A4-overexpressing cells to either pancreatic cancer cell line PANC-1 or primary colon cancer cells, and by indirect co-culture (CC) of liver and cancer cells in a shared medium compartment. Experimental proteasome inhibitor MG-132 was used as test substance as it is detoxified by CYP3A4. RESULTS: Cancer cells showed higher viabilities in the first-pass simulation by CMT and CC formats when compared to monocultures indicating effective detoxification of MG-132 by HepG2 CYP3A4-overexpressing cells. HepG2-CYP3A4 cells showed reduced viabilites after treatment with MG-132. CONCLUSIONS: We successfully established two different culture systems to simulate the liver first-pass effect in vitro. Such systems easily allow to study drug effects simultaneously on liver and on target cancer cells. They are of great value in pre-clinical cancer research, pharmaceutical research and drug development. [ABSTRACT FROM AUTHOR]

BACKGROUND: Drug induced liver injury (DILI) is the most frequent cause for failure of new drugs in clinical studies. Thus, toxicity studies are indispensable during drug development. The proliferative human liver cell strain HepaFH3 with promising primary-like cellular properties might be a suitable liver model for such studies, but its cytochrome-P450 (CYP) expression is still in low ranges compared to freshly isolated primary human hepatocytes. OBJECTIVE: We aimed to optimize the induction protocol for CYP1A2 and CYP3A4 in HepaFH3 to obtain a physiologically relevant in vitro liver model. METHODS: CYP1A2 and CYP3A4 were induced by omeprazole and rifampicin, respectively. Induction of the two CYPs was measured by qRT-PCR, immunofluorescence and by P450 Glo enzyme activity assays. RESULTS: The optimized protocol made the experimental design six days shorter than the original procedure. CYP1A2 mRNA levels were induced 118-fold, CYP3A4 levels 36-fold. This result was also reflected at protein level. Enzymatic activity of CYP1A2 increased 3.7-fold and CYP3A4 activity increased 9.8-fold after induction. CONCLUSIONS: We succeeded in optimizing the induction protocol for HepaFH3 to such an extent that CYP1A2 and CYP3A4 are expressed in sufficient amounts that the cell strain can be used as a physiological relevant human liver model for in vitro toxicology studies. [ABSTRACT FROM AUTHOR]

Increased expression levels of KCNJ3 have been correlated with lymph node metastases and poor prognosis in patients with breast cancer, suggesting a prognostic role of KCNJ3 We aimed to establish protocols for the detection of KCNJ3 in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue. Several antibodies were tested for sensitivity and specificity by western blot, followed by optimisation of the immunohistochemistry (IHC) procedure and establishment of KCNJ3 mRNA in situ hybridisation (ISH). Methods were validated by processing 15 FFPE breast cancer samples for which microarray data were available. Spearman's rank correlation analysis resulted in borderline significant correlation for IHC versus ISH (r S : 0.625; p<0.05) and IHC versus microarray (r S : 0.668; p<0.01), but in significant correlation for ISH versus microarray (r S : 0.861; p<0.001). The ISH method was superior to IHC, regarding robustness, sensitivity and specificity and will aid to further study expression levels of KCNJ3 in both malignant and physiological conditions., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Aminopeptidase inhibitors are receiving attention as combination chemotherapeutic agents for the treatment of refractory acute myeloid leukemia. However, the factors determining therapeutic efficacy remain elusive. Here we identified the molecular basis of acquired resistance to CHR2863, an orally available hydrophobic aminopeptidase inhibitor prodrug with an esterase-sensitive motif, in myeloid leukemia cells. CHR2863 enters cells by diffusion and is retained therein upon esterase activity-mediated conversion to its hydrophilic active metabolite drug CHR6768, thereby exerting amino acid depletion. Carboxylesterases (CES) serve as candidate prodrug activating enzymes given CES1 expression in acute myeloid leukemia specimens. We established two novel myeloid leukemia sublines U937/CHR2863(200) and U937/CHR2863(5uM), with low (14-fold) and high level (270-fold) CHR2863 resistance. The latter drug resistant cells displayed: (i) complete loss of CES1-mediated drug activation associated with down-regulation of CES1 mRNA and protein, (ii) marked retention/sequestration of the prodrug, (iii) a substantial increase in intracellular lipid droplets, and (iv) a dominant activation of the pro-survival Akt/mTOR pathway. Remarkably, the latter feature coincided with a gain of sensitivity to the mTOR inhibitor rapamycin. These finding delineate the molecular basis of CHR2863 resistance and offer a novel modality to overcome this drug resistance in myeloid leukemia cells.