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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 15  |  Issue : 2  |  Page : 85-91

Bcl-2, active caspase-9 and inactive caspase-9 levels as markers of apoptosis in the serum and tissue of vitiligo patients


1 Department of Dermatology, Venereology and Andrology, Faculty of Medicine, Assiut University, Assuit, Egypt
2 Department of Pathology, Faculty of Medicine, Assiut University, Assuit, Egypt
3 Department of Biochemistry, Faculty of Medicine, Assiut University, Assuit, Egypt

Date of Submission21-May-2017
Date of Acceptance03-Jul-2017
Date of Web Publication21-Nov-2017

Correspondence Address:
Ayman M.M Mahran
Department of Dermatology, Venereology and Andrology, Assiut University Hospital, Assiut, 71515
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AZMJ.AZMJ_27_17

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  Abstract 


Background Vitiligo is the most common depigmentation disorder of the skin. It is considered a multifactorial disorder. However, its exact etiology is unknown. As the acute inflammatory response is not a general finding in vitiligo, it was hypothesized that melanocytes may die in a controlled manner via apoptosis.
Aim This study was done to evaluate Bcl-2, active caspase-9, and inactive caspase-9 levels in vitiligo in order to establish the role of apoptosis in the etiopathogenesis of vitiligo.
Patients and methods We examined Bcl-2 (serum and tissue), active caspase-9 (serum) and inactive caspase-9 (tissue) levels of 48 vitiligo patients and 30 healthy volunteers by ELISA using commercial kits and immunohistochemically using the avidin–biotin immunoperoxidase complex technique. The collected data were analyzed by SPSS version 16.
Results The mean age of vitiligo patients was 32.6±12.4 years. Of these, 65% of patients were women and 33% of patients have a positive family history. The mean vitiligo area scoring index was 5.84±6.57. Bcl-2 levels in the serum and tissues of patients were significantly lower than in controls. Bcl-2 expression in lesional skin was significantly lower than in the perilesional and unaffected skin. On the other hand, active caspase-9 level in patients’ serum was significantly higher than in controls. Inactive caspase-9 expression in lesional skin was significantly lower than in controls. Inactive caspase-9 expression in lesional skin was insignificantly lower than in the perilesional and unaffected skin.
Conclusion Our data strongly support apoptosis rather than necrosis as a mechanism for melanocytes’ destruction in vitiligo.

Keywords: active caspase-9, apoptosis, Bcl-2, inactive caspase-9, vitiligo


How to cite this article:
Hofny ER, Hasan HI, Mohamed NA, Bamatraf MS, Mahran AM. Bcl-2, active caspase-9 and inactive caspase-9 levels as markers of apoptosis in the serum and tissue of vitiligo patients. Al-Azhar Assiut Med J 2017;15:85-91

How to cite this URL:
Hofny ER, Hasan HI, Mohamed NA, Bamatraf MS, Mahran AM. Bcl-2, active caspase-9 and inactive caspase-9 levels as markers of apoptosis in the serum and tissue of vitiligo patients. Al-Azhar Assiut Med J [serial online] 2017 [cited 2018 Dec 17];15:85-91. Available from: http://www.azmj.eg.net/text.asp?2017/15/2/85/218852




  Introduction Top


Vitiligo is a chronic pigmentary disorder of the skin and mucous membranes, which is characterized by persistent well-circumscribed, depigmented macules, and patches caused by selective melanocytes, destruction [1],[2],[3]. It is a multifactorial disorder related to both genetic and nongenetic factors [4]. The published prevalence of vitiligo is 0.5–1% [5]. All races and both sexes are affected [6].

The one and the only way to understand the etiology is to determine the mechanism by which melanocytes are destroyed. Two known mechanisms for this cell loss are recognized, necrosis and apoptosis [7]. The histological data, and some laboratory data, support apoptosis, rather than necrosis in the etiopathogenesis of melanocytes’ destruction in vitiligo [8].

The induction and execution of apoptosis are complex but well-controlled processes. The Bcl-2 family of proteins constitutes one of the most relevant classes of apoptosis-modifying gene products. Bcl-2 can protect any cell from a wide range of apoptotic stimuli [9] and its importance in melanocytes’ survival is emphasized by the accelerated disappearance of melanocytes in Bcl-2-deficient mice [10],[11].

Normally, exposure of cells to ultraviolet radiation results in increased cell death through apoptosis by increasing the activities of both the mitochondrial-derived intrinsic and plasma membrane-derived extrinsic apoptotic pathways [12]. The caspases family has been found to have an important role in this process that leads to organized destruction of the cell by a restricted cleavage of a variety of crucial cellular substrates [13].

It was reported that infiltrating T-cells and macrophages can be observed adjacent to the remaining perilesional melanocytes in generalized vitiligo and it was shown that cytotoxic T-cells and macrophages are both capable of inducing apoptosis of melanocytes in vitro. Therefore, it is feasible that in-vivo melanocytes’ apoptosis in vitiligo can be induced by autoreactive T-cell and macrophages [14].

Previous reports stated that the melanocytic cell death in vitiligo, particularly the generalized type, can occur in the absence of neutrophil influx and erythema, suggesting apoptosis. Apoptosis is induced when the antiapoptotic factors are overwhelmed by lethal stimuli or by loss of their natural defenses [7],[15].


  Aim Top


To evaluate the Bcl-2 (serum and tissue), active caspase-9 (serum), and inactive caspase-9 (tissue) levels as markers of apoptosis in vitiligo patients upper Egypt.


  Patients and methods Top


Patients

Forty-eight (31 women, 17 men) patients with vitiligo and 30 (17 women, 13 men) age-matched and sex-matched healthy volunteers participated in this study from the Dermatology Department in Assiut University Hospitals, Assiut, Egypt.

The study was approved by the research ethics committee for experimental and clinical studies at Faculty of Medicine, Assiut University, Assiut, Egypt. Informed consent was obtained from all participants.

Exclusion criteria

  1. Patients and controls less than or equal to 16 and more than or equal to 55 years old.
  2. Patients on topical or systemic medications during the last month.
  3. Patients and controls with concomitant systemic or dermatological disease.
  4. Pregnant women.
  5. Smokers.


Methods

All study participants were subjected to complete history taking plus general and dermatological examinations.

The total body vitiligo area scoring index (VASI) was calculated using Hamzavi et al. [16] formula; VASI=€ (hand units in all body sites)×residual depigmentation.

The notion of stable disease is subject to interpretation, and the clinical definition of ‘stability’ for many authors varies greatly. In our study, we identified the stability according to the Vitiligo Global Issues Consensus Conference Statements (2012) [3]. Therefore, we identified any patient who showed neither extension of existing lesions nor occurrence of new lesions within the last year as a stable case.

Venous blood samples

Six millilitre of venous blood was collected in plain tubes by venipuncture, after a rest of 30 min in sitting position by a 25 G needle through the antecubital vein. Samples were then centrifuged at 1500 rpm for 15 min at room temperature. Sera were divided into 1.5 ml aliquots using sterile plastic transfer pipettes and frozen at −70°C until future analysis.

Skin biopsies

Three 5 mm punch biopsies were taken from each patient after local anesthesia from the lesional, perilesional, and unaffected skin. The perilesional biopsies were taken after carefully marking with ink the pigmented side bordering the vitiliginous area to facilitate the precise cutting of biopsies, resulting in sections that contain both lesional and nonlesional skin. The biopsy from unaffected skin was taken 10 cm away from the lesion. Each biopsy was fixed in 10% formalin. Paraffin-embedded tissues were made from the specimens. A 5 µm section of the paraffin-embedded tissue was mounted on coated glass slides and processed for immunohistochemical evaluations.

Measurement of Bcl-2 and active caspase-9 in serum

Bcl-2

Bcl-2 was measured using Human Bcl-2 ELISA kit (catalog number KN2069Hu; Kono Biotech Co. Ltd, Jiaxing, Zhejiang, China). The kit uses a double-antibody sandwich ELISA to assay the level of Bcl-2 in samples. Standard, test sample, and HRP-labeled Bcl-2 antibodies were added to enzyme wells that were precoated with the Bcl-2 antibody, and then incubation and washing were done to remove the uncombined enzyme. Upon adding chromogen solution A and B, the color of the liquid changed to blue, and the reaction with the acid caused the color to become yellow. The depth of color and the concentration of the Bcl-2 sample were positively correlated. The measurement unit of serum Bcl-2 is ng/ml.

Active caspase-9

Active caspase-9 was measured using Human Caspase-9 ELISA kit (catalog number KN2070Hu; Kono Biotech Co. Ltd). The kit uses a double-antibody sandwich ELISA to assay the level of active caspase-9 in samples. Standard, test sample, and HRP-labeled caspase-9 antibodies were added to enzyme wells that were precoated with the caspase-9 antibody, and then incubation and washing were done to remove the uncombined enzyme. Upon adding chromogen solutions A and B, the color of the liquid changed to blue, and the reaction with the acid caused the color to become yellow. The depth of color and the concentration of the caspase-9 sample were positively correlated. The measurement unit of serum caspase-9 is pg/ml.

Measurement of Bcl-2 and inactive caspase-9 in tissue

A panel of Bcl-2 and inactive caspases-9 proteins was analyzed by immunohistochemical staining using the avidin–biotin immunoperoxidase complex technique (Thermo Scientific Corporation, Fairmont, California, USA). Any endogenous peroxidase activity was quenched by incubating the specimen for 5–10 min with dual endogenous enzyme block. The specimen was then incubated with an appropriately characterized and diluted mouse or rabbit primary antibody, followed by incubation with the labeled polymer using 30 min incubation for each. Staining was completed by 5–10 min incubation with 3,3′-diaminobenzidine substrate chromogen, which results in a brown-colored precipitate at the antigen site.

Statistical analysis

The data were tested for normality using the Anderson–Darling test and for homogeneity variances prior to further statistical analysis. Categorical variables were described as number and percent, where continuous variables were described as the mean±SD. χ2-Test and Fisher’s exact test were used to compare between categorical variables. But, comparisons between continuous variables were done by t-test and analysis of variance. The Pearson correlation coefficient (r) was used to assess the association between continuous variables. A P value of less than 0.05 was considered statistically significant. All analyses were performed with the IBM SPSS 20.0 software (Armonk, New York, USA).


  Results Top


Characteristics of the study population

The study included 48 vitiligo patients and 30 age-matched and sex-matched healthy volunteers. The mean age of vitiligo patients was 32.6±12.4 years and of control cases was 31.9±7.13. Sixty-five percent of patients were women. The mean duration of the disease was 8.21±6.49 years. Thirty-three percent of patients have a positive family history. Vitiligo was unstable in 71% of patients. The most frequent subtype was generalized vitiligo in 60% of patients, followed by the acrofacial type in 40%. The mean of VASI was 5.84±6.57 ([Table 1]).
Table 1 Demographic and clinical data of patients

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Bcl-2

The Bcl-2 level was insignificantly lower in the serum of patients (1.61±0.32) than controls (1.87±1.08) ([Table 2]). However, the Bcl-2 expression in lesional skin of patients (0.2±0.4) was significantly lower (P<0.001) than in the skin of controls (2.6±0.5) ([Table 2]).
Table 2 Levels of Bcl-2 (serum and tissues), active caspase-9 (serum), and inactive caspase-9 (tissues) in patients and controls

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In patients, the expression of Bcl-2 in lesional skin (0.17±0.45) was significantly lower than in perilesional (1.19±0.89, P<0.001) and unaffected skin (1.44±0.77, P<0.001). However, the expression of Bcl-2 in the perilesional skin (1.19±0.89) was insignificantly lower than unaffected skin (1.44±0.77). We found that Bcl-2 expression in unaffected skin, (1.44±0.77) was insignificantly lower than that in serum (1.61±0.32). However, its expression in the lesional and perilesional skin was significantly lower than in serum (P<0.001 and 0.009 respectively; [Table 3] and [Figure 1]).
Table 3 Levels of Bcl-2 (serum and tissues) and inactive caspase-9 (tissues) of patients

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Figure 1 The expression of Bcl-2 protein throughout the basal layer of epidermis showing a relatively higher level of expression in the control skin (A) and slightly lower level in the unaffected skin (B), gradual loss in the perilesional skin (C) and maximal decrease in the lesional skin (D) (IP X 400).

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No significant correlations could be detected between the levels of Bcl-2 in serum and tissue with age, disease duration, clinical type and stability and VASI ([Table 4],[Table 5],[Table 6],[Table 7]).
Table 4 Correlation of levels of Bcl-2 (serum and tissues), active caspase-9 (serum), and inactive caspase-9 (tissues) with age and disease duration

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Table 5 Levels of Bcl-2 (serum and tissues), active caspase-9 (serum), and inactive caspase-9 (tissues) in different clinical types

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Table 6 Levels of Bcl-2 (serum and tissues), active caspase-9 (serum), and inactive caspase-9 (tissues) in stable and unstable vitiligo

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Table 7 Correlations of levels of Bcl-2 (serum and tissues), active caspase-9 (serum), and inactive caspase-9 (tissues) with vitiligo area scoring index

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Active caspase-9 and inactive caspase-9

The serum level of active caspase-9 in patients (2.81±0.81) was significantly higher (P=0.047) than in controls (2.48±0.44) ([Table 2]).

No significant relations could be detected between the levels of active caspase-9 in serum with age, disease duration, clinical types, stability, and VASI ([Table 4],[Table 5],[Table 6],[Table 7]).

The expression of inactive caspase-9 level in lesional skin (1±0.87) was significantly lower (P<0.001) than in the skin of controls (1.9±0.8) ([Table 2]).

In patients, inactive caspase-9 expression was insignificantly lower in lesional skin (1±0.87) than in perilesional skin (1.05±0.8) and lower in the perilesional skin (1.16±0.87) than in unaffected skin ([Table 3] and [Figure 2]).
Figure 2 The expression of inactive caspase-9 protein throughout epidermal layers showing a higher level of expression in the control skin (A) and lower level in the unaffected skin (B), gradual loss in the perilesional skin (C) and maximal decrease in the lesional skin (D) (IP X 400).

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Regarding clinical type, inactive caspase-9 level in lesional skin was significantly higher (P=0.046) in the acrofacial type (1.06±0.66) than in generalized type (0.85±0.93) ([Table 5]).

No significant relations could be detected between the levels of inactive caspase-9 in tissue with age, disease duration, stability, and VASI ([Table 4], [Table 6] and [Table 7]).


  Discussion Top


According to the view of the apoptotic pathway, it is accepted that the initial cell stress activates the proapoptotic members of the Bcl-2 family. These proteins are localized in the cytosol, where they act as probes for cell damage or stress; once activated, they relocate to the mitochondrial surface, thereby leading to the formation of transmembrane pores. These pores promote the release of many proapoptotic proteins, such as cytochrome c and various molecules from the intermembrane space [17]. Once released into the cytosol, these proteins promote the activation of caspase-9, the initiator caspase [18], which can, in turn, activate the effector caspases, such as caspase-3 and caspase-6. Both are responsible for the cleavage of key cellular proteins, such as cytoskeletal proteins, leading to the typical morphological changes of apoptosis [19].

In our study, we found no significant difference in the serum Bcl-2 levels between patients and controls; however, it was slightly lower in patients. No correlations were detected between serum Bcl-2 levels and age, disease duration, clinical type, stability, or VASI. To the best of our knowledge, no previous researches were done to dispute these findings.

At the tissue level, we found a significantly lower expression of Bcl-2 in lesional skin than in perilesional and unaffected skin, and Bcl-2 expression in the unaffected skin was the highest. In accordance with earlier reports, the in-situ analysis revealed high melanocytic Bcl-2 expression in normal skin [20],[21]. Equally, low Bcl-2 expression was observed in lesional skin compared with the normally pigmented epidermis [15],[22]. The Bcl-2 level in lesional skin was even significantly lower than in the serum. These results support previous studies about the specific relation of Bcl-2 with melanocytes. So, absence of melanocytes becomes reflected by the low Bcl-2 levels in the vitiliginous skin [21],[23],[24].

We found that active caspase-9 level in patients’ serum was significantly higher than in controls. This indicates that vitiligo patients have more apoptotic process than healthy controls as a result or a cause of the disease process. However, Gulbahar et al. [25] showed no such significant difference. In our study, the serum level of active caspase-9 did not show any correlation with age, disease duration, clinical type, stability, or VASI. However, Gulbahar et al. [25] showed that active caspase-9 in serum was significantly inversely correlated only with disease duration.

Our study showed that inactive caspase-9 protein expression in lesional skin of patients was significantly lower than in the skin of controls. In addition, in patients, the inactive caspase-9 expression in lesional skin was insignificantly lower than in the perilesional and unaffected skin. Besides, it was lower in the perilesional skin than in the unaffected skin.

Caspases exist in cells as inactive zymogens and are activated by proteolytic cleavage during apoptosis. A decrease of the inactive zymogens occurs along with their activation [24]. Therefore, this means that there is much activation of caspase-9 protein in patients than in controls and in the lesional skin than in the perilesional and unaffected skin. This activation indicates apoptosis. Our finding is in accordance with that of Lee et al. [24], where they found that the expression of inactive caspase-9 was lower in the depigmented epidermis compared with the normally pigmented epidermis.

Moreover, we found that inactive caspase-9 level in lesional skin was significantly higher in the acrofacial type than in the generalized type. No significant relations between inactive caspase-9 levels with age, disease duration, stability, and VASI were observed. No previous studies support or disprove our findings.


  Conclusion Top


Our data strongly support apoptosis rather than necrosis as the proposed mechanism for the elimination of melanocytes in vitiligo. Bcl-2 deficiency can be considered as a marker for melanocyte damage rather than a cause. Further research about the role and the cause of apoptosis as an important etiopathogenic factor of vitiligo is highly recommended in order to give the dermatological community more clues about the etiology and hence, the treatment of such a disfiguring illness.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Kyriakis KP, Palamaras I, Tsele E, Michailides C, Terzoudi S. Case detection rates of vitiligo by gender and age. Int J Dermatol 2009; 48:328–329.  Back to cited text no. 1
    
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Ezzedine K, Lim HW, Suzuki T, Katayama I, Hamzavi I, Lan CC et al. Revised classification/nomenclature of vitiligo and related issues: the Vitiligo Global Issues Consensus Conference. Pigment Cell Melanoma Res 2012; 25:E1–E13.  Back to cited text no. 3
    
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Ortonne JP. Vitiligo and other disorders of hypopigmentation. In: Bolognia J, Jorizzo J, Rapini R, editors. Dermatology. Philadelphia, PA: Mosby Elsevier; 2008. p. 928.  Back to cited text no. 4
    
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Yamamura K, Kamada S, Ito S, Nakagawa K, Ichihashi M, Tsujimoto Y. Accelerated disappearance of melanocytes in bcl-2-deficient mice. Cancer Res 1996; 56:3546–3550.  Back to cited text no. 11
    
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van den Wijngaard RM, Aten J, Scheepmaker A. Expression and modulation of apoptosis regulatory molecules in human melanocytes: significance in vitiligo. Br J Dermatol 2000; 143:573–581.  Back to cited text no. 14
    
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Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY et al. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 2001; 410:549–554.  Back to cited text no. 18
    
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21.
Plettenberg A, Ballaun C, Pammer J. Human melanocytes and melanoma cells constitutively express the Bcl-2 proto-oncogene in-situ and in cell culture. Am J Pathol 1995; 146:651–659.  Back to cited text no. 21
    
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Abdel-Aal AM, Kasem MA, Abdel-Rahman AH. Evaluation of the role of apoptosis in vitiligo: immunohistochemical expression of P53, Bcl-2 and MART-1 antigens. Egypt J Hosp Med 2002; 8:1–11.  Back to cited text no. 22
    
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Lee AY, Youm YH, Kim NH, Yang H, Choi WI. Keratinocytes in the depigmented epidermis of vitiligo are more vulnerable to trauma (suction) than keratinocytes in the normally pigmented epidermis, resulting in their apoptosis. Br J Dermatol 2004; 151:995–1003.  Back to cited text no. 24
    
25.
Gulbahar O, Eren C, Tatlican S, Ergun MA, Bukan N. Caspase levels in the evaluation of apoptosis in vitiligo patients. Gazi Med J 2010; 21:81–83.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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