Walter Berger

KP1019, A New Redox‐Active Anticancer Agent – Preclinical Development and Results of a Clinical Phase I Study in Tumor Patients

The promising drug candidate indazolium trans‐[tetrachlorobis(1H‐indazole)ruthenate(III)] (KP1019) is the second Ru‐based anticancer agent to enter clinical trials. In this review, which is an update of a paper from 2006 (Hartinger et al., J. Inorg. Biochem. 2006, 100, 891–904), the experimental evidence for the proposed mode of action of this coordination compound is discussed, including transport into the cell via the transferrin cycle and activation by reduction. The results of the early clinical development of KP1019 are summarized in which five out of six evaluated patients experienced disease stabilization with no severe side effects.

Green tea extract and (–)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities

Green tea is the most widely consumed beverage. It has attained high reputation as a health‐promoting dietary component ascribed to the antioxidant activity of (−)‐epigallocatechin‐3‐gallate (EGCG), its main polyphenolic constituent. Evidence is increasing that tea constituents can be cell damaging and pro‐oxidant themselves. These effects were suggested to be due to spontaneous H2O2 generation by polyphenols in solution. In the present study, we investigated the oxidant and antioxidant properties of green tea extracts (GTE) and of EGCG by means of the rodent macrophage‐like RAW 264.7 and human promyelocytic leukemic HL60 cell lines. The results obtained show that both under cell‐free conditions and in the presence of cells the oxidant activities of GTE and EGCG exceeded those of spontaneously generated H2O2 (FOX assay). Increase of intracellular oxidative stress was indicated by 2′,7′‐dichlorofluorescin probing, and the enhanced genotoxicity was demonstrated by the alkaline comet assay and by the micronucleus assay (cytokinesis block). Time‐ and dose‐dependent induction of cell death was monitored by trypan blue exclusion, MTT assay, and Hoechst staining. Furthermore, in our systems in vitro, EGCG neither directly scavenges H2O2 nor mediates other antioxidant activities but rather increased H2O2‐induced oxidative stress and DNA damage. In conclusion, our data suggest that detailed mechanistic studies on the effects of GTE and EGCG should be performed in vivo before excessive intake and/or topical application of green tea products can be recommended to healthy and/or diseased persons.

Resistance against novel anticancer metal compounds: Differences and similarities

The platinum antitumor drugs cisplatin, carboplatin and oxaliplatin are widely used components of modern cancer chemotherapy. However, their success is limited by severe adverse effects and because of the impact of intrinsic and acquired resistance mechanisms on tumor responses. Consequently, intense efforts have been made to develop new metal compounds that either display enhanced tumor specificity or are less prone to the development of resistance. Despite the synthesis of thousands of compounds during the last decades only very few novel metal drugs have successfully reached clinical development and/or approval so far. In this review we summarize the current knowledge on drug resistance mechanisms against novel metal compounds (including platinum, arsenic, ruthenium, gallium, titanium, copper, and lanthanum drugs), and address the question whether there might exist a general metal-drug resistance phenotype.

Impact of Metal Coordination on Cytotoxicity of 3-Aminopyridine-2-carboxaldehyde Thiosemicarbazone (Triapine) and Novel Insights into Terminal Dimethylation

The first metal complexes of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine) were synthesized. Triapine was prepared by a novel three-step procedure in 64% overall yield. In addition, a series of related ligands, namely, 2-formylpyridine thiosemicarbazone, 2-acetylpyridine thiosemicarbazone, 2-pyridineformamide thiosemicarbazone, and their N(4)-dimethylated derivatives (including the N(4)-dimethylated analogue of Triapine) were prepared, along with their corresponding gallium(III) and iron(III) complexes with the general formula [M(L)(2)](+), where HL is the respective thiosemicarbazone. The compounds were characterized by elemental analysis, (1)H and (13)C NMR, IR and UV-vis spectroscopies, mass spectrometry, and cyclic voltammetry. In addition, Triapine and its iron(III) and gallium(III) complexes were studied by X-ray crystallography. All ligands and complexes were tested for their in vitro antiproliferative activity in two human cancer cell lines (41M and SK-BR-3), and structure-activity relationships were established. In general, the coordination to gallium(III) increased the cytotoxicity while the iron(III) complexes show reduced cytotoxic activity compared to the metal-free thiosemicarbazones. Selected compounds were investigated for the capacity of inhibiting ribonucleotide reductase by incorporation of (3)H-cytidine into DNA.

Multidrug resistance markers P-glycoprotein, multidrug resistance protein 1, and lung resistance protein in non-small cell lung cancer : prognostic implications

PurposeThe aim of this retrospective study was to comparatively investigate the expression of the three drug-resistance genes P-glycoprotein (P-gp), multidrug-resistance protein 1 (MRP1), and lung resistance protein (LRP), in non-small cell lung cancer (NSCLC) tissues, and to assess possible associations with clinicopathologic features.MethodsTumor specimens from 126 patients were analyzed by immunohistochemistry and, in selected cases, by reverse transcriptase polymerase chain reaction (RT-PCR), and data were statistically analyzed by SPSS.ResultsThe mean expression levels of tumor tissues in the case of P-gp and LRP did not exceed the one of normal epithelia, while MRP1 was significantly enhanced in NSCLC. A weak association was observed between higher grading and P-glycoprotein expression ( p <0.08) as well as lower grading and MRP1 expression in the case of adenocarcinoma ( p <0.05). MRP1 levels were highest in TNM stage I and declined with advanced stage ( p <0.03). A significant association was found between high MRP1 levels and longer overall survival ( N =115, p <0.04), which was highly significant in the patient group never treated with chemotherapy ( N =77; p <0.007). P-gp expression was enhanced in those patients who had received chemotherapy before surgery ( p <0.05).ConclusionsOur data point towards a major role of MRP1 in the intrinsic treatment resistance of NSCLC and suggest, in addition, a significant activation of P-gp expression during chemotherapy.

Structure-activity relationship analysis of novel derivatives of narciclasine (an Amaryllidaceae isocarbostyril derivative) as potential anticancer agents.

Narciclasine (1) is a plant growth regulator that has been previously demonstrated to be proapoptotic to cancer cells at high concentrations (> or = 1 microM). Data generated in the present study show that narciclasine displays potent antitumor effects in apoptosis-resistant as well as in apoptosis-sensitive cancer cells by impairing the organization of the actin cytoskeleton in cancer cells at concentrations that are not cytotoxic (IC(50) values of 30-90 nM). The current study further revealed that any chemical modification to the narciclasine backbone generally led to compounds of variable stability, weaker activity, or even the complete loss of antiproliferative effects in vitro. However, one hemisynthetic derivative of narciclasine, compound 7k, demonstrated by both the intravenous and oral routes higher in vivo antitumor activity in human orthotopic glioma models in mice when compared to narciclasine at nontoxic doses. Narciclasine and compound 7k may therefore be of potential use to combat brain tumors.

Expression of the major vault protein LRP in human non‐small‐cell lung cancer cells: Activation by short‐term exposure to antineoplastic drugs

Non‐small‐cell lung cancer (NSCLC) cells are characterised by resistance to the toxic impact of antineoplastic drugs both in vivo and in vitro. The lung resistance‐related protein (LRP), identical with the human major vault protein, is over‐expressed in a variety of tumour cells characterised by intrinsic or acquired chemoresistance. We investigated the expression and cellular localisation of LRP in 16 unselected NSCLC cell lines, immortalised bronchial epithelial cells and embryonic lung fibroblasts. All cell lines analysed expressed LRP mRNA, while protein expression ranged from undetectable up to high levels. Cell fractionation and immunofluorescence staining in selected cell lines localised LRP almost exclusively to the cytoplasm. LRP was contained in the 100,000 g pellet and absent in the soluble, cytosolic fraction and nuclei. A small proportion of LRP, however, was shown to be loosely associated with the outside of the nuclei. Sucrose gradient equilibrium centrifugation revealed presence of LRP exclusively in the fraction known to accumulate purified vault particles. Short‐term exposure (16 hr) to subtoxic daunomycin (DM)‐, and bleomycin (BM)‐concentrations significantly (up to 4‐fold) enhanced LRP expression in 2/4 cell lines tested. Cisplatin (CDDP) had a minor effect while vinblastine (VBL) was ineffective. At cytotoxic conditions all drugs rather decreased than increased LRP expression. When basic LRP expression was compared with chemosensitivity, a significant correlation was detected for resistance to CDDP but not DM, doxorubicin (DOX), etoposide (VP‐16), VBL and BM. Summing up, our data suggest a role of vaults both in basic CDDP resistance and, additionally, in an short‐term defensive response of NSCLC cells against several other drugs. Int. J. Cancer 88:293–300, 2000.

Evidence for a role of FGF-2 and FGF receptors in the proliferation of non-small cell lung cancer cells

Basic fibroblast growth factor (FGF‐2) has been implicated in the progression of human tumours via both autocrine and paracrine (angiogenic) activities. We investigated the expression of FGF‐2 and FGF receptors (FGFR‐1 to ‐4) in NSCLC cell lines (N = 16), NSCLC surgical specimens (N = 21) and 2 control cell lines. Our data show that almost all NSCLC cells produce elevated levels of FGF‐2 and FGFR in vitro and in vivo. FGF‐2 expression did correlate with a short doubling time as well as with potent anchorage‐independent growth of NSCLC cell lines. In contrast with control cells, NSCLC cells did not secrete considerable amounts of FGF‐2 into the extracellular space. Expression levels of FGFR‐1 and ‐2 in NSCLC cell lines correlated with FGF‐2 production. FGFR were located at the plasma membranes in some low FGF‐2‐producing NSCLC and control cell lines. These cells were sensitive to the proliferative effect of recombinant FGF‐2 (rFGF‐2). In NSCLC cell lines with an enhanced FGF‐2 production, representing the majority studied, FGFR localisation was predominantly intracellular. These cells were insensitive to both the proliferative effect of rFGF‐2 and growth inhibition by FGF‐2‐neutralising antibodies. In contrast, several agents antagonised FGF‐2 intracellularly impaired growth of almost all NSCLC cell lines. Our data suggest a role of FGF‐2 and FGFR in the growth stimulation of NSCLC cells possibly via an intracrine mechanism. Int. J. Cancer 83:415–423, 1999.

Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular growth and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant growth of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = 16) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD166866) significantly reduced growth, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal growth factor receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal growth factor receptor are involved in growth and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches. [Mol Cancer Ther 2008;7(10):3408–19]

Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes

The poor prognosis of glioma patients is partly based on the minor success obtained from chemotherapeutic treatments. Resistance mechanisms at the tumor cell level may be, in addition to the blood–brain barrier, involved in the intrinsic chemo-insensitivity of brain tumors. We investigated the expression of the drug-transporter proteins P-glycoprotein (P-gp) and multidrug-resistance protein 1 (MRP1) in cell lines (N=24) and primary cell cultures (N=36) from neuroectodermal tumors, as well as in brain tumor extracts (N=18) and normal human astrocytes (N=1). We found that a considerable expression of P-gp was relatively rare in glioma cells, in contrast to MRP1, which was constitutively overexpressed in cells derived from astrocytomas as well as glioblastomas. Also, normal astrocytes cultured in vitro expressed high amounts of MRP1 but no detectable P-gp. Meningioma cells frequently co-expressed P-gp and MRP1, while, most of the neuroblastoma cell lines express higher P-gp but lower MRP1 levels as compared to the other tumor types. Both, a drug-exporting and a chemoprotective function of P-gp as well as MRP1 could be demonstrated in selected tumor cells by a significant upregulation of cellular 3H-daunomycin accumulation and daunomycin cytotoxicity via administration of transporter antagonists. Summing up, our data suggest that P-gp contributes to cellular resistance merely in a small subgroup of gliomas, but frequently in neuroblastomas and meningiomas. In contrast, MRP1 is demonstrated to play a constitutive role in the intrinsic chemoresistance of gliomas and their normal cell counterpart.