Characterisation of enzyme prodrug gene therapy combinations in coated spheroids and vascular networks in vitro

Journal of Gene Medicine - Tập 14 Số 1 - Trang 62-74 - 2012
Michelle Hunt1, Dan Li2, Michael P. Hay2, Margaret J. Currie1, Bridget Robinson1, Adam V. Patterson2, Gabi U. Dachs1,3
1Angiogenesis and Cancer Research Group, Department of Pathology, University of Otago, Christchurch, New Zealand
2Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand
3G. Dachs, Angiogenesis and Cancer Research Group, Department of Pathology, University of Otago, Christchurch, PO Box 4345, Christchurch 8140, New Zealand.

Tóm tắt

AbstractBackgroundEnzyme prodrug gene therapy is designed as a targeted cancer treatment, destroying gene‐modified and bystander cells via exogenous enzyme‐generated cytotoxins. Targeting of tumour blood vessels using gene therapy is attractive, although optimal enzyme prodrug combinations have yet to be identified.MethodsSeven enzyme prodrug combinations were ranked in two endothelial (HUVEC, HMEC‐1) and one tumour cell line (T24) for their ability to reduce proliferation and viability. The ability to destroy bystander cells in two dimensions (2D) and three dimensions (3D), mode of cell kill, and the ability to disrupt vascular networks were measured.ResultsEndothelial cell proliferation (bromodeoxyuridine uptake) was reduced most effectively by Herpes simplex virus thymidine kinase (TK) with ganciclovir (GCV), followed by Escherichia coli nitroreductase NfsB (NTR) with CB1954; viability [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay] was reduced most efficiently by NTR/CB1954 followed by TK/GCV. Of the seven combinations, only NTR/CB1954 displayed measurable bystander effects in 2D monolayers, and none demonstrated bystander killing in coated spheroids, a 3D spatially distinct model with tissue‐like cell density. NTR‐expressing endothelial cells displayed increased apoptosis, necrosis and caspase‐3 activity after CB1954 treatment. Despite good antiproliferative activity, TK/GCV was ineffective at disrupting vascular network‐like structures of endothelial cells, whereas NTR/CB1954 was efficient. NTR/metronidazole and the vascular disrupting agent, combretastatin A‐4 phosphate, were the only other effective agents.ConclusionsCollectively, these data demonstrate that cytotoxic rather than cytostatic activity is necessary for efficient vascular disruption in vitro, and bystander killing is not essential. We identify NTR/CB1954 and NTR/metronidazole as candidates for in vivo investigation of vascular‐targeted gene therapy. Copyright © 2012 John Wiley & Sons, Ltd.

Từ khóa


Tài liệu tham khảo

10.1056/NEJM197111182852108

10.1016/j.ejim.2009.07.009

10.1016/j.semradonc.2004.04.009

Cooney MM, 2006, Phase II study of combretastatin A4 phosphate (CA4P) in patients with advanced anaplastic thyroid carcinoma (ATC), J Clin Oncol, 24, 5580, 10.1200/jco.2006.24.18_suppl.5580

10.1111/j.1365-2710.2007.00800.x

10.1158/1078-0432.CCR-09-0575

10.1038/nature10144

10.1097/00001813-200504000-00001

10.1089/104303400750038499

10.1038/sj.cgt.7700382

Ram Z, 1993, In situ retroviral‐mediated gene transfer for the treatment of brain tumors in rats, Cancer Res, 53, 83

Benouchan M, 2006, Delivery of the bacterial nitroreductase gene into endothelial cells prolongs the survival of tumour‐bearing mice by bystander mechanisms, Int J Oncol, 28, 457

10.1038/sj.gt.3301981

10.1038/sj.gt.3301883

10.4161/cbt.8.5.7589

10.1002/jgm.662

10.1038/sj.gt.3301483

Pramudji C, 2001, In situ prostate cancer gene therapy using a novel adenoviral vector regulated by the caveolin‐1 promoter, Clin Cancer Res, 7, 4272

Rancourt C, 1998, Endothelial cell vehicles for delivery of cytotoxic genes as a gene therapy approach for carcinoma of the ovary, Clin Cancer Res, 4, 265

10.1158/1535-7163.MCT-09-0110

10.1159/000164219

10.1038/mt.2009.80

Freeman SM, 1993, The ‘bystander effect’: tumor regression when a fraction of the tumor mass is genetically modified, Cancer Res, 53, 5274

10.2165/00002512-199914020-00001

Chen L, 1996, Sensitization of human breast cancer cells to cyclophosphamide and ifosfamide by transfer of a liver cytochrome P450 gene, Cancer Res, 56, 1331

10.1038/sj.cgt.7700200

10.1038/sj.gt.3301557

10.1007/BF00046365

10.3390/molecules14114517

Mesnil M, 2000, Bystander effect in herpes simplex virus‐thymidine kinase/ganciclovir cancer gene therapy: role of gap‐junctional intercellular communication, Cancer Res, 60, 3989

10.1089/hum.1997.8.6-709

10.1021/jm0498699

Wilson WR, 2002, Quantitation of bystander effects in nitroreductase suicide gene therapy using three‐dimensional cell cultures, Cancer Res, 62, 1425

10.1002/dvdy.21100

10.1667/RR0807.1

10.1007/s002800050886

10.1093/jnci/djj306

10.1071/CH08240

10.1021/tx034116v

10.1016/S0006-2952(03)00199-0

10.1021/jm030399c

10.1006/bbrc.1998.9646

10.1038/sj.bjc.6601612

10.1158/1535-7163.MCT-08-1209

Hunt MA, 2010, Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents, J Biomol Tech, 21, 66

10.2174/1381612033454108

10.1038/bjc.1993.59

Pettit GR, 1998, Antineoplastic agents 389. New syntheses of the combretastatin A‐4 prodrug, Anticancer Drug Des, 13, 183

10.1172/JCI24586

10.1111/1523-1747.ep12613748

10.1590/S0100-879X2006000700009

10.1038/sj.cgt.7701088

10.1038/sj.gt.3300784

10.1016/j.canlet.2008.06.002

10.1016/j.mvr.2003.11.002

10.1152/ajpheart.00265.2005

10.1038/sj.gt.3300806

10.1002/1097-0320(20011201)45:4<237::AID-CYTO10024>3.0.CO;2-J

10.1080/003655299750025156

Nishihara E, 1998, Treatment of thyroid carcinoma cells with four different suicide gene/prodrug combinations in vitro, Anticancer Res, 18, 1521

10.1038/sj.gt.3302492

Trinh QT, 1995, Enzyme/prodrug gene therapy: comparison of cytosine deaminase/5‐fluorocytosine versus thymidine kinase/ganciclovir enzyme/prodrug systems in a human colorectal carcinoma cell line, Cancer Res, 55, 4808

10.1038/sj.cgt.7700102

10.1083/jcb.107.4.1589

10.1007/978-1-60761-968-0_24

10.1038/sj.gt.3300367

10.1002/glia.1061

10.1002/neu.1026

10.1002/jnr.21223

10.1016/j.jbiotec.2010.08.007

10.1038/sj.bjc.6601211

10.1023/A:1011154616992

10.4049/jimmunol.178.3.1573

10.1038/sj.bjc.6601606

10.1089/hum.1997.8.15-1807

10.1038/sj.cgt.7700215

10.1007/s00280-009-1188-1

10.1093/jac/31.1.9

10.1158/1535-7163.MCT-05-0365

10.1186/1471-2407-9-301

Grove JI, 2003, Generation of Escherichia coli nitroreductase mutants conferring improved cell sensitization to the prodrug CB1954, Cancer Res, 63, 5532

10.1038/sj.gt.3302919

10.1016/j.bcp.2009.07.025

Thông tin
Thông tin xuất bản

Journal of Gene Medicine

Tập 14 Số 1

62-74

Thông tin tác giả