Charakterisierung der Wirkmechanismen des neuartigen, dual wirksamen HDAC-Inhibitors „Animacroxam“ am Beispiel testikulärer Keimzelltumore

Die meistvorkommende maligne Erkrankung bei Männern zwischen 20 und 45 Jahren ist der testikuläre Keimzelltumor (TKZT). Mit platinbasierter Chemotherapie können bis zu 80 Prozent aller Patienten erfolgreich behandelt werden. Bei den verbleibenden 20 Prozent der Fälle hingegen kommt es häufig zur Entwicklung von Platinresistenzen, wodurch sich die Fünf-Jahres-Überlebensrate drastisch senkt. Für diese sehr junge Patientengruppe ist die Entwicklung neuer Therapieansätze dringend notwendig. Als vielversprechender Ansatz in der Tumortherapie hat der Einsatz von Dualinhibitoren in den letzten Jahren an Bedeutung gewonnen. In dieser Arbeit wurde Animacroxam, eine dual wirksame Substanz bestehend aus einer Histonedeacetylase- (HDAC) und einer Zytoskelett-inhibierenden Pharmacophore, auf ihre Wirksamkeit in testikulären Keimzelltumoren untersucht. Die Wirkung von Animacroxam wurde dabei mit der Wirksamkeit des Goldstandards in der TKZT-Therapie Cisplatin und dem klinisch zugelassenen HDAC-Inhibitor (HDACi) Vorinostat verglichen. Animacroxam zeigte dabei eine dosisabhängige Proliferationshemmung in TKZT-Zellen von bis zu 95 Prozent, ungeachtet der Cisplatinsensitivität der untersuchten Zelllinien. Dabei konnte keine unspezifische Zytotoxizität in den verschiedenen Zelllinien festgestellt werden. Zellzyklusuntersuchungen ergeben, dass Animacroxam zu einer ausgeprägten Akkumulation von Zellen in der G0/G1-Phase der untersuchten TKZT-Zellen führt. Zusätzlich konnte gezeigt werden, dass Animacroxam die Bildung von Aktin-Stressfasern induziert. Dies bestätigt, dass der Imidazolanteil das Zytoskelett der untersuchten TKZT-Zellen direkt angreift. Zusätzlich wurde auch die Migration von TKZT-Zellen durch Animacroxam signifikant gehemmt. Außerdem konnte gezeigt werden, dass Animacroxam dosisabhängig zu einem Zusammenbruch des intrazellulären Glukosestoffwechsels in TKZT-Zellen führt und die Angioge- nese von Endothelzellen in vitro hemmt. Zusätzlich kam es zu einer Reduktion von Connexin 43 (Cx43) und zu einer Inhibition der gapjunktionalen Zell-Zellkommunikation. Die antineoplastischen und antiangiogenen Effekte von Animacroxam wurden in einem erweiterten Chorioallantoismembran (CAM)-Modell befruchteter Hühnereier in vivo untersucht. Moderne Magnetresonanztomographie-Bildgebung (MRT) und Positron-Emissions-Tomographie-Bildgebung (PET) wurden zur Bestimmung des Tumorvolumens und zur Messung der Aufnahme von radioaktiv markierter Glukose (2-Fluor-2-desoxy-D-glucose oder F18-FDG) verwendet. So konnte ein deutlich reduziertes Tumorvolumen sowie eine signifikant reduzierte Aufnahme von Glukose in auf der CAM inokulierten TKZT nach Behandlung mit Animacroxam festgestellt werden. Die vorliegende Arbeit zeigt, dass der Dualinhibitor Animacroxam einen vielversprechenden Ansatz zur Behandlung von TKZT darstellt. Um zu einer abschließenden Bewertung der Wirksamkeit von Animacroxam zu gelangen, werden weitere In-vivo-Untersuchungen benötigt. Die vorliegenden Daten können als Grundlage dazu dienen.The most common malignancy in males between 20 and 45 years of age is the testicular germ cell tumor (TGCT). Using platin-based chemotherapy, up to 80 percent of the patients can be cured. However, the remaining 20 percent often develop cisplatin resistance, which reduces the five-year survival rate considerably. Therefore, this young patient group urgently needs the development of new treatment strategies. As a promising approach in tumor therapy, the development of dual-mode inhibitors has gained increased attention over the last years. In this study, the new dual-mode inhibitor animacroxam, comprising of a histonedeacetylase- (HDAC) and a cytoskeleton-inhibitory pharmacophore, is investigated for the first time to assess the effectiveness in TGCT. The effect of animacroxam is compared with the effects of the gold standard on TGCT therapy, namely cisplatin and the established HDAC-inhibitor (HDACi) vorinostat. Animacroxam shows a dose-dependent proliferation inhibition in TGCT cells up to 95 percent, irrespective of the cisplatin sensitivity of the investigated cell lines. Additionally, animacroxam does not induce unspecific cytotoxicity. Cell-cycle investigations reveal a pronounced accumulation of cells in the G0/G1-phase of the cell cycle after incubation with animacroxam in the investigated TGCT cell lines. Animacroxam is also able to induce the formation of actin-stressfibers, thereby directly targeting the cytoskeleton of the TGCT cells. Furthermore, the migration of TGCT cells is significantly inhibited after incubation with animacroxam. Investigations on the glucose metabolism of TGCT cells revealed that animacroxam causes a dose-dependent breakdown of cellular glycolysis. Animacroxam is also able to inhibit angiogenesis in vitro and cause a significant reduction of connexin 43 (cx43) and a concomitant inhibition of the gap-junctional intercellular communication in endothelial cells. The antineoplastic and antiangiogenic effects in vivo were investigated using an advanced chorioallantoic membrane (CAM) model of fertilized chicken eggs. State-of-the-art magnet resonance imaging (MRI) and positron-emission tomography (PET) were used to non-invasively determine the tumor volume and the uptake of radioactive labeled glucose (18F-FDG) of inoculated TGCT on the CAM. Treatment with animacroxam induces a significantly reduced tumor volume and glucose uptake in inoculated TGCTs. This study shows that the dual-mode inhibitor animacroxam is a promising candidate for the treatment of TGCT. Further in vivo investigations are warranted for a final evaluation of the effectiveness of animacroxam. The presented data may serve as a basis for these investigations

The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells

The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-kappa B and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4

Antitumor and antiangiogenic activity of the novel chimeric inhibitor animacroxam in testicular germ cell cancer

Chimeric inhibitors, which merge two drug pharmacophores in a single molecule have become a prominent approach for the design of novel anticancer compounds. Here, we examined animacroxam, which combines histone deacetylase (HDAC) inhibitory and cytoskeleton-interfering pharmacophores, in testicular germ cell tumors (TGCT). The effectiveness of animacroxam was compared to that of the commonly applied chemotherapeutic cisplatin as well as the clinically approved HDAC inhibitor vorinostat. The antineoplastic and antiangiogenic effects of animacroxam on TGCT in vivo were assessed through exploratory animal studies and a modified chorioallantoic membrane assay, revealing that animacroxam has significant antitumor activity in TGCT. A novel positron emission tomography/MR-imaging approach was applied to determine tumor volume and glucose [2-fluoro-2-deoxy-d-glucose (18F-FDG)] uptake in TGCT tumors, revealing reduced glucose uptake in animacroxam-treated TGCTs and showing a dose-dependent suppression of glycolytic enzymes, which led to a breakdown in glycolytic energy production. Furthermore, the observed antiangiogenic effects of animacroxam were related to its ability to inhibit endothelial cell-cell communication, as the expression of gap junction-forming connexin 43 was strongly suppressed, and gap-junctional intercellular mass transport was reduced. Our data suggest that the chimeric HDAC inhibitor animacroxam may become a promising candidate for the treatment of solid cancers and may serve as an interesting alternative to platinum-based therapies