Matthias U. Kassack

Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines.

Cell viability assays are important tools in oncological research and clinical practice to assess the tumor cell sensitivity of individual patients. The purpose of this study was to demonstrate the comparability of 3 widely used assays (MTT, ATP, calcein assays) by principal component analysis. The study included 4 different cytostatics (cisplatin, docetaxel, doxorubicin, vinblastine) and 3 different human cancer cell lines (MCF-7, A2780, doxorubicin resistant A2780adr). Ninety-three percent of the total variance of all variables included in the principal component analysis (resulting from 3 cell lines and 3 assays) could be explained by 1 principal component. Factor loadings were > 0.937 except for the variable MTT-A2780adr, which was 0.872. These results indicate the similarity of the 3 assays. A 2nd principal component analysis included literature data and showed accordance of data from this study and the literature. The MTT assay was further improved as a high-throughput screening–capable assay. The ATP assay is able to detect effects of cytostatics already after 1 h incubation. The determination of resistance factors allowed to differentiate cytostatics into P-gp or non-P-gp substrates. In conclusion, this study provides improved microplate reader-based cell viability assays and sets a statistically solid basis for a future comparison of data obtained in different laboratories by any of the 3 assays.

Common variants in P2RY11 are associated with narcolepsy

Growing evidence supports the hypothesis that narcolepsy with cataplexy is an autoimmune disease. We here report genome-wide association analyses for narcolepsy with replication and fine mapping across three ethnic groups (3,406 individuals of European ancestry, 2,414 Asians and 302 African Americans). We identify a SNP in the 3′ untranslated region of P2RY11, the purinergic receptor subtype P2Y11 gene, which is associated with narcolepsy (rs2305795, combined P = 6.1 × 10−10, odds ratio = 1.28, 95% CI 1.19–1.39, n = 5689). The disease-associated allele is correlated with reduced expression of P2RY11 in CD8+ T lymphocytes (72% reduced, P = 0.003) and natural killer (NK) cells (70% reduced, P = 0.031), but not in other peripheral blood mononuclear cell types. The low expression variant is also associated with reduced P2RY11-mediated resistance to ATP-induced cell death in T lymphocytes (P = 0.0007) and natural killer cells (P = 0.001). These results identify P2RY11 as an important regulator of immune-cell survival, with possible implications in narcolepsy and other autoimmune diseases.

Structure-activity studies on suramin analogues as inhibitors of NAD+-dependent histone deacetylases (sirtuins).

Suramin is a symmetric polyanionic naphthylurea originally used for the treatment of trypanosomiasis and onchocerciasis. Suramin and diverse analogues exhibit a broad range of biological actions in vitro and in vivo, including, among others, antiproliferative and antiviral activity. Suramin derivatives usually target purinergic binding sites. Class III histone deacetylases (sirtuins) are amidohydrolases that require nicotinamide adenine dinucleotide (NAD+) as a cofactor for their catalytic mechanism. Deacetylation of the target proteins leads to a change in conformation and alters the activity of the proteins in question. Suramin was reported to inhibit human sirtuin 1 (SIRT1). We tested a diverse set of suramin analogues to elucidate the inhibition of the NAD+‐dependent histone deacetylases SIRT1 and SIRT2 and discovered selective inhibitors of human sirtuins with potency in the two‐digit nanomolar range. In addition, the structural requirements for the binding of suramin derivatives to sirtuins were investigated by molecular docking. The recently published X‐ray crystal structure of human SIRT5 in complex with suramin and the human SIRT2 structure were used to analyze the interaction mode of the novel suramin derivatives.

Hyperactivation of the Insulin-like Growth Factor Receptor I Signaling Pathway Is an Essential Event for Cisplatin Resistance of Ovarian Cancer Cells

Platinum plays a central role in the therapy of ovarian cancer, and the emergence of platinum resistance is a major obstacle for clinical management of the disease. We treated A2780 ovarian cancer cells by weekly cycles of cisplatin over a period of 6 months and unveiled that enhanced insulin-like growth factor I receptor (IGF-IR) expression and autocrine IGF-I are associated with hyperactivation of the IGF-IR and phosphatidylinositol-3-OH kinase (PI3K) pathways in cisplatin-resistant cells. IGF-IR expression levels increased during treatment cycles and correlated with cisplatin resistance. Purified IGF-I induced cisplatin resistance in diverse ovarian cancer cell lines, and small molecule inhibitors proved that IGF-IR and PI3K are essential for cisplatin resistance. Similar results were obtained with BG-1 ovarian cancer cells. Cytogenetic and array comparative genomic hybridization analyses revealed selection and de novo formation of chromosomal alterations during resistance development. An analysis of gene expression profiles of primary ovarian carcinomas identified the regulatory subunit PIK3R2 of PI3-kinase as a significant negative prognosis factor for ovarian cancer. We conclude that targeting the IGF-IR and the PI3K pathways is a promising new strategy to treat cisplatin-resistant ovarian carcinomas.

Discovery of Potent and Selective Agonists for the Free Fatty Acid Receptor 1 (FFA1/GPR40), a Potential Target for the Treatment of Type II Diabetes

A series of 4-phenethynyldihydrocinnamic acid agonists of the free fatty acid receptor 1 (FFA(1)) has been discovered and explored. The preferred compound 20 (TUG-424, EC(50) = 32 nM) significantly increased glucose-stimulated insulin secretion at 100 nM and may serve to explore the role of FFA(1) in metabolic diseases such as diabetes or obesity.

Extracellular NAD+ Is an Agonist of the Human P2Y11 Purinergic Receptor in Human Granulocytes

Micromolar concentrations of extracellular β-NAD+ (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document}) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document} generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document}-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y11 receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y11-transfected 1321N1 astrocytoma cells: micromolar \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document} promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y11 but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y11, and the down-regulation of P2Y11 expression by short interference RNA prevented \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document}-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\beta}\mathrm{-}\mathrm{NAD}_{e}^{+}\) \end{document} is an agonist of the P2Y11 purinoceptor and that P2Y11 is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.

Epidermal Growth Factor Receptor Pathway Analysis Identifies Amphiregulin as a Key Factor for Cisplatin Resistance of Human Breast Cancer Cells

The use of platinum complexes for the therapy of breast cancer is an emerging new treatment modality. To gain insight into the mechanisms underlying cisplatin resistance in breast cancer, we used estrogen receptor-positive MCF-7 cells as a model system. We generated cisplatin-resistant MCF-7 cells and determined the functional status of epidermal growth factor receptor (EGFR), MAPK, and AKT signaling pathways by phosphoreceptor tyrosine kinase and phospho-MAPK arrays. The cisplatin-resistant MCF-7 cells are characterized by increased EGFR phosphorylation, high levels of AKT1 kinase activity, and ERK1 phosphorylation. In contrast, the JNK and p38 MAPK modules of the MAPK signaling pathway were inactive. These conditions were associated with inactivation of the p53 pathway and increased BCL-2 expression. We investigated the expression of genes encoding the ligands for the ERBB signaling cascade and found a selective up-regulation of amphiregulin expression, which occurred at later stages of cisplatin resistance development. Amphiregulin is a specific ligand of the EGFR (ERBB1) and a potent mitogen for epithelial cells. After exposure to cisplatin, the resistant MCF-7 cells secreted amphiregulin protein over extended periods of time, and knockdown of amphiregulin expression by specific short interfering RNA resulted in a nearly complete reversion of the resistant phenotype. To demonstrate the generality and importance of our findings, we examined amphiregulin expression and cisplatin resistance in a variety of human breast cancer cell lines and found a highly significant correlation. In contrast, amphiregulin levels did not significantly correlate with cisplatin resistance in a panel of lung cancer cell lines. We have thus identified a novel function of amphiregulin for cisplatin resistance in human breast cancer cells.

Inhibition of Neutrophil Apoptosis by ATP Is Mediated by the P2Y11 Receptor

Neutrophils undergo rapid constitutive apoptosis that is delayed by a range of pathogen- and host-derived inflammatory mediators. We have investigated the ability of the nucleotide ATP, to which neutrophils are exposed both in the circulation and at sites of inflammation, to modulate the lifespan of human neutrophils. We found that physiologically relevant concentrations of ATP cause a concentration-dependent delay of neutrophil apoptosis (assessed by morphology, annexin V/To-Pro3 staining, and mitochondrial membrane permeabilization). We found that even brief exposure to ATP (10 min) was sufficient to cause a long-lasting delay of apoptosis and showed that the effects were not mediated by ATP breakdown to adenosine. The P2 receptor mediating the antiapoptotic actions of ATP was identified using a combination of more selective ATP analogs, receptor expression studies, and study of downstream signaling pathways. Neutrophils were shown to express the P2Y11 receptor and inhibition of P2Y11 signaling using the antagonist NF157 abrogated the ATP-mediated delay of neutrophil apoptosis, as did inhibition of type I cAMP-dependent protein kinases activated downstream of P2Y11, without effects on constitutive apoptosis. Specific targeting of P2Y11 could retain key immune functions of neutrophils but reduce the injurious effects of increased neutrophil longevity during inflammation.

Purinergic Receptors Influence the Differentiation of Human Mesenchymal Stem Cells

Adult stem cells, including adipose tissue-derived mesenchymal stem cells (MSCs) or ectomesenchymal dental follicle cells (DFCs), attract considerable attention for their potential to differentiate into lineages, which are of major interest in the field of Regenerative Medicine. Purinergic receptors exert a wide range of biological actions in many cell and tissue types through extracellular nucleotides. Little is known about P2 receptors in adult stem cells and changes in their expression levels during differentiation. All known P2 receptors have been investigated, and a variety of P2X and P2Y receptor subtypes were detected in MSCs. Studies investigating intracellular calcium levels on receptor stimulation demonstrated that the found P2 receptors are metabolically active. Interestingly, up- or downregulation of several P2 receptor subtypes at gene and protein level was observed during adipogenic and osteogenic differentiation, and the effect on differentiation was directly influenced by both the application of agonists/antagonists and apyrase-induced nucleotide cleavage. Here, we show for the first time that the combination of several P2 receptors plays a role in the differentiation of adult stem cells. The expression pattern of the P2 receptors, as well as their fate in differentiation, varies in stem cells of mesenchymal origin if compared with stem cells of ectomesenchymal origin. The subtypes P2X6, P2Y4, and P2Y14 seem to be pivotal regulators in MSC commitment, as they are regulated in both adipogenic and osteogenic differentiation of adipose tissue-derived stem cells and DFCs. These findings provide new insights into the differentiation processes and might reveal novel options to influence stem cell fate in future applications.

Functional Screening of G Protein—Coupled Receptors by Measuring Intracellular Calcium with a Fluorescence Microplate Reader

Ligand binding studies reveal information about affinity to G protein—coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca2+ appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca2+ signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca2+ fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested—Gq-coupled P2Y2, Gs-coupled dopamine D1 and D5, Gi-coupled dopamine D2L, and Gq/11-coupled muscarinic acetylcholine M1—yielded a significant rise in intracellular free [Ca2+] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y2 receptors (EC50 = 1 μM), SKF38393 stimulation of hD1 and hD5 (EC50 = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC50 = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca2+ response. Furthermore, the Ca2+ assay served to screen suramin analogs for antagonistic activity at P2Y2 receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca2+ fluxes.