EGFR is not a major driver for osteosarcoma cell growth in vitro but contributes to starvation and chemotherapy resistance

Background Enhanced signalling via the epidermal growth factor receptor (EGFR) is a hallmark of multiple human carcinomas. However, in recent years data have accumulated that EGFR might also be hyperactivated in human sarcomas. Aim of this study was to investigate the influence of EGFR inhibition on cell viability and its interaction with chemotherapy response in osteosarcoma cell lines. Methods We have investigated a panel of human osteosarcoma cell lines regarding EGFR expression and downstream signalling. To test its potential applicability as therapeutic target, inhibition of EGFR by gefitinib was combined with osteosarcoma chemotherapeutics and cell viability, migration, and cell death assays were performed. Results Osteosarcoma cells expressed distinctly differing levels of functional EGFR reaching in some cases high amounts. Functionality of EGFR in osteosarcoma cells was proven by EGF-mediated activation of both MAPK and PI3K/AKT pathway (determined by phosphorylation of ERK1/2, AKT, S6, and GSK3). The EGFR-specific inhibitor gefitinib blocked EGF-mediated downstream signal activation. At standard in vitro culture conditions, clinically achievable gefitinib doses demonstrated only limited cytotoxic activity, however, significantly reduced long-term colony formation and cell migration. In contrast, under serum-starvation conditions active gefitinib doses were distinctly reduced while EGF promoted starvation survival. Importantly, gefitinib significantly supported the anti-osteosarcoma activities of doxorubicin and methotrexate regarding cell survival and migratory potential. Conclusion Our data suggest that EGFR is not a major driver for osteosarcoma cell growth but contributes to starvation- and chemotherapy-induced stress survival. Consequently, combination approaches including EGFR inhibitors should be evaluated for treatment of high-grade osteosarcoma patients.(VLID)486733

Landomycins as Glutathione-Depleting Agents and Natural Fluorescent Probes for Cellular Michael Adduct-Dependent Quinone Metabolism

Landomycins are angucyclines with promising antineoplastic activity produced by Streptomyces bacteria. The aglycone landomycinone is the distinctive core, while the oligosaccharide chain differs within derivatives. Herein, we report that landomycins spontaneously form Michael adducts with biothiols, including reduced cysteine and glutathione, both cell-free or intracellularly involving the benz[a]anthraquinone moiety of landomycinone. While landomycins generally do not display emissive properties, the respective Michael adducts exerted intense blue fluorescence in a glycosidic chain-dependent manner. This allowed label-free tracking of the short-lived nature of the mono-SH-adduct followed by oxygen-dependent evolution with addition of another SH-group. Accordingly, hypoxia distinctly stabilized the fluorescent mono-adduct. While extracellular adduct formation completely blocked the cytotoxic activity of landomycins, intracellularly it led to massively decreased reduced glutathione levels. Accordingly, landomycin E strongly synergized with glutathione-depleting agents like menadione but exerted reduced activity under hypoxia. Summarizing, landomycins represent natural glutathione-depleting agents and fluorescence probes for intracellular anthraquinone-based angucycline metabolism

Characterization of fibroblast growth factor receptor 4 as contributor to oncogenesis in Glioblastoma multiforme

Mit 45-55% aller malignen Gliomen ist das Glioblastom (GBM) der häufigste Tumor des zentralen Nervensystems (ZNS) in Erwachsenen. Das GBM ist der aggressivste primäre Gehirntumor und gehört zur Gruppe der hochgradigen Astrozytome, welche sich durch eine besonders niedrige Überlebenszeit von 14 Monaten auszeichnet. Besonders charakteristisch für das GBM sind seine heterogene Zellpopulation, starke Vaskularisierung und strichförmige Nekrosen, welche die wichtigsten histo- pathologischen Merkmale des Tumors darstellen. In Bezug auf die molekular-diagnostischen Eigenschaften dieser Entität sind besonders Mutationen in den Genen der Isozitrat-Dehydrogenase 1/2 (IDH1/2), des Telomerase reverse Transkriptase (TERT) Promoters, des O6-Methylguanin Methyltransferase (MGMT) Promoters und Amplifikationen des epidermalen Wachstumsfakor-Rezeptor (EGFR) Gens nennenswert. Trotz intensiver Forschung im Bereich der zielgerichteten Krebstherapie, wurden bisher keine GBM-spezifischen Biomarker identifiziert und auch die zuvor genannten molekularen Marker konnten nicht dafür genutzt werden. Frühere Beobachtungen in unsere Arbeitsgruppe ergaben gesteigerte Zellproliferation und Tumorwachstum in vivo welche stark von fibroblastischen Wachstumsfaktoren (FGFs) beeinflusst wurden. Basierend auf diesen vorherigen Ergebnissen konzentriert sich diese Studie auf FGF Rezeptor 4 (FGFR4), die Unterschiede zwischen seinen Varianten (Arg388 und Gly388), und dessen Rolle in GBM -Wachstum, -Aggressivität, -migration und 3D-Wachstum in vitro. Um FGFR4 in FGFR4 niedrig exprimierende GBM Zelllinien über zu exprimieren, wurden U251MG und HU-MI mit FGFR4 Arg388 oder Gly388 Expressionsvektoren transfiziert. Klonformation, 3D-Wachstum, und Migrationspotential wurden in den transfizierten Zellmodellen untersucht. FGFR4 Arg388 und Gly388 Überexprimierung hat induzierte Migration in U251MG zur Folge. Dieser Effekt konnte jedoch nicht in transfizierten HU-MI Zellen nachgewiesen werden. Bemerkenswerterweise zeigen alle transfizierten Zellmodelle außer HU-MI FGFR4 Gly388 gesteigertes Klon- und 3D-wachstum. Zusammenfassend ist wichtig zu erwähnen, dass induzierte FGFR4 (Arg388 und Gly388) Expression in U251MG zu verstärkter Migration, Klon- und 3D-Wachstum führt. Weiterführend wird die Funktion von FGFR4 noch in vivo untersucht werden.Glioblastoma multiforme (GBM) is a high-grade astrocytoma and accounts for 45-55% of all malignant gliomas in adults. GBM represents the most aggressive primary brain tumor and survival rates are very low with a median overall survival of 14 months under treatment and a 5-year survival of only 5.5%. A heterogenous cell population, high vascularization and pseudopalisading necrosis are the main histopathological characteristics typifying GBM tumor tissue. Molecular features such as isocitrate dehydrogenase 1/2 (IDH 1/2) mutations, telomerase reverse transcriptase (TERT) promoter mutations, O6-methylguanine DNA methyltransferase (MGMT) promoter methylation and epidermal growth factor receptor (EGFR) amplification are parameters used in for the diagnosis of GBM. Despite intensive research, GBM-specific biomarkers -to develop targeted therapies- are still not identified and therefore GBM remains complicated to treat. Previous studies performed in our group revealed the influence of fibroblast growth factors (FGFs) on GBM proliferation and tumor growth in vivo. In the present study, we aimed to dissect the role of both FGF receptor tyrosine kinase 4 (FGFR4) Arg388 and Gly388 single nucleotide polymorphism (SNP) variants on GBM growth aggressiveness, 3D-neurosphere formation and migration. To do so, we transfected two FGFR4 low expressing GBM cell lines, the primary GBM cell line U251MG and the GBM primo-culture HU-MI, to ectopically over-express both FGFR4 Arg388 and Gly388 SNP variants. All cell models were analyzed on their colony forming capacity, 3D-neurosphere growth and migratory potential in relation to GFP transfected controls. Upon transfection with both FGFR4 Arg388 and Gly388 constructs, distinct pro-migratory effects were visible in U251MG cells in contrast to HU-MI transfected cells. Additionally, colony formation and 3D-growth were significantly enhanced in U251MG FGFR4 Arg388 and Gly388 cell models and HU-MI FGFR4 Arg388. Introduction of FGFR4 Gly388 into the HU-MI cell model was not efficient enough to obtain results strongly differing from the parental cell line. Summarizing, ectopic expression of FGFR4 Arg388 or Gly388 in U251MG resulted in enhanced migration, 3D-growth and clone formation. To analyze the role of FGFR4 in these processes into more detail, further biological and molecular analyses with focus on migratory markers are of high interest. Additionally, altered tumorigenicity of the U251MG FGFR4 transfected sublines requires examination in in vivo xenograft models. In conclusion, these data suggest a prominent role of FGFR4 in GBM migratory and proliferative characteristics as well as 3D-neurosphere formation and clonogenicity

Distinct activity of the bone-targeted gallium compound KP46 against osteosarcoma cells - synergism with autophagy inhibition

Abstract Background Osteosarcoma is the most frequent primary malignant bone tumor. Although survival has distinctly increased due to neoadjuvant chemotherapy in the past, patients with metastatic disease and poor response to chemotherapy still have an adverse prognosis. Hence, development of new therapeutic strategies is still of utmost importance. Methods Anticancer activity of KP46 against osteosarcoma cell models was evaluated as single agent and in combination approaches with chemotherapeutics and Bcl-2 inhibitors using MTT assay. Underlying mechanisms were tested by cell cycle, apoptosis and autophagy assays. Results KP46 exerted exceptional anticancer activity at the nanomolar to low micromolar range, depending on the assay format, against all osteosarcoma cell models with minor but significant differences in IC50 values. KP46 treatment of osteosarcoma cells caused rapid loss of cell adhesion, weak cell cycle accumulation in S-phase and later signs of apoptotic cell death. Furthermore, already at sub-cytotoxic concentrations KP46 reduced the migratory potential of osteosarcoma cells and exerted synergistic effects with cisplatin, a standard osteosarcoma chemotherapeutic. Moreover, the gallium compound induced signs of autophagy in osteosarcoma cells. Accordingly, blockade of autophagy by chloroquine but also by the Bcl-2 inhibitor obatoclax increased the cytotoxic activity of KP46 treatment significantly, suggesting autophagy induction as a protective mechanism against KP46. Conclusion Together, our results identify KP46 as a new promising agent to supplement standard chemotherapy and possible future targeted therapy in osteosarcoma

The Natural Fungal Metabolite Beauvericin Exerts Anticancer Activity In Vivo: A Pre-Clinical Pilot Study

Recently, in vitro anti-cancer properties of beauvericin, a fungal metabolite were shown in various cancer cell lines. In this study, we assessed the specificity of this effect by comparing beauvericin cytotoxicity in malignant versus non-malignant cells. Moreover, we tested in vivo anticancer effects of beauvericin by treating BALB/c and CB-17/SCID mice bearing murine CT-26 or human KB-3-1-grafted tumors, respectively. Tumor size and weight were measured and histological sections were evaluated by Ki-67 and H/E staining as well as TdT-mediated-dUTP-nick-end (TUNEL) labeling. Beauvericin levels were determined in various tissues and body fluids by LC-MS/MS. In addition to a more pronounced activity against malignant cells, we detected decreased tumor volumes and weights in beauvericin-treated mice compared to controls in both the allo- and the xenograft model without any adverse effects. No significant differences were detected concerning percentages of proliferating and mitotic cells in tumor sections from treated and untreated mice. However, a significant increase of necrotic areas within whole tumor sections of beauvericin-treated mice was found in both models corresponding to an enhanced number of TUNEL-positive, i.e., apoptotic, cells. Furthermore, moderate beauvericin accumulation was detected in tumor tissues. In conclusion, we suggest beauvericin as a promising novel natural compound for anticancer therapy