Reduced Expression of GPX3 in Breast Cancer Patients in Correlation with Clinical Significance

Glutathione peroxidase 3 (GPX3) is the main antioxidant enzyme in plasma. Its biological roles are to protect cells from oxidative stress-induced damage. Several studies have been reported the association between GPX3 expression and its correlation with cancer carcinogenesis including breast cancer. The aim of this research was to investigate the GPX3 messenger ribonucleic acid (mRNA) expression in 82 breast tumors and paired normal breast tissues by SYBR green quantitative real-time reverse transcription-polymerase chain reaction and the association with clinicopathological data. Our results show that GPX3 reduced expression was found significantly associated with number of metastatic lymph nodes (odds ratio [OR] = 3.41, 95% confidence interval [CI] = 1.35–8.64, p  = 0.01), no distant metastasis (OR = 5.52, 95% CI = 3.74–11.89, p  = 0.04), and nonhormone usage breast cancer patients (OR = 0.19, 95% CI = 0.04–0.93, p  = 0.04). This finding suggested that GPX3 plays a role in breast carcinogenesis, and might serve as a prognostic biomarker in breast cancer patients.


Introduction
Glutathione peroxidase 3 (GPX3), a tumor suppressor gene that is located on chromosome 5q23, is the major antioxidant enzyme in plasma and plays an important role in detoxifying hydrogen peroxide and other oxygen-free radicals, protecting cell from oxidative stress-induced damage. 1- 3 Inactivation of GPX3 results in the accumulation of an elevated amount of hydrogen peroxide and other reactive oxygen species (ROS) that may involve breast carcinogenesis via induction of oxidative deoxyribonucleic acid (DNA) damage, genetic alterations, and neoplastic transformation. 4,5 Several studies have reported the association between GPX3 expression and its correlation with cancer carcinogenesis such as gastric cancer, 3,6 cervical cancer, 7 thyroid cancer, 8 nonsmall cell lung cancer, 2 prostate cancer, 9 hepatocellular carcinoma (HCC), 10 including breast cancer 11,12 however, the mechanisms in breast tumorigenesis remain unclear.
Previously, our data from microarray analysis (data not shown) was identified for GPX3 expression, which is one of the consistently downregulated genes in breast tumor. In the current study, we studied the role of GPX3 in breast carcinogenesis by determined GPX3 mRNA expression in 82 breast tumors and paired normal breast tissues by SYBR green quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and correlation with clinicopathological characteristics including overall survival.

GPX3 mRNA Expression Analysis by SYBR Quantitative Real-Time Reverse Transcription-PCR
Alterations in GPX3 mRNA expression levels were analyzed by LightCycler Instrument (Roche Diagnostics GmbH, Mannheim, Germany). The reaction components were 20 ng of template cDNA, 1x LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics GmbH, Mannheim, Germany), 4 mM MgCl 2 and 0.5 μM forward and reverse primers in 10 μL of a total volume. The primer sequences were designed by Primer-BLAST, forward F-GPX3 (5′-AGTGCTGGACAGTGA-CAACC-3′) and reverse R-GPX3 (5′-GGCCCCAAGGTTGAGG-TATC-3′. β-globin housekeeping gene was used as an endogenous reference to obtain relative expression values. PCR was started at 95°C for 5 minutes (to activate the FastStart Taq), followed by 40-cycle amplification (95°C for 10 seconds, 62°C for 30 seconds, and 72°C for 30 seconds. After the PCR, each amplification reaction was checked using a dissociation curve. PCR product purity was checked by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV light. Relative gene expression level was determined as previously described by Livak and Schmittgen. 13 Median expression levels were used for the cutoff values for gene expression that were adopted from median expression levels. Gene expression <0.3 was assigned as reduced expression.

Statistical Analysis
The association between GPX3 mRNA reduced expression level and clinicopathological characteristics-age at diagnosis; tumor size; histological grade; axillary lymph-node status; number of lymph nodes; staging; triple-negative breast tumor; immunohistochemistry staining of ER, PR, and HER2; treatment; metastasis; hormone usage; cancer family history; alcohol consumption; and smoking statuswas examined statistically by chi-squared test. Overall survival was analyzed by Kaplan-Meir method and p-value < 0.05 was considered a significant correlation.

Reduced Expression of GPX3 mRNA in Breast Cancer Patients
Previously, our data from microarray analysis was identified for GPX3 expression, which is one of the consistently downregulated genes. In this research, we verified GPX3 mRNA expression level in 82 breast tumors and corresponding normal breast tissues by SYBR quantitative real-time RT-PCR. The results show that GPX3 reduced expression was detected in 41.50% (34/82) that is consistent with microarray data that shown downregulated gene. In addition, we also found that GPX3 reduced expression was significantly associated with number of metastatic lymph nodes (more than 2 lymph nodes), (OR ¼ 3.41, 95% CI ¼

Survival Analysis
The association between GPX3 reduced expression and survival was analyzed by Kaplan-Meir method. The results show that there was no correlation between GPX3 reduced expression and overall survival (p ¼ 0.44) as shown in ►Fig. 1.

Discussion
Glutathione peroxidase (GPx) is a major antioxidative damage enzyme family, which comprises eight submembers (GPx 1-8). GPX3 is the only extracellular enzyme in the GPx family that removing ROS products during cellular metabolism or oxidative damage. 1,14 GPX3 has been reported to be downregulated in several types of cancers such as GPX3 downregulation that can promote the proliferation, motility, and invasion of melanoma cells in vitro. 15 Qi et al found that GPX3 low expression was significantly associated with advanced tumor stage, venous infiltration, and poor overall survival in HCC patients 10 as well as in gallbladder cancer 16 that has been shown poor prognosis.
Furthermore, Mohamed et al 11 demonstrated that downregulation of GPx3 levels was found in aggressive inflammatory breast cancer carcinoma tissues when comparing to noninflammatory breast cancer tissues as well as Lou et al 12 reported that GPX3 mRNA and protein expression level in breast cancer tissues was expressed less than corresponding Global Medical Genetics Vol. 7 No. 3/2020 © 2020. The Author(s). normal tissues, suggesting the involvement of GPX3 in breast pathogenesis.
In the present study, we found that GPX3 reduced expression correlated with number of metastatic lymph nodes (more than 2 lymph nodes) (p ¼ 0.01), as in accordance with previous research, the downregulation of GPX3 expression was significantly associated with lymph node metastasis in gastric cancer and cervical cancer 3,6,7 as well as reduced GPX3 protein levels were correlated with tumor size and lymph node metastasis in thyroid cancer. 8 In addition, several studies has been reported GPX3 downregulation was promoted tumor invasion, motility. 10,15 However, our findings were found that reduced expression of GPX3 was significantly relevant to no distant metastasis in breast cancer patients (p ¼ 0.04); this demonstrated the uncomplete inactivation of GPX3 expression was found in this study. Furthermore, we analyzed the association between GPX3 reduced expression and clinical data of hormone usage, cancer family history, alcohol consumption, and smoking status; we also found on the first time that GPX3 reduced expression was significantly correlated with nonhormone usage in breast cancer patients (p ¼ 0.04).
In conclusion, our finding showed that GPX3 reduced expression was significantly correlated with number of metastatic lymph nodes, no distant metastasis, and nonhormone usage of breast cancer patients; this finding suggested that GPX3 plays an important role in breast carcinogenesis, and might serve as a prognostic biomarker in breast cancer patients.

Funding
This work was supported by a grant from the Fiscal Budget of the Royal Thai Government. Abbreviations: -, no reduced expression; þ, reduced expression; CI, confidence interval; GPX3, glutathione peroxidase 3. Fig. 1 Survival was analyzed by Kaplan-Meir method and log rank test was used to compare between glutathione peroxidase 3 reduced expression and nonreduced expression, p ¼ 0.44.