INTRODUCTION

Oxidative stress, associated with the formation of reactive oxygen species (ROS), appears when antioxidant levels decrease and plays an important role in the pathogenesis of various deleterious processes in humans such as inflammation, immunosuppression, aging and carcinogenesis [1,2].

ROS play a critical role in programmed cell death (PCD), which can occur via two pathways: apoptosis and autophagic death. Apoptosis is characterized by caspase activation, cell shrinkage, nuclear and cytoplasmic condensation, DNA fragmentation, and formation of the apoptosome [3,4]. Autophagy is characterized by the formation of vesicles in the cytoplasm, loss of the cytoplasmic material, and pyknosis of nuclear material within an intact membrane [5]. This mechanism is important for maintaining cell homeostasis and performs a prosurvival function under stress conditions [3,4]. Therefore, autophagy has gained much attention recently.

H₂O₂ produces oxygen radicals that cause direct oxidative stress to human keratinocytes, making it an ideal reagent for studying oxidative damage [6]. H₂O₂-induced cell injury may be prevented through antioxidants such as Chios Gum Mastic (CGM), a natural resin extracted from the leaves of Pistacia lentiscus, a plant endemic to the Greek island of Chios [7,8]. CGM has been used by traditional healers for the relief of upper abdominal discomfort, stomach aches, dyspepsia, and peptic ulcer [9-11] and it has antibacterial and antifungal properties and therapeutic benefits for the skin because of its softening, soothing, and healing properties [2]. CGM also reduces the formation of dental plaque and bacterial growth in oral saliva; therefore, it is used in many organic toothpastes and mouthwash solutions to promote fresh breath and prevent dental caries [12,13]. Recent studies have been demonstrated an antioxidant activity in CGM [8].

TNatural products were the main source of health care products in ancient times. In modern medicine, they are still major sources of new drug development [14,15]. Although CGM has been widely investigated, its protective effect against oxidative damage to keratinocytes, as well as the relationship between CGM and autophagy, has not been investigated. The aim of this study was to assess the protective effect of CGM against H₂O₂-induced oxidative stress and to evaluate the autophagic features induced by CGM in human keratinocytes.

Materials and Methods

Reagents

Chios gum mastic was obtained from mastic korea (Seoul, Korea). The following reagents were obtained commercially: 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT), acridine orange, monodansylcadaverine (MDC) were purchased from Sigma (St. Louis, MO, USA). 3-Methyladenine (3-MA, class III PI3K inhibitor) was obtained from Calbiochem (La Jolla, CA, USA).

Antibodies against the cleaved form of caspase-3, caspase-8, PARP and Beclin-1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against LC3 (Sigma) were also used. The caspase-9, p62/SQSTM1, ATG5-ATG12 complex, GAPDH, mouse anti-actin antibody, mouse anti-rabbit IgG antibody, and rabbit anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals and reagents were purchased from Sigma unless otherwise specified.

Cell culture

Human keratinocytes (HaCaT) were purchased from the ATCC (Rockville, MD, USA). They were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% penicillinstreptomycin (GIBCO-BRL, Rockville, MD, USA).

Twenty four hours after HaCaT cells were subcultured, the original medium was removed. The cells were washed with phosphate-buffered saline (PBS) and then incubated in the same fresh medium. Cells were cultured at 37°C in a humidified 5% CO₂ incubator.

Treatment of CGM

CGM was dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 mM and was kept frozen at -20°C until use. The stock was diluted to the required concentration with DMEM when needed. Prior to CGM treatment cells were grown to about 80% confluence and then exposed to CGM at different concentrations (0 - 50 µ M) for a different periods of time (0 - 24 h). Cells grown in medium containing an equivalent amount of DMSO without CGM served as control. For autophagy control, cells were grown in Earle's Balanced Salt Solution (EBSS, GIBCO-BRL). The concentrations of DMSO, 0.002 - 0.1% (vol/vol) used in this study, both as a vehicle for CGM and as a control, had no effect on HaCaT cells proliferation in our preliminary studies.

Cell viability assay

Cells were placed in a 96-well plates and then incubated for different time in the presence of various doses of CGM (0 - 50 µM) or H₂O₂ (0 - 500 µM). At the required time point, the medium was removed and 100 µl of MTT (500 mg/mL) was added to each well. The plates were incubated at 37°C for 4 h. After incubation, the medium was removed from all the wells. The formazan crystals that formed were then solubilized in 100 µl of DMSO. The coloured solution was quantified at 570 nm using a ELISA reader (Tecan, Mnnedorf, Switzerland). Cell viability was determined as percent of the control.

Hoechst staining

Cells were harvested and cytocentrifuged onto a clean, fat-free glass slide with a cytocentrifuge. Cells were stained in 4 µg/ml Hoechst 33342 for 10 min at 37°C in the dark and were washed twice in PBS. The slides were mounted with glycerol. The samples were observed and photographed under an epifluorescence microscope (Carl Zeiss, Goettingen, Germany). The number of cells that showed condensed or fragmented nuclei was determined by a blinded observer from a random sampling of 3 × 10 ² cells per experiment.

Flow cytometer analysis

To quantify the development of acidic vesicular organelles (AVOs), the cells were stained with acridine orange (1 µg/mL) for 15 min, removed from the plate with trypsin-EDTA (GIBCO-BRL), and analyzed using a FACScan flow cytometer.

For autophagy inhibition, cells were pretreated with 1 mM 3-MA for 1 h and incubated with 10 µM CGM for 24 h. In order to quantification of DNA hypoploidy, cells were harvested by trypsinization, and ice cold 95% ethanol with 0.5% Tween 20 was added to the cell suspensions to a final concentration of 70% ethanol. After overnight fixed cells were washed in 1% Bovin serum albumin (BSA)-PBS solution, and then resuspended in PBS containing 40 mg/mL PI, 0.5 mg/mL RNase A. After 30 min at 37°C in the dark, the cells were analyzed with a flow cytometer system (Beckman Coulter, FL, CA, USA) and data was analyzed using the Multicycle software which allowed a simultaneous estimation of cell-cycle parameters and apoptosis.

MDC and AO staining

Cells were grown on coverslips and stained with 0.05 mM MDC at 37°C for 1 h. The cellular fluorescence changes were observed using a fluorescence microscope (Axioskop, Carl Zeiss, Germany). As an autophagy control, cells were starved using EBSS. For further detection of the acidic cellular compartment, we used acridine orange, which emits bright red fluorescence in acidic vesicles but fluoresces green in the cytoplasm and nucleus. Cells were stained with 1 µg/mL acridine orange for 15 min and washed with PBS. Pictures were obtained with a fluorescence microscope (Axioskop, Carl Zeiss, Germany).

Western blot analysis

Cells (2 × 10⁶) were washed twice in ice-cold PBS, resuspended in 200 µl ice-cold solubilizing buffer [300 mM NaCl, 50 mM Tris-Cl (pH 7.6), 0.5% Triton X-100, 2 mM PMSF, 2 µl/ml aprotinin and 2 µl/ml leupeptin] and incubated at 4°C for 30 min. The lysates were centrifuged at 14,000 revolutions per min for 15 min at 4°C. Protein concentrations of cell lysates were determined with Bradford protein assay (Bio-Rad, Richmond, CA, USA) and 20 µg of proteins were resoved by 12.5% SDS/PAGE. The gels were transferred to Polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and reacted with appropriate primary antibodies. Immunostaining with secondary antibodies was detected using SuperSignal West Femto (Pierce, Rockford, IL, USA) enhanced chemiluminescence substrate and detected with Alpha Imager HP (Alpha Innotech, Santa Clara, USA).

Statistical analysis

Three independent experiments were performed for each experimental group and each experiment was performed in triplicate. The results of the experimental and control groups were compared for statistical significance (p<0.01 and 0.05) using paired T-test statistical method by SPSS for Win 12.0 for summary data.

Results

CGM protected HaCaT cells against H2O2-induced cell death

Quantitative assessment of H₂O₂ cytotoxicity in HaCaT cells was conducted by exposing cells to different concentrations of H₂O₂. After 2 h, the cells were assayed for cell viability. As shown in Fig. 1A, H₂O₂ dose-dependently damaged the cells, as exposure to different concentrations of H₂O₂ (50, 100, 250, and 500 µ M), reduced cell viability to 94.8, 80.3, 62.1, and 34.8 %, respectively. The effect of H₂O₂ cytotoxicity on HaCaT cells was also examined by exposing the cells to H₂O₂ (250 µM) for different durations (1, 2, 4, and 6 h). Time-dependent increases in cytotoxicity were observed (Fig. 1B).

The effect of CGM on HaCaT cells was examined by treating the cells with different concentrations of CGM (0 - 50 µM). After 24 h, the cells showed viabilities of 102.7 and 105.2 %, at 5 and 10 µM CGM, respectively (Fig. 1C). Exposure of the cells to CGM (10 µM) for different durations (24, 48, and 72 h) resulted in increased cell viability to 104.9, 112.1, and 117.2 %, respectively (Fig. 1D).

The protective effect of CGM against H₂O₂-induced cell death was examined by treating HaCaT cells with 10 µM CGM for 24 h, and then exposing the cells to H₂O₂ (250 µM) for 2 h. Pretreatment of cells with 10 µM CGM significantly increased the cell viability compared to the viability of cells exposed to H₂O₂ alone (Fig. 1E).

CGM treatment decreased H2O2-induced apoptosis in HaCaT cells

Apoptosis in HaCaT cells was examined morphologically after Hoechst staining. Exposure to H₂O₂ for 2 h caused a change in nuclear morphology, but pretreatment of cells with CGM prevented that cell damage. Pretreatment with CGM significantly reduced apoptosis in H₂O₂-exposed HaCaT cells (Fig. 2). The protective effect of CGM against apoptotic cell death was further confirmed at the molecular level by investigation of the effect of H₂O₂ and CGM on the protein levels of markers of apoptosis, including caspase-3, caspase-8, caspase-9, and PARP. Western blotting of extracts from HaCaT cells treated with 10 µM CGM for 24 h, followed by exposure to H₂O₂ for 2 h, revealed that the cleavage of caspase-3 was prevented in cells pretreated with CGM indicating that CGM has the capability of suppressing the activation of caspase-3. CGM pretreatment also promoted the degradation of caspase-8, caspase-9 and induced the formation of the processed PARP 85 kDa (Fig. 3).

Inhibition of CGM-induced autophagy increased apoptosis in HaCaT cells

We clarified the role of CGM-induced autophagy in HaCaT cells by investigating the consequences of treatment with 3-methyladenine (3-MA), a selective autophagy inhibitor. The inhibitory effects on acidic vesicular organelle (AVO) formation by 3-MA were confirmed by quantitatively measurement of the red-to-green fluorescence ratio after AO staining (Fig. 4A). We validated whether CGM-induced autophagy was attributable to a reduction in apoptosis by blocking autophagy by 3-MA or by inducing autophagy by serum starvation. Flow cytometry analysis showed that a single CGM treatment caused 4.3% apoptosis, while 3-MA pretreatment increased the percentage of apoptosis to 9.6% while the serum starvation condition showed 1.7% apoptosis (Fig. 4B).

CGM treatment leads to induction of autophagy in HaCaT cells

We evaluated autophagy morphologically using the autofluorescent drug monodansylcadaverine (MDC) to detect autophagic vacuoles. When cells were stained with MDC, a selective fluorescent marker of autophagic vesicles, CGM-treated cells exhibited strong staining compared to the untreated control group (Fig. 5A). We also used acridine orange (AO) staining as another method to detect autophagic vacuoles. Treatment with CGM caused an increase in vesicle formation compared to control group (Fig. 5B), indicating significantly induced autophagy.

We determined whether CGM activated or repressed autophagy factors in HaCaT cells by measuring the amounts of these factors by western blotting. The level of p62/SQSTM1, a protein that is degraded by autophagy, was reduced and conversion of LC3-I to LC3-II was increased in CGM treated HaCaT cells. Treatment with CGM increased ATG5-ATG12 complex (Fig. 5C).

Discussion

Oxidative stress has drawn considerable attention in the field of dermatology. It is believed that H₂O₂-induced oxidative stress of keratinocytes can cause many skin disease, such as vitiligo and skin aging [16,17]. H₂O₂ is a form of ROS that is responsible for a variety of neurodegenerative and aging-related diseases [18]. This molecule is a signal transducer that elicits autophagy and/or apoptosis when present at high levels [19,20]. Previous studies have shown that autophagy plays an important role in promoting cell survival against apoptosis [21]. Some experiments have revealed a positive side to autophagy. Autophagy mostly functions as a survival mechanism while apoptosis leads to cell death. However, because of simultaneous activation of autophagy and apoptosis during stress conditions, their functions integrate with each other's, making it difficult to determine their role [22]. Thus, we conducted an experiment in which both apoptotic and autophagic factors were active in order to determine the action of CGM in the presence of oxidative stress. We used H₂O₂ as the inducer of oxidative stress.

We found that H₂O₂-induced cell death and that CGM had a protective effect in HaCaT cells (Fig. 1A, 1B, 1C, & 1D). In addition, CGM protected HaCaT cells against H₂O₂-induced cell death (Fig. 1E). Cell staining with Hoechst in general confirmed that pre-treatment with CGM reduced apoptosis. Hoechst staining discriminates live cells from dead ones by showing DNA fragmentation and condensation of chromatin, which cause the cells to brightly fluoresce. CGM protected keratinocytes against H₂O₂-induced apoptosis, detected by Hoechst staining as a decrease in chromatin condensation (Fig. 2).

We explored this process in more detail by western blot analysis. We measured activation of caspase-3, caspase-8, caspase-9 and PARP, which are associated with apoptosis. Caspases are major players in apoptotic cell death, and activated caspase-3 is responsible for initiation of apoptosis. When the cells were pretreated with at 10M CGM, the levels of these apoptotic factors decreased (Fig. 3), confirming a protective role of CGM.

The specific feature of autophagy is autophagosome formation. We detected autophagic vesicles by using MDC, a fluorescent dye that specifically labels autophagic vesicles. This autofluorescent drug is believed to be a selective marker for the autophagolysosome [23]. This assay allows autophagy to be monitored easily and accurately by measuring the fluorescence of MDC incorporated by the cells under different experimental conditions [24, 25]. MDC incorporation points to the occurrence of autophagy [24]. The results of MDC staining are shown in Fig. 5A. We then investigated the progression of autophagy by AO staining, which showed acidic compartments containing hydrolytic enzymes [26]. AO is a permeable weak base dye which shows green fluorescence in neutral conditions but fluoresces red in acidic compartments of cells. The appearance of red fluorescence indicates dye aggregation in acidic vesicular organelles. When more acidic vacuoles are formed in cell, more red fluorescence will be detected [27,28]. Exposure of cells to CGM significantly increased autophagy (Fig. 5A & B).

Autophagy is characterized by the formation and promotion of AVOs. We next determined whether 3-MA could inhibit CGM-induced autophagy by examining the accumulation of AVOs. Our results showed that the CGM treated group showed a greater accumulation of AVOs than did the 3-MA pretreated group (Fig. 4A). We also analyzed the percentage of apoptosis to validate whether CGM-induced autophagy contributed to the reduction in apoptosis. The percentage of cells undergoing apoptosis in the 3-MA pre-treated group was higher than in the group treated with CGM alone. The cells in the serum starvation condition, a positive control for autophagy, had the lowest percentage of apoptosis (Fig. 4B). Therefore, CGM-induced autophagy is related to the reduction in apoptosis, indicating that autophagy had a survival function.

The process of autophagosome formation depends on several autophagy proteins [29,30], so we measured the quantity of p62/SQSTM1, ATG5-ATG12 complex, Beclin-1, and LC3, which are associated with autophagy (Fig. 5C). Translational modification of LC3 causes it to localize exclusively on the autophagosomal membranes; thus, it is considered as the chief autophagy marker [31]. Autophagy mediates a nonspecific bulk degradation pathway responsible for degradation of the majority of long lived proteins and some organelles. ATG5-ATG12 conjugation systems are necessary for the formation of the autophagosome [32]. Several recent studies using different cell types and stimuli have also described that caspase-mediated cleavage of Beclin-1 and ATG proteins enhances apoptosis [33-36]. In our case, CGM suppressed p62 expression, induced ATG5-ATG12, and enhanced the LC3-I to LC3-II conversion in HaCaT cells.

In conclusion, our data suggest that CGM-induced autophagy is a pro-survival mechanism in HaCaT cells exposed to H₂O₂. Also CGM helps cells to survive under stress conditions by preventing apoptosis and enhancing autophagy. Therefore, the present investigation provides evidence to support the antioxidant potential of CGM in vitro and opens up a new horizon for future experiments.