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Table of Contents   
ORIGINAL RESEARCH  
Year : 2013  |  Volume : 24  |  Issue : 2  |  Page : 255-260
Immunolocalization of Bcl-2 oncoprotein in amlodipine-induced gingival overgrowth


1 Department of Periodontics, Thai Moogambigai Dental College, Golden George Nagar, Chennai, India
2 Department of Periodontics, Faculty of Dental Sciences, Sri Ramachandra University, Porur, Chennai, India

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Date of Submission23-Nov-2011
Date of Decision28-Feb-2012
Date of Acceptance30-Mar-2012
Date of Web Publication20-Aug-2013
 

   Abstract 

Background: Drug-induced gingival overgrowth (DIGO) is one of the unwanted side effects of amlodipine therapy, but the pathogenesis still remains unclear. Apoptosis, which plays a ubiquitous role in tissue homeostasis, including gingiva, may be involved in the development of gingival enlargement.
Aims and Objectives: (i) To study the distribution of Bcl-2 in healthy and overgrown gingival tissues. (ii) To compare and correlate the Bcl-2 expression in gingival samples from subjects on amlodipine therapy to the findings in healthy controls.
Materials and Methods: A total of 25 subjects were recruited for the study - 15 hypertensive patients and 10 systemically healthy subjects. Both the groups were analyzed for Bcl-2 expression using immunohistochemistry.
Results: Few of the control specimens showed weak positivity to Bcl-2 antibody, with the distribution limited to the basal cell layers alone, whereas 10 hyperplastic specimens expressed Bcl-2 and, unlike the control group, the distribution pattern was seen in both basal and suprabasal layers.
Conclusion: The results indicate that the pathogenesis of amlodipine-induced gingival overgrowth might involve inhibition of apoptosis, especially with morphogenesis of hyperplastic gingival epithelia.

Keywords: Amlodipine, apoptosis, Bcl-2, gingival hyperplasia, immunohistochemistry

How to cite this article:
Arunachalam LT, Rao S. Immunolocalization of Bcl-2 oncoprotein in amlodipine-induced gingival overgrowth. Indian J Dent Res 2013;24:255-60

How to cite this URL:
Arunachalam LT, Rao S. Immunolocalization of Bcl-2 oncoprotein in amlodipine-induced gingival overgrowth. Indian J Dent Res [serial online] 2013 [cited 2020 May 25];24:255-60. Available from: http://www.ijdr.in/text.asp?2013/24/2/255/116695
Clinically detectable fibrotic overgrowth of gingiva is caused by a variety of etiological factors and is exacerbated by local bacterial plaque accumulation. Drug-induced gingival overgrowth is a well-documented side effect associated with chronic usage of anticonvulsants like phenytoin, immunosuppressants like cyclosporine, and calcium channel blockers like nifedipine, and amlodipine. It is characterized by epithelial hyperplasia and accumulation of collagenous components within the connective tissue. The degree of inflammation, fibrosis, and cellularity depends upon the duration, dose, and identity of the drug, on the quality of the oral hygiene, and on individual susceptibility that stems from genetic factors and environmental influences. Hassell, [1] referred drug-induced gingival hyperplasia as "Gingival Overgrowth," as the lesion is characterized by an increase in '"normal growth,''' as opposed to cellular hypertrophy or hyperplasia.

Amlodipine, a dihydropyridine, is a long-acting vasodilator, widely prescribed for cardiovascular diseases, including acute and chronic cardiac insufficiencies, essential hypertension, and chronic stable angina pectoris. Amlodipine was first reported for causing gingival overgrowth as a side effect, by Seymour et al. [2] An incidence of 3.3% in patients taking 5 mg/day of amlodipine has been reported. [3] Although the relationship between calcium channel blockers and gingival overgrowth is a widely accepted concept, the pathogenesis still remains an enigma. One of the recent hypotheses is the inhibition of apoptosis by these drugs.

Apoptosis, first introduced by Kerr et al., [4] a genetically programmed process for cells to commit suicide, plays a ubiquitous role in tissue homeostasis, including gingiva. It plays a key role in oral development, the progression of oral disease, bone resorption, immunological response to inflammation, and wound healing. Apoptosis occurs in the cells of periodontium as part of normal turnover and remodeling, and may be more prevalent than necrosis in periodontal disease. Its aberrant activation may contribute to a number of diseases like cancer and AIDS. There are multiple cellular pathways triggering apoptosis, two of which, the extrinsic and intrinsic pathways, are better characterized. The extrinsic pathway is activated by cell surface death receptors, while the intrinsic pathway is initiated by formation of the cytosolic apoptosome (composed of Apaf-1, procaspase 9, and the cytochrome c released from mitochondria). The apoptotic process is executed by a family of cysteine proteases, known as caspases, which are activated through extrinsic and/or intrinsic pathways. In the intrinsic pathway, mitochondrion is the central organelle and is governed by pro- and anti-apoptotic Bcl-2 family member.

Bcl-2 is the acronym for the B-cell lymphoma/leukemia-2 gene. It was first discovered because of its involvement in B-cell malignancies. Bcl-2 resides in the nuclear envelope, parts of the endoplasmic reticulum (ER), and outer mitochondrial membrane, but not in a variety of other intracellular membrane compartments including the plasma membrane. Vaux et al., [5] were the first to report that Bcl-2 can prolong cell survival. In addition to delaying cell death, over-expression of Bcl-2 can prevent or markedly reduce cell killing induced by a wide variety of stimuli [Table 1]. It is established that there are calcium changes taking place during apoptosis - enhanced calcium influx or intracellular calcium relocalization and the calcium in the mitochondria also varies. One of the mechanisms of Bcl-2 in inhibiting apoptosis is by influencing the redistribution of intracellular calcium. Considering the connection between Bcl-2 and calcium regulation, this study was undertaken to evaluate the expression of Bcl-2 in amlodipine-treated gingival overgrowth and study the distribution pattern of Bcl-2 in healthy and overgrown gingival tissues by immunohistochemistry.
Table 1: Causes of cell death that Bcl-2 has been documented to block


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   Materials and Methods Top


Subject population

The present study received approval from the Ethics Committee, Sri Ramachandra Medical College and Research Institute, Porur. The purpose and nature of the study, including sample collection, were explained to all potential participants and they were asked to sign informed consent forms, with which they acknowledged their willingness to participate.

A total of 25 subjects were recruited for this study, of which 15 (10 females, and 5 males; age group 32-67 years) were from the group of patients seeking treatment in hypertensive clinic, Sri Ramachandra Medical College and Research Institute, Porur. Ten healthy subjects (5 males, and 5 females; age group 26-56 years) were chosen from those, who attended Department of Periodontics, Sri Ramachandra Dental College, Porur.

The subjects who satisfied the entry criteria were included in the study. The inclusion criteria for the drug-induced gingival overgrowth (DIGO) group were the presence of six of the eight anterior upper and lower teeth (first premolar to first premolar), and under amlodipine therapy for at least a minimum period of 6 months, with no change in the drug regimen till the time of examination. All the 15 subjects had not received any form of periodontal treatment in the past 6 months, other than professional supragingival plaque removal. The 10 healthy individuals had no history of treatment with agents known to cause drug-induced gingival overgrowth.

The exclusion criteria applied were the presence of any other systemic disease, and receiving antibiotic therapy, or other medications that would affect periodontal health.

Clinical parameters and sample collection

The degree of gingival overgrowth was classified into four categories based on the criteria of Angelopoulos and Goaz, [6] which were later modified by Pernu et al. [7] The patients with no signs of gingival overgrowth were categorized as score 0, and scores (I-III) were assigned to those with signs of overgrowth. Other parameters included were the Plaque Index and the Gingival Index. [8] The initial treatment included scaling, and the tissue samples from both the groups were collected at the second visit. The tissues were removed under local anesthesia. The samples were immediately fixed in 10% neutral formalin at room temperature. They were embedded in paraffin wax and 4 μm serial sections were prepared on gelatin-coated slides. One section was used for morphological examination, based on Hematoxylin and Eosin (H and E) staining, and the other for immunohistochemistry.

Immunohistochemistry

The gelatin-coated slides were dewaxed in xylene, dehydrated in graded alcohol. The epitope retrieval was done by pressure cooking in citrate buffer for 10 min. After heating, the slides were permitted to cool down to room temperature over a period of 30-60 min. Sections were then washed in distilled water and soaked in phosphate-buffered saline (PBS) and 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. The sections were incubated with prediluted monoclonal antibody (DAKO Corporation, Carointeria, CA, USA) for 1 h at room temperature. After washing with PBS, the slides were sequentially incubated with biotinylated secondary link antibody (DAKO Corporation) for 30 min. After washing with PBS, they were incubated with AEC substrate solution (DAKO Corporation) for 10 min, and later immersed in a bath of Lille Meyer's hematoxylin for 2-5 min and mounted in DePex mounting medium, viewed, and photographically recorded. The following procedure was repeated for all the gingival overgrowth samples as well as the control specimens.

Evaluation of Bcl-2 staining

The staining pattern with each monoclonal antibody was assessed for each section, as given by Ioffe et al. [9]

  • No staining of cells = (-) negative
  • Slight staining of cells = (+) 10-20%
  • Moderate staining in most of the cells = (++) 20-50%
  • Strong staining in the cells = (+++) >50%

   Results Top


The demographic details of both the DIGO and control groups are shown in [Table 2]. In the group of 15 overgrowth patients studied, 10 were females and most of them were in the fourth decade when compared to the third, fifth, and sixth decade patients.
Table 2: Demographic data of patients


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The duration of therapy and the gingival overgrowth score for the DIGO group are shown in [Table 3]. Among the 15 patients on amlodipine treatment, 9 subjects were given score I of gingival overgrowth, 4 patients were assigned score II, while 2 patients exhibited overgrowth covering more than two-thirds of the anatomic crown and were scored III.
Table 3: Duration of therapy and the gingival overgrowth score


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Correlation between the duration of therapy and gingival overgrowth index scores is shown in [Table 4]. 100% of the cases in the 6 months to 2 years duration period showed an overgrowth score of I, 80% of the cases in the 2-4 year period of therapy showed a score of II, whereas 100% of cases over the 5 year category had gingival overgrowth index score of III. Graph 1 [Additional file 1] shows the distribution of Bcl-2 in both DIGO and control groups, and the intensity of staining is shown in [Table 5].
Table 4: Correlation between duration of therapy and the gingival overgrowth score


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Table 5: Intensity of staining in overgrowth cases


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The H and E sections of few hyperplastic gingival specimens showed mild inflammatory infiltration in the connective tissue, predominantly of perivascular location and elongation of rete pegs. In some specimens, thickening of the epithelium was noted. Acanthosis and parekeratinization were also observed, and a few specimens showed increased number of capillaries in the connective tissue.

Considering the expression pattern of Bcl-2, five of the control specimens showed positivity and the other five revealed negative immunoreactions against the ligand, and distribution was limited to the basal cell layers alone [Figure 1] and [Figure 2]. Of the hyperplastic samples, 10 specimens expressed Bcl-2. However, the distribution pattern unlike the control group was seen in the basal and the suprabasal layers [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7] and [Figure 8].
Figure 1: Bcl-2 positivity in the basal cells of the normal oral epithelium (LSAB x157)

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Figure 2: Bcl-2 positivity in the basal cells of the normal oral epithelium (LSAB x252)

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Figure 3: DIGO showing Bcl-2 positivity (+) (LSAB x157)

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Figure 4: DIGO showing Bcl-2 positivity (++) (LSAB x157)

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Figure 5: DIGO showing Bcl-2 positivity (++) (LSAB x157)

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Figure 6: DIGO showing Bcl-2 positivity (+++) (LSAB x157)

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Figure 7: Tangential section showing Bcl-2 positivity (LSAB x252)

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Figure 8: Tangential section showing Bcl-2 positivity (LSAB x252)

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   Discussion Top


Gingival hyperplasia is a known side effect associated with the usage of calcium channel blockers, especially dihydropyridines. Despite the numerous studies available on nifedipine-induced gingival hyperplasia, relatively less research has been done on amlodipine-induced gingival overgrowth. To our knowledge, this is the first study to have explored the effect of amlodipine on apoptosis of hyperplastic gingival epithelial cells.

In this study, 80% of the cases in the 2-4 year period of therapy showed a score of II, whereas 100% of cases over the 5 year category had gingival overgrowth index score of III. This finding is in concurrence with that of Tavassoli et al., [10] who showed that those on dihydropyridine medication for more than 4 years were likely to have an increased severity of gingival overgrowth.

The H and E sections showed characteristic findings of overgrowth, which is in accordance with the histologic findings of Jones et al., [11] who have reported marked epithelial hyperplasia with varying degree of fibrosis and infiltration of inflammatory cells. Few specimens showed increased number of capillaries in the connective tissue, similar to the results shown by Thomasson et al., [12] who reported that increased vascularization and vasodilatation in the connective tissue were due to the effect of calcium channel blockers on the vasculature.

From the yester-years till date, various hypotheses have been put forward regarding the pathogenesis of drug-induced gingival overgrowth. Initially more attention was directed toward the alterations in the connective tissue homeostasis, including the effect of overgrowth-inducing drugs on collagen, non-collagenous matrix, and collagenous matrix degradation. In respect to epithelium, though epithelial hyperplasia (test tube appearance) characteristic of gingival overgrowth was studied, importance on the mechanism of epithelial hyperplasia in the pathogenesis was not emphasized. The recent studies on epithelial-mesenchymal transition (EMT) showed the transition of epithelial cells to smooth muscle fibroblast, which in the process of migration into connective tissue may stretch the epithelium giving a test tube like appearance (unpublished data). Epithelial hyperplasia can be considered in two aspects - increase in the proliferation rate of the cells or prolongation of their lifespan which is tightly regulated by various transmembrane proteins including Bcl-2 oncoprotein. In this study, the distribution pattern of Bcl-2 was seen in the basal and suprabasal layers, which is in par with the results shown by the above-mentioned authors.

All the implicated drugs in the hyperplasia of the gingival tissue have a common mechanism - inhibition of intracellular uptake of calcium. Calcium has long been recognized as a participant in the apoptotic pathway. Major organelles involved in calcium homeostasis are the ER and mitochondria. The Bcl-2 family members have been found to have effects on the calcium dynamics of ER and mitochondrial membrane integrity and function, especially calcium release from the ER and its uptake into mitochondria, which is pivotal in triggering the release of cytochrome c and apoptosome formation leading to oligonucleosomal DNA fragmentation. [13] At the cellular level, the demonstrated effects of calcium channel blockers and calcium on apoptosis are complex as both increases and decreases in intracellular calcium can be linked to apoptosis. Nifedipine and verampamil have been reported to inhibit apoptosis of renal epithelial cells and human aortic smooth muscle cells in vitro. [14] Therefore, it is possible that apoptosis of gingival keratinocytes is inhibited under low calcium conditions as sustained elevation of calcium is necessary to activate degradative enzymes such as calcium-dependent protease and endonucleases responsible for DNA degradation. [15] According to Shimuzu et al., [16] gingival keratinocytes grown under low levels of calcium express Bcl-2, which inhibits apoptosis. In contrast, keratinocytes grown under high levels of calcium express Bax, which induces apoptosis.

Therefore, in this scenario, it is reasonable to speculate that Bcl-2 regulates the calcium fluxes by inhibition of release of calcium by the ER which, rather than the calcium concentration in the ER, controls the amplitude of "death signal" reaching the mitochondria, or by prevention of caspase activation, possibly through inhibition of mitochondrial cytochrome c release.

Bcl-2 oncoprotein impedes the commitment to terminal differentiation as well as apoptosis of epithelial cells. The distribution of the Bcl-2 oncoprotein in the suprabasal cell layers may be involved with the DNA damage by genotoxic stress of the causative drug, amlodipine. Therefore, the Bcl-2 over-expression is implicated in parakeratinization in cornified call layers of hyperplastic epithelia. Bcl-2 over-expression may provide prolonged life-span of epithelial cells and the inactivated renewal of epithelia in hyperplastic gingiva by inhibition of terminal differentiation and leads to cellular accumulation in the suprabasal cell layers, resulting in acanthosis of gingival hyperplasia. [17] Nimmi et al., [18] have shown that drug-induced gingival overgrowth tissues exhibited less DNA polymerase in the basal keratinocytes, suggesting epithelial acanthosis may be due to enhanced lifespan rather than an increase in keratinocyte proliferation.

Saito et al., [19] showed synergistic overexspression of Bcl-2 and C-myc oncoprotein in gingival hyperplasia induced by nifedipine and phenytoin, suggesting their role in pathogenesis, especially the development of hyperplastic epithelia. They have also shown that Bcl-2 abrogates the apoptotic effects of c-myc oncoprotein, which commits diverse cell populations, including epithelial cells to apoptosis. We speculate that the same scenario is possible in amlodipine-induced gingival overgrowth resulting in increased epithelial thickness.


   Conclusion Top


Considering the results, the epithelial hyperplasia in amlodipine-induced overgrowth is not due to increase in keratinocyte proliferation, but is caused by prolongation of cell life through inhibition of keratinocyte apoptosis. Within the limitations of the study, the results suggest two salient findings, namely, the role of the epithelium in the pathogenesis of gingival overgrowth and inhibition of apoptosis by Bcl-2 oncoprotein towards the morphogenesis of hyperplastic gingival epithelia. Further studies with larger sample size are required to elucidate the role of calcium channel blockers in the development of overgrown gingiva.

 
   References Top

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Correspondence Address:
Lalitha Tanjore Arunachalam
Department of Periodontics, Thai Moogambigai Dental College, Golden George Nagar, Chennai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.116695

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

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

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