Cancer Letters

Cancer Letters

Volume 343, Issue 2, 28 February 2014, Pages 190-199
Cancer Letters

Peroxiredoxin 2 knockdown by RNA interference inhibits the growth of colorectal cancer cells by downregulating Wnt/β-catenin signaling

https://doi.org/10.1016/j.canlet.2013.10.002Get rights and content

Highlights

  • Prdx2 knockdown led to increased apoptosis and decreased proliferation in CRC cells.

  • Inhibition of growth in CRC cells was mediated through increased endogenous ROS production.

  • Inhibition of growth in CRC cells was mediated through negative regulation of Wnt/ß-catenin signaling.

  • Prdx2 knockdown led to a reduction in xenograft tumor growth in vivo.

Abstract

Peroxiredoxin 2 (Prdx2) has been shown to act as an antioxidant whose main function is to reduce hydrogen peroxide (H2O2) in cells, and Prdx2 is abnormally elevated in colorectal cancer (CRC). However, the functional significance of this up-regulation and the detailed molecular mechanism behind the regulatory effect of Prdx2 on the growth of CRC cells have not been elucidated. In this study, we demonstrated that Prdx2 knockdown using a lentiviral vector-mediated specific shRNA inhibited cell growth, stimulated apoptosis, and augmented the production of endogenous reactive oxygen species (ROS). Further, silencing of Prdx2 resulted in an altered expression of proteins associated with the Wnt signaling pathway. Finally, Prdx2 knockdown contributed to attenuated CRC growth in BALB/c nude mice. In conclusion, these findings demonstrate that the regulatory effects of Prdx2 can be partially attributed to Wnt/β-catenin signaling.

Introduction

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second most common in females, with over 1.2 million new cancer cases worldwide and 608,700 deaths due to CRC estimated in 2008. Despite significant advances in CRC treatment, it remains one of the leading causes of cancer-related death worldwide [1]. Although traditional treatments for CRC such as surgical resection and chemotherapy have allowed great progress, the recurrence rates for this type of cancer remain high and new therapeutic strategies are needed.

Reactive oxygen species (ROS) such as H2O2 and superoxide are produced by cells as by-products of normal cellular metabolism [2], and elevated levels of ROS are associated with many disease states such as neurodegenerative diseases and cancer [3], [4]. Furthermore, members of a recently discovered antioxidant enzyme family, the peroxiredoxins (Prdxs), are important scavengers of ROS [5].

Prdx enzymes, which were initially characterized in yeast, constitute a family of antioxidants that share no homology with other conventional antioxidant proteins [6]. Prdxs reduce ROS using thioredoxin as an intermediate electron donor [7]. Prdx2 is a member of this family, which regulates ROS in the cellular environment and is up-regulated in many cancers, including cancers of the brain, breast, cervix, and prostate as well as mesothelioma [8], [9], [10], [11], [12], suggesting a possible role for this peroxiredoxin in cancer cell maintenance. Several studies have shown that Prdx2 plays a crucial role in multiple cellular functions, including the protection of proteins and lipids against oxidative injury [13], [14], [15], cell proliferation, differentiation [16], and mediation of intracellular signaling pathways involved in apoptosis [17]. Cultured cells over-expressing Prdx2 are more resistant to apoptosis caused by serum deprivation, hydrogen peroxide, and exposure to ceramide or etoposide [18]; in particular, Prdx2 over-expression protects thyroid cells from H2O2-induced apoptosis [17]. Moreover, down-regulation of Prx2 sensitizes head and neck cancer cells to radiation [19] and gastric carcinoma cells to cisplatin [20]. In addition, Parmigiani and colleagues [21] suggested that Prdx2 may serve as a therapeutic target for cancers, as elevated levels of Prdx2 were associated with resistance to cancer therapy and shown to promote aggressive survival phenotypes of cancer cells. Regarding CRC, Prdx2 over-expression has also been related to clinical staging, tumor progression, and lymph node metastasis [22], but the mechanism by which Prdx2 achieves its effects remains elusive.

Wnt/β-catenin signaling is described as part of the canonical Wnt pathway and plays a key role during normal animal development [23]. The stability of the Wnt pathway transcription factor β-catenin is tightly regulated by a multi-subunit destruction complex. Dysregulation of the Wnt pathway has been implicated in many cancers, making this pathway an attractive target for anticancer therapies [24]. Moreover, stabilized β-catenin affects a transcriptional response that is thought to be critical in tumorigenesis [26]. Loss of function of the adenomatous polyposis coli (APC) protein occurs in more than 80% of CRCs [25], and APC is a component of the β-catenin destruction complex. β-catenin is a key element in driving Wnt/β-catenin signaling; when β-catenin is phosphorylated and ubiquitinated, the intranuclear level of β-catenin protein becomes quite low. Consequently, the interaction of this protein with the lymphoid enhancer factor/T-cell factor (LEF/TCF) and the transcription of target genes are inhibited [27]. Therefore, constitutive activation of Wnt/β-catenin signaling following mutation of the APC gene has been considered to be an important event for carcinogenesis and progression in colon cancer [28].

In recent years, ROS have been implicated in the regulation of Wnt signaling [29]. For example, it has been demonstrated that nucleoredoxin (Nrx, a thioredoxin (Trx) family member) is a redox-sensitive negative regulator of canonical Wnt signaling via its interaction with Dvl [30]. In addition, a number of studies have shown that treatment of cells with H2O2 to induce ROS-dependent signaling inhibits β-catenin/TCF transcriptional activity [31], [32]. Furthermore, a recent study indicated that ROS are involved in arsenic-induced cell transformation and tumor formation, possibly through the Wnt/β-catenin pathway, in the human colorectal adenocarcinoma cell line DLD1 [33]. In light of these observations, we speculated that the regulatory role of Prdx2, which uses thioredoxin as an immediate electron donor, in the growth of CRC cells may be associated with the Wnt/β-catenin pathway. Therefore, using in vitro and in vivo approaches, we examined whether Prdx2 plays a role in the survival and maintenance of CRC cells and whether these effects are mediated by the Wnt/β-catenin pathway.

Section snippets

Cell culture and antibodies

The human normal colorectal epithelial cell line FHC and the human CRC cell lines SW480 and SW620 were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). These cell lines were cultured in Leibovitz L-15 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Shanghai, China) and 2% penicillin/streptomycin (Beyotime, Jiangsu, China) and maintained at 37 °C in a humidified atmosphere. The following antibodies were used for

Expression of Prdx2 and β-catenin in CRC tissues and cell lines

Prdx2 and β-catenin protein levels were examined in 35 CRC tissues and their corresponding normal colorectal mucosal tissues using immunohistochemistry. Prdx2 and β-catenin expression was predominantly located in the cytoplasm and nucleus of CRC cells (Fig. 1Ab–Ad and Af–Ah), and this expression was weak in normal colorectal mucosa tissues (p < 0.01; Fig. 1Aa and Ae). The proportions of CRC samples that were positive for Prdx2 and β-catenin were 80.00% (28/35) and 82.86% (29/35), respectively.

Discussion

Several recent studies have reported over-expression of Prdx2 in numerous types of cancers [8], [9], [10], [11], [12], [22]. Furthermore, Prdx2 expression has been associated with various cellular phenomena, including cell proliferation and growth control, differentiation, immune responses, tumorigenesis, and apoptosis [34]. Because cancer cells are known to produce large amounts of ROS [35], it is readily appreciated that the over-expression of Prdx antioxidant enzymes such as Prdx2 could

Conflict of interest

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

Acknowledgement

This work was supported by a Grant from National Natural Science Foundation of China (No. 81172295).

References (53)

  • M. Almeida et al.

    Oxidative stress antagonizes Wnt signaling in osteoblast precursors by diverting beta-catenin from T cell factor to forkhead box O-mediated transcription

    J. Biol. Chem.

    (2007)
  • S.Y. Shin et al.

    Involvement of glycogen synthase kinase-3beta in hydrogen peroxide-induced suppression of Tcf/Lef-dependent transcriptional activity

    Cell. Signal.

    (2006)
  • Z. Zhang et al.

    Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/β-catenin pathway in human colorectal adenocarcinoma DLD1 cells

    Toxicol. Appl. Pharmacol.

    (2011)
  • K.W. Lee et al.

    Peroxiredoxin II restrains DNA damage-induced death in cancer cells by positively regulating JNK-dependent DNA repair

    J. Biol. Chem.

    (2011)
  • Y. Yan et al.

    The antioxidant enzyme Prdx1 controls neuronal differentiation by thiol-redox-dependent activation of GDE2

    Cell

    (2009)
  • D. Qu et al.

    Role of Cdk5-mediated phosphorylation of Prx2 in MPTP toxicity and Parkinson’s disease

    Neuron

    (2007)
  • F. Takahashi-Yanaga et al.

    The Wnt/β-Catenin signaling pathway as a target in drug discovery

    J. Pharmacol. Sci.

    (2007)
  • Y. Gao et al.

    Inhibition of cytoplasmic GSK-3β increases cisplatin resistance through activation of Wnt/β-catenin signaling in A549/DDP cells

    Cancer Lett.

    (2013)
  • Y.S. Lim et al.

    Removals of hydrogen peroxide and hydroxyl radical by thiol-specific antioxidant protein as a possible role in vivo

    Biochem. Biophys. Res. Commun.

    (1993)
  • A. Jemal et al.

    Global cancer statistics

    CA Cancer J. Clin.

    (2011)
  • B. Halliwell et al.

    Free Radicals in Biology and Medicine

    (1999)
  • A. Sun et al.

    Oxidative stress and neurodegenerative disorders

    J. Biomed. Sci.

    (1998)
  • D. Gius et al.

    Redox signaling in cancer biology

    Antioxid. Redox Sign.

    (2006)
  • S.Y. Shim et al.

    Oxidative stress and the antioxidant enzyme in the developing brain

    Korean J. Pediatr.

    (2013)
  • S. Järvelä et al.

    Specific expression profile and prognostic significance of peroxiredoxins in grade II–IV astrocytic brain tumors

    BMC Cancer

    (2010)
  • D.Y. Noh et al.

    Overexpression of peroxiredoxin in human breast cancer

    Anticancer Res.

    (2001)
  • Cited by (64)

    • The beginning of GPX2 and 30 years later

      2022, Free Radical Biology and Medicine
    • Wnt/β-catenin signaling in colorectal cancer: Is therapeutic targeting even possible?

      2022, Biochimie
      Citation Excerpt :

      Deletion of SH3BP4 increases stem cell numbers and accelerates tumor development through hyperactivation of Wnt signaling. The antioxidant enzyme Peroxiredoxin 2 (PRDX2) increases the levels of β-catenin phosphorylation by elevating GSK3β activity, altering nuclear transport [100]. While the guanine nucleotide exchange factor RAPGEF5 regulates β-catenin nuclear translocation by activating nuclear GTPase [101], it is unclear if similar mechanism is used in colorectal cancer.

    View all citing articles on Scopus
    View full text