Gene therapy for hepatocellular carcinoma using non-viral vectors composed of bis guanidinium-tren-cholesterol and plasmids encoding the tissue inhibitors of metalloproteinases TIMP-2 and TIMP-3

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.

Inhibition of tumor growth and induction of apoptosis in prostate cancer cell lines by overexpression of tissue inhibitor of matrix metalloproteinase-3

“…Western blot and immunohistochemical assays showed that TIMP-3 is expressed at low levels both in human primary prostate cancer tissues and in prostate cancer cell lines. These data are consistent with other studies 7,11,21 that have shown loss or low expression of TIMP-3 in a variety of human tumors including prostate cancer. RT-PCR and western blot analyses showed that TIMP-3 is expressed at low levels in prostate cancer cell line PC-3M.…”

“…4 Earlier data have shown that the absence of TIMP-3 in the host enhances tumor growth and angiogenesis. 5 The TIMP-3 protein can inhibit invasion and only TIMP-3 has the ability to induce apoptosis in select cell lines such as melanoma cells, 6 hepatocellular carcinoma, 7 breast cancer cells, 8 and lung cancer cells. 9 It has also been reported to suppress tumor growth through inhibition of endothelial cell migration and angiogenesis.…”

Tissue Inhibitor of Metalloproteinase-3 Expression from an Oncolytic Adenovirus Inhibits Matrix Metalloproteinase Activity In vivo without Affecting Antitumor Efficacy in Malignant Glioma

“…In addition, TIMP-3 overexpression showed inhibitory effects on tumor growth and angiogenesis in neuroblastoma, melanoma, and hepatocellular carcinoma tumor models (27)(28)(29)(30). Moreover, evidence is accumulating for a role of TIMP-3 in inducing apoptosis in various cell types by stabilization of death receptors such as Fas on the cell surface (31)(32)(33).…”

Inhibition of human leukemia xenograft in nude mice by adenovirus-mediated tissue inhibitor of metalloproteinase-3

“…A volume of 500 ml CM from infected/uninfected K562 cells was placed in the lower chambers. 10 5 HUVECs, resuspended in serum-free RPMI-1640 containing 0.5% BSA, were placed in the upper chambers. After 4 h of incubation at 371C, cells in the lower chambers were collected, and counted by FACS at least three times.…”

“…TIMP-3 can inhibit all MMPs tested so far and its overexpression can drastically retard tumor angiogenesis, invasion and metastasis in mice model. [7][8][9][10] Recently, additional physiological functions have been attributed to TIMP-3, including inhibition of angiogenesis, 11 promotion of apoptosis 7,8,12,13 and the modulation of tumor necrosis factor a (TNF-a) signal transduction. 14 TIMP-3 gene is also considered to be a tumor suppressor gene, as its expression is silenced in different types of cancer due to the promoter hypermethylation.…”