Tumor-targeted gene therapy using Adv-AFP-HRPC/IAA prodrug system suppresses growth of hepatoma xenografted in mice

Abstract

Clinical efficacy of current therapies for hepatocellular carcinoma (HCC) treatment is limited. Indole-3-acetic acid (IAA) is non-toxic for mammalian cells. Oxidative decarboxylation of IAA by horseradish peroxidase (HRP) leads to toxic effects of IAA. The purpose of this study was to investigate the effects of a novel gene-targeted enzyme prodrug therapy with IAA on hepatoma growth in vitro and in vivo mouse hepatoma models. We generated a plasmid using adenovirus to express HRP isoenzyme C (HRPC) with the HCC marker, alpha-fetoprotein (AFP), as the promoter (pAdv-AFP-HRPC). Hepatocellular cells were infected with pAdv-AFP-HRPC and treated with IAA. Cell death was detected using MTT assay. Hepatoma xenografts were developed in mice by injection of mouse hepatoma cells. The size and weight of tumors and organs were evaluated. Cell death in tumors was assessed using hematoxylin and eosin-stained tissue sections. HRPC expression in tissues was detected using Reverse Transcriptase-Polymerase Chain Reaction. IAA stimulated death of hepatocellular cells infected with pAdv-AFP-HRPC, in a dose-and time-dependent manner, but not in control cells. Growth of hepatoma xenografts, including the size and weight, was inhibited in mice treated with pAdv-AFP-HRPC and IAA, compared with that in control group. pAdv-AFP-HRPC/IAA treatment induced cell death in hepatoma xenografts in mice. HRPC gene expressed only in hepatoma, but not in other normal organs of mice. pAdv-AFP-HRPC/IAA treatment did not cause any side effects on normal organs. These findings suggest that pAdv-AFP-HRPC/IAA enzyme/prodrug system may serve as a strategy for HCC therapy.

Hydrogen peroxide-independent generation of superoxide by plant peroxidase: hypotheses and supportive data employing ferrous ion as a model stimulus

“…Based on the views that formation of enzyme-substrate complexes such as [POX-IAA-O 2 ] results in release of O •− 2 (Kawano et al, 2001), medical application of HRP-labeled antibodies and IAA has been proposed as a novel O •− 2 -generating system for cancer cell-targeted and controlled cell death induction, by designing the HRP-conjugated immuno-labeling of cancer-related molecules or expression of recombinant HRP in mammalian cells (Folkes and Wardman, 2001; Folkes et al, 2002; Kawano, 2003b; Dai et al, 2012). Although the IAA-induced O •− 2 in HRP reaction mixture is very intense, the IAA-induced oxidative burst likely lasts only for few seconds (Kawano et al, 2001).…”

Recombinant horseradish peroxidase variants for targeted cancer treatment

“…Potential strategies to overcome this challenge are antibody‐directed enzyme prodrug therapy (ADEPT) and gene‐directed enzyme prodrug therapy (GDEPT) which allow the selective release of a cytotoxic agent from a non‐toxic prodrug at the site of the tumor (e.g., ). In this respect numerous studies investigated the cytotoxic effect of the prodrug indole‐3‐acetic acid (IAA) after oxidation with the heme‐containing enzyme horseradish peroxidase (HRP, EC 1.11.7.1; ). In 1998, Folkes et al.…”

Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

“…20 The formal oxidation states of the heme within the peroxidase enzyme are indicated by numbers in the small brackets. [27][28][29][30] Furthermore, we have previously proposed our view that nitric oxide (NO) is also one of candidate chemicals for reducing native plant peroxidase into ferrous intermediate to initiate the oxygenase-like cycle of plant peroxidases. 2 As summarized in Figure 1A, after completion of the oxygenase-like cycle, O 2 ¡ which could be converted to H 2 O 2 via disproportionation can be released, suggesting that H 2 O 2 -dependent cycle of peroxidase reaction can be concomitantly achieved depending on the types and combination of the substrates or chemicals added to the system (Fig.…”