Z Gastroenterol 2004; 42(12): 1363-1370
DOI: 10.1055/s-2004-813310
Originalarbeit

© Karl Demeter Verlag im Georg Thieme Verlag KG Stuttgart · New York

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

Adenoviral Vector Expressing the TRAIL Gene Driven by the hTERT PromoterD. Jacob1 , J. Davis2 , G. Schumacher1 , M. Bahra1 , P. Neuhaus1 , B. Fang2
  • 1Klinik für Transplantations-, Viszeral- und Allgemeinchirurgie, Charité Campus-Virchow, Humboldt-Universität zu Berlin, Deutschland
  • 2Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, and the Program in Gene Therapy and Virology, The University of Texas Graduate School of Biomedical Sciences, Houston, USA
Weitere Informationen

Publikationsverlauf

Manuskript eingetroffen: 16.4.2004

Manuskript akzeptiert: 26.5.2004

Publikationsdatum:
09. Dezember 2004 (online)

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.

  • 1 Yeo C J, Cameron J L, Sohn T A. et al . Six hundred fifty consecutive Pancreaticoduodenectomies in the 1990 s: Pathology, Complications, and Outcomes.  Ann Surg. 1997;  226 248-257
  • 2 Yeo T P, Hruban R H, Leach S D. et al . Pancreatic Cancer.  Curr Probl Cancer. 2002;  4 176-275
  • 3 Ghung-Faye G A, Kerr D J, Young L S. et al . Gene therapy strategies for colon cancer.  Mol Med Today. 2000;  6 82-87
  • 4 Huang X, Lin T, Gu J. et al . Long-term tumor-free survival from treatment with the GFP-TRAIL fusion gene expressed from the hTERT promoter in breast cancer cells.  Int J Oncol. 2003;  22 1241-1245
  • 5 Zhang L, Gu J, Huang X. et al . Mechanisms involved in development of resistance to adenovirus-mediated proapoptotic gene therapy in DLD1 human colon cancer cell line.  Gene Ther. 2002;  9 1262-1270
  • 6 Kagawa S, He C, Gu J. et al . Antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene.  Cancer Res. 2001;  61 3330-3338
  • 7 Lin T, Gu J, Zhang L. et al . Targeted expression of green fluorescent potein/tumor necrosis factor-related apoptosis-inducing ligand fusion protein from human telomerase reverse transcriptase promoter elicits antitumor activity without toxic effects on primary human hepatocytes.  Cancer Res. 2002;  62 3620-3625
  • 8 Voelkel-Johnson C, King D L, Norris J S. Resistance of prostate cancer cells to soluble TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) can be overcome by doxorubicin or adenoviral delivery of full-length TRAIL.  Cancer Gene Ther. 2002;  9 164-172
  • 9 Jacob D, Schumacher G, Bahra M. et al . Targeted expression of GFP/TRAIL fusion protein from hTERT promoter elicits apoptosis in human lung cancer cells.  J Cancer Res Clin Oncol. 2004;  130 (Supp 1) S160
  • 10 Nitsch R, Bechmann I, Deisz R A. et al . Human brain-cell death induced by tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL).  Lancet. 2000;  356 (9232) 827-828
  • 11 Jo M, Kim T H, Seol D W. et al . Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand.  Nat Med. 2000;  6 564-567
  • 12 Gu J, Kagawa S, Takakura M. et al . Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the bax gene to cancer.  Cancer Res. 2000;  60 5359-5364
  • 13 Koch P, Guo Z S, Kagawa S. et al . Augmenting transgene expression from carcinoembryonic antigen (CEA) promoter via a GAL4 gene regulatory system.  Mol Ther. 2001;  3 278-283
  • 14 Bergelson J M, Krithivas A, Celi L. et al . The murine CAR homolog is a receptor for coxsackie B virus and adenovirus.  J Virol. 1998;  72 415-419
  • 15 Pearson A S, Koch P E, Atkinson. et al . Factors limiting adenovirus-mediated gene transfer into human lung and pancreatic cancer cell lines.  Clin Cancer Res. 1999;  5 4208-4213
  • 16 Jee Y S, Lee S G, Lee J C. et al . Reduced expression of coxsackievirus and adenovirus receptor (CAR) in tumor tissue compared to normal epithelium in head and neck squamous cell carcinoma patients.  Anticancer Res. 2002;  22 2629-2634
  • 17 Dehari H, Ito Y, Nakamura T. et al . Enhanced antitumor effect of RGD fiber-modified adenovirus for gene therapy of oral cancer.  Cancer Gene Ther. 2003;  10 75-85
  • 18 Wu H, Seki T, Dimitriev et al. Double modification of adenovirus fiber with RGD and polylysine motifs improves coxsackievirus-adenovirus receptor-independent gene transfer efficiency.  Human Gene Ther. 2002;  13 1647-1653
  • 19 Kanerva A, Wang M, Bauerschmitz G J. et al . Gene transfer to ovarien cancer versus normal tissues with fiber-modified adenovirus.  Mol Ther. 2002;  5 695-704
  • 20 Nakamura T, Sato K, Hamada H. Effective gene transfer to human melanomas via integrin-targeted adenoviral vectors.  Human Gene Ther. 2002;  13 613-626
  • 21 Koizumi N, Mizuguchi H, Hosono T. et al . Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide.  Biochim Biophys Acta. 2001;  1568 13-20
  • 22 Fang B, Ji L, Bouvet M. et al . Evaluation of GAL4/TATA in vivo. Induction of transgene expression by adenovirally mediated gene codelivery.  J Biol Chem. 1998;  27 4972-4975
  • 23 Zhu H, Zhang L, Wu S. et al . Induction of S-phase arrest and p21 overexpression by a small molecule 2[[3-(2,3-dichlorophenoxy)propyl] amino]ethanol in correlation with activation of ERK.  Oncogene. 2004;  May (online publication)
  • 24 Kim M, Sumerel L A, Belousova N. et al . The coxsackevirus and adenovirus receptor acts as a tumor suppressor in malignant glioma.  Br J Cancer. 2003;  88 (9) 1411-1416
  • 25 Lin T, Zhang L, Davis J. et al . Combination of TRAIL gene therapy and chemotherapy enhances antitumor and antimetastasis effects in chemosensitive and chemoresistant breast cancer.  Mol Therapy. 2003;  8 441-448
  • 26 Uchida H, Shinoura N, Kitayama J. et al . 5-Fluorouracil efficiently enhanced apoptosis induced by adenovirus-mediated transfer of caspase-8 in DLD-1 colon cancer cells.  J Gene Medicine. 2003;  5 287-299
  • 27 Sung M W, Yeh H C, Thung S N. et al . Intratumoral adenovirus-mediated suicide gene transfer for hepatic metastases from colorectal adenocarcinoma: results of a phase I clinical trial.  Mol Therapy. 2001;  4 182-191
  • 28 Swisher G S, Roth J A, Komaki R. et al . Induction of p53-regulated genes and tumor regression in lung cancer patients after intratumoral delivery od adenoviral p53 (INGN 201) and radiation therapy.  Clin Cancer Res. 2003;  9 93-101
  • 29 Roth J A. Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer.  Nat Med. 1996;  2 985-991
  • 30 Habib N A, Sarraf C E, Mitry R R. et al . E1B-deleted adenovirus (dl1520) gene therapy for patients with primary and secondary liver tumors.  Human Gene Therapy. 2001;  12 219-226

Dr. med. Dietmar Jacob

Klinik für Transplantations-, Viszeral- und Allgemeinchirurgie, Charité Campus-Virchow

Humboldt-Universität zu Berlin

Augustenburger Platz 1

13353 Berlin

Telefon: ++ 49/30/4 50-55 20 01

Fax: ++ 49/30/4 50-55 29 00

eMail: dietmar.jacob@charite.de