Gentherapie durch einen RGD-modifizierten, hTERT-regulierten, TRAIL-exprimierenden adenoviralen Vektor in malignen Tumoren

Zusammenfassung

Hintergrund: Die Modifikation adenoviraler Vektoren mit einer Arg-Gly-Asp-(RGD-)Sequenz kann Resistenzentwicklungen umgehen und zu einer erhöhten Transfektion der Zelle führen. Wir konstruierten daher einen adenoviralen Vektor mit einer RGD-Sequenz, der das TRAIL-Gen, gesteuert von einem Human-Telomerase-Reverse-Transcriptase-(hTERT-)Promoter, exprimiert, und untersuchten seine zelltötende Aktivität in vitro und in vivo, wozu ein orthotopes Pankreastumormodel in Nacktmäusen etabliert wurde. Material und Methoden: Die Apoptoseinduktion des Vektors Ad/TRAIL-F/RGD wurde in humanen Zelllinien von hepatozellulären Karzinomen (Hep G2, Hep 3b), Pankreaskarzinomen (Panc-1, Capan-1) und Kolonkarzinomen (LOVO, SW 620) untersucht. Die Hemmung der Zellproliferation wurde mit einem XTT-Assay bestimmt, die GFP-Expression und Apoptoseinduktion mittels Durchflusszytometrie sowie TRAIL- und Caspase-8-Expression durch Western Blot-Analysen. In-vivo-Untersuchungen wurden in einem orthotopen Pankreastumormodel an Nu/nu-Nacktmäusen durchgeführt. Ergebnisse: Die Behandlung mit Ad/TRAIL-F/RGD und Ad/gTRAIL zeigte eine signifikant reduzierte Zellproliferation und deutliche Apoptoseinduktion im Vergleich zu den Kontrollgruppen in allen getesteten Zelllinien. Zusätzlich zeigten die mit Ad/TRAIL-F/RGD behandelten Tiere ein signifikant geringeres Tumorwachstum (p < 0,05) als die mit PBS oder einem Kontrollvektor behandelten Tiere. Schlussfolgerung: Unsere Ergebnisse zeigen bei der Verwendung des adenoviralen Vektors Ad/TRAIL-F/RGD in vitro eine signifikante Proliferationshemmung und deutliche Apoptoseinduktion in humanen Tumorzelllinien sowie eine signifikante Tumorwachstumshemmung in orthotop implantierten Pankreastumoren im Pankreasschwanz. Der Einsatz des adenoviralen Vektors Ad/TRAIL-F/RGD bei der Behandlung maligner Tumoren könnte in Zukunft eine Therapieoption darstellen.

Abstract

Background: Resistance can be overcome by modified adenoviral vectors containing an Arg-Gly-Asp (RGD) sequence. We constructed an adenoviral vector with RGD-modified fibers, expressing the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter (designated Ad/TRAIL-F/RGD), and evaluated its antitumor activity in vitro and in vivo. Methods: The induction of apoptosis by the new vector Ad/TRAIL-F/RGD was evaluated in human carcinoma cells derived from hepatocellular carcinoma (Hep G2, Hep 3b), pancreatic carcinoma (Panc-1, Capan-1), and colon carcinoma (LOVO, SW 620). Cell viability was measured by the XTT assay and GFP expression and apoptosis induction by fluorescence-activated cell sorting (FACS) and Western blot. In vivo experiments were performed in an orthotopic pancreas tumor model in nu/nu nude mice. Results: Treatment with Ad/TRAIL-F/RGD and Ad/gTRAIL resulted in significantly reduced cell viability in comparison to PBS and Ad/CMV-GFP treatment in all examined human carcinoma cell lines. In addition, mice treated with Ad/TRAIL-F/RGD showed a significantly decreased tumor growth than both control groups. Conclusions: Our results suggest that Ad/TRAIL-F/RGD may become a potent therapeutic agent for the treatment of different human solid carcinomas.

Schlüsselwörter

Apoptose - Gentherapie - TRAIL - RGD - Adenovirus - Gentransfer

Key words

Apoptosis - gene therapy - TRAIL - RGD - adenovirus

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