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Moguntinones—New Selective Inhibitors for the Treatment of Human Colorectal Cancer

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Moguntinones—New Selective Inhibitors for the Treatment of Human Colorectal Cancer Published OnlineFirst April 17, 2014; DOI: 10.1158/1535-7163.MCT-13-0224 Molecular Cancer Small Molecule Therapeutics Therapeutics Moguntinones—New Selective Inhibitors for the Treatment of Human Colorectal Cancer Annett Maderer1, Stanislav Plutizki2, Jan-Peter Kramb2, Katrin Gopfert€ 1, Monika Linnig1, Katrin Khillimberger1, Christopher Ganser2, Eva Lauermann2, Gerd Dannhardt2, Peter R. Galle1, and Markus Moehler1 Abstract 3-Indolyl and 3-azaindolyl-4-aryl maleimide derivatives, called moguntinones (MOG), have been selected for their ability to inhibit protein kinases associated with angiogenesis and induce apoptosis. Here, we characterize their mode of action and their potential clinical value in human colorectal cancer in vitro and in vivo. MOG-19 and MOG-13 were characterized in vitro using kinase, viability, and apoptosis assays in different human colon cancer (HT-29, HCT-116, Caco-2, and SW480) and normal colon cell lines (CCD-18Co, FHC, and HCoEpiC) alone or in combination with topoisomerase I inhibitors. Intracellular signaling pathways were analyzed by Western blotting. To determine their potential to inhibit tumor growth in vivo, the human HT-29 tumor xenograft model was used. Moguntinones prominently inhibit several protein kinases associated with tumor growth and metastasis. Specific signaling pathways such as GSK3b and mTOR downstream targets b were inhibited with IC50 values in the nanomolar range. GSK3 signaling inhibition was independent of KRAS, BRAF, and PI3KCA mutation status. While moguntinones alone induced apoptosis only in concentra- tions >10 mmol/L, MOG-19 in combination with topoisomerase I inhibitors induced apoptosis synergistically at lower concentrations. Consistent with in vitro data, MOG-19 significantly reduced tumor volume and weight in combination with a topoisomerase I inhibitor in vivo. Our in vitro and in vivo data present significant proapoptotic, antiangiogenic, and antiproliferative effects of MOG-19 in different human colon cancer cells. Combination with clinically relevant topoisomerase I inhibitors in vitro and xenograft mouse model demon- strate a high potency of moguntinones to complement and improve standard chemotherapy options in human colorectal cancer. Mol Cancer Ther; 13(6); 1399–409. Ó2014 AACR. Introduction ing structural features of 3 natural compounds (combre- Targeting of tyrosine kinases in angiogenesis and other tastatin, rebeccamycin, staurosporin). They showed inhi- bition of the protein kinases VEGFR-2/3, PDGFRb, FLT3, cellular signaling pathways has proved to be effective in b advanced or metastatic human cancer (1). Inhibition of and GSK3 , antiangiogenic efficacy, and antiproliferative VEGFR1–3 and its ligands in the angiogenic pathway by activity. In addition, they induce apoptosis in FLT3-ITD– different classes of inhibitors is an integral part of the dependent acute myeloblastic leukemia (AML) cell lines clinical armamentarium (2). VEGFs induce angiogenesis and patient blasts at low micromolar concentrations (7). by binding to its cognate receptors VEGFR1–3. Intracel- While key function of VEGF and platelet-derived growth factor (PDGF) receptors may be clearly attributed to lular signal transduction results in endothelial cell pro- b liferation, migration, and new vessel formation (3). angiogenesis, GSK3 regulates proliferation in a cell- Recently, we have developed and characterized a novel type–specific manner. One of the important functions of GSK3b is the regulation of transcription factor b-catenin. class of selective VEGFR-2/3 inhibitors (4–6). These novel b b indolyl and azaindolyl-aryl-maleimides, called mogunti- Phosphorylation of -catenin by a GSK3 protein complex nones, are synthetically designed small molecules, includ- leads to its ubiquitination and proteosomal degradation, thereby abolishing its active role in cell proliferation. GSK3b can be inactivated by protein Disheveled if the Authors' Affiliations: Departments of 1Internal Medicine I and 2Pharmacy Wnt signaling pathway is activated resulting in cell pro- and Biochemistry, Johannes Gutenberg University of Mainz, Mainz, b Germany liferation (8). However, until now, the role of GSK3 inhibition (9) is still controversial in a multiplicity of Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). indications and is under investigation (10, 11). FLT3, also known as CD135, binds FLT3 ligands and in turn activates Corresponding Author: Markus Moehler, University Medical Centre of the Johannes Gutenberg University Mainz, First Department of Internal Medicine, signal transduction molecules regulating cell survival and Langenbeckstrasse 1, Mainz 55101, Germany. Phone: 49-6131-176076; proliferation in a cell-type–specific manner. While func- Fax: 49-6131-176621; E-mail: [email protected] tion of FLT3 in colorectal cancer remains unclear, clinical doi: 10.1158/1535-7163.MCT-13-0224 interest for treatment is based on its immunologic func- Ó2014 American Association for Cancer Research. tion (12). Injection of FLT ligands was demonstrated to www.aacrjournals.org 1399 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst April 17, 2014; DOI: 10.1158/1535-7163.MCT-13-0224 Maderer et al. A MOG-19 MOG-13 H H O N O O N O O O N N N O Figure 1. Characterization of MOG- H O H O O 19 and MOG-13. A, chemical structures of MOG-19 and MOG- 13. B, HET-CAM assay showed a B Control MOG-19 (82%) MOG-13 (61%) significant inhibition of vessel growth for MOG-19 and MOG-13 in comparison with the control. mobilize dendritic cells resulting in antitumor activity of ferent specificity and the possibility of combination with the immune system in patients with colon cancer. chemotherapy (20). We have selected moguntinones for their potential to In this article, we describe the effects of moguntinones effectively serve a dual role: inhibition of VEGFR-2/3 as on cell signaling and apoptosis in different human colon well as cell signaling downstream of GSK3b. On the basis cancer cell lines versus normal human epithelial cells, as of our preselection process of more than a hundred sub- well as in vivo significant antiangiogenic properties in stances (4–6), 2 moguntinones (MOG-13 and MOG-19) subcutaneously grown human colon tumors. For summa- were chosen for further characterization in vitro and in vivo ry of key features, see Fig. 1 and Table 1. with respect to their potential to treat human colorectal cancer. Materials and Methods Other tested small-molecule tyrosine kinase inhibitors Cell culture and authentication of cell lines (TKI) to VEGFR and PDGFR like sunitinib or sorafenib, The human colorectal cancer cell lines HT-29, HCT-116, although being effective in some indications, cause side Caco-2, and SW480 were obtained from the German effects as hand–foot syndrome and general fatigue (13). Resource Centre for Biological Material (DSMZ, Braunsch- Such side effects were also observed with, for instance, the weig, Germany, in 2006–2009). Caco-2 was cultured in novel multikinase inhibitor regorafinib (VEGFR, TIE-2) in colorectal cancer (14–16). Especially in this indication, effective treatment of met- Table 1. IC50 values of MOG-19 and MOG-13 astatic disease has also been shown by blockage of intra- cellular signaling pathways promoting cell growth, for MOG-19 MOG-13 example, by inhibiting EGF receptor (EGFR), KRAS, and TargetAV (nmol/L) (nmol/L) PI3K/mTOR-dependent signaling cascades (17). Howev- er, EGFR blocking by monoclonal antibody, for example, Receptors cetuximab or panitumumab, is limited in clinical use for FLT3 18 132 KRAS wild-type patients and also by severe side effects VEGFR-2 70 37 (18). The third approved targeted therapy for colorectal Signaling cascade b > cancer is bevacizumab, a VEGF-binding antibody. Recent- GSK3 71 10.000 ly, continuation of bevacizumab after first progression NOTE: IC50 values of MOG-19 and MOG-13 were carried out plus standard second-line chemotherapy proved the clin- at Millipore (http://www.millipore.com/life_sciences/flx4/ ical efficiency of continuous angiogenesis inhibition (19). ld_kinases) by a kinase assay (in nmol/L). MOG-19 is known With respect to the work presented here, it is of special as a potent VEGFR-2/3 inhibitor (IC50: 70/5 nmol/L) as well as interest that several studies on combination of chemo- FLT3 and GSK3b inhibitor and a moderate PDGFRb inhibitor therapy and different TKI therapy have confirmed a lack (IC50, 680 nmol/L). With the exception of GSK3b, the same is of efficacy or increased toxicity. Taken together, this true for MOG-13. demonstrates the need of novel TKI inhibitors with dif- 1400 Mol Cancer Ther; 13(6) June 2014 Molecular Cancer Therapeutics Downloaded from mct.aacrjournals.org on October 2, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst April 17, 2014; DOI: 10.1158/1535-7163.MCT-13-0224 Moguntinones—New Selective Inhibitors McCoy’s 5A medium (Invitrogen) supplemented with 20% Apoptosis detection was done by nuclear staining with heat-inactivated fetal calf serum (FCS; PAA), 100 units/mL propidium iodide (PI; ref. 22) to measure the sub-G1 cells. penicillin, and 100 mg/mL streptomycin (1%, Cambrex). Cells were incubated with MOG-19 or MOG-13 at the The other cell lines were cultured in RPMI-1640 (Invitro- required dose alone and in combination with cytostatic gen) supplemented with 10% FCS and 1% penicillin/ drugs such as Irinotecan (0.8 mg/mL) and topotecan (20 streptomycin. The normal human epithelial colon cell lines nmol/L) for 7 days. Stained cells were measured by flow FHC and HCoEpiC were obtained from American Type cytometric analysis (FACS) by a BD FACS Calibur. Each Culture Collection (ATCC; CRL-1831) and Sciencell experiment was performed in triplicate. Research Laboratories (2950). FHC was cultured in DMEM:F12 Medium (Invitrogen) supplemented with 10 Caspase assay mmol/L HEPES, 10 ng/mL cholera toxin, 0.005 mg/mL Assays were performed in white 96-well microtiter insulin, 0.005 mg/mL transferrin, 100 ng/mL hydrocorti- plates (Nunc) with the Caspase-Glo 3/7 Assay of Pro- sone, 10% FCS, and 1% penicillin/streptomycin.
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