Assessment of salivary thioredoxin levels in oral lichen planus and oral squamous cell carcinoma

Abstract Objective Oral lichen planus (OLP) is a chronic mucocutaneous inflammatory disease, which is considered as a potentially malignant condition and could transformed into oral squamous cell carcinoma (OSCC). Squamous cell carcinmoma is the most common oral cancer. This study aimed to compare salivary thioredoxin levels as an antioxidant protein among patients with OSSC, OLP and healthy subjects. Materials and methods Twenty‐eight patients with OLP, 20 patients with OSCC and 40 healthy people enrolled in this observational study. Saliva samples were collected from all subjects and salivary thioredoxin levels were evaluated by Elisa test. The data were recorded in the check lists and analyzed using SPSS (ver.17). Results Thioredoxin levels of healthy controls were insignificantly higher than OLP and SCC patients (p = 0.135). There was a statistically remarkable indirect relationship between thioredoxin levels and severity of the lesions determined by Thongprasom criteria among OLP patients. The thioredoxin concentration was significantly higher in the keratotic OLP. Among the OSCC patients, the highest levels of thioredoxin were found among patients aged more than 65 years. Salivary thioredoxin levels based on other variables were not significantly different between the studied groups. Conclusion In this study, there was not any significant difference between salivary thioredoxin levels in the OLP and OSCC patients, though it was unremarkable higher in the healthy group compared to the patients; therefore, the role of thioredoxin in the cellular oxidation–reduction status could be suggested; however, further studies are recommended.

aged adults (30-70 years), and this disease is more common in women with 3:2 ratio (M. Carrozzo & Thorpe, 2009). The articles reported the prevalence rates ranging from 0.5% to 2.2% for oral lichen planus (M. , oral lichen planus (OLP) lesions are considered as a potentially malignant condition; the rate of these malignant transformation has been reported to be 0.2%-5.3% (Carrozzo & Thorpe, 2009); however, dysplastic changes even in 10% of samples were reported (Cawson, 1968).
The etiology of lichen planus is not known. Research results suggests that identifying a specific etiological factor for this condition is difficult, though it seems that the immune system plays a significant role in the incidence of this disease. T lymphocytes, which function against the epithelium, have been propounded as the first important factor for the development of OLP. Also, other factors such as inflammatory cytokines, imbalance of antioxidants are also involved in the pathogenicity of disease (Ismail et al., 2007). Nevertheless, the role of factors such as stress, local trauma, infection, and lifestyle cannot be neglected (Ismail et al., 2007).
Oxidative stress leads to impair in the balance between oxidants and antioxidants, resulting in increased oxidative damage to lipids, DNA molecules and proteins in patients with OLP and could act as a trigger in the initiation or progression of many diseases (Ergun et al., 2011). Among mucosal disorders, the role of oxidative stress has been proven in some diseases including psoriasis, lichen planus, and pemphigus vulgaris (Yesilova et al., 2013). Since the human body is constantly exposed to different endogenous and exogenous reactive oxygen, it is equipped with any non-enzymatic and enzymatic antioxidants such as vitamin C, vitamin E, catalase, super oxidase dismutase, and many others that are essential for the human health and cell survival (Kurutas, 2015). The thioredoxin is one of the major oxidoreductases of cells composed of thioredoxin-thioredoxin reductase system, and NADPH. The role of this system has been discussed extensively in the health and disease (Kiafar et al., 2020;Sobhani et al., 2016;Yaghoubi et al., 2019).
SCC is a multifactorial disease; no single etiological factor has been clearly defined or accepted. It is possible that more than one factor is required for development of this malignancy (co-carcinogenesis) (Kumar et al., 2016). Human thioredoxin system plays a significant role in establishing the homeostasis of cellular oxidation-reduction status.
In some studies, a paradoxical role has been discussed for thioredoxin; it prevents cancer; on the other hand, it acts in its development. This system prevents cancer through removing carcinogen antioxidants and repairing mutated DNA. On the other side, it has been found that thioredoxin is involved in the growth of cancer cells. In addition, redox system is associated with tumor angiogenesis through inducing vascular endothelial growth factor. Thioredoxin-thioredoxin reductase system plays a key role in development of cancer cells. Some studies have also suggested that this system plays a significant role in the growth of tumors and resistance against some chemotherapeutic agents in many malignancies (Iwasawa et al., 2011;Mohammadi et al., 2019).
Although the role of thioredoxin system in the proliferation of tumoral cells and resistance to chemotherapeutic drugs has been found in some neoplasms, its role in oral SCC has remained unclear.
Only some limited studies including Iwasawa et al. have reported elevated thioredoxin reductase in OSCC, whereby they found it to be related to metastasis of tumor to the lymph nodes (Sobhani et al., 2016).
Considering the mechanisms mentioned for thioredoxin in SCC, as well as; its possible role in lichen planus disease, this research was performed to assess the salivary antioxidant levels through measuring thioredoxin in patients with oral SCC, as a malignant lesion, and compare with OLP patients, as a premalignant lesion, and healthy individuals. A checklist was completed for all subjects including demographic characteristics (age, gender, and medical history), lesion characteristics (site of lesion, type and severity of lesion for OLP patients based on the Thongprasom criteria (Thongprasom et al., 1992), clinical features:

| Experimental methods for assessing thioredoxin
For all of the three groups, the patients with OSCC, OLP, and healthy individuals, saliva sampling was performed. The subjects were recommended not to drinking, eating, or smoking for 90 min before collecting unstimulated saliva through spitting method. Saliva samples were collected between 9 a.m. and 11 a.m. The patients were asked to sit in a convenient position and gather the saliva in their mouth and then spit it in a falcon sterilized tube. This was performed every 60 s for 5-15 min. Saliva samples were kept on ice and sent to the laboratory for processing (Shirzaiy et al., 2017). Until the time of investigation, the samples were kept at −20 C. In order to separate squamous cells and debris and mucous, the saliva samples were centrifuged immediately after the de-frizzing for 15 min (3000 g at 25 C). We placed the samples in the test tubes with the volume of 10 cc. Next, in order for the mucin present in the saliva to be lysed (mucolysis), the samples were placed inside a fridge for 24 h at −20 C. After 24 h, the samples were centrifuged (3000 g for 10 min). Eventually, the watery part of the saliva was transferred to micro tubes with a volume of 1.5 cc. Next, the level of thioredoxin in the watery part of saliva was measured by the kits of ZellBio Co., Germany (Human TRX ELISA kit with the product code of ZB-11452) and based on the protocol presented by the kit manufacturer's company.
First, the reagents, samples, and standards were prepared. Then, 40 μl of the sample(s) and 10 μl of thioredoxin antibody were added to 50 μl of standard and 50 μl of streptavidin-HPR, and placed at 37 C for 60 min. Next, the containers were washed five times with 300 μl diluted buffer. Thereafter, 50 μl of chromogen A solution and 50 μl of chromogen B solution were added and placed at 37 C for 10 min for color detection until incubation. Then, 50 μl of stop solution was added. Finally, the optical density (OD) was measured at the wavelength of 450 nm.

| Statistical analysis
The information collected for each patient was introduced into a special form and then statistical analysis was performed. The data was entered into SPSS software (version 17)  The levels of thioredoxin was measured in OLP and OSCC and healthy individuals groups and there was not any significant different between them (p = 0.135), though it was higher in the healthy subjects as compared to other groups ( Table 2). The levels of thioredoxin did not differ significantly between the healthy and OLP subjects in terms of age and gender. However, thioredoxin levels in patients with OSCC, with the age above 65 years were significantly higher than other ones (gender: p = 0.130; age: p = 0.001) (Table 1). Furthermore, investigation of the thioredoxin levels in terms of the clinical features of lesions indicated that in keratotic OLP patients, thioredoxin levels were significantly higher than the non-keratotic features (p = 0.003) ( Figure 1).
In the patients with OLP, when the severity of lesions increased, thioredoxin levels decreased, and this difference was only significant between severities of 1 and 3 (in terms of Thongprasom criteria) (p = 0.013) ( Table 3).
In patients with OSCC, the thioredoxin levels in the exophytic type were insignificantly greater than other presentations (p = 0.383)

| DISCUSSION
Oral cancer is a complex and multidimensional process, and in most cases it begins with epithelial dysplasia. Nevertheless, effective interventional measures for treating or delaying conversion of potentially malignant to malignant lesions are limited. Specific rise of oxidative stress levels in cancerous cells is a result of an imbalance between production and removal of reactive oxygen species (ROS). It has been reported that oxidative stress contributes to transformation of dysplasia to oral cancer. Human thiordoxin-1 (Trx-1) belongs to a family of small redox proteins reduced by Trx reductase (TrxR) and NADPH.
Trx-1 plays a significant role in maintaining redox balance, which is essential for cell survival, tumor growth, and angiogenesis (Chen et al., 2018;S. I. Hashemy, 2011).

A study was performed by Zhu et al. to investigate Trx-1 and
Trx-1 reductase in the histopathological samples of patients with lingual squamous cell carcinoma. Elevation of Trx-1 in this study had a significant relationship with the degree of differentiation of tumoral cells and five-year survival of patients. In this study, it was found that Trx-1 elevation predicts a poor prognosis for patients with lingual SCC (Zhu et al., 2011).
In other study, HU et al. used matrix-assisted laser desorption/ ionizationmass spectrometry (MALDI-TOF MS) analysis to compare oral fluid samples from 20 oral cancer and 20 control subjects and indicated that the mean level of thioredoxin in cancer samples was higher than normal samples (Hu et al., 2007). In our study, Trx-1 levels were compared between SCC and healthy subjects. Unlike the study by Zhu and HU, Trx levels were found to be lower in SCC patients than in healthy individuals. Further, in the present study, which was performed on saliva samples unlike the research by Zhu, it did not  gluthardoxin-1 and proxyredoxin-2, were also effective in this process (Chen et al., 2018). In our study, it was found that thioredoxin levels did not differ significantly across the healthy, OLP, and OSCC samples. However, in OLP lesions, the thioredoxin levels had grown significantly in keratotic types with a less severe clinical severity (based on Thongprasom criteria Amirchaghmaghi, Hashemy, et al., 2016).
One of the strong points of this study was concurrent investigation of thioredoxin in patients with OLP, as a premalignant lesion and OSCC, as the most common malignancy of the oral cavity, as well as use of saliva as a noninvasive and convenient method for investigating thioredoxin.
There is not any information regarding the correlation between serum and salivary thioredoxin; however, thioredoxin is a small protein (12 KD) and it is expected to be found in saliva. Therefore, it seems logical to claim that any change in the blood proteins levels could reflect in the patients' saliva. Moreover, serum and salivary thioredoxin has been studied in previous studies (Soyama et al., 2020).
It is suggested to design a comprehensive study for concurrent assessment of thioredoxin reductase and thioredoxin levels and investigation of the correlation between the levels of thioredoxin and response to treatment, metastasis, and different subtypes of OSCC and dysplasia of OLP.

| CONCLUSION
Based on the results of the present study, salivary thioredoxin was lower in the patients with OLP or OSCC as compared to healthy individuals; however, it is not statistically significant. Furthermore, a significant relationship was observed between thioredoxin levels and the severity of OLP based on Thongprasom criteria. Thus, it is possible that thioredoxin may be involved in the pathogenesis of these two diseases. Moreover, in patients with OSCC, thioredoxin was significantly correlated with older age, which can be considered for further detailed studies.