Parecoxib decreases cellular growth and Bcl‐2 protein levels in primary cultures of keloid fibroblasts

Abstract

Keloids seem to overexpress cyclo‐oxygenase‐2 (COX‐2), suggesting a role in its deregulated pathway in inducing an altered epithelial‐mesenchymal interaction, which may be responsible for the overgrowth of dermal components resulting in scars or keloid lesions. This study aimed to evaluate the effect of Parecoxib, a COX‐2 inhibitor, on cell growth in fibroblast primary cultures obtained from human keloid tissues. Tissue explants were obtained from patients who underwent intralesional excision of untreated keloids; central fractions were isolated from keloid tissues and used for establishing distinct primary cultures. Appropriate aliquots of Parecoxib, a COX‐2 inhibitor were diluted to obtain the concentration used in the experimental protocols in vitro (1, 10 or 100 μM). Treatment with Parecoxib (at all concentrations) caused a significant decrease in cellular growth from 24 hours onwards, and with a maximum at 72 hours (P < .02). Moreover, at 72 hours Parecoxib significantly reduced cellular vitality. Parecoxib treatment also induced an increase in fragmented nuclei with a maximum effect at 100 μM and a significant decrease in Bcl‐2 and an increase in activated caspase‐3 protein levels at 72 hours compared with control untreated cultures. Our findings suggest a potential use of the COX‐2 inhibitor, Parecoxib, as the therapy for keloids.

1. INTRODUCTION

Keloids and hypertrophic scars originate from an aberrant wound healing process. ¹ , ² , ³ Children and young adults are more often affected, with a greater propensity for black people, particularly African Americans, but it is also not uncommon among Hispanics and Orientals.

Hypertrophic scars and keloids initially present a similar clinical appearance: red, raised, and firm possess a smooth, shiny surface. Keloids differ from hypertrophic scars by extending beyond the confines of the original wound and usually prominently above the surrounding skin, whereas hypertrophic scars remain confined in the margins of the wound. ⁴ , ⁵ , ⁶ , ⁷ Moreover, hypertrophic scars tend to flatten out spontaneously in one or more years, whereas keloids do not regress over time.

The pathogenesis of keloid formation is still unknown. Many studies conducted to investigate the nature of keloid, indicate that there are three important factors involved in keloid development: cytokine production, cell growth, and apoptosis. Several studies assert that keloids are the result of an aberrant or exaggerated release of cytokines and growth factors such as interleukin (IL)‐6 and vascular endothelial growth factor (VEGF). ⁸ , ⁹

Indeed IL‐6 gene and protein have been found to increase in keloid fibroblasts derived, ¹⁰ , ¹¹ VEGF could play a central role in keloid formation by impairing extracellular matrix homeostasis toward a condition of reduced degradation and excessive accumulation. ⁹

Granulation tissue conversion to neodermis requires a reduction in cellularity by apoptosis of endothelial cells, fibroblasts, and myofibroblasts, meanwhile, cell growth and death are regulated by cytokines and growth factors release. ¹² , ¹³ Some evidence indicates that the processes of proliferation and apoptosis are deregulated in keloid fibroblasts, which may be the reason for the overproduction of collagen and extracellular matrix in keloid tumours. ¹⁴ , ¹⁵ , ¹⁶

In other studies, ¹⁷ we reported a significant overexpression of the enzyme cyclo‐oxygenase‐2 (COX‐2) in the dermis of keloidal lesions in comparison with hypertrophic scar tissues as well with normal skin.

Our research supports the thesis that both COXs play a role in the genesis of scar lesions by different mechanisms. In particular, keloids seem to overexpress COX‐2, suggesting a role in its deregulated pathway in inducing an altered epithelial‐mesenchymal interaction, ¹⁸ , ¹⁹ , ²⁰ that may be responsible for the overgrowth of dermal components resulting in scars or keloid lesions.

Moreover, the evidence of reduced scar inflammation in patients using non‐steroidal anti‐inflammatory drugs or a COX‐2 inhibitor highlights the importance of COXs in the pathogenesis of scar lesions. ²¹

To date, there aren't studies that verify the efficacy of COX‐2 selective antagonist in the reduction of cellular growth in keloid derivate tissue. This study aimed to prove the effect of Parecoxib, a COX‐2 inhibitor, on cell growth in primary cultures of fibroblasts derived from human keloids.

2. MATERIALS AND METHODS

2.1. Study population

The study was approved by the Ethical Committee of Our Institution (28 February 2015), and it complies with the Declaration of Helsinki. Human keloid tissue was derived from 37 Caucasian patients (25 female, 12 male), with an average age of 41 (29‐60) at our Plastic and Reconstructive Surgery Unit, from September 2015 to September 2018, by intralesional biopsy. Inclusion criteria were: not previously treated keloids. Exclusion criteria were: keloids previously treated with standard therapy; hypertrophic scars.

2.2. Cell cultures and treatments

Specimens taken were splintered and incubated directly in different culture dishes in a humidified tissue incubator (37°C, 5% CO₂) with the appropriate growth medium. The middle fraction was isolated from keloid tissue and used for establishing distinct primary cultures, by Luo et al and Giugliano et al. ¹⁴ , ²²

Fibroblasts proliferation was promoted in Dulbecco modified Eagle medium (DMEM) supplemented with L‐glutamine, 10% foetal bovine serum (FBS) and antibiotics (Life Technology, Milan, Italy). Cellular cultures were utilised in our experimental protocol, in the next step. A stock solution of 10 mM Na‐Parecoxib (Dynastat, Pfizer, New York, New York) was obtained by dissolving it in 0.9% NaCl by the manufacturer's instructions. Appropriate aliquots of this preparation were diluted in DMSO to obtain the concentration used in the experimental protocols in vitro (1, 10 or 100 μM).

2.3. Cellular growth curve

Cellular replication was analysed in 2 × 10³ cells of 60 mm in DMEM, supplemented with 10% FBS. At preset intervals (24‐48‐72 hours), the culture dishes were trypsinized in triplicate for each experimental set and the cellular amount was counted in a Neubauer Chamber.

2.4. Cell proliferation assay

Cellular growth was estimated by the tetrazolium salt (MTT) method. A colorimetric assay for the non‐radioactive quantification of viable cells was used. This proliferation kit (Roche Diagnostics GmBh, Mannheim, Germany) allows the reduction of MTT only by metabolically active cells to form a formazan dye (UV absorbance spectrum is between 550 and 600 nm). Cells were seeded in microtiter plates into a final volume of 100 μL complete culture medium at a concentration of 2 × 10³ cell/well and grown for 24 hours at 37°C in 5% CO₂. Starving cells in modified Eagle medium (MEM) without FBS for 24 hours, were incubated in 1% FBS‐supplemented MEM with Parecoxib (1, 10 or 100 μM) or solvent (control cells) for 72 hours. Then the 10 μL MTT solution was added to each well and the plates were incubated for 4 hours. Ten microlitres of solubilisation solution were added to each well and plates were kept overnight in the incubator. Absorbance was read at 550 nm using a microtiter plate reader.

2.5. Apoptosis detection

The cell death detection kit (TUNEL, Roche Diagnostics GmbH) was used to evaluate cell apoptosis and quantify DNA strand breaks in individual cells. The cellular monolayers were grown on sterilised slides (Superfrost, Carlo Erba, Milan, Italy), starved for 24 hours in MEM without FCS, and then incubated in 1% FCS‐supplemented with Parecoxib (1, 10 or 100 μM) or solvent (control cells) for 96 hours. The slides were then fixed in buffered paraformaldehyde, permeabilized with Triton‐x, and labelled with a TUNEL reaction mixture in accordance with the manufacturer's instructions. Samples were analysed using a Leitz Diaplan microscope (Leica, Milan, Italy) equipped with epifluorescence. A negative control (obtained by incubating a slide with a labelled solution without terminal transferase) and positive control (obtained by treating a slide with Dnase I solution) were included in each assay run.

2.6. Western blot analyses

Western blot analyses were performed as previously described by Rossi et al. ²³ , ²⁴ Polyclonal antibodies against Casp‐3‐3‐3 (H‐277) and BCL‐2 (∆C21) (Santa Cruz Biotechnology, Inc., Santa Cruz, California) were used. Caspase‐3 and Bcl‐2 protein levels were evaluated by western blotting analyses of the protein extracts from cultured fibroblasts. For electrophoresis and immunoblotting analyses, the cells were harvested by scraping in PBS containing 0.2 mmol/L EDTA and centrifuged. Afterward, the pellets were measured and resuspended in 2× denaturing lysis buffer (1:1, vol/vol) containing 0.25 mol/L Tris‐HCl (pH 6.8), 5% sodium dodecyl sulfate (SDS), 8 mol/L urea, 10 mmol/L EDTA, and 0.1 mol/L dithiothreitols in accordance with the manufacturer's instructions. The visualisation of the immunosignal was obtained through the autoradiography of the reaction of the secondary immunoperoxidase reaction with the luminescent substrate (ECL, Amersham).

2.7. Statistical analysis

Statistical analysis of results was performed by analysis of variance for repeated measures with Bonferroni correction. A value of P < .05 was considered significant.

3. RESULTS

The effects of Parecoxib on the growth of fibroblasts derived from the central part of keloids are depicted in Figure 1. Treatment with Parecoxib (at all concentrations) caused a significant decrease in cellular growth from 24 hours onwards, and with a maximum at 72 hours (P < .02). Cell counts are expressed as mean ± SD of three determinations in duplicate. Significant differences at 72 hours of incubation between the treated and control cultures are indicated (**P < .02) (Figure 1). MTT assay for cell viability, expressed as optical density (OD) arbitrary units, after 72 hours of incubation with vehicle (c) or parecoxib at different concentrations. The OD demonstrated the relative cell number in cultures at 72 hours. Each data point is the mean ± SD of four determinations in duplicate. Significant differences from the control cultures are indicated as **P < .02. At 72 hours Parecoxib cellular vitality reduced significantly (Figure 2). Percentage of fragmented nuclei by TUNEL at 72 hours of incubation with vehicle (c) or Parecoxib. Each data point is the mean ± SD of four determinations in triplicate. Significant differences from the control cultures are indicated as *P < .05, **P < .02. Parecoxib treatment for 72 hours induced an increase in fragmented nuclei with a maximum effect at 100 μM (Figure 3). Treatment with Parecoxib also induced a significant decrease in Bcl‐2 and an increase in activated caspase‐3 protein levels at 72 hours compared with control untreated cultures (Figure 4).

FIGURE 1.

Figure showing growth of fibroblasts treated with vehicle (c) or Parecoxib at different concentrations. Treatment with Parecoxib (at different doses) caused a significant decrease in cellular growth. Significant differences at 72 hours of incubation between the treated and control cultures are indicated (**P < .02)

FIGURE 2.

MTT assay for cell viability, expressed as optical density (OD) arbitrary units, after 72 hours of incubation with vehicle (c) or parecoxib at different concentrations. Significant differences from the control cultures are indicated as **P < .02

FIGURE 3.

Percentage of fragmented nuclei by TUNEL at 72 hours of incubation with vehicle (c) or parecoxib. Significant differences from the control cultures are indicated as *P < .05, **P < 0.02

FIGURE 4.

The expression level of Bcl‐2 and Caspase‐3 after 72 hours incubation of fibroblasts with vehicle (c) or parecoxib at different concentrations

4. DISCUSSION

Despite the continuous improvement of the treatment techniques and the new information on available technology, the management of keloids and hypertrophic scars remains a serious problem for aesthetics, plastic surgeons and dermatologist.

Currently, there is no standard therapy for their treatment, but several studies and progress have been made in this field. ²⁵ The most effective treatment remains the combination of different therapies such as intralesional steroids (triamcinolone), surgical excision, silicone gel and others. ²⁵ , ²⁶ , ²⁷ , ²⁸ , ²⁹ , ³⁰

In this study, we demonstrated that the COX‐2 inhibitor Parecoxib is an effective tool by inhibiting the growth of primary fibroblast cultures in vitro. The action of Parecoxib was evident in both cell proliferation inhibition and apoptosis induction, resulting in a reduced cell number in treated cultures. Moreover, we demonstrated that these effects were linked with the reduction of proliferative signals and the increase of apoptotic pathways, respectively.

The effects of Parecoxib are dose‐dependent and appear significant at 10 μM. Parecoxib does not have cytotoxic effects, as demonstrated by the MTT assay. In a previous study, our group reported overexpression of COX‐1 and COX‐2 in altered wound healing conditions characterised by excessive extracellular matrix deposition and fibroblast proliferation, such as hypertrophic scars and keloids. ¹⁷ In particular, keloids seem to overexpress COX‐2, suggesting a role in its deregulated pathway in inducing an altered epithelial‐mesenchymal interaction, ¹⁸ , ¹⁹ , ²⁰ that may be responsible for the overgrowth of dermal components resulting in scars or keloid lesions. COXs are important enzymes in the conversion of arachidonic acid into prostaglandins. After an inflammatory stimulation, the induction of COX‐2 represents an early response to the lesion, which promotes the conversion of arachidonic acid to prostaglandins. ³¹ The inflammation causes the release of arachidonic acid from membrane phospholipids by activating phospholipases and promotes COX‐2 enzyme formation. ³¹

These substances along with other proinflammatory cytokines are involved in the pathogenesis of wound healing disorders. The importance of COXs is also highlighted by the evidence of reduced scar inflammation in patients receiving non‐steroidal anti‐inflammatory drugs or a COX‐2 inhibitor. ³¹

Parecoxib is a preferential inhibitor of COX‐2 that is more important than COX‐1 in the development of keloids. Indeed, COX‐2 appears overexpressed in keloidal lesions.

5. CONCLUSIONS

In this study, we demonstrated clear negative effects of Parecoxib on keloids derived from fibroblast proliferation, even if we did not test these cells for relative COX‐1 and COX‐2 expression. The COX‐2 inhibitor is effective in inhibiting the proliferation of cancer cells, displaying COX‐2 overexpression, like colon, breast, bladder and prostate cancer cells. ³² , ³³ , ³⁴ , ³⁵ , ³⁶ Our findings suggest a potential use of the COX‐2 inhibitor, Parecoxib, as the therapy for keloids.

Grella R, Lanzano G, Faenza M, Ferraro G, Pieretti G. Parecoxib decreases cellular growth and Bcl‐2 protein levels in primary cultures of keloid fibroblasts. Int Wound J. 2024;21(3):e13946. doi: 10.1111/iwj.13946

DATA AVAILABILITY STATEMENT

Research data are not shared.