Silencing peroxiredoxin-2 sensitizes human colorectal cancer cells to ionizing radiation and oxaliplatin

doi:10.1016/j.canlet.2016.12.009

Cancer Letters

Volume 388, 1 March 2017, Pages 312-319

https://doi.org/10.1016/j.canlet.2016.12.009 Get rights and content

Highlights

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Prx2 oxidation was associated with radiation response in colorectal cancer (CRC) cells.
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Radioresistant cells have higher monomer/dimer Prx2 ratio than radiosensitive cells.
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Prx2 inhibition sensitizes CRC cells to irradiation plus oxaliplatin in vitro.
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Prx2 inhibition sensitizes CRC cells to irradiation in vitro and in vivo.

Abstract

Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. Antioxidant enzymes decrease the generation of ionizing radiation (IR)-induced free radicals and therefore are associate to radioresistance. The main goal of this work is to study the involvement of peroxiredoxin-2 (Prx2) in the radio and chemoradiotherapy response in CRC cells in vitro and in vivo. We found that Prx2 oxidation state is associated to differential response to ionizing radiation in CRC cell lines. HCT116 radioresistant CRC cell line have lower ROS levels and a higher monomer/dimer Prx2 ratio, compared to halfway resistant Caco-2 and T84, and radiosensitive LoVo cell line. Constitutive and transient Prx2 silencing in CRC cells increase ROS levels, and most importantly, enhance in vitro radiation sensitivity. In addition, we showed that administration of IR plus oxaliplatin in down regulated Prx2 HCT116 cells has higher citotoxic effect than in control cells. Finally, radiosensitizing effect of Prx2 depletion was confirmed in vivo. These results suggest that Prx2 is an important component in tumoral radiation response, and their inhibition could improve radio and chemoradiotherapy protocols in patients with CRC.

Introduction

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females. Nowadays, despite significant advances in CRC treatment, it remains as one of the leading causes of cancer-related death worldwide [1]. Pre-surgical radiochemotherapy is commonly used to improve CRC treatments outcome; however, only a few percentage of patients have shown a complete response to this treatment [2], [3]. Intrinsic cellular resistance is one of the most important constraints that yet remain to be solved and new therapeutic strategies should be explored to overcome this barrier. Oxidative stress generated by IR damages cells and induces the activation of antioxidant signal transduction pathways associated with radioresistant phenotypes [4]. Thus, antioxidant enzymes decrease the generation of IR-induced free radicals and therefore reduce radiation damage. This phenomenon has been established as one of the main mechanism of cancer radioresistance [5].

Peroxiredoxins (Prxs) enzymes constitute a family of antioxidants that consist of six isoforms (Prx1–Prx6) encoded by distinct genes in mammals, which are classified into three mechanistic subgroups (typical 2-Cys, atypical 2-Cys, and 1-Cys) [6]. All Prxs contain a conserved NH2-terminal cysteine residue that serves as the active site for catalysis and typical 2-Cys Prxs, (Prx1–Prx4) have an additional conserved C-terminal cysteine residue responsible for resolving the oxidized active site cysteine [7]. Prxs protect cells by removing constitutive levels of H₂O₂, hydroperoxides and peroxynitrite [8], [9]. In this process, the conserved catalytic cysteine of typical 2-Cys, named peroxidatic cysteine (Cys–SPH) is oxidized to a cysteine sulfenic acid (Cys–SOH), which is attacked by the resolving cysteine (Cys–SRH) located in the C terminus of the other subunit [10]. This condensation reaction results in the formation of a stable inter-subunit disulfide bond [11]. This modification is completely reversible by the subsequent reduction that regenerates the active cysteine [12].

Prxs are up-regulated in many cancers, including cancers of the lung [13], thyroid [14], breast [15], and mesothelioma [16], suggesting a possible role of these enzymes in cancer cell maintenance. Particularly in CRC has been reported Prx2 overexpression in biopsies tumor tissue versus normal tissue [17]. So, here we studied the role of Prx2 in the intracellular ROS levels regulation and in the tumor response to IR and chemotherapeutic drugs as oxaliplatin (OXLP). We show that Prx2 knocking down through RNAi sensitizes CRC cells to IR and IR plus OXLP, in addition to the strong decrease of cell proliferation and tumorigenicity of this type of cells.

Section snippets

Cell culture

Human CRC cell lines HCT116, Caco-2, T84 and LoVo were was kindly given by Dr Podhajcer (Fundación Instituto Leloir, Argentina). Cell lines were cultivated in complete Dulbecco's modified Eagle's medium/F12 (DMEM/F12, Invitrogen Argentina SA) supplemented with 10% (vol/vol) fetal bovine serum (FBS, NatoCor, Argentina), 50 units/mL of penicillin G and 50 μg/mL of streptomycin sulfate, at 37 °C and at 5% (vol/vol) of CO₂ in humidified atmosphere. Subcultures were performed following standard

Doses response curves of CRC cells to γ-radiation

The doses response curves of CRC cells to γ-radiation were obtained from clonogenic assays and the survival curves were fitted to the linear quadratic model (S = exp − (αD + βD²)) (Fig. 1). The parameters α, β and FS2 are detailed in Table 1. According to statistical difference between α parameters, CRC cell lines were arranged in descending order of radioresistance as following: HCT116 > T84 ≈ Caco-2 > LoVo; being HCT116 the most radioresistant cell line in this model (p < 0.05).

Differential intracellular ROS level and Prx2 redox status is associated to radioresistance in CRC cells

DCF

Discussion

In this work, we showed novels findings about intracellular ROS levels, Prx2 state and its association with cellular radioresistance in CRC: 1 – HCT116 radioresistant CRC cell line has lower ROS levels and a higher monomer/dimer Prx2 ratio, compared to halfway resistant Caco-2 and T84 and radiosensitive LoVo cell line, indicating a possible association between radioresistance and Prx2 redox status in CRC; 2 – the inhibition of Prx2 expression sensitizes CRC cells to IR and to IR combined with

Conflict of interest

The authors declare no financial or other conflict of interest with regard to this work.

Acknowledgement

This work was supported by the National Agency for Promotion of Science (ANPCyT) grants PICT-2013/1751 and 2014/2436 and grants from Roemmers Foundation. Cerda MB was the recipient of the Ph.D. fellowship from CONICET and National Institute of Cancer, Argentina.

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Oxidative stress is one of the hallmarks of cancer. Tumorigenesis and progression are accompanied by elevated reactive oxygen species (ROS) levels and adaptive elevation of antioxidant expression levels. Peroxiredoxins (PRDXs) are among the most important antioxidants and are widely distributed in a variety of cancers. PRDXs are involved in the regulation of a variety of tumor cell phenotypes, such as invasion, migration, epithelial-mesenchymal transition (EMT) and stemness. PRDXs are also associated with tumor cell resistance to cell death, such as apoptosis and ferroptosis. In addition, PRDXs are involved in the transduction of hypoxic signals in the TME and in the regulation of the function of other cellular components of the TME, such as cancer-associated fibroblasts (CAFs), natural killer (NK) cells and macrophages. This implies that PRDXs are promising targets for cancer treatment. Of course, further studies are needed to realize the clinical application of targeting PRDXs. In this review, we highlight the role of PRDXs in cancer, summarizing the basic features of PRDXs, their association with tumorigenesis, their expression and function in cancer, and their relationship with cancer therapeutic resistance.
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Moreover, an increase in the level of Prdxs in the cells led to an increase in their radioresistance. In contrast, a decrease in PRDX1, PRDX2, and PRDX3 gene expression in cancer cells has been associated with a loss of radioresistance [9,14,45–47]. In line with these results, we observed that inhibition of endogenous PRDX6 expression by siRNA in 3T3 cells also decreased their radioresistance by 30–50%, and this was accompanied by caspase-3 activation and an increase of apoptotic cell death (Fig. 4).
Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with multi-substrate peroxidase and phospholipase activities that is involved in cell redox homeostasis and regulates intracellular processes. Previously, recombinant Prdx6 was shown to exert a radioprotective effect during whole-body exposure to a lethal dose of X-ray radiation. Moreover, a mutant form Prdx6-C47S, which lacks peroxidase activity, also had a radioprotective effect, and this indicates that the mechanism of radioprotection is unknown. The present study was aimed to test the hypothesis that the radioprotective effect of Prdx6 and Prdx6-C47S may be mediated through the TLR4/NF-κB signaling pathway.
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The Role of Peroxiredoxins in Cancer Development
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Effects of Antioxidant Gene Overexpression on Stress Resistance and Malignization In Vitro and In Vivo: A Review
2022, Antioxidants

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Original ArticleSilencing peroxiredoxin-2 sensitizes human colorectal cancer cells to ionizing radiation and oxaliplatin

Highlights

Abstract

Introduction

Section snippets

Cell culture

Doses response curves of CRC cells to γ-radiation

Differential intracellular ROS level and Prx2 redox status is associated to radioresistance in CRC cells

Discussion

Conflict of interest

Acknowledgement

Cancer Lett.

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

Trends biochem. Sci.

J. Biol. Chem.

Cancer Lett.

Mol. Ther.

Cell Stem Cell

Cell Stem Cell

Biochim. Biophys. Acta

Free Radic. Biol. Med.

Mol. Cells

Free Radic. Biol. Med.

Cancer Lett.

Global cancer statistics, 2012

CA Cancer J. Clin.

Preoperative versus postoperative chemoradiotherapy for rectal cancer

N. Engl. J. Med.

Preoperative versus postoperative chemoradiotherapy in stage T3, N0 rectal cancer

Int. J. Clin. Oncol.

Original Article
Silencing peroxiredoxin-2 sensitizes human colorectal cancer cells to ionizing radiation and oxaliplatin