Katharina Mahal

Gold(I)-NHC complexes of antitumoral diarylimidazoles: structures, cellular uptake routes and anticancer activities.

Five new heterocyclic gold carbene complexes were prepared, four chlorido-[1,3-dimethyl-4,5-diarylimidazol-2-ylidene]gold complexes 6a-d and a chlorido-[1,3-dibenzylimidazol-2-ylidene]gold complex 11, and three of them were characterised by X-ray single crystal analyses. They were tested for cytotoxicity against a panel of four human cancer cell lines and non-malignant fibroblasts, for tubulin interaction, and for the pathways of their uptake into 518A2 melanoma cells. All complexes showed cytotoxic activity in the micromolar IC(50) range with distinct selectivities for certain cell lines. In stark contrast to related metal-free 1-methyl-4,5-diarylimidazoles, the complexes 6 and 11 did not noticeably inhibit the polymerisation of tubulin to give microtubules. The cellular uptake of complexes 6 occurred mainly via the copper transporter (Ctr1) and the organic cation transporters (OCT-1/2). Complex 11 was accumulated preferentially via the organic cation transporters and by Na(+)/K(+)-dependent endocytosis. The new gold carbene complexes seem to operate by a mechanism different from that of the parent 1-methylimidazolium ligands.

Effects of chrysin, apigenin, genistein and their homoleptic copper(II) complexes on the growth and metastatic potential of cancer cells

The (iso-)flavonoids chrysin 1, apigenin 2, genistein 3 and their homoleptic copper(II) complexes 4-6 were compared for general cancer cell growth inhibition and for antimetastatic effects on rapidly proliferating and metastasizing 518A2 melanoma cells. The complexes 4-6 were three to five times more active than the free flavonoids in cytotoxicity assays with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] against 518A2 melanoma, HCT-116 colon, KB-V1/Vbl cervix, and MCF-7/Topo breast carcinoma cells. This activity correlated with an arrest of the cell cycle of 518A2 melanoma cells at the G2/M transition. The complexes also diminished the migration propensity of these cells in wound healing assays more distinctly than the flavonoid ligands. By fluorescent staining of F-actin and beta-catenin the antimetastatic effects of the Cu(II) genistein complex 6 were shown to originate from a remodeling of the actin cytoskeleton and an increase in cadherin-catenin complex formation, factors that favor cell-cell adhesion. Complex 6 also attenuated the expression and secretion of the metastasis-relevant matrix metalloproteinases MMP-2 and MMP-9. In summary, coordination of apigenin and genistein to Cu(II) greatly enhances the antitumoral properties of these flavonoids and potentiates their mechanistic diversity.

Ferrocene and (arene)ruthenium(II) complexes of the natural anticancer naphthoquinone plumbagin with enhanced efficacy against resistant cancer cells and a genuine mode of action

A series of ferrocene and (arene)ruthenium(II) complexes attached to the naturally occurring anticancer naphthoquinones plumbagin and juglone was tested for efficacy against various cancer cell lines and for alterations in the mode of action. The plumbagin ferrocene and (p-cymene)Ru(II) conjugates 1c and 2a overcame the multi-drug drug resistance of KB-V1/Vbl cervix carcinoma cells and showed IC50 (72 h) values around 1 μM in growth inhibition assays using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT). They were further investigated for their influence on the cell cycle of KB-V1/Vbl and HCT-116 colon carcinoma cells, on the generation of reactive oxygen species (ROS) by the latter cell line, for their substrate character for the P-glycoprotein drug eflux pump via the calcein-AM efflux assays, and for DNA affinity by the electrophoretic mobility shift assay (EMSA). The derivatives 1c and 2a increased the number of dead cancer cells (sub-G0/G1 fraction) in a dose- and time-dependent manner. ROS levels were significantly increased upon treatment with 1c and 2a. These compounds also showed a greater affinity to linear DNA than plumbagin. While plumbagin did not affect calcein-AM transport by P-glycoprotein the derivatives 1c and 2a exhibited a 50% or 80% inhibition of the P-glycoprotein-mediated calcein-AM efflux relative to the clinically established sensitizer verapamil.

Effects of the Tumor‐Vasculature‐Disrupting Agent Verubulin and Two Heteroaryl Analogues on Cancer Cells, Endothelial Cells, and Blood Vessels

Two analogues of the discontinued tumor vascular‐disrupting agent verubulin (Azixa®, MPC‐6827, 1) featuring benzo‐1,4‐dioxan‐6‐yl (compound 5 a) and N‐methylindol‐5‐yl (compound 10) residues instead of the para‐anisyl group on the 4‐(methylamino)‐2‐methylquinazoline pharmacophore, were prepared and found to exceed the antitumor efficacy of the lead compound. They were antiproliferative with single‐digit nanomolar IC50 values against a panel of nine tumor cell lines, while not affecting nonmalignant fibroblasts. Indole 10 surpassed verubulin in seven tumor cell lines including colon, breast, ovarian, and germ cell cancer cell lines. In line with docking studies indicating that compound 10 may bind the colchicine binding site of tubulin more tightly (Ebind=−9.8 kcal mol−1) than verubulin (Ebind=−8.3 kcal mol−1), 10 suppressed the formation of vessel‐like tubes in endothelial cells and destroyed the blood vessels in the chorioallantoic membrane of fertilized chicken eggs at nanomolar concentrations. When applied to nude mice bearing a highly vascularized 1411HP germ cell xenograft tumor, compound 10 displayed pronounced vascular‐disrupting effects that led to hemorrhages and extensive central necrosis in the tumor.

Improved anticancer and antiparasitic activity of new lawsone Mannich bases

Substituted lawsone Mannich bases 2a-e, 3a-e and 4a-e were prepared and tested for their biological activities. The new fatty alkyl substituted compounds 2a-c exhibited strong and selective growth inhibitory activities in the low one-digit micromolar and sub-micromolar range against a panel of human cancer cell lines associated with ROS formation. In addition, compounds 2a-c revealed sub-micromolar anti-trypanosomal activities against parasitic Trypanosoma brucei brucei cells via deformation of the microtubule cytoskeleton. The N-hexadecyl compound 2c was also highly active against locally isolated Entamoeba histolytica parasite samples exceeding the activity of metronidazole.

Cellular localisation of antitumoral 6-alkyl thymoquinones revealed by an alkyne-azide click reaction and the streptavidin-biotin system.

The subcellular distribution and accumulation of thymoquinone 1, a natural anticancer agent, has hitherto been unknown. We prepared 6‐(dec‐9‐ynyl)thymoquinone 3, an alkyne‐labelled derivative with anticancer activity similar to that of its parent compound 1. Alkyne 3 was seen, after a Huisgen‐type click reaction with 3‐azido‐7‐hydroxycoumarin, to accumulate in distinct compartments of the nuclei of PtK2 potoroo kidney cells, and in adjoining regions that were stained with an antibody specific for the Golgi apparatus. In contrast, a biotinlabelled thymoquinone 4 seemed to accumulate across the entire cell nucleus upon visualisation with streptavidin; but this was less easily traceable because of co‐staining of other structures such as mitochondria. In conclusion, for small drug‐like molecules, visualisation by alkyne–azide cycloaddition seems to be superior to conventional visualisation by the biotin–streptavidin system.

4-(1-Ethyl-4-anisyl-imidazol-5-yl)-N-hydroxycinnamide – A new pleiotropic HDAC inhibitor targeting cancer cell signalling and cytoskeletal organisation

Histone deacetylases (HDAC) which play a crucial role in cancer cell proliferation are promising drug targets. However, HDAC inhibitors (HDACi) modelled on natural hydroxamic acids such as trichostatin A frequently lead to resistance or even an increased agressiveness of tumours. As a workaround we developed 4-(1-ethyl-4-anisyl-imidazol-5-yl)-N-hydroxycinnamide (etacrox), a hydroxamic acid that combines HDAC inhibition with synergistic effects of the 4,5-diarylimidazole residue. Etacrox proved highly cytotoxic against a panel of metastatic and resistant cancer cell lines while showing greater specificity for cancer over non-malignant cells when compared to the approved HDACi vorinostat. Like the latter, etacrox and the closely related imidazoles bimacroxam and animacroxam acted as pan-HDACi yet showed some specificity for HDAC6. Akt signalling and interference with nuclear beta-catenin localisation were elicited by etacrox at lower concentrations when compared to vorinostat. Moreover, etacrox disrupted the microtubule and focal adhesion dynamics of cancer cells and inhibited the proteolytic activity of prometastatic and proangiogenic matrix metalloproteinases. As a consequence, etacrox acted strongly antimigratory and antiinvasive against various cancer cell lines in three-dimensional transwell invasion assays and also antiangiogenic in vivo with respect to blood vessel formation in the chorioallantoic membrane assay. These pleiotropic effects and its water-solubility and tolerance by mice render etacrox a promising new HDACi candidate.

Synergistic "gold effects" of anti-vascular 4,5-diarylimidazol-2-ylidene gold(I) carbene complexes.

The natural cis-stilbene combretastatin A-4 (CA-4) is a vascular disrupting agent (VDA) that leads to a vascular shutdown in solid tumors. Its activity originates from an inhibition of the polymerization of tubulin and of the functionality of the VE-cadherin/ β-catenin complex which is crucial for cell– cell adhesion [1]. However, the insufficient cytotoxicity of CA-4 and its chemical instability in solution limits its clinical usability. A more water-soluble phosphate prodrug of CA-4 is currently undergoing clinical trials [2]. Derivatives of CA-4 with improved chemical stability and retained anti-vascular effect were obtained by incorporation of the Z-alkene in heterocycles such as imidazoles [3]. Since structurally simple imidazolium salts had already been employed as ligand precursors for anticancer N-heterocyclic carbene (NHC) complexes [4], we prepared gold carbene complexes that combine the intrinsic anti-vascular activity of CA-4 analogous imidazoles 1 (Figure 1A) with the known anticancer properties of gold fragments [5]. Previously, we reported on the cytotoxicity and the cellular uptake of such new gold(I)-NHC complexes [5]. Now we present first insights into the mechanism of action of two such complexes, 2a and 3a (Figure 1A).

Biological evaluation of 4,5-diarylimidazoles with hydroxamic acid appendages as novel dual mode anticancer agents

AbstractPurpose New (4-aryl-1-methylimidazol-5-yl)cinnamoylhydroxamic acids were prepared as potential dual mode anticancer agents combining the antivascular effect of the 4,5-diarylimidazole moiety and the histone deacetylases (HDAC) inhibition by the cinnamoyl hydroxamate. MethodsTheir antiproliferative activity against a panel of primary cells and cancer cell lines was determined by MTT assays and their apoptosis induction by caspase-3 activation. Their ability to reduce the activity of HDAC was measured by enzymatic assays and Western blot analyses of cellular HDAC substrates. Additional effects on cancer cell migration were ascertained via immunofluorescence staining of cytoskeleton components and three-dimensional migration assays. The chorioallantoic membrane assay was used as an in vivo model to assess their antiangiogenic properties.ResultsThe 4-phenyl- and 4-(p-methoxyphenyl)-imidazole derivatives had a greater antiproliferative and apoptosis inducing effect in a variety of cancer cell lines when compared with the approved HDAC inhibitor SAHA, and most distinctly so in non-malignant human umbilical vein endothelial cells. Like SAHA, both compounds acted as pan-HDAC inhibitors. In 518A2 melanoma cells, they led to hyperacetylation of histones and of the cytoplasmic HDAC6 substrate alpha-tubulin. As a consequence, they inhibited the migration and invasion of these cells in transwell invasion assays. In keeping with its pronounced impact on endothelial cells, the 4-phenyl-imidazole derivative also inhibited the growth and sprouting of blood vessels in the chorioallantoic membrane of fertilized hen eggs.ConclusionsThe 4-phenyl- and 4-(p-methoxyphenyl)-imidazole compounds combine the antivascular effects of 4,5-diarylimidazoles with HDAC inhibition by cinnamoyl hydroxamates and show additional antimetastatic activity. They are promising candidates for pleiotropic HDAC inhibitors.

Role of JNK and NF-κB in mediating the effect of combretastatin A-4 and brimamin on endothelial and carcinoma cells.

PurposeThe 4,5-diarylimidazole brimamin is an analog of the natural vascular-disrupting agent combretastatin A-4 (CA-4) with improved water solubility, tolerance by animals and efficacy in multidrug-resistant tumors. Here, we aimed at identifying the major mechanisms underlying the in vitro and in vivo actions of brimamin on endothelial and carcinoma cells, including vascularization.MethodsThe contribution of specific signaling kinases to the effects of brimamin on cytoskeleton organization and the viability and differentiation of endothelial cells was assessed by MTT and tube formation assays in the presence or absence of specific kinase inhibitors. Changes in DNA affinity and expression of NF-κB in endothelial and carcinoma-derived cells and their solid tumors (xenografts) treated with brimamin were ascertained by electrophoretic mobility shift assays and Western blotting. The anti-vascular effect of brimamin in solid tumors was verified by CD31 immunostaining.ResultsWe found that brimamin can inhibit tubulin polymerization and cause a reorganization of F-actin in Ea.hy926 endothelial cells. Its inhibitory effect on tube formation was found to depend on functional Rho kinase and JNK. JNK inhibition was found to suppress the induction of endothelial cell apoptosis by brimamin. In CA-4-refractory human BxPC-3 pancreas carcinoma-derived and triple-negative MDA-MB-231 breast carcinoma-derived cells brimamin was found to inhibit growth and to induce apoptosis at low nanomolar concentrations by blocking NF-κB activation in a dose-dependent manner. Brimamin was also found to reduce the in vivo growth rate and vascularization of MDA-MB-231 xenografts in mice. Residual tumor cells of these treated xenografts showed a relatively low expression of the p65 subunit of NF-κB.ConclusionsOur data indicate that cellular JNK and Rho kinase activities are crucial for the cytotoxic and cytoskeleton reorganizing effects of brimamin on endothelial cells. In addition, we found that in resistant carcinoma cells and xenografts brimamin can induce down-regulation of anti-apoptotic NF-κB expression and signaling. Its chemical properties and efficacy against clinically relevant cancer entities make brimamin a promising candidate vascular-disrupting agent.