Sheremet, Shidlovskyy, Sydorchuk, and Sydorchuk: Caspase-3 and Caspase-8 in Patients with Nodular Goiter and Autoimmune Thyroiditis Caspase-3 and Caspase-8 in Patients with Nodular Goiter and Autoimmune Thyroiditis



Problem statement and analysis of the recent research

Cells are exposed to many damaging factors of endogenous and exogenous nature in the process of their vital activity. Various toxic influences or metabolic disorders undoubtedly lead to the development of oxidative stress, and in this case the future of the cell is determined by a balance of various adaptive metabolic processes induced by a pathological factor as well as by genetic and constitutional features of its biochemical systems [1-5]. One of cell responses in case of not only the toxic effects action, but also imbalance of growth factors, hormones, cytokines, when DNA is damaged, other structural elements of the cell or when the cell cycle course is interrupted, is activation of the genetic program of the cell death, that is apoptosis [6-15].

According to the literature, the earliest stage of apoptosis is oxidation of proteins and cell membranes, which is influenced by an excessive production of reactive oxygen species (ROS) [3, 5]. The processes that allow the cell to adapt to the negative impacts and determine the possibility of its further existence or death, are believed to depend on the characteristics of the mechanisms of induction and regulation of apoptosis and its implementation [10].

Mechanisms of apoptotic death in autoimmune thyroid diseases have been a subject of an active study for the last decades [8, 10-14].

Intensity of studies on apoptosis in recent years has been connected with a number of circumstances. First of all, methodological possibility of registering various manifestations of apoptosis and analysis of its molecular mechanisms are closely related to mechanisms other current events (for example, activation of peroxidation) [1-4]. In addition, the study of apoptosis has been very productive for understanding a number of important processes, including immune homeostasis and oncogenesis [14]. Finally, due to apoptosis, it became necessary to reconsider a number of conceptual bases of pathophysiology [10].

Nowadays, a number of mechanisms of thyrocytes apoptosis induction have been found. Normally, apoptosis occurs in 2 major ways: internal way, mediated by mitochondria, resulting from activation of caspase 9, while the outer way is mediated by activation of the Fas-receptors (CD95) and includes the activation of caspase 8 [10, 14]. Both ways converge to the activation of effectors’ caspases, executors of apoptosis (caspase 3, 6, 7) leading to DNA degradation and cell death [15, 16].

Proteins whose degradation causes irreversible changes in the cell are targeted by effectors caspase [10-12]. Caspases action is specific: under their influence only certain proteins degrade to fragments of a certain length [15]. Therefore, caspases make up a central component of apoptosis program, their activation leads to the final stage of the cell death, namely, to DNA fragmentation and degradation of structural proteins of the cytoskeleton and cell membranes as well as to inactivation of other proteins ensuring normal functioning of the cells [16]. The appearance of such protein fragments serves as a biochemical marker of apoptosis [14-16]. However, the literature does not give any serious data on the relationship of lipid peroxidation processes and activation of caspase cascade in patients with NGAIT.

The objective of the research was to study pro- and anti-oxidative activity, caspase-dependent mechanisms of apoptosis’ induction in the thyroid tissue of patients suffering from autoimmune thyroiditis.

Materials and methods

75 women complaining of discomfort in the neck were examined during 2013-2015. We evaluated the hormonal status (with thyroid stimulating hormone (TSH), free T4 thyroxin, free  triiodothyronine T3) levels of antibodies to thyroglobulin (AB-TG) and to thyroid peroxidase (AB-TPO), the volume and structure of the thyroid gland (TG) according to ultrasound examination.

25 women (group I) were diagnosed with thyroid adenoma after the surgery according to the ultrasound, fine-needle aspiration biopsy (FNAB) and histological findings. We identified this group due to the fact that this pathology is one of the most common forms of nodular goiter.

50 women were diagnosed with NGAIT (group II). The indications for the surgery in this group were: enlargement of the thyroid gland with symptoms of compression and narrowing of the trachea and esophagus; some nodes compressed organs of the neck; progressive growth of goiter, despite ongoing conservative therapy for 1.5 years; suspected malignant degeneration, based on FNAB findings.

The study did not involve the patients with hyperthyroidism, those manifesting hypothyroidism, hypertension and cardio-vascular diseases, as well as with severe somatic pathology and after the onset of menopause.

The patients of groups I and II were comparable in age (34.2 ± 10.33 and 38.0 ± 10.62 years respectively, p = 0.12), anthropometric data (body mass index - BMI under 23.5 ± 2.71 and 24.3 ± 4.88 kg/m2, p = 0.43) and the level of free T3 (4.4 ± 0.91 and 4.4 ± 0.93 ng / L, p = 0.93) but differed in terms of free T4 (16.6 ± 2.02 and 12.9 ± 3.42 mmol / L, p<0.0001), TSH (1.9 ± 0.76 and 4.93 ± 51 mU / L, p<0.0001) and AT-TPO (11.9 ± 13.92 and 255.7 ± 340.58 mU / L, p = 0.0009). In general, the differences between the groups were naturally determined and confirmed autoimmune destruction and tendency towards depression of the function on the background of NGAIT in patients from group II.

All the patients underwent surgery. Surgical interventions ranged from hemithyroidectomy to thyroidectomy. After the intervention the tissue was taken in the operating room no later than 30 minutes after the operation. In patients of group I we isolated separately macroscopically unchanged (paranodular) tissue, which served as the control one for both comparison groups, and adenomatous tissue. In patients with NGAIT we took the tissue from the left, right lobes and from the isthmus. The pieces of tissue weighing 100-300 mg were transported to the laboratory on ice and immediately cut into 4-6 pieces weighing an average of 50-70 mg each. After the partition they were closed in a special plastic container and stored at -70°C before basic research with them.

In addition, we investigated pro- and antioxidant activity in 5% of thyroid tissue homogenates determining the activity of glutathione peroxidase (GP mmol / min • g tissue), Glutathione-S-transferase (GST, umol / min • g tissue) and degree of oxidative modification of proteins (OMP, optical density unit / g protein) by means of accepted methods.

In order to study the activity of caspases 3 and 8 thyroid tissue was crushed in homogenizer “WiseTis” HG-15 series (“Daihan Scientific”, South Korea) with 8mm rotor at a speed of 4500 rev / min. For this purpose, the isolating medium (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT) was used to which a cocktail of protease inhibitors (104 mM AEBSF, 0.08 mM aprotinin, 1.5 A pepstatin mM, 2 mM leptin 4 bestatin mM, 1.4 mM E-64) was added at a ratio of 100: 1 (all reagents were manufactured by “Sigma”, USA). The homogenates were centrifuged at microcentrifuge “Heraeus fresco 17” (“Thermo Electron LED GmbH”, Germany) at 1500 revolutions for 30 minutes at a temperature of +4°C. The resulting supernatant was used to assess the activity of caspase-3 and caspase-8. Specific activity of the effectors caspase-3 and initiator caspase-8 in the tissue was studied using colorimetric method with enzyme-linked immunosorbent (ELISA) “Sanrise ™ -Tecan” (Austria), at a wavelength of 405 nm, with the speed of splitting the synthetic substrate N-Acetyl-Asp-Glu -Val-Asp-nitroanilin (Ac-DEVD-NHA) and N-Acetyl-Ili-Glu-Asp-Tre-nitroaniline (Al-IETD-PNA) respectively. All reagents used in this study were produced by the company “Sigma” (USA). Caspase activity was assessed in (mmol of paranitro-aniline/ [h • mg of protein]).

Results and discussion

The study of peroxidation processes in the thyroid tissue established that there was a significant increase in OMP parameters in the modified tissue at the same time, the activity of antioxidant enzymes (AOE) was significantly reduced, especially in patients with NGAIT (Fig. 1).

Fig. 1.
gmj-23-gmj.2016.3.50-g1.jpg

For instance, the activity of GP in patients of group I decreased by almost 15% compared to the paranodular tissue and by 18% in patients of group II. ST-T level decreased by 49.5% in patients of group I and by 56.8% in patients of group II. POM degree was 24% higher in patients of group I and by 33.4% in those of group II.

In the course of the study caspase-3 activity in the tissue having signs of NGAIT was found to be twice higher than in the unchanged thyroid tissue (Fig. 2) indicating the activation of caspase-dependent way of apoptosis under these conditions. In this case, the activity of caspase-8 increased significantly compared to both that of intact thyroid tissue and to patients of group I.

Fig. 2.
gmj-23-gmj.2016.3.50-g2.jpg

Such imbalance between the activity of peroxidation processes and antioxidant defense systems created the conditions for damaging action of peroxidation processes on the thyroid structures and for ROS impact on pro- and antiapoptic targets and mechanisms directly or indirectly through the intracellular redox-dependent signal-conveying systems. We consider these structures to be the elements of the thyrocytes, namely cell membrane, intracellular structures, which cause launching of apoptotic signal, the indication of which is likely activation of both initiator and effectors caspases.

Additionally, several molecular paths interacting with each other can be activated in the cell. The findings of different sources confirm the role of antiapoptotic protein Bcl-2 under the condition of NGAIT development and this protein is among the main factors in the regulation of apoptotic function of mitochondria [10-13].

Oxidative stress leads to the formation of cell membranes of oxidated lipids which are also apoptotic factors. Caspase oxidative modification (including caspases-3), which are sensitive to cells redox status, depending on the type and location of such changes may cause their activation.

Considering this, as well as the findings obtained while performing the work we can assume that basic mechanisms of apoptosis are triggered in patients with NGAIT due to excessive activation of peroxidation aimed at attracting external receptor mechanism of initiation and increased activity of caspase-3, and can also occur as a result of caspase-8, the indicative of which is probable high increase in the activity of this index both as compared to that in unchanged tissue in the thyroid gland and in patients with thyroid adenoma.

The research of caspase signaling pathways in apoptosis of thyroid cells has recently started and requires further study. Discovery of physiological regulators of apoptosis in caspase activity shows the inexhaustible possibilities of cells to maintain homeostasis and the natural end of life cycle. Tracking ways causing cell death may contribute to the development of new approaches to the prevention and treatment of autoimmune thyroid disease.

Conclusions

  1. Processes of protein peroxidation are activated and the systems of antioxidant defense become weaker in the thyroid tissue of patients with autoimmune thyroiditis (NGAIT).

  2. Induction of thyrocytes apoptosis in patients with NGAIT on external mechanism is associated with increased activity of caspase-8, which significantly predominates in patients with thyroid adenoma and in practically healthy people by 56.18% and 49.46%, with an implementation through effectors caspase-3 the activity of which grew almost twice.

Prospects for further research

Detection of pathogenic factors and mechanisms of apoptosis deregulation in case of NGAIT will allow determining the additional causes of their onset and formulate pathogenetically grounded methods of immunopathological changes correction.

References

1 

J Ai, JM Leonhardt, WR Heymann. Autoimmune thyroid diseases: Etiology, pathogenesis, and dermatologic manifestations. J Am Acad Dermatol. 2003;48:641-659. doi:10.1067/mjd.2003.257

2 

CL Burek. Autoimmune thyroiditis and ROS. Autoimmun Rev. 2008;7 (7):530-537. doi:10.1016/j.autrev.2008.04.006

3 

JT Hancock, R Desikan, SJ Neill. Role of reactive oxygen species in cell sig-naling pathways. Biochem Soc Trans. 2001;29:345-350. doi:10.1042/bst0290345

4 

H Erdamar, H Demirci, H Yaman, et al. The effect of hypothyroidism, hyperthyroidism, and their treatment on parameters of oxidative stress and antioxidant status. Clin Chem Lab Med. 2008;46(7):1004-1010. doi:10.1515/CCLM.2008.183

5 

HM Shen, CF Yang, WX Ding, et al. Superoxide radical-initiated apoptotic signaling pathway in selenite-treated HepG(2) cell: mitochondria serve as main target. Free Radic Biol Med. 2001;30:9-21. doi:10.1016/S0891-5849(00)00421-4

6 

LP Sydorchuk, AR Sydorchuk, K Amosova, et al. Caspases and TNF-alfa in hypertension: response to combination therapy and genes mutations. J Hypertension. 2012;30(е-Suppl):367.

7 

SA Levitska, LP Sydorchuk, VV Kostenko. C-511T polymorphism of interleukin 1β gene in patients with chronic inflammation of the sinuses. Buk Med Herald. 2011;3(59):51-54.

8 

JD Bretz, JR Baker. Apoptosis and autoimmune thyroid disease: following a TRAIL to thyroid destruction?. Clin Endocrinol. 2001;55(1):1-11. doi:10.1046/j.1365-2265.2001.01345.x

9 

JD Bretz, M Rymaszeweski. TRAIL death pathway expression and induction in thyroid follicular cells. J Biolog Chemistr. 1999;274:23627-23632. doi:10.1074/jbc.274.33.23627

10 

N Mitsiades, V Poulaki, V Kotoula, et al. Fas/Fas ligand up-regulation and bcl-2 down regulation may be significant in the pathogenesis of Hashimoto's thyroiditis. J Clin Endocrinol Metab. 1998;83:2199-2203. doi:10.1210/jcem.83.6.4853

11 

K Łącka, M Maciejewski. Współczesne poglądy na temat etiopatogenezy autoimmunologicznego zapalenia tarczycy, (chvoroby Hashimoto). Pol Merk Lek. 2011;30(176):132-138.

12 

K Łącka, A Maciejewski. Rola procesu apoptozy w etiopatogenezie autoimmunologicz-nego zapalenia tarczycy. Pol Merk Lek. 2012;32 (188):87-92.

13 

C Giordano, G Stassi, Maria R De. Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto's thyroiditis. Science. 1997;275:960-963.

14 

J Yanagisawa, M Takahashi, H Kanki, et al. The molecular interaction of Fas and FAP-1: a tripeptide blocker of human Fas interaction with FAP-1 promotes Fas-induced apoptosis. J Biol Chem. 1997;272:8539-8545. doi:10.1074/jbc.272.13.8539

15 

F Vendrame, M Segni, D Grassetti, et al. Impaired Caspase-3 Expression by Peripheral T Cells in Chronic Autoimmune Thyroiditis and in Autoimmune Polyendocrine Syndrome-2. J Clin Endocrinol Metab. 2006;91(12):5064-5068. doi:10.1210/jc.2006-1358

16 

M Nakahara, Y Nagayama, O Saitoh, et al. Expression of Immunoregulatory Molecules by Thyrocytes Protects Nonobese Diabetic-H2h4 Mice from Developing Autoimmune Thyroiditis. Endocrinol. 2009;150:1545-1551. doi:10.1210/en.2008-0702



Copyright (c) 2017 M. I. Sheremet, V. O. Shidlovskyy, L. P. Sydorchuk, R. I. Sydorchuk

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