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Abstract

OBJECTIVE:

This study was designed to evaluate the relation between GPx2 (glutathione peroxidase 2) expressions and clinicopathological features as well as prognosis of patients with nasopharyngeal carcinoma (NPC).

METHODS:

A total of 89 cases of NPC were investigated to examine the immunohistochemical expression of GPx2. Fourteen pairs of NPC and the control samples were analyzed respectively by qRT-PCR and Western blot. The correlations of GPx2 expressions with the clinicopathologic features and the prognosis of NPC patients were also analyzed.

RESULTS:

The expression of GPx2 in NPC tissues was elevated immunohistochemically when compared with normal nasopharyngeal tissues ( $P<$ 0.05). The mRNA expression of GPx2 in carcinoma tissues was highly elevated compared with the control tissues ( $P<$ 0.05). GPx2 protein in carcinoma tissues was also over expressed than in control tissues ( $P<$ 0.05). Also GPx2 expression was significantly higher in the late clinical stage ( $P=$ 0.02). While there was no significant association between GPx2 expression and patient age, sex, T-stage, N-stage and the metastasis.

CONCLUSIONS:

GPx2 may play an important role in the development of nasopharyngeal carcinoma. Furthermore, GPx2 may serve as a prognostic biomarker for NPC patient.

Keywords

Nasopharyngeal carcinoma GPx2 prognosis

1. Introduction

Nasopharyngeal carcinoma (NPC) is a malignant neoplasm arising from the nasopharynx epithelium. It has obvious geographic and racial distribution. It is mainly reported in southern China, Southeast Asia, North and East Africa, the Middle East and Alaska [1, 2]. In southern China, the incidence rate is approximately 25–50 per 100,000 people, which is 100-fold higher than the western world [3, 4]. The development of NPC is related to both genetic and environmental factors. It has been confirmed that Epstein Barr Virus (EBV) infection is a risk factor for NPC as well as genetically controlled biological progresses such as oxidative stress [5, 6].

Oxidative stress is defined as the imbalance of free radicals, reactive oxygen species (ROS) and their elimination by protective mechanisms, such as antioxidants. It has been confirmed that cancer initiation and progression were linked to oxidative stress by increasing DNA mutations or inducing DNA damage, genome instability and cell proliferation [7]. As one member of antioxidants, glutathione peroxidase (GPX) can react with H ${}_{2}$ O ${}_{2}$ and fatty acid hydroperoxides. GPx2, the gastrointestinal glutathione peroxidase (GI-GPX), is a selenium-dependent glutathione peroxidase belonging to the GPx family. It is mainly expressed in the whole gastrointestinal tract including the epithelium of the colon, small intestine, esophagus and, in humans, also the liver [8]. GPx2 is suggested to play an important role in cellular redox control. Previous researches demonstrated GPx2 was upregulated in colorectal cancer [9, 10, 11], Barrett’s esophagus [12], squamous cell carcinoma [13], or lung adenocarcinoma of smokers [14]. The putative physiological function of GPx2 in cancer is regarded as carcinogenesis. Further experiments revealed GPx2 appeared to facilitated tumor growth by suppression of hydroperoxide-mediated apoptosis [15, 16]. Also, GPx2 proved to abrogate invasiveness and mobility of cancer cells [16, 17]. However, the expression of GPx2 within NPC is rarely reported, and the relationship between GPx2 expression and NPC prognosis is still unclear.

In this study, we detected the expressions of GPx2 both in NPC tissues and the normal nasopharyngeal tissues. Also, we analyzed the associations between expressions of GPx2 and clinicopathologic features, as well as metastasis of NPC.

Table 1
Association of GPx2 expression and clinicopathologic features of 89 NPC patients

Clinicopathological $n$ (%) GPx2 protein $P$ -value

variable expression (%)

Low High

Age (y)

$\leqslant$ 50 55 (61.8) 19 (34.5) 36 (65.5) 0.149

$>$ 50 34 (38.2) 17 (50.0) 17 (50.0)

Sex

Male 59 (66.3) 25 (42.4) 34 (57.6) 0.604

Female 30 (33.7) 11 (36.7) 19 (63.3)

Clinical stage

1–2 18 (20.2) 13 (72.2) 5 (27.8) 0.020

3–4 71 (79.8) 23 (32.4) 48 (67.6)

T stage

$\leqslant$ T2 54 (60.7) 23 (42.6) 31 (57.4) 0.609

$>$ T2 35 (39.3) 13 (37.1) 22 (62.9)

N stage

N0 13 (14.6) 6 (46.2) 7 (53.8) 0.650

$\geqslant$ N1 76 (85.4) 30 (39.5) 46 (60.5)

Clinicopathological	$n$ (%)	GPx2 protein	$P$ -value
variable		expression (%)
		Low	High
Age (y)
$\leqslant$ 50	55 (61.8)	19 (34.5)	36 (65.5)	0.149
$>$ 50	34 (38.2)	17 (50.0)	17 (50.0)
Sex
Male	59 (66.3)	25 (42.4)	34 (57.6)	0.604
Female	30 (33.7)	11 (36.7)	19 (63.3)
Clinical stage
1–2	18 (20.2)	13 (72.2)	5 (27.8)	0.020
3–4	71 (79.8)	23 (32.4)	48 (67.6)
T stage
$\leqslant$ T2	54 (60.7)	23 (42.6)	31 (57.4)	0.609
$>$ T2	35 (39.3)	13 (37.1)	22 (62.9)
N stage
N0	13 (14.6)	6 (46.2)	7 (53.8)	0.650
$\geqslant$ N1	76 (85.4)	30 (39.5)	46 (60.5)

2. Materials and methods

2.1 Patients and materials

A total of 119 archived paraffin-embedded tissues were used for immunohistochemical analysis, including 89 NPC patients and 30 control cases. The case with chronic nasopharyngeal inflammation was defined as normal control. All tissues were obtained from the Pathology Department, the First Affiliated Hospital of Chongqing Medical University. The samples were collected by biopsy before any treatment was performed. Tumor and clinical stages were defined according to the criteria of the 2002 TNM Classification [18]. After confirmed as NPC, the patients received radiotherapy with or without chemotherapy from March 2006 to March 2013. The last follow-up of all patients ended at October 2013. Clinical features of patients and characteristics of tumor were shown in Table 1. For qRT-PCR and Western blot analysis, fourteen paired fresh specimens were collected randomly, frozen in liquid nitrogen immediately following biopsy and then maintained at $-$ 80 ${{}^{\circ}}$ C until use. The study was approved by ethics committee of the First Affiliated Hospital of Chongqing Medical University.

2.2 Immunohistochemistry

Table 2
Results of GPx2 expression determined by immunohistochemistry

Group GPx2 protein expression (%) $P$ -value

Low High

NPC patient 36 (40.40) 53 (59.60) $<$ 0.05

Control 28 (93.33) 2 (6.67)

Group	GPx2 protein expression (%)	$P$ -value
	Low	High
NPC patient	36 (40.40)	53 (59.60)	$<$ 0.05
Control	28 (93.33)	2 (6.67)

Before immunohistochemistry analysis, HE staining was performed to identify the NPC cancer cells. Immunohistochemistry analysis was performed by 2-step plus Poly-HRP anti-mouse/rabbit IgG detection system (Zhongshan Golden Bridge Bio-technology, Beijing, China). The slides were incubated successively with GPx2 antibody (dilution 1:30, sc-54604, Santa Cruz Biotechnology, CA, USA) for overnight. Sections were evaluated by two pathologists blindly. The following scoring system based on the percentage of positive cells and intensity was used to evaluate the GPx2 expressing. The percentage was scored as 0 (0–9% cells stained), 1 (10%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The intensity scored as follows: 0 $=$ negative, 1 $=$ weak, 2 $=$ moderate and 3 $=$ strong. The final scores were defined as multiplying of the percentage and intensity. Strong staining (scored 5–12) was considered as high expression, whereas light staining (scored 0–4) was considered as low expression.

2.3 Western blot

Figure 1.

HE staining for NPC tissues (A) and normal nasopharyngeal tissues (B). Representative immunohistochemistry for NPC (high expression (C); light expression (D)) and normal nasopharyngeal tissues (E).

Total proteins from 7 pairs of fresh nasopharyngeal carcinoma and inflammation tissues were extracted using RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China). The concentrations of protein were detected by BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Nantong, China). The 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to separate protein samples. Then proteins on the gel were transferred to PVDF membranes. Membranes were incubated with GPx2 antibody (dilution 1:500, sc-54604, Santa Cruz Biotechnology, CA, USA). $\beta$ -actin was used as the control. Membranes were scanned by ChemiDoc Touch Imaging System (Bio-Rad) and analyzed with the Image Lab software.

2.4 Real-time quantitative RT-PCR

QRT-PCR was performed by One-Step SYBR PrimeScript RT-PCR Kit II (Takara Biotechnology, Dalian, China) following the supplied protocol. Total RNA from 7 pairs of specimens was extracted by Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA was then reverse transcribed into cDNA. GAPDH was used as the internal standard in qRT-PCR system. Then GPx2 and GADPH expressions were analyzed in a LightCycler 480 qRT-PCR system (Bio-RAD CFX96, Hercules, CA, USA) with SYBR Premix Ex Taq (Takara Biotechnology). The PCR primers for GPx2 were: 5 ${}^{\prime}$ -GTGAGGTGAATGGGCAGAAC-3 ${}^{\prime}$ (forward), 5 ${}^{\prime}$ -ACAGGGCTCCAAATGATGAG-3 ${}^{\prime}$ (reverse). The primers for GAPDH were: 5 ${}^{\prime}$ -ACCTG ACCTGCCGTCTAGAA-3 ${}^{\prime}$ (forward), 5 ${}^{\prime}$ -TCCACCA CCCTGTTGCTGTA-3 ${}^{\prime}$ (reverse). The results were analyzed by the $\Delta\Delta$ Ct method. Each experiment was repeated three times.

2.5 Statistic analysis

All statistical analyses were performed by SPASS 21.0 statistics. Data shown as $P<$ 0.05 was considered statistically significant. Student’s t-test, chi-square test and logistic regression were used to evaluate the associations among categorical variables. Survival data were evaluated using univariate and multivariate Cox regression analysis.

Table 3
Results of logistic regression exploring the effects of covariates on clinical stage

Predictors $\beta$ SE Wals Exp ( $\beta$ ) 95% confidence intervals $P$

Age 0.137 0.178 0.038 1.147 0.287 4.59 0.997

Sex 0.259 0.712 0.132 1.295 0.321 5.227 0.716

GPx2 expre $-$ 1.934 0.665 8.448 0.145 0.039 0.533 0.004

T stage $-$ 20.576 6335.383 0.000 0.000 0.000 0.997

Predictors	$\beta$	SE	Wals	Exp ( $\beta$ )	95% confidence intervals	$P$
Age	0.137	0.178	0.038	1.147	0.287	4.59	0.997
Sex	0.259	0.712	0.132	1.295	0.321	5.227	0.716
GPx2 expre	$-$ 1.934	0.665	8.448	0.145	0.039	0.533	0.004
T stage	$-$ 20.576	6335.383	0.000	0.000	0.000		0.997

Table 4

Prognosic factors for metastasis of 89 patients with NPC by Cox regression analysis

Variables	HR	95% CI	$P$ -value
Univariate analysis
Age, years ( $\leqslant$ 50 vs. $>$ 50)	1.954	0.736–5.190	0.179
Sex (male vs. female)	1.688	0.475–6.000	0.419
Clinical stage (1–2 vs. 3–4)	0.232	0.030–1.764	0.158
T stage (1–2 vs. 3–4)	0.421	0.156–1.137	0.088
GPx2 expression (neg vs. pos)	4.042	0.916–17.841	0.065
Multivariate analysis
Age, years ( $\leqslant$ 50 vs. $>$ 50)	1.938	0.696–5.400	0.206
Sex (male vs. female)	1.612	0.445–5.839	0.467
Clinical stage (1–2 vs. 3–4)	0.581	0.061–5.573	0.638
T stage (1–2 vs. 3–4)	0.608	0.206–1.789	0.366
GPx2 expression (neg vs. pos)	3.950	0.804–19.399	0.091

3. Results

Expression of GPx2 in nasopharyngeal carcinoma as determined by immunohistochemistry

Totally 89 nasopharyngeal carcinoma patients and 30 nasopharyngeal inflammation tissues were analyzed. Results were shown in Table 2. Representative examples of staining are shown in Fig. 1, indicating that nasopharyngeal carcinoma samples have strong cytoplast GPx2. Among 89 cases of nasopharyngeal carcinoma, 53 cases had strong staining and 36 cases had light staining. While in 30 control cases, 28 cases had light staining and 2 cases had strong staining, which demonstrated that GPx2 was over expressed in nasopharyngeal carcinoma ( $P<$ 0.05).

Figure 2.

GPx2 mRNA expression in NPC patients and the controls (* $P<$ 0.05).

Figure 3.

GPx2 protein expression in NPC patients and the controls (* $P<$ 0.05).

3.1 Elevated GPx2 mRNA by qRT-PCR and protein by Western blot

Seven pairs of nasopharyngeal carcinoma and the control samples were analyzed respectively. Mean age of patients for qRT-PCR analysis was (42.9 $\pm$ 10.4) years old and (44.6 $\pm$ 9.1) years old for the control group. And Mean age of patients for Western blot analysis was (50.6 $\pm$ 12.3) years old and (46.6 $\pm$ 13.1) years old for the control group. No significant difference was shown in the age and sex ( $P<$ 0.05). The mRNA expression of GPx2 in carcinoma tissues was highly elevated when compared with the control tissues ( $P<$ 0.05, Fig. 2). Gpx2 protein in carcinoma tissues was also over expressed than in control tissues ( $P<$ 0.05, Fig. 3).

3.2 Relation between GPx2 expression and clinicopathologic features

Table 1 shows the association between GPx2 expression and the clinicalpathologic features of nasopharyngeal carcinoma patients. Statistical analysis showed GPx2 expression was significantly higher in the late clinical stage ( $P=$ 0.02). While there was no significant association between GPx2 expression and patient age ( $P=$ 0.149), sex ( $P=$ 0.604), T-stage ( $P=$ 0.609), N-stage ( $P=$ 0.65).

Logistic regression analysis showed that GPx2 expression was the only statistically significant predictor for clinical stage (Table 3).

To investigate whether GPx2 expression was associated with metastasis, Cox regression analysis was performed. Both univariate analysis and multivariate analysis revealed that there was no significant relation between GPx2 expression and the metastasis (Table 4).

4. Discussion

The present study showed that GPx2 was clearly detected in the cytoplasm of cancer cells of NPC tissues immunohistochemically. Also, $q$ RT-PCR and western blot showed elevated expression of GPx2 in NPC tissues when compared with the normal nasopharyngeal tissues.

The shift in the balance between ROS and biological antioxidative response plays a vital role in tumor progression. ROS such as superoxide anion (O ${}_{2}^{-}$ ), hydrogen peroxide (H ${}_{2}$ O ${}_{2}$ ), hydroxyl radical (OH $\cdot$ ) are generated from reduction oxidation reactions. ROS have been identified as important chemical mediators in the regulation of signal transduction processes involved in cell growth and differentiation [19, 20, 21, 22]. ROS also have connection with multiorgan carcinogenesis and cancer progression by causing oxidative damage to macromolecules and by inducing various expression of transcriptional factors involved in neoplastic transformation [23, 24, 25]. The most abundant endogenous ROS is H ${}_{2}$ O ${}_{2}$ , one type of hydroperoxides. Researches had shown hydroperoxides can cause DNA damage, genome instability and cell proliferation [7]. It has been reported hydroperoxides were involved in various cancers including nasopharyngeal carcinoma. Segawa et al. [26] found the expression of iNOS induces oxidative stress in NPC. Tan et al. [27] showed that higher concentration and longer durations of H ${}_{2}$ O ${}_{2}$ were required to induce apoptosis in normal nasopharyngeal epithelial cells (NP69) compared with NPC cells (HK1), as the higher genomic instability in cancer cells than normal cells. Then the enzymatic antioxidants are function as to protect organism against these pro-oxidants, including glutathione peroxidase (GPX) [28]. GPX is a selenium-dependent enzyme that is ubiquitously expressed and protects cells against oxidative damage by reducing hydrogen peroxide. Lower GPX expression causes H ${}_{2}$ O ${}_{2}$ accumulation, resulting in higher production of OH $\cdot$ radicals. So far, it is shown that changes of the GPX activities may have connection with the development of cancer [29]. In human, the GPX family has eight known glutathione peroxidases (GPX 1-8) [30]. Within the family of GPX, GPx2 ranks highest in the hierarchy, followed by GPx4, GPx3 and GPx1 [31]. GPx2 is a tetrameric enzyme localized in cytosol. Several studies had shown an involvement of GPx2 in cancer. Naiki-Ito et al. [32] reported that GPx2 played a prominent role in mammary carcinogenesis in both rodent and human. Also, study found upon overexpression, GPx2 alleviated the apoptotic response of MCF7 cells to oxidative stresses [33]. Banning [34] reported that GPx2 knockdown HT-29 cells showed substantially less colony forming capacity in soft agar than controls and formed much smaller tumors in nude mice, revealing that GPx2 has the to facilitate tumor growth indeed. Therefore, the proliferation of tumor may be obtained by suppression of hydroperoxide-mediated apoptosis. Our study shows GPx2 is highly expressed in NPC tissues. Previous study has demonstrated GPx activities were significantly increased in NPC patients’ plasm [35], in line with our results. On the other hand, previous studies indicated ROS contribute to the development of NPC [26, 36]. Through these positive results, we propose GPx2 may promote tumor growth in nasopharyngeal carcinoma by neutralizing the overproduction of ROS that is associated with tumor development. Our study also demonstrates that high expression of GPx2 was associated with late clinical stage, suggesting that GPx2 may serve as a prognostic biomarker for NPC patient.

GPx2 can act as the prevention of metastasis in tumor. It was reported that GPx2 knockdown cells had a higher capability to migrate and invade than the GPx2-expressing controls [34]. Interestingly, the GPx2 promoter is activated by $\beta$ -catenin/TCF [37]. On the other hand, proliferation and differentiation of epithelial mucosa cells are regulated by the Wnt pathway [38], in which $\beta$ -catenin is the main signal transducer. Jiang R [39] found that the tumor growth was markedly suppressed by silencing of $\beta$ -catenin and $\beta$ -catenin may function as an essential protein for the maintenance of migration and proliferation abilities of NPC cells. We may speculate through these results that there might be some connections between GPx2 and Wnt pathway/ $\beta$ -catenin in NPC metastasizing carcinogenesis. However, our results showed no significant connection between GPx2 expression and T class, N class, metastases and recurrence.

In conclusion, our data demonstrated that GPx2 was elevated in NPC and may play an important role in the development of NPC through the regulation of ROS. Furthermore, higher GPx2 expression was associated with late stage of tumor, indicating that GPx2 may serve as a prognostic biomarker for NPC patient.

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