Caffeine Protects Human Skin Fibroblasts From Acute Reactive Oxygen Species-Induced Necrosis

November 2012 | Volume 11 | Issue 11 | Original Article | 1342 | Copyright © November 2012

Oxidative damage by reactive oxygen species (ROS) plays a major role in aging and carcinogenesis. Little is known about either the effects of acute ROS in necrosis and inflammation of skin or the therapeutic agents for prevention and treatment. Previously, our laboratory identified caffeine as an inhibitor of hydrogen peroxide (H2O2)-generated lipid peroxidation products in human skin fibroblasts. Here, we study effects of caffeine on acute ROS-mediated necrosis. Human skin fibroblasts were incubated with caffeine, followed by H2O2 challenge. Flow cytometry was used to analyze cell morphology, counts, apoptosis and necrosis, and ROS. We found that caffeine protects from H2O2 cell damage at lower (0.01 mM) and intermediate (0.1 mM) doses. The beneficial effects of caffeine appear to be mediated by a mechanism other than antioxidant function.

J Drugs Dermatol. 2012;11(11):1342-1346.


Ultraviolet (UV) radiation generates hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) that may result in damage to DNA, RNA, lipids, and proteins as well as cell death.1-4 Reactive oxygen species are an important inducer of human skin aging, carcinogenesis, and inflammation5,6 and are generated both exogenously, including environmental (pollution, smoking, UV radiation) factors, and endogenously, including normal metabolic processes, such as mitochondria-based cytochrome P450 cycling, which is linked to intrinsic aging.7 Oxidative damage to cells that is induced by ROS is highly regulated by a variety of factors, including cellular metabolism and repair in addition to environmental, hormonal, nutritional, and toxicological contributions.8
With increasing evidence suggesting a central role of ROS in cell death, it is logical to investigate novel antioxidant agents that protect skin against ROS-induced toxicity. Little is known regarding the role of ROS in human skin cell necrosis or the ability of caffeine to protect against necrosis. Caffeine is a naturally occurring nutraceutical described to have antioxidant and carcinogenic effects.9-14
The goal of this study was to evaluate the ability of caffeine to prevent normal human skin fibroblast cell death in an in vitro model of acute ROS exposure. We found that caffeine prevents necrosis in human skin fibroblasts independent of antioxidant mechanisms.


Caffeine Preparation

Caffeine was prepared as a 1 mM stock solution as previously described,15 followed by 1:10 serial dilutions to 0.1 and 0.01 mM final concentrations.

Cell Culture

Human skin fibroblasts (AG14135; Coriell Institute for Medical Research, Camden, NJ) were grown and treated in culture, as previously described.16 Briefly, plated cells were washed twice with Dulbecco's modified Eagle medium ([DMEM], Gibco; Invitrogen, Carlsbad, CA), followed by a 4-hour incubation with various concentrations of caffeine (1, 0.1, and 0.01 mM) in a 95:5% air:carbon dioxide (CO2.) humidified incubator at 37°C. After incubation, plated cells were washed twice with DMEM, followed by incubation with H2O2 (1.2 mM) for 30 or 120 minutes, or no H2O2 for controls. After incubation, plated cells were prepared for mitochondrial membrane potential or apoptosis/necrosis assays.

Morphology and Analysis of Absolute Cell Counts

Determination of cell morphology after H2O2 challenge was performed using defined gates based on forward and side scatter, as previously described.16 Determination of absolute cell counts after H2O2 challenge was performed using Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA), as previously described.16

Apoptosis and Necrosis

Flow cytometry was used to analyze apoptotic and necrotic cell death, as previously described.16 Briefly, apoptosis and necrosis were distinguished by the combination of labeling of annexin V (AV) and propidium iodide ([PI]; BD Biosciences Pharmingen, San Jose, CA). AV-PI- was defined as viable cells; AV+PI- was defined as early apoptosis; AV-PI+ was defined as early necrosis; and AV+PI+ was defined as late stage cell death, either by apoptosis or necrosis.

Reactive Oxygen Species

Intracellular ROS were labeled by mitochondrial uptake of dihydrorhodamine 123 and measured by flow cytometry, as