Analysis of the horseradish peroxidase/indole-3-acetic acid combination in a three-dimensional tumor model

Abstract

Horseradish peroxidase has previously been shown to catalyze the conversion of indole-3-acetic acid (IAA) to a potent cytotoxin in a gene therapy setting. A three-dimensional spheroid model composed of a human head and neck carcinoma cell line, has been used to mimic the tumor microenvironment, such as regions of hypoxia. Exposure of intact spheroids to 0.05-5 mM concentrations of IAA and the halogenated indole, 5-bromoindole-3-acetic acid (5Br-IAA), resulted in decreased cell survival, and demonstrates that this combination is effective under tumor-simulated conditions. In addition, 5Br-IAA, displayed selectivity for spheroids with a large hypoxic fraction following short exposure times. Keywords: horseradish peroxidase; hypoxia; indoles; spheroids G ene-directed enzyme prodrug therapy (GDEPT) is a multistep strategy designed to specifically target tumor tissue and reduce normal tissue side effects. The gene coding for an enzyme is delivered to the tumor, followed by the administration of a nontoxic prodrug which can be converted to a cytotoxin by the enzyme, 1 thereby killing surrounding tumor cells.A variety of GDEPT combinations have been proposed, including the horseradish peroxidase/indole-3-acetic acid (HRP/IAA) combination. 2 HRP is a plant enzyme capable of converting the auxin IAA, and other indoles, to cytotoxins via a one-electron oxidation followed by subsequent nonenzymatic reactions. 3 An advantage of the HRP/IAA combination over several other enzyme/ prodrug combinations is that it is effective under hypoxia; a tumor-specific condition and an adverse prognostic factor in a variety of human tumors. 4 When put into a GDEPT setting, the combination is effective even under catalyst-induced anoxia, 5 and also when the expression of the enzyme is placed under the control of hypoxia response elements. 6 In this study, we used a multicellular spheroid model composed of human squamous cell carcinoma cells (FaDu), to mimic the microenvironmental conditions seen in human tumors. This allows evaluation of the HRP/IAA combination under tumor-simulated conditions prior to in vivo investigations. The more complex three-dimensional nature of spheroids compared with monolayers allows for a more heterogeneous cell population. Regions within the spheroid experience different environments, ranging from well oxygenated via hypoxic regions to the necrotic core, 7 accompanied by alterations in the nutrient composition. Oxygen has been shown to have a diffusion range through metabolizing cells of about 150 mm, which results in a diffusion limited, chronically hypoxic cell population in the spheroids.In this study, the efficacy of the HRP gene therapy system was analyzed in a multicellular spheroid system using a cell line with dysfunctional p53 activity. 8 Small and large spheroids with different hypoxic fractions were compared with monolayers. The parent compound IAA was also evaluated against a halogenated indole derivative, 5Br-IAA, which previously showed enhanced anoxic toxicity. 5 Materials and methods Cell cultur...

In vivo characterization of horseradish peroxidase with indole-3-acetic acid and 5-bromoindole-3-acetic acid for gene therapy of cancer

“…In vitro enzyme activity was lower in transiently transfected FaDu cells (0.008 ± 0.2 U mg -1 protein, unpublished data) than stable transfectants 18 (HRP8: 0.08±0.003 U mg -1 protein). Yet, in vitro effectiveness of the HRP/IAA system in stable transfectants 11,18 was less than in transient transfectants of the same cell line. 4 Enzyme activity per cell may be more important than enzyme activity per population of cells.…”

“…GFP1 cells, when grown as spheroids, did not have any growth disadvantage compared with parental or HRP8 cells. 11 GFP and the modified enhanced green fluorescent protein have been reported to produce a strong T cell-mediated immune response, 21 which can lead to both growth delay and spontaneous regression of tumors. However, SCID mice are unable to mount an effective immune response, as they are hypogammaglobulinemic and lack both B and T cells, 22 and thus the immunogenicity of GFP was unlikely to account for the reduced tumor growth rate.…”

“…Cells were kept in a humidified incubator at 37 1C and 5% CO 2 /air. FaDu cells stably transfected with either the HRP gene, clone HRP8, or the marker green fluorescent protein, clone GFP1, were generated earlier 11 and were used throughout the experiments.…”

“…This is in contrast to growth in vitro in which all three clones grew at similar rates. 11 The expression of transgenes in tumors was preserved in vivo. GFP1 tumors showed diffuse, often punctate expression of GFP throughout the tumor.…”

“…11 In particular, 5Br-IAA showed specificity against larger spheroids, which contain significant regions of hypoxia. Although monolayers and spheroids can be manipulated to approximate the conditions seen in solid tumors, they are far less complex than the neoplastic conditions seen in patients.…”