Molecular mechanisms underlying the anticancer activity of the new lanthanum compound KP772

Platinhältige Chemotherapeutika gehören zu den am häufigsten in der Krebstherapie eingesetzten Medikamenten. Leider ist ihr Einsatz auf Grund hoher Nebenwirkungen und dem Auftreten von Chemotherapieresistenzen nicht immer erfolgreich. Deswegen ist die Entwicklung von neuen, verbesserten metallhältigen Medikamenten weiterhin ein aktuelles Thema. In den letzten Jahrzehnten wurden zu diesem Zweck tausende neue Metallverbindungen entwickelt, von denen es aber nur ein Bruchteil bis zur klinischen Anwendung geschafft hat. Außerdem ist trotz der Menge neuer Substanzen das Wissen über ihre chemischen und biologischen Wirkungsmechanismen im Körper und im Tumor immer noch begrenzt. Besonders der Einfluss und die Entwicklung von Resistenzmechanismen gegen diese Verbindungen sind weitgehend unerforscht (Manuskript 1). [Tris(1,10-phenanthroline)lanthanum(III)]trithiocyanate (KP772) ist eine neue Lanthanverbindung mit vielversprechender Wirksamkeit gegen ein großes Spektrum von Tumorzelllinien in vitro und ein Dickdarmkrebsmodell in vivo. Ziel der hier präsentierten Arbeit war es die molekularen und zellbiologischen Mechanismen zu erforschen, die der Wirksamkeit dieser neuen Substanz gegen Krebszellen zugrunde liegen. Schon in einer frühen Phase des Projekts wurde entdeckt, dass die Behandlung mit KP772 die DNS-Synthese in Tumorzellen blockiert und die Zellteilung in der G0/G1-Phase stoppt. Dieser Zellzyklusarrest, der nicht durch DNS-Schäden hervorgerufen wurde, wurde von apoptotischem Absterben der Zellen begleitet (Manuskript 2, Manuskript 3). Zusätzlich wurde die Zytotoxizität von KP772 gegen 60 Zelllinien am National Cancer Institute (NCI) getestet und das Ergebnis in einer Datenbankanalyse mit anderen Chemotherapeutika verglichen. Dieses Screening zeigte, dass die Wirksamkeit von KP772 nur schwach mit der von anderen Substanzen korrelierte. Interessanterweise waren mehrere Antimetaboliten (z.B. Hydroxyharnstoff) unter jenen Zytostatika, die die höchsten Korrelationswerte erreichten. Da der Hydroxyharnstoff ein allgemein bekannter Ribonukleotidreduktase (RR)-Hemmstoff ist, wurde als nächstes die Hemmwirkung von KP772 auf dieses Enzym untersucht. Die Behandlung mit KP772 führte zu einer signifikanten Reduktion des Cytidineinbaus und dem Abfall der zellulären dNTP-Level. Außerdem zeigten die in diesem Projekt durchgeführten Experimente, dass KP772 die R2 Untereinheit der RR durch Eisenchelatierung und Störung des Tyrosylradikals hemmt (Manuskript 4).Zusätzlich wurde in diesem Projekt der Einfluss von resistenzvermittelnden Transportproteinen auf die Wirksamkeit von KP772 untersucht (Manuskript 5). Interessanterweise stellte sich heraus, dass die Expression dieser Resistenzproteine mit einer erhöhten Sensitivität gegen KP772 einhergingen. Außerdem führte eine längere Behandlung mit subtoxischen KP772 Konzentrationen zum kompletten Verlust der Transportpumpen, was zu einer Wiederherstellung der Sensitivität gegen Chemotherapie führt. Schließlich wurde das mögliche Auftreten von Resistenz gegen KP772 nach mehreren Behandlungszyklen untersucht. Im Gegensatz zu herkömmlichen Medikamenten konnten Krebszellen keine Resistenz gegen KP772 entwickeln. Zusammenfassend deuten die hier präsentierten Untersuchungen daraufhin, dass KP772 besonders vielversprechend für die Behandlung von PatientInnen sein könnte, die an chemotherapieresistenten Tumortypen leiden. Darüber hinaus könnte es sogar ein Wiederansprechen dieser Tumorerkrankungen auf herkömmliche Chemotherapie bewirken.Platinum drugs are essential components of modern cancer chemotherapy. However, their success is limited due to severe adverse effects and/or drug resistance. Thus, several research groups focus on the synthesis of novel metal drugs with reduced adverse side effects and less propensity to induce drug resistance (Manuscript 1). Although a large number of new metal compounds has been developed, in many cases the knowledge on their actual chemical and biological interactions within the human body, especial with regard to mechanisms which lead to resistance of tumor cells is still limited. [Tris(1,10-phenanthroline)lanthanum(III)] trithiocyanate (KP772) is a new compound exerting potent activity against a wide range of tumor cell lines in vitro and a colon carcinoma xenograft model in vivo. Aim of the here presented thesis was the investigation of the molecular and cell biological mechanisms underlying the anticancer activity of KP772. Treatment with KP772 was found to block DNA synthesis and cell cycle progression in G0/G1 phase which was not based on radical- or intercalation-induced DNA damage. This was accompanied by apoptotic cell death via the mitochondrial pathway. Additionally, KP772 was evaluated against a panel of 60 cell lines as part of the in vitro anticancer-screening services provided by the NCI (Manuscript 2, Manuscript 3). When this NCI profile was compared to that of other drugs, only moderate correlations with other known chemotherapeutics were observed. Notably, several antimetabolic drugs including hydroxyurea (HU) were amongthose reaching significant correlations. Since HU is a known ribonucleotide reductase (RR) inhibitor, the next step was the investigation of the RR inhibitory potential of KP772. The respective experiments revealed that KP772 treatment led to significant reduction of cytidine incorporation and decrease of all dNTP pools. Moreover, the iron center of the R2 subunit of RR might be targeted by due to the iron chelating and radical scavenging properties of KP772 (Manuscript 4). With regard to the impact of common resistance mechanisms, overexpression of resistancemediating efflux pumps (ABC-transporter) was found to significantly sensitise against KP772. This hypersensitivity was demonstrated to be based on stronger apoptosis induction and/or cell cycle arrest at unaltered cellular drug accumulation. Moreover, long-term KP772 treatment of transporter-overexpressing cells at subtoxic concentrations led within 20 passages to a complete loss of drug resistance. Additionally, when exposing chemosensitive cells to stepwise increasing KP772 concentrations, we observed, in contrast to several other metal drugs, no acquisition of resistance (Manuscript 5). Taken together, these data indicated that KP772 might be especially promising for treatment of patients suffering from chemotherapy-resistant tumors based on ABC transporter-mediated resistance or as second line treatment after standard chemotherapy failure

Identifying new topoisomerase II poison scaffolds by combining publicly available toxicity data and 2D/3D-based virtual screening

Molecular descriptor (2D) and three dimensional (3D) shape based similarity methods are widely used in ligand based virtual drug design. In the present study pairwise structure comparisons among a set of 4858 DTP compounds tested in the NCI60 tumor cell line anticancer drug screen were computed using chemical hashed fingerprints and 3D molecule shapes to calculate 2D and 3D similarities, respectively. Additionally, pairwise biological activity similarities were calculated by correlating the 60 element vectors of pGI50 values corresponding to the cytotoxicity of the compounds across the NCI60 panel. Subsequently, we compared the power of 2D and 3D structural similarity metrics to predict the toxicity pattern of compounds. We found that while the positive predictive value and sensitivity of 3D and molecular descriptor based approaches to predict biological activity are similar, a subset of molecule pairs yielded contradictory results. By simultaneously requiring similarity of biological activities and 3D shapes, and dissimilarity of molecular descriptor based comparisons, we identify pairs of scaffold hopping candidates displaying characteristic core structural changes such as heteroatom/heterocycle change and ring closure. Attempts to discover scaffold hopping candidates of mitoxantrone recovered known Topoisomerase II (Top2) inhibitors, and also predicted new, previously unknown chemotypes possessing in vitro Top2 inhibitory activity

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

Thiosemicarbazone Derivatives Developed to Overcome COTI-2 Resistance

COTI-2 is currently being evaluated in a phase I clinical trial for the treatment of gynecological and other solid cancers. As a thiosemicarbazone, this compound contains an N,N,S-chelating moiety and is, therefore, expected to bind endogenous metal ions. However, besides zinc, the metal interaction properties of COTI-2 have not been investigated in detail so far. This is unexpected, as we have recently shown that COTI-2 forms stable ternary complexes with copper and glutathione, which renders this drug a substrate for the resistance efflux transporter ABCC1. Herein, the complex formation of COTI-2, two novel terminal N-disubstituted derivatives (COTI-NMe2 and COTI-NMeCy), and the non-substituted analogue (COTI-NH2) with iron, copper, and zinc ions was characterized in detail. Furthermore, their activities against drug-resistant cancer cells was investigated in comparison to COTI-2 and Triapine. These data revealed that, besides zinc, also iron and copper ions need to be considered to play a role in the mode of action and resistance development of these thiosemicarbazones. Moreover, we identified COTI-NMe2 as an interesting new drug candidate with improved anticancer activity and resistance profile

Maleimide-functionalised platinum(IV) complexes as synthetic platform for targeted drug delivery

Maleimide-functionalised Pt(IV) complexes with highly selective binding properties to thiol groups were synthesised as precursors for binding of thiol-containing tumour targeting molecules like human serum albumi

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

Aim To use the antioxidant compounds (sodium selenite, selenomethionine, D-pantethine) for modulation of cytotoxic effect of doxorubicin and cisplatin toward wild type and drug-resistant mutants of several human tumor cells. Similar treatments were applied in vivo toward adult male Wistar rats. Methods Human tumor cells of different lines (HCT-116, Jurkat and HL-60) with various mechanisms of drug-resistance were treated with doxorubicin or cisplatin, alone or in combination with sodium selenite, selenomethionine, or D-pantethine. Cell viability, induction of apoptosis, and production of O2 - radicals were measured. Activity of redox potential modulating enzymes was measured in the liver and blood plasma of adult male Wistar rats subjected to similar treatments. Results All antioxidants used in physiologically harmless concentration inhibited cytotoxic action of doxorubicin toward tumor cells sensitive to chemotherapy treatment by 15%-30%, and slightly enhanced cytotoxic effect of this medicine toward drug-resistant malignant cells. At the same time, there was no significant effect of these antioxidants on cisplatin action. Such effects were accompanied by a complete inhibition of production of superoxide radicals induced by doxorubicin. The results of in vivo study in adult male Wistar rats were in agreement with the results of in vitro study of human tumor cells. Conclusion Protective effect of specific antioxidant agents during cytotoxic action of doxorubicin was demonstrated in vitro in drug-sensitive human tumor cells and in adult male Wistar rats, while there was no protective effect in drug-resistant sub-lines of these tumor cells during action of doxorubicin and cisplatin