Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

Non-viral gene delivery of the oncotoxic protein NS1 for treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcomes due to lack of efficient treatment options and emerging resistance mechanisms. The aim of the present study is to exploit a non-viral gene therapy enabling the expression of the parvovirus-derived oncotoxic protein NS1 in HCC. This anticancer protein interacts with different cellular kinases mediating a multimodal host-cell death. Lipoplexes (LPX) designed to deliver a DNA expression plasmid encoding NS1 are characterized using a comprehensive set of in vitro assays. The mechanisms of cell death induction are assessed and phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential predictive biomarker for a NS1-LPX-based gene therapy. In an HCC xenograft mouse model, NS1-LPX therapeutic approach results in a significant reduction in tumor growth and extended survival. Data provide convincing evidence for future studies using a targeted NS1 gene therapy for PDK1 overexpressing HCC.

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.

TERT expression is susceptible to BRAF and ETS-factor inhibition in BRAFV600E/TERT promoter double-mutated glioma.

The BRAF gene and the TERT promoter are among the most frequently altered genomic loci in low-grade (LGG) and high-grade-glioma (HGG), respectively. The coexistence of BRAF and TERT promoter aberrations characterizes a subset of aggressive glioma. Therefore, we investigated interactions between those alterations in malignant glioma. We analyzed co-occurrence of BRAFV600E and TERT promoter mutations in our clinical data (n = 8) in addition to published datasets (n = 103) and established a BRAFV600E-positive glioma cell panel (n = 9) for in vitro analyses. We investigated altered gene expression, signaling events and TERT promoter activity upon BRAF- and E-twenty-six (ETS)-factor inhibition by qRT-PCR, chromatin immunoprecipitation (ChIP), Western blots and luciferase reporter assays. TERT promoter mutations were significantly enriched in BRAFV600E-mutated HGG as compared to BRAFV600E-mutated LGG. In vitro, BRAFV600E/TERT promoter double-mutant glioma cells showed exceptional sensitivity towards BRAF-targeting agents. Remarkably, BRAF-inhibition attenuated TERT expression and TERT promoter activity exclusively in double-mutant models, while TERT expression was undetectable in BRAFV600E-only cells. Various ETS-factors were broadly expressed, however, only ETS1 expression and phosphorylation were consistently downregulated following BRAF-inhibition. Knock-down experiments and ChIP corroborated the notion of a functional role for ETS1 and, accordingly, all double-mutant tumor cells were highly sensitive towards the ETS-factor inhibitor YK-4-279. In conclusion, our data suggest that concomitant BRAFV600E and TERT promoter mutations synergistically support cancer cell proliferation and immortalization. ETS1 links these two driver alterations functionally and may represent a promising therapeutic target in this aggressive glioma subgroup.

Synthesis and biological evaluation of biotin-conjugated anticancer thiosemicarbazones and their iron(III) and copper(II) complexes.

Triapine, the most prominent anticancer drug candidate from the substance class of thiosemicarbazones, was investigated in >30 clinical phase I and II studies. However, the results were rather disappointing against solid tumors, which can be explained (at least partially) due to inefficient delivery to the tumor site. Hence, we synthesized the first biotin-functionalized thiosemicarbazone derivatives in order to increase tumor specificity and accumulation. Additionally, for Triapine and one biotin conjugate the iron(III) and copper(II) complexes were prepared. Subsequently, the novel compounds were biologically evaluated on a cell line panel with different biotin uptake. The metal-free biotin-conjugated ligands showed comparable activity to the reference compound Triapine. However, astonishingly, the metal complexes of the biotinylated derivative showed strikingly decreased anticancer activity. To further analyze possible differences between the metal complexes, detailed physico- and electrochemical experiments were performed. However, neither lipophilicity or complex solution stability, nor the reduction potential or behavior in the presence of biologically relevant reducing agents showed strong variations between the biotinylated and non-biotinylated derivatives (only some differences in the reduction kinetics were observed). Nonetheless, the metal-free biotin-conjugate of Triapine revealed distinct activity in a colon cancer mouse model upon oral application comparable to Triapine. Therefore, this type of biotin-conjugated thiosemicarbazone is of interest for further synthetic strategies and biological studies.

Altered membrane rigidity via enhanced endogenous cholesterol synthesis drives cancer cell resistance to destruxins.

Destruxins, secondary metabolites of entomopathogenic fungi, exert a wide variety of interesting characteristics ranging from antiviral to anticancer effects. Although their mode of action was evaluated previously, the molecular mechanisms of resistance development are unknown. Hence, we have established destruxin-resistant sublines of HCT116 colon carcinoma cells by selection with the most prevalent derivatives, destruxin (dtx)A, dtxB and dtxE. Various cell biological and molecular techniques were applied to elucidate the regulatory mechanisms underlying these acquired and highly stable destruxin resistance phenotypes. Interestingly, well-known chemoresistance-mediating ABC efflux transporters were not the major players. Instead, in dtxA- and dtxB-resistant cells a hyper-activated mevalonate pathway was uncovered resulting in increased de-novo cholesterol synthesis rates and elevated levels of lanosterol, cholesterol as well as several oxysterol metabolites. Accordingly, inhibition of the mevalonate pathway at two different steps, using either statins or zoledronic acid, significantly reduced acquired but also intrinsic destruxin resistance. Vice versa, cholesterol supplementation protected destruxin-sensitive cells against their cytotoxic activity. Additionally, an increased cell membrane adhesiveness of dtxA-resistant as compared to parental cells was detected by atomic force microscopy. This was paralleled by a dramatically reduced ionophoric capacity of dtxA in resistant cells when cultured in absence but not in presence of statins. Summarizing, our results suggest a reduced ionophoric activity of destruxins due to cholesterol-mediated plasma membrane re-organization as molecular mechanism underlying acquired destruxin resistance in human colon cancer cells. Whether this mechanism might be valid also in other cell types and organisms exposed to destruxins e.g. as bio-insecticides needs to be evaluated.

Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer.

BACKGROUND: Studying the intracellular distribution of pharmacological agents, including anticancer compounds, is of central importance in biomedical research. It constitutes a prerequisite for a better understanding of the molecular mechanisms underlying drug action and resistance development. Hyperactivated fibroblast growth factor receptors (FGFRs) constitute a promising therapy target in several types of malignancies including lung cancer. The clinically approved small-molecule FGFR inhibitor nintedanib exerts strong cytotoxicity in FGFR-driven lung cancer cells. However, subcellular pharmacokinetics of this compound and its impact on therapeutic efficacy remain obscure. METHODS: 3-dimensional fluorescence spectroscopy was conducted to asses cell-free nintedanib fluorescence properties. MTT assay was used to determine the impact of the lysosome-targeting agents bafilomycin A1 and chloroquine combined with nintedanib on lung cancer cell viability. Flow cytometry and live cell as well as confocal microscopy were performed to analyze uptake kinetics as well as subcellular distribution of nintedanib. Western blot was conducted to investigate protein expression. Cryosections of subcutaneous tumor allografts were generated to detect intratumoral nintedanib in mice after oral drug administration. RESULTS: Here, we report for the first time drug-intrinsic fluorescence properties of nintedanib in living and fixed cancer cells as well as in cryosections derived from allograft tumors of orally treated mice. Using this feature in conjunction with flow cytometry and confocal microscopy allowed to determine cellular drug accumulation levels, impact of the ABCB1 efflux pump and to uncover nintedanib trapping into lysosomes. Lysosomal sequestration - resulting in an organelle-specific and pH-dependent nintedanib fluorescence - was identified as an intrinsic resistance mechanism in FGFR-driven lung cancer cells. Accordingly, combination of nintedanib with agents compromising lysosomal acidification (bafilomycin A1, chloroquine) exerted distinctly synergistic growth inhibitory effects. CONCLUSION: Our findings provide a powerful tool to dissect molecular factors impacting organismal and intracellular pharmacokinetics of nintedanib. Regarding clinical application, prevention of lysosomal trapping via lysosome-alkalization might represent a promising strategy to circumvent cancer cell-intrinsic nintedanib resistance.

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.

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

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.

Rapid generation of hydrogen peroxide contributes to the complex cell death induction by the angucycline antibiotic landomycin E.

Landomycin E (LE) is an angucycline antibiotic produced by Streptomyces globisporus. Previously, we have shown a broad anticancer activity of LE which is, in contrast to the structurally related and clinically used anthracycline doxorubicin (Dx), only mildly affected by multidrug resistance-mediated drug efflux. In the present study, cellular and molecular mechanisms underlying the anticancer activity of landomycin E towards Jurkat T-cell leukemia cells were dissected focusing on the involvement of radical oxygen species (ROS). LE-induced apoptosis distinctly differed in several aspects from the one induced by Dx. Rapid generation of both extracellular and cell-derived hydrogen peroxide already at one hour drug exposure was observed in case of LE but not found before 24h for Dx. In contrast, Dx but not LE induced production of superoxide radicals. Mitochondrial damage, as revealed by JC-1 staining, was weakly enhanced already at 3h LE treatment and increased significantly with time. Accordingly, activation of the intrinsic apoptosis pathway initiator caspase-9 was not detectable before 12h exposure. In contrast, cleavage of the down-stream caspase substrate PARP-1 was clearly induced already at the three hour time point. Out of all caspases tested, only activation of effector caspase-7 was induced at this early time points paralleling the LE-induced oxidative burst. Accordingly, this massive cleavage of caspase-7 at early time points was inhibitable by the radical scavenger N-acetylcysteine (NAC). Additionally, only simultaneous inhibition of multiple caspases reduced LE-induced apoptosis. Specific scavengers of both H2O2 and OH• effectively decreased LE-induced ROS production, but only partially inhibited LE-induced apoptosis. In contrast, NAC efficiently blocked both parameters. Summarizing, rapid H2O2 generation and a complex caspase activation pattern contribute to the antileukemic effects of LE. As superoxide generation is considered as the main cardiotoxic mechanism of Dx, LE might represent a better tolerable drug candidate for further (pre)clinical development.

CD44 drives aggressiveness and chemoresistance of a metastatic human osteosarcoma xenograft model.

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumor with a 5 year survival rate of up to 70%. However, patients with metastatic disease have still a very poor prognosis. Osteosarcoma metastasis models are essential to develop novel treatment strategies for advanced disease. METHODS: Based on a serial transplantation approach, we have established a U-2 OS osteosarcoma xenograft model with increased metastatic potential and compared it to other metastatic osteosarcoma models from international sources. Subclones with differing invasive potential were compared for genomic gains and losses as well as gene expression changes by several bioinformatic approaches. Based on the acquired results, the effects of a shRNA-mediated CD44 mRNA knockdown on migration, invasion and chemosensitivity were evaluated. RESULTS: The CD44 gene was part of an amplified region at chromosome 11p found in both U-2 OS subclones with enhanced metastatic potential but not in parental U-2 OS cells, corresponding with distinct CD44 overexpression. Accordingly, shRNA-mediated CD44 knockdown significantly attenuated osteosarcoma cell migration, invasion, and viability especially in the metastatic subclones of U-2 OS and Saos-2 cells. Metastatic subclones generally were hypersensitive against the integrin inhibitor cilengitide paralleled by alterations in integrin expression pattern following CD44 knock-down. Additionally, attenuation of CD44 expression sensitized these cell models against osteosarcoma chemotherapy with doxorubicin but not methotrexate and cisplatin. CONCLUSIONS: The osteosarcoma xenograft models with increased metastatic potential developed in this study can be useful for identification of mechanisms driving metastasis and resistance towards clinically used and novel therapeutic regimens.

Loss of phosphodiesterase 4D mediates acquired triapine resistance via Epac-Rap1-Integrin signaling.

Triapine, an anticancer thiosemicarbazone, is currently under clinical investigation. Whereas promising results were obtained in hematological diseases, trials in solid tumors widely failed. To understand mechanisms causing triapine insensitivity, we have analysed genomic alterations in a triapine-resistant SW480 subline (SW480/tria). Only one distinct genomic loss was observed specifically in SW480/tria cells affecting the phosphodiesterase 4D (PDE4D) gene locus. Accordingly, pharmacological inhibition of PDE4D resulted in significant triapine resistance in SW480 cells. Hence, we concluded that enhanced cyclic AMP levels might confer protection against triapine. Indeed, hyperactivation of both major downstream pathways, namely the protein kinase A (PKA)-cAMP response element-binding protein (Creb) and the exchange protein activated by cAMP (Epac)-Ras-related protein 1 (Rap1) signaling axes, was observed in SW480/tria cells. Unexpectedly, inhibition of PKA did not re-sensitize SW480/tria cells against triapine. In contrast, Epac activation resulted in distinct triapine resistance in SW480 cells. Conversely, knock-down of Epac expression and pharmacological inhibition of Rap1 re-sensitized SW480/tria cells against triapine. Rap1 is a well-known regulator of integrins. Accordingly, SW480/tria cells displayed enhanced plasma membrane expression of several integrin subunits, enhanced adhesion especially to RGD-containing matrix components, and bolstered activation/expression of the integrin downstream effectors Src and RhoA/Rac. Accordingly, integrin and Src inhibition resulted in potent triapine re-sensitization especially of SW480/tria cells. In summary, we describe for the first time integrin activation based on cAMP-Epac-Rap1 signaling as acquired drug resistance mechanism. combinations of triapine with inhibitors of several steps in this resistance cascade might be feasible strategies to overcome triapine insensitivity of solid tumors.

Trabectedin Is Active against Malignant Pleural Mesothelioma Cell and Xenograft Models and Synergizes with Chemotherapy and Bcl-2 Inhibition In Vitro.

Malignant pleural mesothelioma (MPM) is characterized by widespread resistance to systemic therapy. Trabectedin is an antineoplastic agent targeting both the malignant cells and the tumor microenvironment that has been approved for the treatment of advanced soft tissue sarcoma and ovarian cancer. In this preclinical study, we evaluated the antineoplastic potential of trabectedin as a single agent and in drug combination approaches in human MPM. Therefore, we utilized an extended panel of MPM cell lines (n = 6) and primary cell cultures from surgical MPM specimens (n = 13), as well as nonmalignant pleural tissue samples (n = 2). Trabectedin exerted a dose-dependent cytotoxic effect in all MPM cell cultures in vitro when growing as adherent monolayers or nonadherent spheroids with IC50 values ≤ 2.6 nmol/L. Nonmalignant mesothelial cells were significantly less responsive. The strong antimesothelioma activity was based on cell-cycle perturbation and apoptosis induction. The activity of trabectedin against MPM cells was synergistically enhanced by coadministration of cisplatin, a drug routinely used for systemic MPM treatment. Comparison of gene expression signatures indicated an inverse correlation between trabectedin response and bcl-2 expression. Accordingly, bcl-2 inhibitors (Obatoclax, ABT-199) markedly synergized with trabectedin paralleled by deregulated expression of the bcl-2 family members bcl-2, bim, bax, Mcl-1, and bcl-xL as a consequence of trabectedin exposure. In addition, trabectedin exerted significant antitumor activity against an intraperitoneal MPM xenograft model. Together, these data suggest that trabectedin exerts strong activity in MPM and synergizes with chemotherapy and experimental bcl-2 inhibitors in vitro Thus, it represents a promising new therapeutic option for MPM. Mol Cancer Ther; 15(10); 2357-69. ©2016 AACR.

Acquired nintedanib resistance in FGFR1-driven small cell lung cancer: role of endothelin-A receptor-activated ABCB1 expression.

Genomically amplified fibroblast growth factor receptor 1 (FGFR1) is an oncogenic driver in defined lung cancer subgroups and predicts sensibility against FGFR1 inhibitors in this patient cohort. The FGFR inhibitor nintedanib has recently been approved for treatment of lung adenocarcinoma and is currently evaluated for small cell lung cancer (SCLC). However, tumor recurrence due to development of nintedanib resistance might occur. Hence, we aimed at characterizing the molecular mechanisms underlying acquired nintedanib resistance in FGFR1-driven lung cancer. Chronic nintedanib exposure of the FGFR1-driven SCLC cell line DMS114 (DMS114/NIN) but not of two NSCLC cell lines induced massive overexpression of the multidrug-resistance transporter ABCB1. Indeed, we proved nintedanib to be both substrate and modulator of ABCB1-mediated efflux. Importantly, the oncogenic FGFR1 signaling axis remained active in DMS114/NIN cells while bioinformatic analyses suggested hyperactivation of the endothelin-A receptor (ETAR) signaling axis. Indeed, ETAR inhibition resensitized DMS114/NIN cells against nintedanib by downregulation of ABCB1 expression. PKC and downstream NFκB were identified as major downstream players in ETAR-mediated ABCB1 hyperactivation. Summarizing, ABCB1 needs to be considered as a factor underlying nintedanib resistance. Combination approaches with ETAR antagonists or switching to non-ABCB1 substrate FGFR inhibitors represent innovative strategies to manage nintedanib resistance in lung cancer.

Chronic arsenic trioxide exposure leads to enhanced aggressiveness via Met oncogene addiction in cancer cells.

As an environmental poison, arsenic is responsible for many cancer deaths. Paradoxically, arsenic trioxide (ATO) presents also a powerful therapy used to treat refractory acute promyelocytic leukemia (APL) and is intensively investigated for treatment of other cancer types. Noteworthy, cancer therapy is frequently hampered by drug resistance, which is also often associated with enhancement of tumor aggressiveness. In this study, we analyzed ATO-selected cancer cells (A2780ATO) for the mechanisms underlying their enhanced tumorigenicity and aggressiveness. These cells were characterized by enhanced proliferation and spheroid growth as well as increased tumorigenicity of xenografts in SCID mice. Noteworthy, subsequent studies revealed that overexpression of Met receptor was the underlying oncogenic driver of these effects, as A2780ATO cells were characterized by collateral sensitivity against Met inhibitors. This finding was also confirmed by array comparative genomic hybridization (array CGH) and whole genome gene expression arrays, which revealed that Met overexpression by chronic ATO exposure was based on the transcriptional regulation via activation of AP-1. Finally, it was shown that treatment with the Met inhibitor crizotinib was also effective against A2780ATO cell xenografts in vivo, indicating that targeting of Met presents a promising strategy for the treatment of Met-overexpressing tumors after either arsenic exposure or failure to ATO treatment.

Another step toward DNA selective targeting: Ni(II) and Cu(II) complexes of a Schiff base ligand able to bind gene promoter G-quadruplexes.

DNA G-rich sequences are able to form four-stranded structures organized in stacked guanine tetrads. These structures, called G-quadruplexes, were found to have an important role in the regulation of oncogenes expression and became, for such a reason, appealing targets for anticancer drugs. Aiming at finding selective G-quadruplex binders, we have designed, synthesized and characterized a new water soluble Salen-like Schiff base ligand and its Ni(II) and Cu(II) metal complexes. UV-Vis, circular dichroism and FRET measurements indicated that the nickel complex can stabilize oncogene promoter G-quadruplexes with high selectivity, presenting no interactions with duplex DNA at all. The same compound exhibited dose-dependent cytotoxic activity in MCF-7 breast cancer cells when combined with lipofectamine as lipophilic carrier.

Differences in protein binding and excretion of Triapine and its Fe(III) complex.

Triapine has been investigated as anticancer drug in multiple clinical phase I/II trials. Although promising anti-leukemic activity was observed, Triapine was ineffective against solid tumors. The reasons are currently widely unknown. The biological activity of Triapine is strongly connected to its iron complex (Fe-Triapine) which is pharmacologically not investigated. Here, novel analytical tools for Triapine and Fe-Triapine were developed and applied for cell extracts and body fluids of treated mice. Triapine and its iron complex showed a completely different behavior: for Triapine, low protein binding was observed in contrast to fast protein adduct formation of Fe-Triapine. Notably, both drugs were rapidly cleared from the body (serum half-life time <1h). Remarkably, in contrast to Triapine, where (in accordance to clinical data) basically no renal excretion was found, the iron complex was effectively excreted via urine. Moreover, no Fe-Triapine was detected in serum or cytosolic extracts after Triapine treatment. Taken together, our study will help to further understand the biological behavior of Triapine and its Fe-complex and allow the development of novel thiosemicarbazones with pronounced activity against solid tumor types.

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 GSK3ß). 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.

Tumor microenvironment in focus: LA-ICP-MS bioimaging of a preclinical tumor model upon treatment with platinum(IV)-based anticancer agents.

The selection of drug candidates for entering clinical development relies on in vivo testing in (solid) tumor animal models. However, the heterogeneity of tumor tissue (e.g. in terms of drug uptake or tissue composition) is rarely considered when testing novel drug candidates. Therefore, we used the murine colon cancer CT-26 tumor model to study the spatially-resolved drug distribution in tumor tissue upon repetitive treatment of animals over two weeks with three investigational platinum(IV)-based anticancer agents, oxaliplatin or satraplatin. A quantitative laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging method revealed a heterogeneous platinum distribution, which correlated well with the histologic features of the tumor and surrounding tissue at the microscopic level. In most of the cases, higher amounts of intratumoral platinum were found in the surrounding tissue than in the malignant parts of the sample. This indicates that determination of average platinum amounts (e.g. by microwave-assisted digestion of the sample followed by analysis with ICP-MS) might overestimate the drug uptake in tumor tissue causing misleading conclusions. In addition, we studied the platinum distribution in the kidneys of treated animals to probe if accumulation in the cortex and medulla predict potential nephrotoxicity. A 10-fold increase of platinum in the cortex of the kidney over the medulla was observed for oxaliplatin and satraplatin. Although these findings are similar to those in the platinum distribution of the nephrotoxic anticancer drug cisplatin, treatment with the compounds of our study did not show signs of nephrotoxicity in clinical use or clinical trials (oxaliplatin, satraplatin) and did not result in the alteration of renal structures. Thus, predicting the side effects based on bioimaging data by LA-ICP-MS should be considered with caution. To the best of our knowledge, this is the first LA-ICP-MS study on spatially-resolved platinum accumulation in tissues after repetitive platinum-based anticancer drug treatment of mice bearing a preclinical tumor model.