Antimetastatische Effizienz synthetischer Serinproteaseinhibitoren

Zusammenfassung

Es gibt kein wirksames adjuvantes Therapeutikum, das die aggressive Ausbreitung von Tumorzellen in fortgeschrittenen Tumorstadien oder bei chirurgischer Entfernung des Primärtumors verhindert. Obwohl Proteasen einschließlich der Serinproteasen als vielversprechende Zielstrukturen für eine antimetastatische Krebstherapie ausgewiesen wurden, schlug die klinische Einführung synthetischer Proteaseinhibitoren (SPIs) fehl. Zusätzlich zu Überlegungen zu klassischen Herangehensweisen bei der Prüfung von SPIs als Krebstherapeutika in vivo, beleuchten wir hier unsere Ergebnisse mit einem einfach handhabbaren, hochsensitiven und schnellen In-vivo-Metastasierungsmodell und seine Bedeutung für die Entwicklung effizienter, antimetastatisch wirksamer SPIs. Die lacZ-Markierung sehr aggressiver T-Zell-Lymphomzellen ermöglicht die hochsensitive und reproduzierbare Detektion von Metastasen innerhalb von sieben Tagen nach Tumorzellinokulation, durch X-Gal-Färbung ganzer Organe. Dieses Modell erlaubt ein kostengünstiges und materialsparendes paralleles Screening einer Serie von SPIs mit unterschiedlichen Spezifitäten für verschiedene Serinproteasen. Durch Korrelationen zwischen Spezifität und antimetastatischer Effizienz identifizierten wir den Gerinnungsfaktor Xa als wichtige Zielstruktur antimetastatischer SPIs und konnten diese Information für das weitere Design und die Optimierung von Faktor-Xaspezifischen Leitstrukturen verwenden. Schlussfolgerung: Wir können exemplarisch zeigen, dass In-vivo-Analysen mit hohem Durchsatz die Optimierung von Leitstrukturen dirigieren können und darüber hinaus zu unerwarteten, neuen Einblicken in die Molekularbiologie der Metastasierung führen.

Summary

To date, no effective adjuvant drug preventing the aggressive spread of tumour cells in late stages of cancer disease or at the time-point of primary tumour removal is available. Although proteases, including members of the large serine protease family, were shown to be promising targets for an anti-metastatic cancer therapy, synthetic protease inhibitors (SPIs) have so far failed to be introduced into the clinic. In addition to considerations in the design of classical in vivo-tests of SPIs as cancer therapy agents, we here review our findings with a straightforward, highly sensitive and very fast in vivo metastasis model and its implications in the development of efficient anti-metastatic SPIs. The lacZ-tagging of tumour cells of this very aggressive T-cell lymphoma model allowed highly sensitive and reproducible detection of metastases within seven days after tumour cell inoculation by X-gal staining of whole organs, allowing cost-effective and material-saving side-by-side screening of a series of SPIs with different specificities for different serine proteases. By establishment of specificity/antimetastatic efficacy correlations we identified coagulation factor Xa as one important target of anti-metastatic SPIs and could use this information for the subsequent design and optimization of factor Xa-specific lead structures. Conclusion: We exemplify the usefulness of high-throughput in vivo analysis to direct optimization of lead structures and how this may allow unexpected insight into the molecular biology of metastasis.

Schlüsselwörter

Tiermodelle - Serinproteasen - Proteaseinhibitoren - Metastasierung - Screening - Lymphom - Fibrosarkom

Keywords

Animal models - serine proteases - protease inhibitors - metastasis - screening - lymphoma - fibrosarcoma

• Literatur

• 1 Arlt M, Kopitz C, Pennington C. et al. Increase in gelatinase-specificity of matrix metalloproteinase inhibitors correlates with antimetastatic efficacy in a T-cell lymphoma model. Cancer Res 2002; 62: 5543-50.
• 2 Atkin G, Chopada A, Mitchell I. Colorectal cancer metastasis: in the surgeon’s hands?. Int Semin Surg Oncol 2005; 24: 5.
• 3 Bachmeier BE, Iancu CM, Jochum M. et al. Matrix metalloproteinases in cancer: comparison of known and novel aspects of their inhibition as a therapeutic approach. Expert Rev Anticancer Ther 2005; 5: 149-63.
• 4 Banke IJ, Arlt MJ, Mueller MM. et al. Effective inhibition of experimental metastasis and prolongation of survival in mice by a potent factor Xa-spe- cific synthetic serine protease inhibitor with weak anticoagulant activity. Thromb Haemost 2005; 94: 1084-93.
• 5 Banke IJ, Arlt MJ, Pennington C. et al. Increase of anti-metastatic efficacy by selectivity- but not affinity- optimization of synthetic serine protease inhibitors. Biol Chem 2003; 384: 1515-25.
• 6 Chen EX, Siu LL. Development of molecular targeted anticancer agents: successes, failures and future directions. Curr Pharm Des 2005; 11: 265-72.
• 7 Dano K, Behrendt N, Hoyer-Hansen G. et al. Plasminogen activation and cancer. Thromb Haemost 2005; 93: 676-81.
• 8 Eccles SA. Basic principles for the study of metastasis using animal models. In: Brooks SA, Schumacher U. (Hrsg). Metastasis Research Protocols, Vol 2, Analysis of Cell Behavior In Vitro and In Vivo. Totowa, New Jersey: Humana Press; 2001: 161-71.
• 9 Fingleton B. Matrix metalloproteinase inhibitors for cancer therapy: the current situation and future prospects. Expert Opin Ther Targets 2003; 7: 385-97.
• 10 Folgueras AR, Pendas AM, Sanchez LM. et al. Matrix metalloproteinases in cancer: from new functions to improved inhibition strategies. Int J Dev Biol 2004; 48: 411-24.
• 11 Folkman J. How is blood vessel growth regulated in normal and neoplastic tissue? G. H. A. Clowes memorial Award lecture. Cancer Res 1986; 46: 467-73.
• 12 Francis JL, Amirkhosravi A. Effects of antihemostatic agents on experimental tumor dissemination. Semin Thromb Hemost 2002; 28: 29-38.
• 13 Groves MD, Puduvalli VK, Hess KR. et al. Phase II trial of temozolomide plus the matrix metalloproteinase inhibitor, Marimastat, in recurrent and progressive glioblastoma multiforme. J Clin Oncol 2002; 20: 1383-8.
• 14 Guy CT, Cardoso G. Transgenic animal models. In: Brooks SA, Schumacher U. (Hrsg). Metastasis Research Protocols. Vol 2. Analysis of Cell Behavior In Vitro and In Vivo. Totowa, New Jersey: Humana Press; 2001: 231-5.
• 15 Günthert U. Biologie der Metastasierung und Tumorangiogenese. In: Ostendorf PC, Seeber S. (Hrsg). Hämatologie und Onkologie. München: Urban und Schwarzenberg; 1997: 160-7.
• 16 Heath EI, Burtness BA, Kleinberg L. et al. Phase II, parallel-design study of preoperative combined modality therapy and the matrix metalloprotease (mmp) inhibitor prinomastat in patients with esophageal adenocarcinoma. Invest New Drugs 2006; 24: 135-40.
• 17 Hejna M, Raderer M, Zielinski CC. Inhibition of metastases by anticoagulants. J Natl Cancer Inst 1999; 91: 22-36.
• 18 Hirota M, Shimada S, Yamamoto K. et al. Pancreatectomy using the no-touch isolation technique followed by extensive intraoperative peritoneal lavage to prevent cancer cell dissemination: a pilot study. JOP 2005; 6: 143-51.
• 19 Jedinak A, Maliar T. Inhibitors of proteases as anticancer drugs. Neoplasma 2005; 52: 185-92.
• 20 Jiang X, Bailly MA, Panetti TS. et al. Formation of tissue factor-factor VIIa-factor Xa complex promotes cellular signalling and migration of human breast cancer cells. J Thromb Haemost 2004; 2: 93-101.
• 21 King J, Zhao J, Clingan P. et al. Randomised double blind placebo control study of adjuvant treatment with the metalloproteinase inhibitor, Marimastat in patients with inoperable colorectal hepatic metastases: significant survival advantage in patients with musculoskeletal side-effects. Anticancer Res 2003; 23: 639-45.
• 22 Koblinski JE, Ahram M, Sloane BF. Unraveling the role of proteases in cancer. Clin Chim Acta 2000; 291: 113-35.
• 23 Kopitz C, Anton M, Gänsbacher B. et al. Reduction of experimental human fibrosarcoma lung metastasis in mice by adenovirus-mediated cystatin C overexpression in the host. Cancer Res 2005; 65: 8608-12.
• 24 Krüger A, Schirrmacher V, Khokha R. The bacterial lacZ gene: an important tool for metastasis research and evaluation of new cancer therapies. Cancer Metastasis Rev 1998–1999 17: 285-94.
• 25 Lah TT, Duran Alonso MB, van Noorden CJ. Antiprotease therapy in cancer: hot or not?. Expert Opin Biol Ther 2006; 6: 257-79.
• 26 Lopez-Otin C, Overall CM. Protease degradomics: a new challenge for proteomics. Nat Rev Mol Cell Biol 2002; 3: 509-19.
• 27 Morris DR, Ding Y, Ricks TK. et al. Protease activated receptor-2 is essential for factor VIIa and Xa-induced signaling, migration and invasion of breast cancer cells. Cancer Res 2006; 66: 307-14.
• 28 Ohta T, Futagami F, Arakawa H. et al. Inhibitory effect of FOY-305 on liver metastasis of the pancreatic cancer. Cancer and Chemotherapy 1996; 23: 1669-72.
• 29 Overall CM, Kleifeld O. Towards third generation matrix metalloproteinase inhibitors for cancer therapy. Br J Cancer 2006; 94: 941-6.
• 30 Overall CM, Kleifeld O. Tumour microenvironment – opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer 2006; 6: 227-39.
• 31 Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer 2004; 4: 448-56.
• 32 Pardee AB, Jacob F, Monod J. The role of the inducible alleles and the constructive alleles in the sythesis of beta-galactosidase in zygotes of Escherichia coli. C R Hebd Seances Acad Sci 1958; 246: 3125-8.
• 33 Paris S, Sesboue R. Metastasis models: the green fluorescent revolution?. Carciogenesis 2004; 25: 2285-92.
• 34 Price JE. Xenograft models in immunodeficient animals, I. Nude mice: spontaneous and experimental metastasis models. In: Brooks SA, Schumacher U. (Hrsg). Metastasis Research Protocols, Volume 2, Analysis of Cell Behavior In Vitro and In Vivo. Totowa, New Jersey: Humana Press; 2001: 205-14.
• 35 Rosenberg B, van Camp L, Trosko JE. et al. Platinum compounds: a new class of potent antitumor agents. Nature 1969; 222: 385-6.
• 36 Sato S, Kopitz C, Schmalix WA. et al. High-affinity urokinase-derived cyclic peptides inhibiting urokinase/urokinase receptor-interaction: effects on tumor growth and spread. FEBS Lett 2002; 528: 212-6.
• 37 Schweinitz A, Steinmetzer T, Banke IJ. et al. Design of novel and selective inhibitors of urokinasetype plasminogen activator with improved pharmacokinetic properties for use as anti-metastatic agents. J Biol Chem 2004; 279: 33613-22.
• 38 Setyono-Han B, Stürzebecher J, Schmalix WA. et al. Suppression of rat breast cancer metastasis and reduction of primary tumour growth by the small synthetic urokinase inhibitor WX-UK1. Thromb Haemost 2005; 93: 779-86.
• 39 Tsuchiya Y, Sawada S, Yoshioka I. et al. Increased surgical stress promotes tumor metastasis. Surgery 2003; 133: 547-55.
• 40 Vihinen P, Ala-aho R, Kahari VM. Matrix metalloproteinases as therapeutic targets in cancer. Curr Cancer Drug Targets 2005; 5: 203-20.