British Journal of Dermatology:Volume 148(3)March 2003p 544-552
Suppression of insulin-like growth factor signalling pathway and collagen expression in keloid-derived fibroblasts by quercetin: its therapeutic potential use in the treatment and/or prevention of keloids
[Original Articles: Therapeutics]
PHAN, T. T.; SEE, P.; TRAN, E.*; NGUYEN, T. T.T.*; CHAN, S. Y.†; LEE, S. T.; HUYNH, H.
Department of Plastic Surgery, Singapore General Hospital, Singapore (PHAN) (SEE) (LEE) (HUYNH)
*Division of Molecular and Cellular Research, National Cancer Centre, Singapore
†Department of Pharmacy, National University of Singapore, Singapore
Correspondence: Hung Huynh, Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre of Singapore, Singapore 169610. E-mail: firstname.lastname@example.org
Accepted for publication 7 August 2002
Background: Keloids are characterized by abnormal proliferation of fibroblasts and overproduction of collagen. Insulin-like growth factor (IGF)-I is mitogenic for fibroblasts and a stimulatory factor for collagen synthesis.
Objectives: We have assessed the in vitro effects of quercetin on proliferation, collagen synthesis and the expression of the IGF system in keloid-derived fibroblasts.
Methods: Fibroblasts were isolated from earlobe keloids and exposed to quercetin at different concentrations. The inhibitory effects of quercetin on fibroblast proliferation were assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western and Northern blot analyses.
Results: Quercetin inhibited keloid fibroblast (KF) proliferation in a dose-dependent manner. Significant growth inhibition was observed on day 2 of culture. The dose required for 50% growth inhibition was approximately 25 µg mL-1. Collagen 1 expression was significantly decreased while collagen 3 was almost undetectable following quercetin treatment. Basal levels of IGF-I receptor (IGF-IR) ß subunits, p85 subunit of phosphatidylinositol 3-kinase, c-Raf, phospho-Raf-1, phospho-MEK 1/2, phospho-mitogen-activated protein kinase, phospho-Elk-1 and phospho-Akt-1 were significantly reduced when KF cells were exposed to quercetin for 24 h. Blocking IGF-IR activity with IGF-IR antibody or neutralizing endogenous IGF-I activity with IGF-I antibody led to significant growth inhibition suggesting the role of IGF-I in regulation of KF proliferation.
Conclusions: Because the IGF system plays an important part in fibroblast cell proliferation and collagen production, the described activities of quercetin on the IGF system and collagen expression may provide a novel approach for the use of quercetin in treatment and/or prevention of hypertrophic scar and keloid.
Keloids are proliferative dermal growths that represent a pathological wound healing response to skin injury in susceptible persons. Keloid scar formation has afflicted humans for centuries 1 and is a disease entity that has, as yet, no animal model. Much has been written about the epidemiology of keloids, 2,3 and much work has been done to elucidate their microstructure. 4-6 Keloid fibroblasts (KFs) overproduce collagen by as much as 20 times more than normal skin fibroblasts. 7,8 The exact pathophysiology, however, remains largely unknown, although tension has been suggested to be one factor involved, another being skin pigmentation. 3 Keloid tissue has long been noted to extend beyond the borders of the original wound, to not regress spontaneously and, notoriously, to be prone to recurrence. 9 The difficulty in treating keloids can be seen by the large number of largely empirical modalities of treatment. 2,10 The most common mode of treatment for keloids is intralesional injection of corticosteroids which is often not very effective. 11 Thus, there is an urgent need for novel treatment strategies of keloids.
The most common flavonoid glycones found in the diet are quercetin, rutin and robinin. 12,13 The richest sources of quercetin are found in onions, apples and red wine. 14 Quercetin is also identified as an active ingredient of medicinal plants. 13 Quercetin has a wide range of biological activities including inhibition of the Na+K+ATPase, 15 protein kinase C, 16 tyrosine kinase, 17 human immunodeficiency virus reverse transcriptase 18 and pp60src kinase. It is a potent inhibitor of enzymes involved in the proliferation of signalling pathways including phosphatidylinositol 3-kinase (PI-3K) 19 and 1-phosphatidylinositol 4-kinase. 20 It causes the cell cycle arrest 21,22 and apoptosis. 23
Insulin-like growth factors (IGF)-I and -II are potent mitogens and inhibitors of apoptosis for cell types. 24 IGF-I has been shown to stimulate fibroblast proliferation and enhances collagen synthesis. 25 Both IGF-I and IGF-II bind type I IGF receptor (IGF-IR). 24,26 Overexpression of IGF-IR made KFs become resistant to ceramide-induced apoptosis. 27 IGF/IGF-IR signal mediates the invasiveness of KFs. 28 IGF binding activates IGF-IR, which in turn phosphorylates PI-3K and Ras/Raf/mitogen-activated protein kinase (MAPK). 29 Ras/Raf/MAPK and PI-3K play important roles in IGF-IR-induced cellular proliferation and the inhibition of apoptosis. 30 IGFs also bind with high affinity to specific IGF-binding proteins (IGFBPs), which modulate their bioactivity. Early studies attributed the growth inhibitory action of IGFBP to the reduction of IGF-I and/or IGF-II bioactivity resulting from competition for somatomedins between IGFBPs and IGF-IR. 24,26
As keloids are characterized by excessive deposition of collagen in the dermis by fibroblasts 11 and IGF-I enhances collagen synthesis, 25,31 we undertook this in vitro study to ascertain if quercetin treatment could inhibit keloid-derived fibroblast proliferation, the IGF signalling pathways and collagen expression. Here we report that treatment of KFs with quercetin led to growth inhibition. Expression of several key proteins involved in IGF signal cascades and their basal phosphorylation as well as collagens were significantly inhibited by quercetin. The data suggest that quercetin can be used either as a single agent or in combination with other agents in the treatment or the prevention of keloid formation.
© 2003 British Association of Dermatologists