Adenovirus-mediated transfer of siRNA against peroxiredoxin I enhances the radiosensitivity of human intestinal cancer

Abstract

Peroxiredoxin I (Prx-I), a key member of the peroxiredoxin family, reduces peroxides and equivalents through the thioredoxin system. Our previous work has shown that expression of Prx-I in mammalian cells increases following ionizing radiation (IR), and suppression of its expression enhances radiation-induced cell death in vitro, suggesting that inhibition of Prx-I might be an important pretreatment for cancer radiotherapy. To test this hypothesis in vivo, we suppressed the expression of Prx-I in the human intestinal cancer cell line SW480 by adenovirus-mediated transfer of siRNA. Our results showed that expression of Prx-I in SW480 cells was dramatically reduced by recombinant Ad-SiPrxI, which resulted in decreased cell growth and increased cell death by IR. Significantly more cell apoptosis was detected by flow cytometry analysis when Prx-I expression was knocked down. To evaluate the effect of recombinant Ad-SiPrxI in vivo, xenografts were pretreated with adenovirus before IR. Tumor growth in mice was inhibited when the xenografts were pretreated with Ad-SiPrxI before IR. Our results suggest that pretreatment with recombinant adenovirus to inhibit Prx-I expression can enhance the radiosensitivity of cancer cells, and thus might be a potential application in clinical therapy.

Introduction

Ionizing radiation (IR) has been well demonstrated to be effective as a therapeutic agent against cancer. However, certain side effects and complications have been encountered which limit its applications in cancer radiotherapy. One of the important reasons for this is that some cancer cells are not sensitive to IR because of their genetic backgrounds. Although some genes related to DNA repair, cell cycle regulation, cell growth and so on, are suspected to have roles in radiation sensitivity, the genetic factors responsible for cancer radiosensitivity remain elusive. IR promotes many important cellular processes, such as DNA damage, apoptosis, signal transduction and oxidative stress [1]. One of the early effects of IR is that it produces reactive oxygen species (ROS). Oxidative stress induced by IR produces a variety of highly reactive free radicals that damage cells, initiate signal transduction pathways and alter gene expression. ROS can induce cellular antioxidant defense enzymes such as superoxide dismutase and glutathione peroxidase [2]. The relationship between IR and antioxidant enzymes has been thoroughly investigated. Antioxidant enzymes can attenuate radiation injuries [3]. Thus, different cancer cells have different radiosensitivities, partially because of different expression of antioxidant enzymes.

Peroxiredoxin (Prx), a newly defined family of highly conserved antioxidant enzymes, has been shown to play a critical role in peroxide detoxification [4], [5]. Six Prx isoforms have been found in mammalian cells, which can be divided into two subgroups based on conserved cysteine residues. Peroxiredoxin I (Prx-I) contains two conserved cysteine residues, and has been found in abundance in the cytoplasm of cells as homodimers. From the results of biochemical and physiological studies, Prx-I has been implicated in a number of cellular functions, such as cell proliferation and differentiation, enhancement of natural killer cell activity and intracellular signaling, in addition to its antioxidant activity [6], [7], [8].

Increased Prx-I expression has been observed in various kinds of cancer cells, indicating it may be involved in cancer development or progression. Noh et al. found by Western immunoblotting analysis that Prx-I, as well as Prx-II and Prx-III, was over-expressed in human breast cancer tissues compared to normal tissues [9]. Using comparative proteome analysis between human normal (BEAS 2B) and malignant (A549) lung epithelial cells, Chang et al. found that Prx-I was reproducibly increased more than two-fold [10]. Additionally, Prx-I expression levels in the follicular neoplasm and thyroiditis groups were significantly higher than those of the control group, although there was not a statistically significant difference compared to those of the papillary carcinoma group [11]. All of these data suggest that Prx-I is involved in tumor development and progression, and might be a new target for gene therapy of tumor cells.

Our previous work demonstrated that the expression of Prx-I was up-regulated by IR in mouse intestinal epithelia and cultured IEC-6 cells [12], [13]. This was consistent with studies from other researchers [14]. We speculated that increased Prx-I expression post-irradiation was just a compensation for increased ROS after IR. We also found knock down of Prx-I expression enhanced radiation-induced cell death in vitro. To further confirm the relationship between Prx-I expression and radiosensitivity in SW480 cancer cells in vivo, in this study we sought to inhibit the expression of Prx-I by RNA interference, and evaluate its effect on IR-induced cell death.

To achieve this goal, we utilized an adenovirus-mediated gene delivery system, as viral delivery systems are the most advanced in terms of preclinical development and clinical potential [15]. Because of their many advantages, adenovirus vectors have now been developed as tools for gene transfer into mammalian cells and for gene therapy applications. The recombinant adenovirus DNA is transfected into 293 cell lines that express the deleted viral genes in trans, so that viral particles can be easily produced and isolated. In this study, we used adenovirus as the gene delivery vehicle to efficiently transfer genetic material to cancer cells.