Research Article Cytoprotective responses in HaCaT keratinocytes exposed to high doses of curcumin Ditte M.S. Lundvig a , Sebastiaan W.C. Pennings a , Katrien M. Brouwer a , Matilda Mtaya-Mlangwa b , Emeria Mugonzibwa b , Anne Marie Kuijpers-Jagtman a , Frank A.D.T.G. Wagener a , Johannes W. Von den Hoff a,n a Department of Orthodontics and Craniofacial Biology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, PO Box 9101, 6500 HB Nijmegen, The Netherlands b Department of Preventive and Community Dentistry, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania article info Article history: Received 19 March 2015 Received in revised form 2 June 2015 Accepted 4 June 2015 Available online 10 June 2015 Keywords: Keratinocytes Curcumin Heme oxygenase-1 Ferritin Cytoprotection ROS abstract Wound healing is a complex process that involves the well-coordinated interactions of different cell types. Topical application of high doses of curcumin, a plant-derived polyphenol, enhances both normal and diabetic cutaneous wound healing in rodents. For optimal tissue repair interactions between epi- dermal keratinocytes and dermal fibroblasts are essential. We previously demonstrated that curcumin increased reactive oxygen species (ROS) formation and apoptosis in dermal fibroblasts, which could be prevented by pre-induction of the cytoprotective enzyme heme oxygenase (HO)-1. To better understand the effects of curcumin on wound repair, we now assessed the effects of high doses of curcumin on the survival of HaCaT keratinocytes and the role of the HO system. We exposed HaCaT keratinocytes to curcumin in the presence or absence of the HO-1 inducers heme (FePP) and cobalt protoporphyrin (CoPP). We then assessed cell survival, ROS formation, and caspase activation. Curcumin induced cas- pase-dependent apoptosis in HaCaT keratinocytes via a ROS-dependent mechanism. Both FePP and CoPP induced HO-1 expression, but only FePP protected against curcumin-induced ROS formation and cas- pase-mediated apoptosis. In the presence of curcumin, FePP but not CoPP induced the expression of the iron scavenger ferritin. Together, our data show that the induction of ferritin, but not HO, protects HaCaT keratinocytes against cytotoxic doses of curcumin. The differential response of fibroblasts and kerati- nocytes to high curcumin doses may provide the basis for improving curcumin-based wound healing therapies. & 2015 Elsevier Inc. All rights reserved. 1. Introduction Normal wound repair depends on the timely progression of the inflammatory, proliferative, and remodeling phases, and on the interactions between different cell types, growth factors, and cy- tokines [1]. The interactions between epidermal keratinocytes and dermal fibroblasts are essential for optimal tissue regeneration and remodeling [2–4], and disruptions may lead to pathological wound healing [5–7]. The topical application of high doses of curcumin, a plant-de- rived polyphenol, enhances both normal and diabetic cutaneous wound healing in rodents [8,9]. In contrast, in vitro studies have demonstrated that high doses of curcumin induce apoptosis in various cell lines via a ROS-dependent mechanism. This can be prevented by antioxidants such as N-acetyl cysteine and bilirubin [10–14]. The cytoprotective enzyme heme oxygenase-1 (HO-1) also exerts potent antioxidant effects by degrading free heme to CO, bililrubin and free iron [15–17]. The downstream induction of ferritin also protects against oxidative stress. Ferritin is induced by HO-1 via IRE-mediated posttranscriptional regulation through in- creased iron levels or via ARE-mediated gene induction [18]. HO-1 is normally expressed at low levels but can be strongly induced by its substrate heme protoporphyrin (FePP) and other cellular stresses, including oxidative stress [19]. Animals with impaired HO-1 function due to transgenic or pharmacological ablation demonstrate delayed wound closure and reduced angio- genesis, which can be restored by HO-1 induction [20]. Ther- apeutic HO-1 induction in skin cells may thus improve cell survival and function, wound healing, and reduce scarring. Pharmacologic Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/yexcr Experimental Cell Research http://dx.doi.org/10.1016/j.yexcr.2015.06.002 0014-4827/& 2015 Elsevier Inc. All rights reserved. Abbreviations: ARE, antioxidant responsive element; BR, bilirubin; CoPP, cobalt protoporphyrin; FePP, heme; H 2 DCFDA, 2′,7′-dicholoro-fluorescin diacetate; HO-1, heme oxygenase-1; IRE, iron responsive element; NAC, N-acetyl cysteine; PI, propidium iodide; ROS, reactive oxygen species n Corresponding author. Fax: þ31 24 3540631. E-mail address: hans.vondenhoff@radboudumc.nl (J.W. Von den Hoff). Experimental Cell Research 336 (2015) 298–307