The activity and tissue distribution of thioredoxin reductase in basal cell carcinoma

Abstract

Purpose

Basal cell carcinoma (BCC) is the most prevalent cancer worldwide. Different mechanisms are proposed to be involved in its pathogenesis such as oxidative stress. Oxidative stress, which is the consequence of the disruption of redox balance in favor of oxidants, is involved in the initiation or progression of many tumors. Thioredoxin reductase (TrxR) is a key enzyme of the thioredoxin (Trx) system, containing Trx and TrxR and NADPH, which is one of the main cellular oxidoreductases with an essential role in cellular health and survival through providing and maintaining redox balance. Therefore, we aimed to study and compare the activity and tissue distribution of TrxR in tumoral tissue and its healthy margin in patients with BCC.

Methods

After biopsy and taking samples from 18 patients, TrxR activity was measured using a commercial kit and its tissue distribution was assessed immunohistochemically.

Results

Both the activity and tissue distribution of TrxR in tumoral tissues were significantly higher compared to their healthy margins. Regarding the tissue distribution, this significant increase in TrxR in tumoral tissues was documented based on both staining intensity and abundance of positive cells in immunohistochemistry.

Conclusions

Based on these results, it is concluded that TrxR is involved in the pathogenesis of BCC; however, more investigations are required to clarify whether this increase is a consequence of BCC or it is an initiating mechanism.

Introduction

Basal cell carcinoma (BCC) is the most prevalent dermal tumor in Caucasians and the most common cancer worldwide with an annual increase in its incidence (Deng et al. 2015; Razi et al. 2015). Different risk factors are defined for BCC, and a number of mechanisms are proposed to be involved in its pathogenesis (Cretnik et al. 2009; Goppner and Leverkus 2011; Iwasaki et al. 2012). Among the risk factors, ionizing radiation exposure can be exemplified (Li and Athar 2016), which leads to cellular damages via different mechanisms such as mitochondrial dysfunction and persistent oxidative stress (Szumiel 2015; Yoshida et al. 2012).

Oxidative stress, which is the consequence of the disruption of the balance between oxidants and antioxidants in favor of the former, is involved in the initiation or progression of many diseases such as autoimmune disorders (Fujii et al. 2015; Kaffe et al. 2015), cardiovascular diseases (Dhalla et al. 2000; Mei et al. 2015) and cancers (Gupta et al. 2014; Milkovic et al. 2014). Among mucocutaneous disorders, the role of oxidative stress is proved in a number of diseases including psoriasis, lichen planus and pemphigus vulgaris (Amirchaghmaghi et al. 2016; Hashemy et al. 2016; Shah and Sinha 2013; Taheri et al. 2016). The role of oxidative stress in the pathogenesis of human skin cancers including BCC is also reported by several researchers (Sander et al. 2003; Vural et al. 1999). A decrease in plasma antioxidants in BCC is shown as an example which is suggested to be due to the long exposure to UV (Vural et al. 1999).

Since human body is persistently exposed to endogenous and exogenous reactive oxygen species, it is equipped with both non-enzymatic and enzymatic antioxidants such as vitamin C, vitamin E, ghrelin, catalase, superoxide dismutase and many more (Cheraskin 1996; Halliwell 1996; Nazoury et al. 2014) to provide the redox balance that is essential for cellular health and survival (Sarsour et al. 2009; Stroes et al. 1998). The thioredoxin (Trx) system is one of the main cellular oxidoreductases that is composed of Trx, thioredoxin reductase (TrxR) and NADPH. The role of this system has been extensively studied in health and diseases (Hashemy 2011; Lillig and Holmgren 2007; Mahmood et al. 2013). The role of this system in pathogenesis and progression of cancers like breast cancer, gastrointestinal cancers and many more is specially a concern for many researchers (Arner and Holmgren 2006; Flores et al. 2012).

Since the role of this system and its possible changes in BCC is not investigated yet, we aimed to study and compare the activity and tissue distribution of TrxR, the key enzyme of this system, in tumoral tissue and its healthy margin in patients with BCC.

Materials and methods

This study was performed on 18 patients who were admitted in the Department of Dermatology in Imam Reza hospital, Mashhad, Iran, with a diagnosis of BCC by a dermatologist and candidate for surgery. The exclusion criteria were the unwilling patients to participate in the study, patients with any systemic disease, such as diabetes or cardiovascular diseases, and patients with a recent history of chemotherapy or radiotherapy. This study was ethically approved by the Research Council of Mashhad University of Medical Sciences (MUMS). An informed consent was obtained from each patient.

The activity of TrxR was measured using TrxR Colorimetric Assay Kit from Cayman Company, USA (Item No 10007892). Antibodies for immunohistochemistry were purchased from Abcam, UK. A Microm HM325 microtome (Thermo Fisher Scientific, Waltham, USA) was utilized to provide tissue sections. All other reagents were provided from Sigma-Aldrich, Germany.

The activity of TrxR was investigated in fresh samples, which were washed with cold phosphate-buffered saline (PBS) and homogenized in 50 mM potassium phosphate, pH 7.4 containing 1 mM EDTA, based on the instructions provided by the manufacturer. This assay is based on a method that has been previously described (Smith and Levander 2002) in which NADPH is used by TrxR to reduce 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid (TNB). TNB production was measured by following the increase in absorbance at 412 nm, and TrxR activity was calculated (µmoles TNB produced/min) using an extinction coefficient of 13.6 × 10³ M⁻¹ for TNB at 412 nm.

The tissue distribution of TrxR was assessed using immunohistochemistry based on the intensity of staining and abundance of positive cells. Five-micrometer tissue sections were provided from both tumoral tissues and their healthy margins that had been fixed in formalin and embedded in paraffin. Tissue sections were immunohistochemically stained for TrxR. Two experienced pathologists evaluated the samples and graded the staining based on a system that was previously described (Shimizu et al. 1990). The intensity of staining and the abundance of positive cells were scored as shown in Table 1. A total immunoreactivity score was obtained for each sample via the multiplication of intensity and abundance of positive cells scores which ranged from a minimum score of 1 (weak staining of ≤25 % of cells) to a maximum score of 12 (strong staining of ≥75 % of cells).

Table 1.

Scoring system for the intensity of staining and the abundance of positive cells in immunohistochemistry of BCC tumoral tissues and their healthy margins

Score	Intensity of staining	Abundance of positive cells
1	Weak	Staining of ≤25 % of cells
2	Moderate	Staining of 26–50 % of cells
3	Strong	Staining of 51–75 % of cells
4	Not applicable	Staining of ≥76 % of cells

All samples were evaluated by two experienced pathologists

Data were analyzed using SPSS 21.0. The results are shown as Mean ± SD, and a p value of ≤0.05 was described statistically significant.

Results

This study was conducted on 18 patients (11 men, 7 women) who were 60.72 ± 13.3 year old.

TrxR activity: As it is shown in Fig. 1, the activity of TrxR in tumoral tissues (0.11 ± 0.04 unit/mg crude extract) was significantly higher compared to the healthy margins (0.05 ± 0.02 unit/mg crude extract) (p value = 0.002).

Fig. 1.

TrxR activity. The activity of thioredoxin reductase in tumoral tissues and healthy margins of patients were 0.11 ± 0.04 and 0.05 ± 0.02 unit/mg crude extract, respectively

TrxR distribution: Immunohistochemically stained samples were scored as mentioned above (Fig. 2; Table 2). Concerning the severity of staining, the scores of 2 and 3 were recorded for 11 and 7 patients, respectively, in their tumoral tissues. For healthy margins, however, 5, 9 and 4 samples had the intensity of 1, 2 and 3, respectively.

Fig. 2.

Immunohistochemical localization of TrxR in tumoral and surrounding healthy tissues of BCC; (a) moderate staining of tumoral cells at ×400 magnification, (b) moderate staining of normal cells and weak staining of tumoral cells at ×100 magnification, (c) strong staining of tumoral cells at ×100 magnification, and (d) strong staining of tumoral cells at ×400 magnification

Table 2.

Tissue distribution of TrxR in BCC samples and their normal margins

	Score	Tumoral tissue (%)	Normal tissue (%)
Intensity of staining	1	0	27.8
	2	61.1	50.0
	3	38.9	22.2
Abundance of positive cells	1	38.9	66.7
	2	44.4	22.2
	3	16.7	11.1
	4	0	0

The percentage of each score is shown for tumoral or healthy samples

Regarding the abundance of positive cells, the scores of 1, 2 and 3 were recorded for 7, 8 and 3 patients, respectively, in their tumoral tissues, and for 12, 4 and 2 patients, respectively, in their healthy tissues.

The final score for tissue distribution of each patient was obtained from the multiplication of two scores of that patient. From 18 tumoral samples, the scores of 3, 4, 6 and 9 were recorded for 4 (22.2 %), 8 (44.4 %), 5 (27.8 %) and 1 (5.6 %) patients, respectively, while, for their normal tissues, the scores of 1, 2, 3 and 4 were recorded for 4 (22.2 %), 7 (38.9 %), 5 (27.8 %) and 2 (11.1 %) patients, respectively.

Based on these results, a significant difference was observed between tumoral and normal tissues regarding the distribution of TrxR (p value = 0.018).

Discussion

Even though BCC is not very invasive and its metastasis is rare, because of its increasing incidence and cost of treatment, it is necessary to further our knowledge about the pathogenesis of this cancer and different factors that are involved in its initiation and progression.

Oxidative stress, which is due to the overwhelming of antioxidant defense mechanisms by excessive free radicals and reactive oxygen species, is shown to be involved in pathogenesis of both melanoma and non-melanoma skin cancers (Sander et al. 2004). However, the role of TrxR that is an essential enzyme for cellular redox balance is not investigated in pathogenesis of BCC.

In this study, we showed that both the activity and tissue distribution of TrxR were higher in tumoral tissue of BCC compared to the healthy controls. This increase in TrxR might be a response of dermal tissue to attenuate the inflammatory and apoptotic responses caused by UV irradiation which is one of risk factors for BCC (Ono et al. 2012). It is previously shown that the exposure to UV light leads to an increase in TrxR synthesis (Didier et al. 2001). Didier et al. 2001 also showed that UV exposure results in a time-dependent increase in intracellular level of Trx, which is another protein of the Trx system. Besides, the overexpression of Trx and TrxR is reported following the topical application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator and tumor promoter (Kumar and Holmgren 1999). Therefore, this increase in TrxR, which is shown in our study, may act as an enhancer of dermal carcinogenesis.

The increase in the Trx system is shown in several human primary tumors such as breast cancer (Kilic et al. 2014) and pancreatic cancer (Nakamura et al. 2000), where this overexpression is associated with increased tumor cell proliferation, decreased apoptosis, aggressive tumor growth and poor prognosis (Nakamura et al. 2000). Concerning skin carcinogenesis, this relationship is studied by Mustacich et al. (2004) who showed increased skin carcinogenesis in a keratinocyte directed thioredoxin-1 transgenic mouse.

Moreover, the overexpression of Trx or TrxR is believed to be involved in resistance of cancers to chemotherapy or radiotherapy via different mechanisms such as preventing the loss of the membrane potential of the mitochondria, the depletion of cellular ATP content and the loss of cell viability (Kim et al. 2005; Niu et al. 2005; Wang et al. 2015). Therefore, the role of Trx and TrxR in a more invasive behavior of BCC needs to be elucidated. Besides, the inhibition of TrxR might work as a therapeutic strategy for BCC, as it is suggested for some other tumors (Marzano et al. 2007; Yan et al. 2012).

We concluded that both the activity and tissue distribution of TrxR in tumoral tissues of patients with BCC were significantly higher compared to the healthy margins of tumors. Therefore, it seems that TrxR is involved in the pathogenesis of BCC; however, more investigations are required to clarify whether this increase is a consequence of BCC or it is an initiating mechanism.

Compliance with ethical standards

Conflict of interest

All authors declare that there is no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards (Ethical code from Mashhad University of Medical Sciences: 901151).