Peroxiredoxin 2 knockdown by RNA interference inhibits the growth of colorectal cancer cells by downregulating 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)
- et al.
Cloning, sequencing, and mutation of thiol-specific antioxidant gene of Saccharomyces cerevisiae
J. Biol. Chem.
(1993) - et al.
Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-α
J. Biol. Chem.
(1998) - et al.
On the protective mechanism of the thiol-specific antioxidant enzyme against the oxidative damage of biomacro-molecules
J. Biol. Chem.
(1994) - et al.
Removal of hydrogen peroxide by thiol-specific antioxidant enzyme (TSA) is involved with its antioxidant properties
J. Biol. Chem.
(1996) - et al.
The type II peroxiredoxin gene family of the mouse: molecular structure, expression and evolution
Gene
(1998) - et al.
Role of peroxideroxins in regulating intracellular hydrogen peroxide and hydrogen peroxide-induced apoptosis in thyroid cells
J. Biol. Chem.
(2000) - et al.
Thioredoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of Bcl-2
J. Biol. Chem.
(1997) Wnt/β-catenin signaling in development and disease
Cell
(2006)The oncogenic activation of beta-catenin
Curr. Opin. Genet. Dev.
(1999)Regulation of the Wnt/β–catenin pathway by redox signaling
Dev. Cell
(2006)
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.
Involvement of glycogen synthase kinase-3beta in hydrogen peroxide-induced suppression of Tcf/Lef-dependent transcriptional activity
Cell. Signal.
Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/β-catenin pathway in human colorectal adenocarcinoma DLD1 cells
Toxicol. Appl. Pharmacol.
Peroxiredoxin II restrains DNA damage-induced death in cancer cells by positively regulating JNK-dependent DNA repair
J. Biol. Chem.
The antioxidant enzyme Prdx1 controls neuronal differentiation by thiol-redox-dependent activation of GDE2
Cell
Role of Cdk5-mediated phosphorylation of Prx2 in MPTP toxicity and Parkinson’s disease
Neuron
The Wnt/β-Catenin signaling pathway as a target in drug discovery
J. Pharmacol. Sci.
Inhibition of cytoplasmic GSK-3β increases cisplatin resistance through activation of Wnt/β-catenin signaling in A549/DDP cells
Cancer Lett.
Removals of hydrogen peroxide and hydroxyl radical by thiol-specific antioxidant protein as a possible role in vivo
Biochem. Biophys. Res. Commun.
Global cancer statistics
CA Cancer J. Clin.
Free Radicals in Biology and Medicine
Oxidative stress and neurodegenerative disorders
J. Biomed. Sci.
Redox signaling in cancer biology
Antioxid. Redox Sign.
Oxidative stress and the antioxidant enzyme in the developing brain
Korean J. Pediatr.
Specific expression profile and prognostic significance of peroxiredoxins in grade II–IV astrocytic brain tumors
BMC Cancer
Overexpression of peroxiredoxin in human breast cancer
Anticancer Res.
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