Assessment of glutathione peroxidase enzyme response and total antioxidant status in oral cancer – Systematic review and meta‐analysis

Abstract Background Oxidative stress induced by free radical accumulation contributes to many pathologies, including cancer. Antioxidant defense system fails to scavenge free radicals when it is excessively accumulated. Assessing individual antioxidant enzymes and total antioxidant capacity could direct the customized therapeutic strategies. Objective Evaluation of total antioxidant status and enzyme glutathione peroxidase activity in the oral cancer group compared to the healthy control group. Method The literature search included databases PubMed, Science Direct, Wiley Online Library, Cochrane and Cross Reference between 1999 and 2021. The database search was completed in the month of August 2022. The extracted data were analyzed by Comprehensive Meta‐Analysis (CMA) version 3 software (Biostat Inc. Englewood, NJ). Based on search strategies, 1435 articles have been retrieved from the database. In the segregated articles, 1365 were excluded due to duplicated articles, animal studies, low‐quality studies, articles unrelated to the research question, and with unmatched objectives. Based on inclusion criteria, 70 articles were selected for full‐text valuation. However, 33 articles were found highly suitable for inclusion and data extraction. Finally, 11 articles were selected for meta‐analysis. Results The meta‐analysis of four included studies of tissue samples showed a significantly (p < .001) increased GPx activity in the oral cancer group, when compared to the control group, whereas three included studies of erythrocyte samples displayed a significantly (p < .001) decreased GPx activity in the oral cancer group than the control group with the pooled standardized mean difference value of −2.766 moles/min/g Hb at 95% CI (−3.297 to −2.234). The meta‐analysis of the included studies depicted an insignificant (p = .947) reduction of salivary TAS levels in the oral cancer group when compared to the control group. Conclusion Our systematic review and meta‐analysis depict antioxidant GPx enzyme activity in the regional tissue samples of the oral cancer group differs from other systemic biological fluid samples compared to the healthy control group.


| INTRODUCTION
Oral Squamous cell carcinoma is a primary orofacial cancer arising from the oral epithelium. The reported worldwide incidence is more than four hundred thousand cases annually. 1 The major etiological factors for OSCC are conventional habits such as smoking or chewing tobacco and consuming alcohol. The interaction of socioeconomic factors, occupational or environmental factors and other local conditions like trauma or sharp teeth, systemic oncogenic viral infections, along with genetic influences such as mutations of oncogenes or tumor suppressor genes (TSG) may play a primary role in oral carcinogenesis. 2 Traditional habits alike tobacco consumption initiate reactive oxygen species (ROS)/pro-oxidants formation. ROS are unstable free radicals present with a single unpaired electron in the peripheral shell and are readily reactive. ROS attacks healthy human cells, causing them to lose their normal morphology and function and transmuting them into malignant ones. 3 ROS includes some radical compounds like superoxide (O 2 À ) anion, hydroxyl radical (OH À ), hydroperoxyl radical (HOO À ) and a non-radical compound, hydrogen peroxide (H 2 O 2 ). 4 Antioxidants inhibit free radicals' formation and propagation and defend against free radical-induced injuries. The human body comprises several endogenously developed enzymatic and non-enzymatic antioxidants that scavenge the reactive species (oxidants), thus being protected from the deleterious effects of ROS. 5 Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), reduced Glutathione (GSH), and Glutathione Reductase (GR) are the primary enzymatic antioxidants. 6 Other antioxidants include ß carotene, vitamins B-complex, C, and E and the mineral selenium. 7 These antioxidants are released by the tumor cell or the body's response to tumor growth. When the generation of the reactive oxygen species exceeds the preferred levels due to excessive accumulation/reduced elimination, the consequent deficient level of antioxidants may lead to a disproportion between oxidants and antioxidant defense systems, leading to oxidative stress (OS). 8 The induced OS results in irreparable cellular/tissue damage, which is vital in cancer initiation, promotion, and progression. The glutathione antioxidant system includes GPx, GR and Glutathione S-Transferases (GST). GPx is a prime antioxidant enzyme that helps to remove reactive species once formed. It reduces lipid hydroperoxides to their analogous alcohols. This scavenging antioxidant enzyme is concentrated in the cell membrane and cytoplasm. 9 In this system, SOD converts superoxide radical (O 2 À ) into H 2 O 2 ; consequently, glutathione peroxidase (GPx) and catalase transform H 2 O 2 into water. 10  Both endogenous and exogenous antioxidants defend the cellular or local tissue microenvironment from oxidative injury and thus prevent cancer initiation and propagation. The depressed activity of the antioxidant defense system plays a primary role in the development of many pathologies. 16 Furthermore, tobacco products and arecaquids were reported to deteriorate the serum level of TAC. 17 The literature has pointed out that a reduced GSH-dependent anti-oxidant system alters the sensitivity of the tumor cells to oxidative or nitrative stress, cytokines, chemotherapeutic drugs and radiation therapy. 18 So, exploring the link between ROS, the antioxidant system and oral cancer is mandatory. These findings will direct the prospective therapeutic options that target ROS and oxidative stress. Thus, the present systematic review assessed the antioxidant enzyme GPx activity and status of total antioxidants in oral cancer and control group to determine alterations of the antioxidant system in the oral cancer group.

| MATERIALS AND METHODS
The present systematic review (SR) follows the recommended PRISMA protocol. The designated PROSPERO registration number for the present SR is CRD42021271018.

| Exclusion criteria
i. Studies with unrelated objectives or abstracts.
ii. Duplicated studies involving the same patients by the same authors.
iii. Being narrated or systematic reviews.
F I G U R E 1 Prisma flow chart explains the selection process of study articles.  vi. The articles provided insufficient data or graphical representation for comparing the control and oral cancer group.
vii. The observational studies analyzed the therapy effect without the healthy control group evaluation.
viii. The studies have not provided adequate data for comparison with other studies.

| Assessment of literature
The

| Data segregation
The examiners analyzed the selected articles, and extracted the data specific to the authors' details, year of publication, sample size, and assessment methodology of GPx antioxidant enzyme activities and TAC values (mean or median) with statistical significance between oral cancer and control group. The values were considered statistically significant if the p value was <.05.

| Meta-analysis
The standard difference in the mean was performed for deriving the forest plot to analyze the data with the help of the CMA version 3 Biostatistics software (Englewood, NJ). The pooled mean difference value of GPx activities and the TAC levels between OSCC and the control group was derived at a confidence interval of 95%. The random-effects model was performed for quantitative synthesis due to the significant heterogeneity of the selected studies. Articles with a similar sample, measurement units and methodology were chosen for the quantitative analysis.
Studies that displayed out-of-range values were excluded from the meta-analysis to reduce the chances of heterogeneity.  Note: The studies that earned scores from 7 to 9 were denoted as high-quality studies, and no studies scored ≤5, indicating high-risk studies. review (n = 1), systematic reviews (n = 2) and reviews (n = 31) were excluded. In article augmentation, 33 articles were found highly suitable for qualitative synthesis. The articles having coherent data and allowed comparison (n = 11) were included for meta-analysis ( Figure 1).

| RESULTS
The Newcastle-Ottawa quality evaluation scale was displayed in Table 1 to assess the features of included studies in the systematic review.
All the segregated data of selected articles were tabulated in Tables 2 and 3 (Tables 5 and 6).

| Heterogeneity
The studies taken for meta-analysis of the activity of GPx enzyme in   The present systematic review depicts that there was a significantly enhanced GPx activity in tissue samples 19,20,26,42,48 and decreased activity in erythrocyte and plasma samples (p < .05) of the OSCC group compared to the normal control group. [21][22][23][24]27,28,32,33,36,38,41,47 Out of the four included studies that analyzed the GPx activity in blood samples except one, 48 the remaining studies displayed diminished enzyme activity in the OSCC group compared to healthy controls (p < .05). 26 The enhanced glutathione peroxidase activity is due to the higher demand against the toxic substance of the tumor cells and simultaneous defense to protect the prevailing unaffected cells. 61 According to the previous statement, another study pointed out increased salivary antioxidant levels due to that a compensatory defense reaction. 40 In contrast, studies have explained that depletion of glutathione peroxidase in overt neoplasias might be due to increased turnover or utilization of antioxidants from the circulation to counteract overwhelming free radicals and resultant oxidative injury. 33,62 One study correlated low plasma antioxidant levels and tumor burden. 22 The levels of GPx expression were positively correlated with favorable prognoses, especially the patients with advanced stage IV tumors in one reported work. 63 The assessment of TAC in various biological samples is a reliable biomarker that comprises the activity of entire enzymatic and non- displayed depleted TAC levels in OSCC group compared to the control group ( p < .05). 29,31 When assessing the plasma samples, three studies depicted reduced TAC values in the OSCC group compared to the normal control group ( p < .05). 43,44,47 Few other studies not included in the systematic review displayed significantly decreased TAC levels in the oral cancer group.
Korde et al. 16 found a diminution of TAC in the serum and tissues of patients with oral cancer compared to controls ( p < .001). Shetty et al. 65  F I G U R E 2 Forest plot expresses the standardized mean difference levels at 95% CI, shows the comparison of glutathione peroxidase activity in tissue samples of OSCC and healthy control group.
The reduction of TAC values might be due to the following facts: (1) accentuated utilization of these antioxidants in the scavenging reac-

| LIMITATIONS
There was high heterogeneity observed between the included studies.
In addition, only a limited number of studies were included in the meta-analysis, which may impact the generalizability of the findings.
Different studies utilized different measurement methods to detect GPx activity and TAC levels, which were reported in different orders of magnitude and affected the level of evidence.

| CONCLUSIONS
Our systematic review and meta-analysis depict that the regional tis-

CONFLICT OF INTEREST STATEMENT
The authors have stated explicitly that there are no conflicts of interest in connection with this article.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.

ETHICS STATEMENT
Not applicable.
F I G U R E 4 The forest plot displayed the standardized mean difference (Std diff in mean) values at 95% confidence intervals, representing a comparison of salivary levels of total antioxidant capacity between OSCC and normal control groups.