In vitro simulation of the liver first-pass effect with biotransformation-competent HepG2 cells to study effects of MG-132 on liver and cancer cells.

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.

Statins markedly potentiate aminopeptidase inhibitor activity against (drug-resistant) human acute myeloid leukemia cells.

Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells. Methods: U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and in vitro non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation. Results: A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents; and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation; this was experimentally confirmed by impaired Rheb prenylation by simvastatin. Conclusion: These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.

Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation-Relevance to Thrombosis.

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.

Alisertib shows negligible potential for perpetrating pharmacokinetic drug-drug interactions on ABCB1, ABCG2 and cytochromes P450, but acts as dual-activity resistance modulator through the inhibition of ABCC1 transporter.

Alisertib (MLN8237), a novel Aurora A kinase inhibitor, is currently being clinically tested in late-phase trials for the therapy of various malignancies. In the present work, we describe alisertib's potential to perpetrate pharmacokinetic drug-drug interactions (DDIs) and/or to act as an antagonist of multidrug resistance (MDR). In accumulation assays, alisertib potently inhibited ABCC1 transporter, but not ABCB1 or ABCG2. The results of molecular modeling suggested a bifunctional mechanism for interaction on ABCC1. In addition, alisertib was characterized as a low- to moderate-affinity inhibitor of recombinant CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 isoenzymes, but without potential clinical relevance. Drug combination studies revealed the capability of alisertib to synergistically antagonize ABCC1-mediated resistance to daunorubicin. Although alisertib exhibited substrate characteristics toward ABCB1 transporter in monolayer transport assays, comparative proliferation studies showed lack of its MDR-victim behavior in cells overexpressing ABCB1 as well as ABCG2 and ABCC1. Lastly, alisertib did not affect the expression of ABCC1, ABCG2, ABCB1 transporters and CYP1A2, CYP3A4, CYP2B6 isozymes on mRNA level in various systemic and tumoral models. In conclusion, our study suggests that alisertib is a drug candidate with negligible potential for perpetrating systemic pharmacokinetic DDIs on ABCB1, ABCG2 and cytochromes P450. In addition, we introduce alisertib as an effective dual-activity chemosensitizer whose MDR-antagonistic capacities are not impaired by efflux or effect on MDR phenotype. Our in vitro findings provide important pieces of information for clinicians when introducing alisertib into the clinical area.

Tepotinib Inhibits Several Drug Efflux Transporters and Biotransformation Enzymes: The Role in Drug-Drug Interactions and Targeting Cytostatic Resistance In Vitro and Ex Vivo.

Tepotinib is a novel tyrosine kinase inhibitor recently approved for the treatment of non-small cell lung cancer (NSCLC). In this study, we evaluated the tepotinib's potential to perpetrate pharmacokinetic drug interactions and modulate multidrug resistance (MDR). Accumulation studies showed that tepotinib potently inhibits ABCB1 and ABCG2 efflux transporters, which was confirmed by molecular docking. In addition, tepotinib inhibited several recombinant cytochrome P450 (CYP) isoforms with varying potency. In subsequent drug combination experiments, tepotinib synergistically reversed daunorubicin and mitoxantrone resistance in cells with ABCB1 and ABCG2 overexpression, respectively. Remarkably, MDR-modulatory properties were confirmed in ex vivo explants derived from NSCLC patients. Furthermore, we demonstrated that anticancer effect of tepotinib is not influenced by the presence of ABC transporters associated with MDR, although monolayer transport assays designated it as ABCB1 substrate. Finally, tested drug was observed to have negligible effect on the expression of clinically relevant drug efflux transporters and CYP enzymes. In conclusion, our findings provide complex overview on the tepotinib's drug interaction profile and suggest a promising novel therapeutic strategy for future clinical investigations.

Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro.

Drug-induced liver injury (DILI) is the major reason for failures in drug development and withdrawal of approved drugs from the market. Two-dimensional cultures of hepatocytes often fail to reliably predict DILI: hepatoma cell lines such as HepG2 do not reflect important primary-like hepatic properties and primary human hepatocytes (pHHs) dedifferentiate quickly in vitro and are, therefore, not suitable for long-term toxicity studies. More predictive liver in vitro models are urgently required in drug development and compound safety evaluation. This review discusses available human hepatic cell types for in vitro toxicology analysis and their usage in established and emerging three-dimensional (3D) culture systems. Generally, 3D cultures maintain or improve primary hepatic functions (including expression of drug-metabolizing enzymes) of different liver cells for several weeks of culture, thus allowing long-term and repeated-dose toxicity studies. Spheroid cultures of pHHs have been comprehensively tested, but also other cell types such as HepaRG benefit from 3D culture systems. Emerging 3D culture techniques include usage of induced pluripotent stem-cell-derived hepatocytes and primary-like upcyte cells, as well as advanced culture techniques such as microfluidic liver-on-a-chip models. In-depth characterization of existing and emerging 3D hepatocyte technologies is indispensable for successful implementation of such systems in toxicological analysis.

Phycocyanin from Arthrospira platensis as Potential Anti-Cancer Drug: Review of In Vitro and In Vivo Studies.

The application of cytostatic drugs or natural substances to inhibit cancer growth and progression is an important and evolving subject of cancer research. There has been a surge of interest in marine bioresources, particularly algae, as well as cyanobacteria and their bioactive ingredients. Dried biomass products of Arthrospira and Chlorella have been categorized as "generally recognized as safe" (GRAS) by the US Food and Drug Administration (FDA). Of particular importance is an ingredient of Arthrospira: phycocyanin, a blue-red fluorescent, water-soluble and non-toxic biliprotein pigment. It is reported to be the main active ingredient of Arthrospira and was shown to have therapeutic properties, including anti-oxidant, anti-inflammatory, immune-modulatory and anti-cancer activities. In the present review, in vitro and in vivo data on the effects of phycocyanin on various tumor cells and on cells from healthy tissues are summarized. The existing knowledge of underlying molecular mechanisms, and strategies to improve the efficiency of potential phycocyanin-based anti-cancer therapies are discussed.

Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors.

Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity.

Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells.

Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPAUPO). 4-OH-CPAUPO-induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPAUPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment.

Ensartinib (X-396) Effectively Modulates Pharmacokinetic Resistance Mediated by ABCB1 and ABCG2 Drug Efflux Transporters and CYP3A4 Biotransformation Enzyme.

Ensartinib (X-396) is a promising tyrosine kinase inhibitor currently undergoing advanced clinical evaluation for the treatment of non-small cell lung cancer. In this work, we investigate possible interactions of this promising drug candidate with ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 biotransformation enzymes (CYPs), which play major roles in multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). Accumulation studies showed that ensartinib is a potent inhibitor of ABCB1 and ABCG2 transporters. Additionally, incubation experiments with recombinant CYPs showed that ensartinib significantly inhibits CYP3A4 and CYP2C9. Subsequent molecular docking studies confirmed these findings. Drug combination experiments demonstrated that ensartinib synergistically potentiates the antiproliferative effects of daunorubicin, mitoxantrone, and docetaxel in ABCB1, ABCG2, and CYP3A4-overexpressing cellular models, respectively. Advantageously, ensartinib's antitumor efficiency was not compromised by the presence of MDR-associated ABC transporters, although it acted as a substrate of ABCB1 in Madin-Darby Canine Kidney II (MDCKII) monolayer transport assays. Finally, we demonstrated that ensartinib had no significant effect on the mRNA-level expression of examined transporters and enzymes in physiological and lung tumor cellular models. In conclusion, ensartinib may perpetrate clinically relevant pharmacokinetic DDIs and modulate ABCB1-, ABCG2-, and CYP3A4-mediated MDR. The in vitro findings presented here will provide a valuable foundation for future in vivo investigations.

HepG2-1A2 C2 and C7: Lentivirus vector-mediated stable and functional overexpression of cytochrome P450 1A2 in human hepatoblastoma cells.

Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min-1 x mg-1 protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B1 (EC50 < 100 nM) when compared to parental HepG2 cells (EC50∼5 µM). Thus, newly established cell lines are an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP1A2.

Brivanib Exhibits Potential for Pharmacokinetic Drug-Drug Interactions and the Modulation of Multidrug Resistance through the Inhibition of Human ABCG2 Drug Efflux Transporter and CYP450 Biotransformation Enzymes.

Brivanib, a promising tyrosine kinase inhibitor, is currently undergoing advanced stages of clinical evaluation for solid tumor therapy. In this work, we investigated possible interactions of this novel drug candidate with ABC drug efflux transporters and cytochrome P450 (CYP450) drug-metabolizing enzymes that participate in cancer multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). First, in accumulation experiments with various model substrates, we identified brivanib as an inhibitor of the ABCB1, ABCG2, and ABCC1 transporters. However, in subsequent combination studies employing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide proliferation assays in both Madin-Darby canine kidney II (MDCKII) and A431 cellular models, only ABCG2 inhibition was revealed to be able to synergistically potentiate mitoxantrone effects. Advantageous to its possible use as MDR antagonist, brivanib's chemosensitizing properties were not impaired by activity of any of the MDR-associated ABC transporters, as observed in comparative viability assay in the MDCKII cell sublines. In incubation experiments with eight recombinant CYP450s, we found that brivanib potently inhibited CYP2C subfamily members and the CYP2B6 isoform. Finally, in induction studies, we demonstrated that brivanib upregulated ABCB1 and CYP1A2 messenger RNA levels in systemic cell models, although this interaction was not significantly manifested at a functional level. In conclusion, brivanib exhibits potential to cause clinically relevant pharmacokinetic DDIs and act as a modulator of ABCG2-mediated MDR. Our findings might be used as an important background for subsequent in vivo investigations and pave the way for the safe and effective use of brivanib in oncological patients.

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies.

BACKGROUND: Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes. OBJECTIVE: For Upcyte® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte® hepatocytes representing proliferating primary-like hepatocytes. METHODS: POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method. RESULTS: The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3(p = 0.0829) might be present. Compared to primary human hepatocyte microsomes, POR activity was considerably lower in all hepatocyte models. CONCLUSION: In summary, our study revealed that POR expression and activity were clearly detectable in all in vitro hepatocyte models with the highest POR expression in cancer cell line HepG2. However, POR activity was lower in tested hepatocyte models when compared to human primary hepatocyte microsomes. Whether this was caused by e.g. polymorphisms or metabolic differences of investigated hepatocyte models will be target for future studies.

Interactions of Alectinib with Human ATP-Binding Cassette Drug Efflux Transporters and Cytochrome P450 Biotransformation Enzymes: Effect on Pharmacokinetic Multidrug Resistance.

Alectinib is a tyrosine kinase inhibitor currently used as a first-line treatment of anaplastic lymphoma kinase-positive metastatic nonsmall cell lung cancer (NSCLC). In the present work, we investigated possible interactions of this novel drug with ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 (P450) biotransformation enzymes that play significant roles in the phenomenon of multidrug resistance (MDR) of cancer cells as well as in pharmacokinetic drug-drug interactions. Using accumulation studies in Madin-Darby canine kidney subtype 2 (MDCKII) cells alectinib was identified as an inhibitor of ABCB1 and ABCG2 but not of ABCC1. In subsequent drug combination studies, we demonstrated the ability for alectinib to effectively overcome MDR in ABCB1- and ABCG2-overexpressing MDCKII and A431 cells. To describe the pharmacokinetic interaction profile of alectinib in a complete fashion, its possible inhibitory properties toward clinically relevant P450 enzymes (i.e., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) were evaluated using human P450-expressing insect microsomes, revealing alectinib as a poor interactor. Advantageously for its use in pharmacotherapy, alectinib further exhibited negligible potential to cause any changes in expression of ABCB1, ABCG2, ABCC1, CYP1A2, CYP3A4, and CYP2B6 in intestine, liver, and NSCLC models. Our in vitro observations might serve as a valuable foundation for future in vivo studies that could support the rationale for our conclusions and possibly enable providing more efficient and safer therapy to many oncological patients.

Metabolic activity testing can underestimate acute drug cytotoxicity as revealed by HepG2 cell clones overexpressing cytochrome P450 2C19 and 3A4.

Preclinical drug safety assessment includes in vitro studies with physiologically relevant cell cultures. As an in vitro system for hepatic toxicology testing, we have been generating cell clones of human hepatoblastoma cell line HepG2 by lentiviral transduction of phase I cytochrome P450 (CYP) enzymes. Here, we present a stable CYP2C19-overexpressing HepG2 cell clone (HepG2-2C19 C1) showing an enzyme activity of approximately 82 pmol x min-1 x mg-1 total cellular protein. The phenotypic stability over several passages of HepG2-2C19 C1 renders them to be a suitable reference cell clone for benchmarking CYP2C19 enzyme activity. In addition, we were interested to analyze acute cytotoxicity of the model drug cyclophosphamide (CPA) metabolized by HepG2-2C19 C1 and by a previously generated CYP3A4-overexpressing HepG2 cell clone. Upon 10 mM CPA exposure, we were able to detect its metabolites 4-hydroxy-cyclophosphamide and acrolein in CYP3A4- and CYP2C19-expressing cell clones, but not in parental HepG2 cell line. XTT and ATP assays showed a modest reduction of cell viability of not more than 50% with high dose (10 mM) CPA treatment. By contrast, dramatic acute cytotoxic effects of CPA were evident by the formation of nuclear γH2AX foci and by increased cell death events. These effects were paralleled by substantial decreases of cell membrane integrity as measured by the trypan blue exclusion test. Our data on CYP enzyme overexpressing HepG2 cell clones clearly show that cytotoxicity of CPA is dramatically underestimated by standard metabolic activity tests. Thus, additional tests to quantitate DNA damage formation and cell death induction might be required to realistically assess cytotoxicity of such compounds.

Impact of resection on overall survival of recurrent Glioblastoma in elderly patients.

OBJECTIVES: Glioblastoma multiforme (GBM) mostly affects elderly patients. Adequate therapy especially in case of tumor recurrence is still under discussion, since most studies focus on patients under 65 years. We evaluated the impact of second surgery in regard to progression free survival (PFS) and overall survival (OAS) in elderly patients. PATIENTS AND METHODS: 59 patients with recurrent glioblastoma multiforme were retrospectively evaluated. Patients were divided into three subgroups: stereotactic biopsy (STX), resection and second resection. Second and third group were pooled as "surgery" after first diagnosis. The median age for all groups was 71 years.The primary endpoint was overall survival. Statistical analysis was calculated using Kaplan-Meier analysis and SPSS. RESULTS: OAS was significantly longer for patients who had undergone surgery compared to the STX group. For patients who underwent reresection OAS was not significantly longer. Age (over 71) had no significant effect on OAS. PFS was significantly increased in patients who underwent surgery compared to those who underwent STX. PFS was not significantly longer in patients who underwent second resection. Furthermore PFS was not significantly different between patients under 71 and over. CONCLUSION: OAS and PFS were significant increased for patients who underwent surgery compared to only STX. Patients showed prolonged survival of almost 4 months after they underwent reresection (p > 0.05). Therapy of elderly patients with GBM remains an individual decision with priority on quality of life. Clinical status, comorbidities and family background) should be taken into account.

ATP-based cell viability assay is superior to trypan blue exclusion and XTT assay in measuring cytotoxicity of anticancer drugs Taxol and Imatinib, and proteasome inhibitor MG-132 on human hepatoma cell line HepG2.

BACKGROUND: Drug-induced liver injury (DILI) is the most common reason for withdrawal of anticancer drugs from the market. To prevent adverse side effects of drugs, it is important to investigate potential toxicity in vitro. However, outcome of cytotoxicity screenings can differ remarkably depending on the method used. OBJECTIVE: We aimed to compare XTT, ATP-based CellTiter-Glo®2.0 and trypan blue exclusion (TBE) assays regarding their sensitivity in detecting acute cytotoxicity on HepG2 cells after incubation with the classical anticancer drugs Taxol and Imatinib or with the proteasome inhibitor MG-132. METHODS: HepG2 cells were treated for 48 h and cell viability was analysed by XTT, CellTiter-Glo®2.0 or TBE assay. RESULTS: All tested compounds showed a reduction of viability of HepG2 cells. However, assay results differed significantly: Both ATP-based and TBE assay showed concentration-dependent cytotoxic effects, but outcomes were less pronounced with TBE. In contrast, the widely used XTT assay did not detect any acute cytotoxicity of Taxol and Imatinib. CONCLUSIONS: Acute cytotoxic effects of tested compounds could be revealed. However, results were significantly different from each other with ATP assay being the most sensitive one under the conditions tested. Thus, acute cytotoxicity can be dramatically underestimated if only standard XTT test is used.

KCNJ3 is a new independent prognostic marker for estrogen receptor positive breast cancer patients.

Numerous studies showed abnormal expression of ion channels in different cancer types. Amongst these, the potassium channel gene KCNJ3 (encoding for GIRK1 proteins) has been reported to be upregulated in tumors of patients with breast cancer and to correlate with positive lymph node status. We aimed to study KCNJ3 levels in different breast cancer subtypes using gene expression data from the TCGA, to validate our findings using RNA in situ hybridization in a validation cohort (GEO ID GSE17705), and to study the prognostic value of KCNJ3using survival analysis. In a total of > 1000 breast cancer patients of two independent data sets we showed a) that KCNJ3 expression is upregulated in tumor tissue compared to corresponding normal tissue (p < 0.001), b) that KCNJ3 expression is associated with estrogen receptor (ER) positive tumors (p < 0.001), but that KCNJ3 expression is variable within this group, and c) that ER positive patients with high KCNJ3 levels have worse overall (p < 0.05) and disease free survival probabilities (p < 0.01), whereby KCNJ3 is an independent prognostic factor (p <0.05). In conclusion, our data suggest that patients with ER positive breast cancer might be stratified into high risk and low risk groups based on the KCNJ3 levels in the tumor.

Critical evaluation of KCNJ3 gene product detection in human breast cancer: mRNA in situ hybridisation is superior to immunohistochemistry.

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 (rS: 0.625; p<0.05) and IHC versus microarray (rS: 0.668; p<0.01), but in significant correlation for ISH versus microarray (rS: 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.

Multifactorial resistance to aminopeptidase inhibitor prodrug CHR2863 in myeloid leukemia cells: down-regulation of carboxylesterase 1, drug sequestration in lipid droplets and pro-survival activation ERK/Akt/mTOR.

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.