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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 5  |  Page : 1062-1066

Thiol-disulfide homeostasis in breast cancer patients


1 Department of General Surgery, Training and Research Hospital, University of Health Sciences, Konya, Turkey
2 Department of Anesthesiology, Training and Research Hospital, University of Health Sciences, Konya, Turkey
3 Department of Family Medicine, Training and Research Hospital, University of Health Sciences, Konya, Turkey
4 Department of Biochemistry, Training and Research Hospital, University of Health Sciences, Konya, Turkey
5 Department of Biochemistry, Yildirim Beyazit University, Ankara Atatürk Training and Research Hospital, Ankara, Turkey

Date of Web Publication4-Oct-2019

Correspondence Address:
Ibrahim Solak
Department of Family Medicine, University of Health Sciences, Training and Research Hospital, Konya
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_553_17

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 > Abstract 


Objective: The aim of our study is to assess thiol-disulfide homeostasis (TDH), which is a biomarker of systemic oxidative stress, in breast cancer patients.
Materials and Methods: Thirty-seven breast cancer patients and 31 age-matched healthy volunteers were enrolled in this study. Serum native thiol, disulfide, and total thiol levels and disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios were analyzed using a novel colorimetric method.
Results: Serum native thiol level was statistically significantly lower in breast cancer patients (350.39 ± 7.15) than in healthy controls (380.60 ± 7.35) (P = 0.008). Serum disulfide level was statistically significantly higher in breast cancer patients (24.96 ± 0.85) than in healthy controls (19.25 ± 1.34) (P = 0.002).
Conclusion: To our knowledge, this study is the first study in the literature that investigated TDH in breast cancer patients. We have concluded that an alteration in TDH due to oxidative stress is likely to have a role in the pathogenesis of breast cancer.

Keywords: Breast cancer, oxidative stress, thiol-disulfide homeostasis


How to cite this article:
Eryilmaz MA, Kozanhan B, Solak I, Çetinkaya &D, Neselioglu S, Erel &. Thiol-disulfide homeostasis in breast cancer patients. J Can Res Ther 2019;15:1062-6

How to cite this URL:
Eryilmaz MA, Kozanhan B, Solak I, Çetinkaya &D, Neselioglu S, Erel &. Thiol-disulfide homeostasis in breast cancer patients. J Can Res Ther [serial online] 2019 [cited 2019 Nov 22];15:1062-6. Available from: http://www.cancerjournal.net/text.asp?2019/15/5/1062/244468




 > Introduction Top


Breast cancer is the most common type of cancer among women.[1] According to estimates from the World Health Organization, it has been reported that 508,000 women died of breast cancer in 2011.[2] Despite the high rate of incidence of breast cancer, early diagnosis and sufficient treatment are important for better prognosis and survival. One of the most frequently encountered mechanisms in the etiopathogenesis of breast cancer is oxidative stress.[3] In the organism, there is a balance between the rate of formation of free radicals and the removal rate of free radicals, and this is defined as the oxidative balance. Increased free radical formation or reduced antioxidant defense mechanism causes oxidative stress. Molecules known as reactive oxygen species (ROS)/metabolites, which are generated due to oxidative stress, act as an agent in cancer formation by damaging cell components such as lipids, proteins, and DNA.[4]

Thiol is an organic compound that consists of sulfur and hydrogen atoms attached to a carbon atom and plays a significant role in preventing oxidative stress formation in cells.[5] Functional sulfhydryl groups (-SH) in thiols act as substrates for antioxidant enzymes and as a free radical scavenger. The vast majority of the plasma thiol pool is composed of albumin and other plasma proteins, while a small portion comprises low-molecular-weight thiols such as cysteine, cysteinylglycine, glutathione, homocysteine, and γ-glutamylcysteine.[6] Under oxidative stress conditions, thiol groups converted into their reversible forms called disulfide bonds (–S–S–).[7] These disulfide bonds can be reduced again to thiol groups, and thereby dynamic thiol-disulfide homeostasis (TDH) is maintained.[8]

Recent studies have revealed that an alteration in TDH plays a role in the pathogenesis of various clinical conditions such as diabetes mellitus,[9] cardiovascular disease,[10] chronic renal disease,[11] and malignancy.[12] Therefore, the determination of dynamic TDH may give valuable information in different physiological, pathological, and biochemical processes. The one side (reductive thiol) of this reversible, dynamic, and active balance has been measured since 1979. However, the another side (reducible disulfide) can now be measured with a new method which has been recently developed by Erel and Neselioglu.[13] Thus, level of oxidizable functional thiol component, level of reducible disulfide, and ratios of each side to system can be evaluated. The easy, inexpensive, and practical method does not require any separation stage, and the tests are performed using a fully automated analyzer. Our study aimed to examine the relationship between TDH and breast cancer and to demonstrate the possible effect of TDH on the development of breast cancer. According to our information, our study is the first study on this subject in the literature.


 > Materials and Methods Top


Subjects

This prospective study was performed between June and November 2016 at Konya Education and Research Hospital, University of Health Sciences. A total of 68 participants, including 37 newly diagnosed breast cancer patients and 31 healthy controls, were enrolled in the study. Those who smoked cigarette and drank alcohol, who had a history of acute or chronic illness, who had an unbalanced diet (such as vegetarians), and who received antioxidant supplements were excluded in the study. The diagnosis of breast cancer was based on clinical and histopathologic examinations. Cancer staging was made according to the tumor, node, and metastasis staging system.[14] Moreover, the presence of hormone receptors (estrogen receptor [ER] and progesterone receptor [PR]), E-cadherin, HER2/c-erbB2, and Ki-67 as well as demographic data such as age and body mass index (BMI) were investigated.

Measurement of thiol-disulfide homeostasis parameters

After 8 h of fasting, venous blood samples were collected into ethylenediaminetetraacetic acid-containing tubes for TDH tests. Blood samples were centrifuged at 1500 rpm for 10 min. Serum and plasma were separated from the blood. Serum samples were stored at −80°C. Blood TDH was analyzed using a newly developed, fully automated, and colorimetric method developed by Erel and Neselioglu [13] as described previously. For this purpose, dynamic and reducible disulfide bonds (-S-S-) in serum samples were first reduced to functional thiol groups (-SH) using sodium borohydride (NaBH4) solution. Then, formaldehyde was added to remove the remaining unreacted reducing agent NaBH4. In the last step, all thiol groups were reacted with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). Disulfide level was calculated by the formula of serum total thiol − serum native thiol/2.

Ethics statement

For conducting the study, an approval was obtained from the University Research Ethics Board. We informed all participants about the study's design. Both oral and written informed consents were obtained from all participants. All procedures of the study were performed in accordance with the standards of the ethical board, under the 1964 Helsinki Declaration and its later amendments.

Statistical analysis

We used the Statistical Packages for Social Science (SPSS) version 22.0 (SPSS Inc., Chicago IL, USA) for statistical analyses. The normality of each variable was assessed with the Shapiro–Wilk test and Kolmogorov–Smirnov test. Chi-square test was used to examine the relationship between two categorical variables. Categorical variables were presented as number and percentages. The independent sample t-test was used to compare the differences in continuous variables between two independent groups if the variables were normally distributed. The Mann–Whitney U-test was used if the variables were not normally distributed. Pearson's or Spearman's correlation coefficient was used to examine the relationship between two continuous variables. Continuous variables were presented as mean ± standard error. P < 0.05 was considered statistically significant.


 > Results Top


In 37 breast cancer patients and 31 healthy controls, serum native thiol, disulfide, and total thiol levels and disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios were determined using a newly developed method. The breast cancer group consisted of 35 patients with invasive ductal cancer, 1 patient with invasive lobular cancer, and 1 patient with invasive papillary cancer. The cancer stages and the presence of ER, PR, E-cadherin, HER2/c-erbB2, and Ki-67 receptor are shown in [Table 1].
Table 1: Clinicopathological status of breast cancer patients

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There were no significant differences between breast cancer patients and healthy controls in terms of age, BMI, and albumin levels [Table 2]. The mean total thiol level of the breast cancer group was lower than that of the control group [Table 2]. The mean disulfide level of the breast cancer group was statistically significantly higher than that of the control group (P = 0.002) [Table 2]. The mean native thiol level of the breast cancer group was statistically significantly lower than that of the control group [Table 2]. In the breast cancer group, when compared with the control group, the disulfide/native thiol and disulfide/total thiol ratios were higher, but the native thiol/total thiol ratio was lower [Table 2]. The correlations between TDH parameters and other clinicopathological factors in the breast cancer patients are shown in [Table 3]. We could not find any relationship between clinicopathological factors and TDH parameters. The mean values of TDH parameters and hormone receptors are shown in [Table 4]. Serum total thiol level was statistically significantly lower in patients with positive PRs compared to patients with negative PRs.
Table 2: Characteristics and thiol-disulfide homeostasis parameters of two groups

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Table 3: Correlations between thiol-disulfide homeostasis and other clinicopathological factors in breast cancer patients

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Table 4: Mean values of thiol-disulfide homeostasis parameters and hormone receptors

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 > Discussion Top


Experimental and epidemiological studies indicate that free oxygen radicals play a role in the etiopathogenesis of cancer development. ROS are synthesized through different metabolic pathways, including aerobic respiration that occurs in the mitochondria.[15] ROS play an important role as a second messenger at low concentrations in the regulation of many cellular functions and enzymatic reactions. Thus, they can increase cell survival and stimulate cell proliferation. However, when ROS rise above physiological levels, they lead to dysregulation and disrupted redox homeostasis in the organism's cellular defense system, resulting in uncontrolled cell growth and cancerogenesis process. They may also cause genomic instability and cancer formation.[16],[17]

Redox homeostasis is controlled by oxidizing and reducing free radicals and thiol-containing proteins in the cell. ROS produce a reversible disulfide bond (RSSR) by forming an oxidation reaction with the sulfur (-SH) group in the thiols (RSH).[7],[18] Dynamic TDH is maintained through these reactions at the cellular level.[8] Dynamic TDH has major roles in antioxidant defense, modulation of enzyme activity, apoptosis, detoxification, cellular signaling mechanisms, and immune response in the organism.[19] Thiol oxidation and disulfide bond formation, which are a dynamic and reversible process, are an early cellular response to oxidative stress.[20],[21] There are some studies showing that this dynamic homeostasis is altered in cardiovascular diseases and cancers where the effect of oxidative stress is prominent.[10],[12] Moreover, these studies have demonstrated that this redox reaction is critical for monitoring intracellular oxidation state. In this study, oxidative stress state and TDH in breast cancer patients were analyzed using a new fully automatic method. The fact that serum native thiol level was significantly reduced, serum disulfide level was significantly increased, and thiols were oxidized to disulfides supports that oxidative stress has a possible role in the etiopathogenesis of breast cancer.

In a recent study investigating thiol levels in platelets in breast cancer patients, free thiol level was found significantly lower in both benign and malign groups than in control group. The researchers conducting this study have suggested that the change in the level of thiol groups in plasma proteins of breast cancer patients may be a consequence of the nitration process induced by ROS such as peroxynitrite.[22] The same researchers investigated in vitro effects of Aronia melanocarpa extract on the amount of low-molecular-weight thiols and the activity of antioxidative enzymes in plasma was obtained from patients with invasive breast cancer during different phases of treatment and compared them with healthy subjects. They found that that low-molecular-weight thiol levels and total thiol levels were lower in breast cancer patients than in control group.[23] Glutathione, in both reduced (GSH) and oxidized (GSSG) states, constitutes the major part of low-molecular-weight thiols in the human redox system and plays a key role in the cellular response to oxidative damage.[19] Reduced glutathione concentration may cause oxidative stress in breast cancer patients.[24] Therefore, an increase in the amount of oxidized glutathione in the cells and in the tissues may be indicative of oxidative stress. Various oxidants can be measured separately in laboratories. However, this measurement requires expensive, complex, and time-consuming techniques.[25]

Although thiol biochemistry is a promptly developing area in basic and applied biosciences in recent years, no any significant colorimetric method has been developed in this field except for a method developed by Ellman and Lysko in 1979 where -SH groups are measured using DTNB.[26] In this study, we evaluated serum dynamic TDH in breast cancer patients using easy and practical spectrophotometric method developed by Erel and Neselioglu.[13] Similar to recent studies, we found that both serum total thiol and native thiol levels were lower in the breast cancer group than in the control group. Serum disulfide levels were also increased and so TDH was shifted to the right side. We also calculated the three ratios (disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol) to further evaluate TDH state. The disulfide/native thiol and disulfide/total thiol ratios were increased, and the redox equilibrium was shifted toward disulfide bond formation. The data of this study have suggested that the change in TDH state in breast cancer patients may occur due to ROS-induced oxidation/reduction reactions.

The existence of ER and PR in breast tumor tissue is a demonstration of the development and progression of breast cancer and guides us to determine patients with lower relapse risk and better survival rate.[27] ER-positive breast cancers have an improved prognosis and are more prone to respond to hormonal therapy compared to ER-negative breast cancers. Moreover, the existence of PR and ER increases the likelihood of clinical response to hormonal therapy. However, oxidative stress can alter the arrangement and function of ER and PR and thus may affect the biology and clinical consequence of ER-positive breast cancers.[28] We could not found a relationship between clinicopathological factors and TDH parameters. However, we found that serum total thiol level was lower in patients with positive PRs compared to patients with negative PRs. Further studies are needed in the field of thiol and hormone receptors due to possible role of receptors in breast cancer prevention and treatment outcomes.


 > Conclusion Top


In this study, we found that some of TDH parameters differed statistically significantly between breast cancer patients and healthy controls. This suggests that oxidative stress may play a role in the etiopathogenesis of the disease. Since breast cancer is a very heterogeneous disease, further studies with a larger sample size which are performed by dividing it into homogeneous subgroups would become more enlightened on this regard.

Acknowledgment

Assistance provided by Ahmet Pekgor with the statistics used in this report was greatly appreciated.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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