Evaluation of Dynamic Serum Thiol-Disulphide Homeostasis in Colorectal Cancer

Introduction

Colorectal cancer (CRC) ranks as the third most common cancer type globally and is a leading cause of cancer-related deaths. In the United States of America (USA) alone, an estimated 101,420 new cases of CRC and 51,020 CRC-related deaths were reported in 2019. The development of colon cancer is influenced by various factors, including genetic predisposition, smoking, high-fat diets, obesity, and reactive oxidative stress (ROS).

Under normal physiological conditions, there is a delicate balance between oxygen free radicals and antioxidant defense systems. However, when this balance is disrupted, oxidative stress can occur. While low levels of ROS have beneficial effects on several physiological processes, high levels can lead to oxidative tissue damage and other harmful effects. Many exogenous and endogenous factors, such as inflammation, ischemia, infection, ultraviolet radiation, drugs, alcohol, and smoking, can increase ROS production.

Thiols are crucial and potent antioxidant molecules containing hydrogen and sulfhydryl groups that play a vital role in neutralizing ROS. They can undergo oxidation reactions and form disulfide bonds, which can later be reduced back to thiol groups, maintaining dynamic thiol-disulfide homeostasis. In physiological conditions, the thiol/disulfide equilibrium remains balanced. Dynamic thiol-disulfide homeostasis plays a crucial role in several functions, including apoptosis, protein oxidation, antioxidant defense, and cellular signal transduction mechanisms. There is growing evidence suggesting that an abnormal thiol-disulfide homeostasis is associated with the pathogenesis of several benign and malignant conditions.

In the past, oxidative and antioxidant status was evaluated using various indirect methods. However, nowadays, direct measurements of oxidant and antioxidant status are possible. A novel automated and spectrophotometric method was developed by Erel and Neselioglu to measure the two-sided dynamic thiol-disulfide homeostasis.

Despite the importance of thiol-disulfide homeostasis in various health conditions, there have been no clinical trials evaluating its relationship with CRC using the new method developed by Erel and Neselioglu. Therefore, the aim of this study was to compare dynamic thiol/disulfide homeostasis between patients with CRC and healthy individuals and investigate thiol and disulfide levels according to tumor stages and localization.

Material and Methods

Study Subjects

The study prospectively analyzed patients diagnosed with CRC at Ankara Yildirim Beyazit University between 2015 and 2017, who had not received any treatment. Healthy subjects were included as a control cohort. Inclusion criteria required participants to be over 18 years old and diagnosed with CRC through pathological examination of colonoscopic biopsy, tru-cut needle biopsy from metastatic lesions, or surgically resected specimens. Patients with renal or liver disease, diabetes, and active inflammatory or infectious diseases were excluded.

Blood Samples Collection and Thiol-Disulfide Homeostasis

Blood samples for thiol-disulfide homeostasis analyses were collected from patients at the time of diagnosis. Serum samples were separated from the collected blood and stored at -80°C until analysis. Thiol/disulfide homeostasis tests were measured using the novel automated and spectrophotometric method developed by Erel and Neselioglu.

Statistical Analysis

Statistical analysis was performed using SPSS 17.0. Differences in categorical factors were determined with Fisher's exact test, and differences in continuous values were assessed using Student's t-test for normally distributed variables and non-parametric Mann-Whitney U tests for non-normally distributed variables. Correlation coefficients were calculated using the Pearson test for normally distributed parameters and the Spearman test for non-normally or ordinal variables. A P value < 0.05 was considered statistically significant.

Results

A total of 198 cases, including 88 patients with CRC and 110 healthy subjects, were enrolled in the study. The median age in the patient and control arms was 60.5 and 59.5 years, respectively. The patient cohort consisted of 64.8% males and 35.2% females, while the control cohort included 57.3% males and 43.7% females. There were no significant differences in sex and age between the patient and control groups. Within the patient arm, there were no significant differences in baseline characteristics, including sex, age, and tumor stage, between right-sided and left-sided tumors.

Comparison of NT, TT, and disulfide levels between the patient and control groups showed that TT, NT, and disulfide levels were significantly lower in the patient group than in the control group. However, the NT/disulfide ratio, reflecting the thiol-disulfide balance, did not significantly differ between the two groups.

In the patient group, the study also investigated NT, TT, and disulfide levels according to tumor stages. Although NT, TT, and disulfide levels showed a decreasing trend as tumor stage progressed from 1 to 4, the differences were not statistically significant. Detailed data are presented in figure 2. Additionally, the disulfide/native thiol ratio did not significantly differ between tumor stages.

When the patient group was divided into stage 4 disease and early stages (stages 1, 2, and 3), NT, TT, and disulfide levels tended to be lower in stage 4 disease compared to early stages, but the differences were not statistically significant.

In the analysis of NT, TT, disulfide levels, and disulfide/NT ratio according to tumor localization in the patient group, NT and TT levels did not significantly differ between right-sided and left-sided tumors. However, disulfide levels were significantly higher in left-sided tumors compared to right-sided tumors. This finding indicates that the balance of dynamic thiol-disulfide homeostasis is disrupted in favor of disulfide in left-sided tumors.

Discussion and Conclusion

This study represents the first investigation of thiol-disulfide homeostasis, which reflects the two-sided oxidant/antioxidant system, in CRC. The results suggest that thiol-disulfide homeostasis might play an important role in the pathogenesis of CRC. The findings indicate that oxidative stress was increased, but total antioxidant capacity was decreased in CRC patients compared to healthy individuals. However, the balance of thiol-disulfide remained maintained between the two arms.

Our study showed that NT, TT, and disulfide levels tended to decrease as tumor stage progressed from 1 to 4, although the differences were not statistically significant. Further studies with larger patient populations are needed to validate these results.

Moreover, we observed that disulfide levels were significantly lower in right-sided tumors compared to left-sided tumors. This finding suggests that there might be distinct features between right-sided and left-sided colon tumors, such as differences in inflammatory and immunogenic features or high tumor turnover. Further investigation is required to clarify this matter.

In conclusion, thiol-disulfide homeostasis might be a crucial factor in the pathogenesis of CRC. This study sheds light on the two-sided oxidant/antioxidant system in CRC, providing valuable insights for future research. However, the limitations of this study, such as the relatively small patient population and the lack of comparison with other oxidative stress parameters, should be addressed in further investigations.

References

1. Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Piñeros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941-53.
2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69(1):7-34.
3. Özdemir N, Başol Tekin S, Kılıçturgay S, Şanlı S, Türker T, Erel Ö. Dynamic serum thiol/disulphide homeostasis and oxidative stress in colorectal cancer patients. Eur J Clin Invest. 2019;49(2):e13042.
4. Erel Ö, Neselioglu S. A novel and automated assay for thiol/disulphide homeostasis. Clin Biochem. 2014;47(18):326-32.
5. Lushchak VI. Free radicals, reactive oxygen species, oxidative stress and its classification. Chem Biol Interact. 2014;224:164-75.
6. Sies H, Jones DP. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol. 2020;21(7):363-83.
7. Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996;273(5271):59-63.
8. Halliwell B, Gutteridge JMC. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 1990;186:1-85.
9. McCord JM. Human disease, free radicals, and the oxidant/antioxidant balance. Clin Biochem. 1993;26(5):351-7.
10. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39(1):44-84.