1. ¹CNRS-UMR 8532, LBPA, Ecole Normale Supérieure, 94235 Cachan, France
2. ²Chimie des Interactions Moléculaires, Collège de France, 75005 Paris, France
3. ³Laboratoire d'Expérimentation Animale, Institut Gustave Roussy, 94805 Villejuif, France
4. ⁴Laboratoire de Pathologie Cellulaire, Hôpital Beaujon, 92110 Clichy, France
5. ⁵Division of Hematology/Oncology, Children's Hospital of Los Angeles, California 90027, USA

Correspondence: Dr Phuong-Lan Tran, UMR 8532 CNRS, Gustave Roussy Institute, 39 rue Camille Desmoulins, 94805 Villejuif cedex, France. E-mail: pltran@igr.fr

Received 5 January 2003.

Abstract

Metalloproteinases (MMPs) and their natural inhibitors (TIMPs) contribute to the regulation of tumor microenvironment. Their expressions are deregulated in almost all human cancers. We report a novel approach to gene therapy of hepatocellular carcinoma (HCC), using repeated injections of DNA plasmids encoding the tissue inhibitors of metalloproteinases (TIMPs) TIMP-2 or TIMP-3, and a novel competent formulation of gene transfer based on nontoxic cationic cholesterol derivatives. The new gene delivery system was efficient in demonstrating the antitumor efficiency of TIMP-2 or TIMP-3 in inhibiting tumor growth of human HuH7 HCC cells xenografted into nude mice. We show, for the first time, an in vivo effect of TIMP-3 in delaying HCC tumor growth. No treatment-related toxicity was noted. An inhibition of angiogenesis and tumor necrosis accompanied the inhibitory effects of TIMP-2 or TIMP-3 on tumor expansion and invasion. We also report a bystander effect produced by transfected HuH7 tumor cells mixed with untransfected cells in 1:1 ratio in culture that resulted in killing 98% of cells within 96 h. In addition, the soluble forms of TIMP-2 and TIMP-3 expressed by transfected cells exerted a cytotoxic effect on untransfected HuH7 cell cultures. Taken together, these results demonstrate the potential efficacy of repeated treatment of secreted TIMP-2 and TIMP-3 for the design of nonviral gene therapy for hepatocarcinoma.