Showing papers by "Walter Berger published in 2014"

NKP-1339, the first ruthenium-based anticancer drug on the edge to clinical application

Abstract: NKP-1339 is the first-in-class ruthenium-based anticancer drug in clinical development against solid cancer and has recently been studied successfully in a phase I clinical trial. Ruthenium compounds such as KP1019 (indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]) and NKP-1339 (the sodium salt analogue of KP1019, sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]) have a high tumour targeting potential based (1) on their strong binding to serum proteins such as albumin and transferrin as well as (2) on their activation in the reductive tumour milieu. The redox activity of ruthenium compounds is believed to represent one major mode of action leading to disturbance of the cellular redox balance and, consequently, induction of G2/M cell cycle arrest, blockage of DNA synthesis, and induction of apoptosis via the mitochondrial pathway. Moreover, potent synergistic activities of NKP-1339 with the clinically approved tyrosine kinase inhibitor sorafenib were recently reported in vitro and in vivo. Taken together, KP1019 and NKP-1339 are promising drug candidates, and especially the very limited side effects observed so far in clinical phase I trials seem to be a major advantage of this class of ruthenium drugs as compared to other chemotherapeutics and targeted anticancer compounds.

Spatio‐temporally precise activation of engineered receptor tyrosine kinases by light

Abstract: Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease Contactless activation of RTKs with spatial and temporal precision is currently not feasible Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET) In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour

Apelin promotes lymphangiogenesis and lymph node metastasis.

Abstract: // Judit Berta 1,2,* , Mir Alireza Hoda 1,* , Viktoria Laszlo 1,3 , Anita Rozsas 1,2 , Tamas Garay 2,3,4 , Szilvia Torok 2 , Michael Grusch 5 , Walter Berger 5 , Sandor Paku 3,6 , Ferenc Renyi-Vamos 1,7 , Bernard Masri 8 , Jozsef Tovari 9 , Marion Groger 10,11 , Walter Klepetko 1 , Balazs Hegedus 1,4 , Balazs Dome 1,2,7 1 Division of Thoracic Surgery, Department of Surgery, Comprehensive Cancer Center, Medical University of Vienna, Austria 2 Department of Tumor Biology, National Koranyi Institute of Pulmonology, Budapest, Hungary 3 Department of Biological Physics, Eotvos Lorand University, Budapest, Hungary 4 MTA-SE Molecular Oncology Research Group, Hungarian Academy of Sciences, Budapest, Hungary 5 Institute of Cancer Research and Comprehensive Cancer Center, Department of Medicine I, Medical University of Vienna, Austria 6 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary 7 Thoracic Surgery, National Institute of Oncology and Semmelweis University, Budapest, Hungary 8 Cancer Research Center of Toulouse, INSERM U1037-Universite Paul Sabatier Toulouse III, Toulouse, France 9 Department of Experimental Pharmacology, National Institute of Oncology, Budapest, Hungary; Skin and Endothelium Research Division (SERD) 10 Department of Dermatology, Medical University of Vienna, Austria 11 Core Facility Imaging, Core Facilities, Medical University of Vienna, Austria * These authors contributed equally to this work ** These authors share last authorship Correspondence: Balazs Dome, email: // Keywords : apelin, APJ, lymphangiogenesis, lymph node metastasis Received : April 8, 2014 Accepted : May 27, 2014 Published : May 28, 2014 Abstract Whereas the role of the G-protein-coupled APJ receptor and its ligand, apelin, in angiogenesis has been well documented, the ability of the apelin/APJ system to induce lymphangiogenesis and lymphatic metastasis has been largely unexplored. To this end, we first show that APJ is expressed in lymphatic endothelial cells (LECs) and, moreover, that it responds to apelin by activating the apelinergic signaling cascade. We find that although apelin treatment does not influence the proliferation of LECs in vitro , it enhances their migration, protects them against UV irradiation-induced apoptosis, increases their spheroid numbers in 3D culture, stimulates their in vitro capillary-like tube formation and, furthermore, promotes the invasive growth of lymphatic microvessels in vivo in the matrigel plug assay. We also demonstrate that apelin overexpression in malignant cells is associated with accelerated in vivo tumor growth and with increased intratumoral lymphangiogenesis and lymph node metastasis. These results indicate that apelin induces lymphangiogenesis and, accordingly, plays an important role in lymphatic tumor progression. Our study does not only reveal apelin as a novel lymphangiogenic factor but might also open the door for the development of novel anticancer therapies targeting lymphangiogenesis.

Water-Soluble, Biocompatible Polyphosphazenes with Controllable and pH-Promoted Degradation Behavior.

Abstract: The synthesis of a series of novel, water-soluble poly(organophosphazenes) prepared via living cationic polymerization is presented. The degradation profiles of the polyphosphazenes prepared are analyzed by GPC, 31P NMR spectroscopy, and UV–Vis spectroscopy in aqueous media and show tunable degradation rates ranging from days to months, adjusted by subtle changes to the chemical structure of the polyphosphazene. Furthermore, it is observed that these polymers demonstrate a pH-promoted hydrolytic degradation behavior, with a remarkably faster rate of degradation at lower pH values. These degradable, water soluble polymers with controlled molecular weights and structures could be of significant interest for use in aqueous biomedical applications, such as polymer therapeutics, in which biological clearance is a requirement and in this context cell viability tests are described which show the non-toxic nature of the polymers as well as their degradation intermediates and products. © 2013 The Authors Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 287–294

Tumor-targeting of EGFR inhibitors by hypoxia-mediated activation.

Abstract: The development of receptor tyrosine-kinase inhibitors (TKIs) was a major step forward in cancer treatment. However, the therapy with TKIs is limited by strong side effects and drug resistance. The aim of this study was the design of novel epidermal growth factor receptor (EGFR) inhibitors that are specifically activated in malignant tissue. Thus, a Co(III) -based prodrug strategy for the targeted release of an EGFR inhibitor triggered by hypoxia in the solid tumor was used. New inhibitors with chelating moieties were prepared and tested for their EGFR-inhibitory potential. The most promising candidate was coupled to Co(III) and the biological activity tested in cell culture. Indeed, hypoxic activation and subsequent EGFR inhibition was proven. Finally, the compound was tested in vivo, also revealing potent anticancer activity.

Quantitative bioimaging by LA-ICP-MS: a methodological study on the distribution of Pt and Ru in viscera originating from cisplatin- and KP1339-treated mice†

Abstract: Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used to study the spatially-resolved distribution of ruthenium and platinum in viscera (liver, kidney, spleen, and muscle) originating from mice treated with the investigational ruthenium-based antitumor compound KP1339 or cisplatin, a potent, but nephrotoxic clinically-approved platinum-based anticancer drug. Method development was based on homogenized Ru- and Pt-containing samples (22.0 and 0.257 μg g−1, respectively). Averaging yielded satisfactory precision and accuracy for both concentrations (3–15% and 93–120%, respectively), however when considering only single data points, the highly concentrated Ru sample maintained satisfactory precision and accuracy, while the low concentrated Pt sample yielded low recoveries and precision, which could not be improved by use of internal standards (115In, 185Re or 13C). Matrix-matched standards were used for quantification in LA-ICP-MS which yielded comparable metal distributions, i.e., enrichment in the cortex of the kidney in comparison with the medulla, a homogenous distribution in the liver and the muscle and areas of enrichment in the spleen. Elemental distributions were assigned to histological structures exceeding 100 μm in size. The accuracy of a quantitative LA-ICP-MS imaging experiment was validated by an independent method using microwave-assisted digestion (MW) followed by direct infusion ICP-MS analysis.

Fibroblast Growth Factor Receptor Inhibition Is Active against Mesothelioma and Synergizes with Radio- and Chemotherapy

Abstract: Rationale: Malignant pleural mesothelioma is an aggressive malignancy characterized by frequent resistance to chemo- and radiotherapy, poor outcome, and limited therapeutic options. Fibroblast growth factors (FGFs) and their receptors are potential targets for cancer therapy, but their significance in mesothelioma has remained largely undefined.Objectives: To investigate the antimesothelioma potential of FGF receptor 1 (FGFR1) inhibition.Methods: Expression of FGFs and their receptors was analyzed in mesothelioma cell lines and tissue specimens. Several cell models were used to investigate FGFR1 inhibition in vitro and in combination with cisplatin and irradiation. Mouse intraperitoneal xenotransplant models were used for in vivo validation.Measurements and Main Results: FGFR1, FGF2, and FGF18 were overexpressed in mesothelioma. Stimulation with FGF2 led to increased cell proliferation, migration, and transition to a more sarcomatoid phenotype in subsets of mesothelioma cell lines. In contrast, inhibition...

A Novel Class of Bis- and Tris-Chelate Diam(m)inebis(dicarboxylato)platinum(IV) Complexes as Potential Anticancer Prodrugs

Abstract: A novel class of platinum(IV) complexes of the type [Pt(Am)(R(COO)2)2], where Am is a chelating diamine or two monodentate am(m)ine ligands and R(COO)2 is a chelating dicarboxylato moiety, was synthesized. For this purpose, the reaction between the corresponding tetrahydroxidoplatinum(IV) precursors and various dicarboxylic acids, such as oxalic, malonic, 3-methylmalonic, and cyclobutanedicarboxylic acid, was utilized. All new compounds were characterized in detail, using 1D and 2D NMR techniques, ESI-MS, FTIR spectroscopy, elemental analysis, TGA, and X-ray diffraction. Their in vitro cytotoxicity was determined in a panel of human tumor cell lines (CH1, SW480 and A549) by means of the MTT colorimetric assay. Furthermore, the lipophilicity and redox properties of the novel complexes were evaluated in order to better understand their pharmacological behavior. The most promising drug candidate, 4b (Pt(DACH)(mal)2), demonstrated low in vivo toxicity but profound anticancer activity against both the L1210 leukemia and CT-26 colon carcinoma models.

Is fibroblast growth factor receptor 4 a suitable target of cancer therapy

Abstract: Fibroblast growth factors (FGF) and their tyrosine kinase receptors (FGFR) support cell proliferation, survival and migration during embryonic development, organogenesis and tissue maintenance and their deregulation is frequently observed in cancer development and progression. Consequently, increasing efforts are focusing on the development of strategies to target FGF/FGFR signaling for cancer therapy. Among the FGFRs the family member FGFR4 is least well understood and differs from FGFRs1-3 in several aspects. Importantly, FGFR4 deletion does not lead to an embryonic lethal phenotype suggesting the possibility that its inhibition in cancer therapy might not cause grave adverse effects. In addition, the FGFR4 kinase domain differs sufficiently from those of FGFRs1-3 to permit development of highly specific inhibitors. The oncogenic impact of FGFR4, however, is not undisputed, as the FGFR4-mediated hormonal effects of several FGF ligands may also constitute a tissue-protective tumor suppressor activity especially in the liver. Therefore it is the purpose of this review to summarize all relevant aspects of FGFR4 physiology and pathophysiology and discuss the options of targeting this receptor for cancer therapy.

Circulating fibrinogen is a prognostic and predictive biomarker in malignant pleural mesothelioma.

Abstract: Circulating fibrinogen is a prognostic and predictive biomarker in malignant pleural mesothelioma

Triapine and a More Potent Dimethyl Derivative Induce Endoplasmic Reticulum Stress in Cancer Cells

Abstract: Triapine (3-AP; 3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a ribonucleotide reductase inhibitor, has been extensively evaluated in clinical trials in the last decade. This study addresses the role of endoplasmic reticulum (ER) stress in the anticancer activity of 3-AP and the derivative N(4),N(4)-dimethyl-triapine (3-AP-Me), differing from 3-AP only by dimethylation of the terminal nitrogen. Treatment of colon cancer cells with 3-AP or 3-AP-Me activated all three ER stress pathways (PERK, IRE1a, ATF6) by phosphorylation of eIF2α and upregulation of gene expression of activating transcription factors ATF4 and ATF6. In particular, 3-AP-Me led to an upregulation of the alternatively spliced mRNA variant XBP1 (16-fold). Moreover, 3-AP and 3-AP-Me activated the cellular stress kinases c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases, and inhibition of JNK activity antagonized the cytotoxic effect of both compounds. Subsequent to induction of the unfolded protein response, a significant upregulation of proapoptotic proteins was detected, including the transcription factor CHOP and Bim, an essential factor for ER stress-related apoptosis. In correlation with the higher degree of ER stress after 3-AP-Me treatment, also a more potent depolarization of mitochondrial membranes was found. These data suggest that 3-AP and 3-AP-Me induce apoptosis via ER stress. This was further corroborated by showing that inhibition of protein biosynthesis with cycloheximide prior to 3-AP and 3-AP-Me treatment leads to a significant reduction of the antiproliferative properties of both compounds. Taken together, this study demonstrates that induction of ER stress contributes to the mode of action of 3-AP and that terminal dimethylation leads to an even more pronounced manifestation of this effect.

Fibroblast growth factor receptor 4: a putative key driver for the aggressive phenotype of hepatocellular carcinoma

Abstract: Recently, we found upregulation of fibroblast growth factor receptor 4 (FGFR4) in a subset of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight into the role of FGFR4-mediated signalling for the aggressive behaviour of HCC cells. To overexpress FGFR4, hepatoma/hepatocarcinoma cells were transfected with a construct coding for FGFR4. For downmodulation of endogenous FGFR4, we used small interfering RNA or adenoviral infection with dominant-negative FGFR4 constructs being either kinase dead (kdFGFR4) or coding for the autoinhibitory soluble domain (solFGFR4). FGFR4 overexpression in non-tumourigenic hepatocarcinoma cells significantly reduced cell-matrix adhesion, enabled cells to grow anchorage-independently in soft agar, to disintegrate the lymph-/blood-endothelial barrier for intra-/extravasation of tumour cells and to form tumours in SCID mice. Transcriptome analysis revealed altered expression of genes involved in cell-matrix interactions. Conversely, in highly tumourigenic cell lines, kdFGFR4 or solFGFR4 lowered the proportion of cells in S phase of the cell cycle, enhanced the G0/G1 and G2/M-phase proportions, reduced anchorage-independent growth in vitro and attenuated disintegration of the lymph-/blood-endothelium and tumour formation in vivo. These findings were confirmed by altered expression profiles of genes being important for late stages of cell division. Deregulated FGFR4 expression appears to be one of the key drivers of the malignant phenotype of HCC cells. Accordingly, blockade of FGFR4-mediated signalling by soluble dominant-negative constructs, like solFGFR4, may be a feasible and promising therapeutic approach to antagonize aggressive behaviour of hepatoma/hepatocarcinoma cells.

Nanoformulation improves activity of the (pre)clinical anticancer ruthenium complex KP1019.

Abstract: Ruthenium anticancer drugs belong to the most promising non-platinum anticancer metal compounds in clinical evaluation. However, although the clinical results are promising regarding both activity and very low adverse effects, the clinical application is currently hampered by the limited solubility and stability of the drug in aqueous solution. Here, we present a new nanoparticle formulation based on polymer-based micelles loaded with the anticancer lead ruthenium compound KP1019. Nanoprepared KP1019 was characterised by enhanced stability in aqueous solutions. Moreover, the nanoparticle formulation facilitated cellular accumulation of KP1019 (determined by ICP-MS measurements) resulting in significantly lowered IC50 values. With regard to the mode of action, increased cell cycle arrest in G2/M phase (PI-staining), DNA damage (Comet assay) as well as enhanced levels of apoptotic cell death (caspase 7 and PARP cleavage) were found in HCT116 cells treated with the new nanoformulation of KP1019. Summarizing, we present for the first time evidence that nanoformulation is a feasible strategy for improving the stability as well as activity of experimental anticancer ruthenium compounds.

Poly(lactic acid) nanoparticles of the lead anticancer ruthenium compound KP1019 and its surfactant-mediated activation

Abstract: Nanoparticle formulations offer besides the advantage of passive drug targeting also the opportunity to increase the stability of drugs. KP1019 is a lead ruthenium(III) compound which has been successfully tested in a clinical phase I trial. However, it is characterized by low stability in aqueous solution especially at physiological pH. To overcome this limitation, poly(lactic acid) (PLA) nanoparticles of KP1019 with two different surfactants (Pluronic F68 and Tween 80) were prepared by a single oil-in-water (o/w) emulsion. Cytotoxicity measurements comparing different aged Tween 80 nanoparticles revealed that the color change from brown to green was associated with an up to 20 fold increased activity compared to “free” KP1019. Further investigations suggested that this is based on the formation of enhanced intracellular reactive oxygen species levels. Additional studies revealed that the origin of the green color is a reaction between KP1019 and Tween 80. Kinetic studies of this reaction mixture using UV-Vis, ESI-MS and ESR spectroscopy indicated on the one hand a coordination of Tween 80 to KP1019, and on the other hand, the color change was found to correlate with a reduction of the Ru(III) center by the surfactant. Together, the results provide a first experimental approach to stabilize a biologically active Ru(II) species of KP1019 in aqueous solution, which probably can be also used to selectively generate this activated species in the tumor tissue via delivery of KP1019 using Tween 80 nanoparticles.

Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and -independent pathways.

Abstract: Glioblastoma multiforme (GBM), which is classified as grade IV astrocytoma by the WHO, is the most common and aggressive primary brain tumor in adults. Despite advances in treatment options combining surgical resection, radiotherapy, and concomitant alkylating chemotherapy, the prognosis for GBM patients still remains dismal with a median survival of 14.2 months.1 The disproportionate malignancy of GBM is due to its invasive growth pattern and high inter-and intratumoral genetic heterogeneity.2,3 Frequent genetic alterations in GBM affect 3 major cancer pathways: (i) the receptor tyrosine kinase (RTK) pathway and its downstream oncogenic signaling by PI3K/AKT and Ras/MAPK, (ii) the p53, and (iii) the retinoblastoma protein (pRb) signaling network.4 Cellular senescence refers to the state of irreversible cell cycle arrest that occurs at the end of cells’ replicative life span or as a physiological response to different types of cellular stress including aberrant oncogenic activation, DNA damage, and oxidative stress. Depending on the type of senescence-inducing stimulus and their genetic background, different cell types display differential senescence responses. In addition to long-term exit from cell cycle, common characteristic features of the senescent phenotype are the induction of senescence-associated β-galactosidase (SA-β-Gal) activity, chromatin remodeling accompanied by the formation of senescence-associated heterochromatin foci, and morphological transformation, including vacuolization and change in morphology to a flat and enlarged cell shape. At molecular level, the senescence response can be triggered through several genetic effectors converging on the activation of tumor suppressor networks p53 and pRb.5,6 During early stages of tumorigenesis, senescence acts as an important anticancer mechanism. Aberrant activation of oncogenes, such as Ras, or the loss of tumor suppressors, as described for PTEN, can trigger senescence in vitro and in vivo. For further progression, tumor cells have to bypass the senescent arrest by additional mutations of tumor suppressor genes, including p53 and p16, or reactivation of telomerase. Thus, re-induction of the senescence program in tumor cells could represent an additional therapeutic approach. Potential intervention targets for prosenescence therapies are telomerase inhibition, modulation of cyclin-dependent kinase (CDK) activities, the reactivation of tumor suppressor genes (eg, p53) and the inactivation of oncogenes, as demonstrated for c-Myc addicted tumors.6–8 The protein kinase D (PRKD) family belongs to a subgroup of serine/threonine kinases within the calcium/calmodulin-dependent protein kinase superfamily and comprises 3 abundantly expressed mammalian isoforms. The 3 isoforms, PRKD1, PRKD2, and PRKD3, contain highly conserved structural motifs within their regulatory domains and are directly activated by phorbolesters, diacylglycerol (DAG), or DAG-activated protein kinase C (PKC) isoforms. By integrating downstream signaling of tyrosine kinase and G protein-coupled receptors, PRKDs have emerged as central players in tumor-promoting processes including proliferation, migration, invasion, and angiogenesis.9 PRKDs have been linked mechanistically to targets that are implicated in the regulation of cell proliferation, survival, and apoptosis including the Ras/MAPK pathway, HDAC, CREB, JNK, and AKT.10,11 Within a large-scale RNAi screen of human kinases, PRKD2, but not PRKD1 and PRKD3, was identified as an essential regulator of cell survival.12 In human cancers, PRKD2 is positively involved in dedifferentiation,13 survival,14 angiogenesis,15 and invasion.16 In astrocytoma, a positive correlation between PRKD2 expression and tumor grading was shown.17 This study further demonstrated that inhibition of PRKD2 results in an apoptosis-independent reduction of glioma cell proliferation in vitro and prevents tumor formation in a chicken chorioallantoic membrane assay. Just recently, we have identified PRKD2 as a potential kinase target to reduce glioblastoma cell motility and invasion.18 The present study aimed to investigate molecular pathways underlying PRKD2-silencing induced cell-cycle inhibition in glioma. We elucidated the effects of PRKD2 depletion on senescence-associated and RTK-mediated signaling in vitro and confirmed the role of PRKD2 in glioma growth in a xenograft model. Here we show for the first time that PRKD2 silencing induces a senescence-like program in p53wt and p53mut glioma cells and may represent a promising approach for prosenescence therapy in glioma.

Specific antioxidant compounds differentially modulate cytotoxic activity of doxorubicin and cisplatin: in vitro and in vivo study.

Abstract: Low selectivity of action of the chemotherapeutic agents is one of their main shortcomings, leading to serious negative side effects in cancer patients. The main reason for this phenomenon is the formation of free radicals during the action of these drugs in both normal and tumor cells. Doxorubicin and cisplatin are among the most commonly used anticancer drugs. They realize the antineoplastic activity by the intercalation into DNA structure and production of the reactive oxygen species (ROS) (1-3). However, these drugs lead to severe cardio- and nephrotoxicity, which significantly limits their use for tumor treatment (4). It was shown that side effects of doxorubicin and cisplatin are mediated by hydroxyl radicals, which are formed in the presence of iron (II) from superoxide anions whose production is induced by these drugs (3,5). Numerous studies indicate that ROS-induced apoptosis of tumor cells takes place only under supraclinical doses of anthracyclines, and ROS production is not critical for realization of their anticancer activity (3). Thus, selective blocking of ROS action by specific antioxidant agents should at least partially reduce the toxicity of doxorubicin and cisplatin toward normal cells, without significant impact on the antitumor action of these drugs. Promising candidates for such role are derivatives of the pantothenic acid, since they possess significant antioxidant effect toward the mammalian cells and are able to protect the cells against toxic effects of free radicals (6). The inorganic and organic selenium derivatives (sodium selenite and selenomethionine) belong to another group of antioxidants that demonstrated a protective effect during cisplatin chemotherapy (7,8). Similar protective effects were also observed for the pantothenic acid (9). However, it remains unknown whether these antioxidants are capable of inhibiting the production of harmful ROS (including superoxide and hydroxyl radicals) due to the action of anticancer agents, and at the same time not interfering with the anti-tumor activity of these drugs. Besides, the effect of D-pantethine, selenomethionine, and sodium selenite used in combination with the anticancer drugs toward tumor cells resistant to chemotherapy has not been studied thoroughly (10). In this study, we aimed to develop new approaches for cancer chemotherapy that would eliminate negative side effects of the anticancer drugs caused by an excessive production of free radicals, which adversely affect normal tissues and organs in cancer patients. A chemotherapy regimen based on a combination of specific antioxidants (sodium selenite, selenomethionine, D-pantethine) and conventional anticancer drugs (doxorubicin, cisplatin), which are known to induce production of ROS, has been proposed. We studied the molecular mechanisms of antitumor activity of doxorubicin and cisplatin combined with the antioxidants toward tumor cell lines possessing different mechanisms of drug resistance. The results obtained in the in vitro study have been verified in experimental animals (rats).

Enhanced anticancer activity and circumvention of resistance mechanisms by novel polymeric/ phospholipidic nanocarriers of doxorubicin.

Abstract: Severe toxic side effects and drug resistance are the major limitations of doxorubicin (Dox), one of the most potent anticancer agents in clinical use. Nanocarrier preparations offer the opportunity to overcome these drawbacks, which is reflected in the clinical approval of two liposomal Dox preparations. Additionally, there are many attempts to enhance the activity of Dox against multi-drug resistant (MDR) cancer cells. However, most of these strategies resulted in the increased uptake of Dox in resistant cells, only, while it remained unchanged in chemo-sensitive cells. Here, we present a new polymeric-phospholipidic hybrid delivery system which distinctly enhanced the accumulation and activity of Dox in all tested cancer cell lines including several MDR cell models. Notably, the resistance levels against Dox were reduced from about 6-fold to about 2-fold. Moreover, the new nanocarriers were shown to rapidly (within 10 min) and effectively transport Dox into resistant as well as sensitive cancer cells. Consequently, treatment with the new Dox-containing nanocarriers resulted in effective cell cycle arrest in G2/M phase and ROS-induced cell death induction. Finally, the new nanocarriers were tested against NK/Ly lymphoma and L1210 leukemia cells in vivo. In both cell models, the nanoformulation of Dox resulted in 100% cured animals already at low concentrations (0.1 mg/kg), while free Dox solely extended survival time. This indicates that the incorporation of phospholipids into PEGylated polymeric nanocarriers is a promising strategy to enhance efficacy and reduce toxicity of Dox treatment against both sensitive and resistant cancer models in vitro and in vivo.

Calpain-Mediated Integrin Deregulation as a Novel Mode of Action for the Anticancer Gallium Compound KP46

Abstract: On the basis of enhanced tumor accumulation and bone affinity, gallium compounds are under development as anticancer and antimetastatic agents. In this study, we analyzed molecular targets of one of the lead anticancer gallium complexes [KP46, Tris(8-quinolinolato)gallium(III)] focusing on colon and lung cancer. Within a few hours, KP46 treatment at low micromolar concentrations induced cell body contraction and loss of adhesion followed by prompt cell decomposition. This rapid KP46-induced cell death lacked classic apoptotic features and was insensitive toward a pan–caspase inhibitor. Surprisingly, however, it was accompanied by upregulation of proapoptotic Bcl-2 family members. Furthermore, a Bax- but not a p53-knockout HCT-116 subline exhibited significant KP46 resistance. Rapid KP46-induced detachment was accompanied by downregulation of focal adhesion proteins, including several integrin subunits. Loss of integrin-β1 and talin plasma membrane localization corresponded to reduced binding of RGD (Arg–Gly–Asp) peptides to KP46-treated cells. Accordingly, KP46-induced cell death and destabilization of integrins were enhanced by culture on collagen type I, a major integrin ligand. In contrast, KP46-mediated adhesion defects were partially rescued by Mg2+ ions, promoting integrin-mediated cell adhesion. Focal adhesion dynamics are regulated by calpains via cleavage of multiple cell adhesion molecules. Cotreatment with the cell-permeable calpain inhibitor PD150606 diminished KP46-mediated integrin destabilization and rapid cell death induction. KP46 treatment distinctly inhibited HCT-116 colon cancer xenograft in vivo by causing reduced integrin plasma membrane localization, tissue disintegration, and intense tumor necrosis. This study identifies integrin deregulation via a calpain-mediated mechanism as a novel mode of action for the anticancer gallium compound KP46. Mol Cancer Ther; 13(10); 2436–49. ©2014 AACR .

MAP kinase activity supported by BRAF V600E mutation rather than gene amplification is associated with ETV1 expression in melanoma brain metastases

Abstract: In primary melanoma, ETV1 transcription factor was suggested to be activated mainly by gene amplification and to promote tumor growth in cooperation with BRAF V600E . Aim of this study was to investigate ETV1 expression in human melanoma with a focus on brain metastases. We investigated ETV1 in 68 human melanoma brain metastases using FISH for ETV1 gene (located at chromosome 7p21) and centromere chromosome 7 and immunohistochemistry for ETV1, BRAF V600E , and ETV1/BRAF associated proteins pMSK1, pRSK1, pp38, pMEK1/2, MAPKAP kinase 2, CIC, HIF-1alpha and Ki-67. We further studied ETV1 copy number variations in 32 melanoma cell lines from primary and metastatic lesions using array CGH. The influence of the MAP kinase pathway activity on ETV1 mRNA and protein expression under BRAF wild-type and BRAF V600E conditions were determined in melanoma cell lines using qRT-PCR and Western Blot. No ETV1 high grade amplifications were observed in tissue samples, but low grade ETV1 gene amplifications were found in 7 (10.3 %) melanoma brain metastases. ETV1 protein expression in tissue samples (15 %) correlated with BRAF V600E status (p = 0.007) and HIF-1alpha expression (p = 0.049), but not with ETV1 gene dose. Application of the BRAFV600E-specific inhibitor vemurafenib and the BRAFV6ooE/V600K-inhibitor dabrafenib revealed predominant regulation of ETV-1 mRNA and protein via MAPK-pathway. ETV1 expression is a rare event in human melanoma and seems to be rather based on hyperactivation of MAPK signals, by BRAF V600E mutation, than on ETV1 gene amplification. Consequently, therapeutic inhibition of BRAF and the downstream MAPK pathway also down-regulates oncogenic ETV1 expression.

The study of reduced versus oxidized glutathione in cancer cell models employing isotopically labelled standards

Abstract: In this work, LC-MS/MS assays for accurate quantification of underivatized glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) were developed based on isotope dilution. Both hydrophilic interaction (HILIC) and reversed phase chromatography (RPC) were implemented. Different protocols dedicated to cancer cell lysis were validated in terms of extraction efficiency, recovery, and unwanted glutathione oxidation. The latter was monitored using isotopologues of GSSG, which were formed upon reaction with isotopically enriched GSH and natural GSH of the sample. Finally, LC-MS/MS was employed for studying the GSH : GSSG ratios in several cancer cells (HCT116, GLC4, and SW480) upon exposure to anticancer metallodrugs. Clinically well-established cis-diamine-dichloro-platinum(II) (cisplatin) and sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339), promising experimental drugs, were addressed. In both cases, a decrease of the GSH : GSSG ratio was observed upon drug exposure. It was more pronounced for cisplatin, where the ratio shifted from 440 : 1 to 240 : 1 and from 160 : 1 to 90 : 1 in HCT116 and GLC4 cells, respectively. For KP1339, a significant decrease was observed in the SW480 cancer cell model, whereas the change was not significant in HCT116 cells. Taken together, this study introduces a new sensitive and robust method for the evaluation of drug-induced changes in the intracellular GSH : GSSG ratio of human cells.

P04.24mutations of the tert promoter correlate with enhanced telomerase activation and predict a worse prognosis in primary glioblastoma patients

Abstract: The stabilization of telomeres by upregulation of telomerase is compulsive for indefinite proliferation and cell immortalization therefore representing a main feature of malignant solid tumors, including gliomas. However, the mechanisms responsible for cancer-associated telomerase activation are not completely understood. Recently, defined mutations in the TERT promoter were identified in a variety of tumors, most frequent in primary glioblastomas (GBM). Presence of the mutations was associated with increased TERT expression. In the present study GBM derived tumor tissue from 126 patients operated at the Wagner Jauregg Hospital were screened for TERT promoter mutations. Subsequently the collected data were correlated with telomere associated parameters (telomerase activity, TERT mRNA expression, telomere lengths), the glioma biomarkers MGMT promoter methylation and IDH1 mutation as well as clinical parameters including patient survival. Using direct sequencing, the TERT promoter mutations (C228T, C250T) were found in 73% of tumors with predominance of C228T (72% of the mutated cases). Thirty-four (27%) samples contained none of the investigated TERT promoter mutations while mutations at both sites occurred in none of the investigated GBM cases. TERT promoter mutations were accompanied by a significant upregulation of telomerase activity (p = 0.0005) and TERT mRNA expression (p = 0.0004). Accordingly, telomere lengths of TERT promoter mutated tumors were significantly shorter compared to the TERT promoter wild-type subgroup (p = 0.001). Moreover, Kaplan-Meier survival analyses revealed a significantly shorter overall survival for GBM patients harbouring mutant tumors (p < 0.0001). In the multivariate Cox regression analysis TERT promoter mutation was found to have independent prognostic power (p = 0.049) but reached elevated significance in the interaction with age (p = 0.007). Accordingly, the prognostic quality of TERT promoter mutations was confined to the subgroup of patients aged younger than 65 years and completely absent in the older patient cohort. Significantly enhanced TERT mRNA expression and reduced telomere lengths in the aged patients were only observed in the subgroup lacking TERT promoter mutations. Summarizing, these results suggest that the negative prognostic value of TERT promoter mutation is restricted to patients of younger age further corroborating age-associated differences in the way of glioblastoma-associated telomere stabilization.

NKP-1339, the First Ruthenium-Based Anticancer Drug on the Edge to Clinical Application

Abstract: NKP-1339 is the first-in-class ruthenium-based anticancer drug in clinical development against solid cancer and has recently been studied successfully in a phase I clinical trial. Ruthenium compounds such as KP1019 (indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]) and NKP-1339 (the sodium salt analogue of KP1019, sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]) have a high tumour targeting potential based (1) on their strong binding to serum proteins such as albumin and transferrin as well as (2) on their activation in the reductive tumour milieu. The redox activity of ruthenium compounds is believed to represent one major mode of action leading to disturbance of the cellular redox balance and, consequently, induction of G2/M cell cycle arrest, blockage of DNA synthesis, and induction of apoptosis via the mitochondrial pathway. Moreover, potent synergistic activities of NKP-1339 with the clinically approved tyrosine kinase inhibitor sorafenib were recently reported in vitro and in vivo. Taken together, KP1019 and NKP-1339 are promising drug candidates, and especially the very limited side effects observed so far in clinical phase I trials seem to be a major advantage of this class of ruthenium drugs as compared to other chemotherapeutics and targeted anticancer compounds.

EGFR mutations in lung adenocarcinoma: Epidemiology and clinical relevance of common versus rare mutations.

Abstract: e19067 Background: In lung adenocarcinoma, oncogenic mutations of the epidermal growth factor receptor (EGFR) are the most frequent and biologically targetable molecular alterations. Classical acti...

New agents for the treatment of cancer

Abstract: The invention relates to prodrug compounds comprising a Co 3+ ion and aquinazoline tyrosine kinase inhibitor moiety, wherein the quinazoline tyrosine kinase inhibitor moiety comprises a Co 3+ -chelating moiety and wherein the quinazoline tyrosine kinase inhibitor moiety is a 4-anilinoquinazoline tyrosine kinase inhibitor moiety. The 4-anilinoquinazoline tyrosine kinase inhibitor moiety has the general formula B,wherein R 1 , R 2 , R 3 and m are as defined herein. The compounds are useful in the treatment of hyperproliferative diseases, such as cancer.