Resveratrol Induces Long-Lasting IL-8 Expression and Peculiar EGFR Activation/Distribution in Human Keratinocytes: Mechanisms and Implications for Skin Administration

et al. (2013) Resveratrol Induces Long-Lasting IL-8 Expression and Peculiar EGFR Activation/ Distribution in Human Keratinocytes: Mechanisms and Implications for Skin Administration. PLoS ONE 8(3): e59632. doi:10.1371/journal.pone.0059632 Resveratrol Induces Long-Lasting IL-8 Expression and Peculiar EGFR Activation/Distribution in Human Keratinocytes: Mechanisms and Implications for Skin Administration Saveria Pastore 0 Daniela Lulli 0 Riccardo Maurelli 0 Elena Dellambra 0 Chiara De Luca 0 Liudmila G. Korkina 0 Andrzej T. Slominski, University of Tennessee, United States of America 0 Lab. Tissue Engineering and Skin Pathophysiology, Dermatology Institute (Istituto Dermopatico dell'Immacolata, IDI IRCCS) , Rome , Italy Anti-inflammatory and skin tumour preventing effects of resveratrol have been extensively studied pre-clinically and resveratrol has been proposed for clinical investigations. To provide a basis or/and limitations for topical administration to human skin, molecular mechanisms underlying resveratrol effects towards normal human epidermal keratinocytes (NHEK) were evaluated. NHEK were challenged by either resveratrol alone or by its combination with TNFalpha or TGFalpha, and time-dependent molecular events were monitored. Interleukin 8 (IL-8) expression and its mRNA stability, ERK1/2, p65/RelA, and EGFR phosphorylation were determined. Intracellular distribution of EGFR/P-EGFR was measured in the membrane, cytoplasmic, and nuclear fractions. Specific DNA binding activity of NFkB (p65/RelA) and AP-1(c-Fos), NHEK proliferation, and molecular markers of apoptosis/cell cycle were detected. Resveratrol induced delayed, long-lasting and steadily growing IL-8 gene and protein over-expression as well as enhanced EGFR phosphorylation, both abrogated by the EGFR kinase inhibitor PD168393. However, resveratrol did not act as a phosphatase inhibitor. ERK phosphorylation was transiently inhibited at early time-points and activated at 6-24 h. Accordingly, c-Fos-specific DNA binding was increased by resveratrol. Cellular distribution of EGFR/P-EGFR was shifted to membrane and nucleus while cytosolic levels were reduced concomitant with enhanced degradation. Notwithstanding high nuclear levels of EGFR/P-EGFR, spontaneous and TGFalpha-triggered cell proliferation was strongly suppressed by resveratrol mainly through cell cycle arrest. Conclusions/Significance: Resveratrol synergized with TNFa in the induction of delayed, long-lasting IL-8 expression through sustained EGFR-ERK axis activation. The time course indicates that resveratrol metabolites could be implicated. Topical administration of Resv to psoriatic patients over-expressing TNFa, IL-8 and EGFR-ERK in the skin should be cautiously considered. Since high nuclear levels of EGFR correspond to increased risk of tumorigenesis, chronic resveratrol application to the skin may be potentially dangerous. Wound healing acceleration by resveratrol could not be envisaged due to its anti-proliferative effects towards normal keratinocytes. - Funding: The work was financed by grant RC-2011-IDI IRCCS from the Italian Ministry for Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors do not have any competing interests. The plant polyphenol resveratrol (Resv, 3,49,5-trihydroxystilbene), naturally occurring in a number of fruits and other food products, has been extensively studied during the last two decades for its cancer chemopreventive and anti-cancer properties. Its chemopreventive potential has first emerged amid inhibition of experimental carcinogenesis at the stages of initiation, promotion, and progression [1]. A kind of Resv addiction (cit. [2]) results in a steadily growing number of in vitro and in vivo studies attempting to provide evidence of numerous health effects of Resv ranging from anti-inflammatory to cardiovascular disease preventive, cancer chemopreventive, and to age delaying activities. On the basis of promising pre-clinical data, Resv was recommended for clinical trials as extensively reviewed in [2,3,4], while its natural and synthetic analogues have been experimented in vitro [5,6]. However, recent systematic review of publications on biological and clinical effects of Resv [4] did not justify its administration to humans, beyond the dose which can be obtained from dietary sources. Taking into account that (i) proposed chronic administration of Resv to the skin as a chemopreventive approach [2,7,8] could affect different cellular and non-cellular components of this complex organ [9,10], (ii) topically applied Resv could interact with numerous environmental factors, such as UV, heavy metals, and organic toxins [1113], and (iii) Resv metabolism/ bioavailability through the skin differs from that in the gut, thorough evaluation of non-dietary ways of Resv use should be warranted. In the widely discussed cancer chemopreventive concept, ideal substance(s), being administered chronically, should prevent, slow down or reverse tumorigenic transformation/tumor formation. At the same time, deregulating or damaging effects towards normal cells/tissues should be minimal. While the majority of mechanistic studies demonstrate that Resv selectively affects aberrant molecular pathways in tumor cells, there is emerging evidence that normal cells such as endotheliocytes, lymphocytes, smooth muscle cells, chondrocytes, tendocytes [2,8,14], adipocytes [15,16], neurons [17], osteoblasts, hepatic cells, and epidermal keratinocytes [1820] are vulnerable to Resv as well. Numerous mechanisms have been proposed as underlying the modulation of tumor cell proliferation and death-versus-survival strategy by this polyphenol [2,3,21]. Thus, Resv interaction with nuclear receptors, such as estrogen, androgen, and aryl hydrocarbon receptors is thought to greatly influence cell proliferation and survival [2225]. It has also been shown that various survival factors (e.g. survival proteins, kinases, and transcription factors) are inhibited by Resv favoring cell death [2] by multiple mechanisms including apoptosis, autophagy, or mitotic block [21,26]. Since pro- versus anti-proliferative effects of Resv towards tumor cells [27] and stem cells [28] depended on its concentration and duration of cell contact with Resv, a warning has been issued on the chronic use of Resv as a chemopreventive agent. In a single publication on strong anti-proliferative effects of Resv to normal keratinocytes [9], a serious concern has also been expressed about feasibility of this plant polyphenol for skin cancer chemoprevention. However, mechanisms, by which Resv exerts its effects on proliferation of normal cells, have never been evaluated. The EGF receptor (EGFR) and its ligands represent one of the most powerful and complex signaling networks in the skin of higher vertebrates. This system exerts a major impact on keratinocyte proliferation (...truncated)