The ethyl acetate fraction of Sargassum muticum attenuates ultraviolet B radiation-induced apoptotic cell death via regulation of MAPK- and caspase-dependent signaling pathways in human HaCaT keratinocytes

Abstract

Context: Our previous work demonstrated that an ethyl acetate extract derived from Sargassum muticum (Yendo) Fenshol (SME) protected human HaCaT keratinocytes against ultraviolet B (UVB)-induced oxidative stress by increasing antioxidant activity in the cells, thereby inhibiting apoptosis.

Objective: The aim of the current study was to further elucidate the anti-apoptotic mechanism of SME against UVB-induced cell damage.

Materials and methods: The expression levels of several apoptotic-associated and mitogen-activated kinase (MAPK) signaling proteins were determined by western blot analysis of UVB-irradiated HaCaT cells with or without prior SME treatment. In addition, the loss of mitochondrial membrane potential (Δψ_m) was detected using flow cytometry or confocal microscopy and the mitochondria membrane-permeate dye, JC-1. Apoptosis was assessed by quantifying DNA fragmentation and apoptotic body formation. Furthermore, cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Results: SME absorbed electromagnetic radiation in the UVB range (280–320 nm) of the UV/visible light spectrum. SME also increased Bcl-2 and Mcl-1 expression in UVB-irradiated cells and decreased the Bax expression. Moreover, SME inhibited the UVB-induced disruption of mitochondrial membrane potential and prevented UVB-mediated increases in activated caspase-9 and caspase-3 (an apoptotic initiator and executor, respectively) levels. Notably, treatment with a pan-caspase inhibitor enhanced the anti-apoptotic effects of SME in UVB-irradiated cells. Finally, SME reduced the UVB-mediated phosphorylation of p38 MAPK and JNK, and prevented the UVB-mediated dephosphorylation of Erk1/2 and Akt.

Discussion and conclusion: The present results indicate that SME safeguards HaCaT keratinocytes from UVB-mediated apoptosis by inhibiting a caspase-dependent signaling pathway.

• Apoptosis
• caspase
• mitochondrial membrane potential
• mitogen-activated protein kinase

Introduction

The epidermis is exposed to various external stimuli throughout the life of an organism. Of these, ultraviolet (UV) radiation is perhaps the most important. While UV light is essential for human life, it is also harmful; it can induce cancer, immunosuppression, photoaging, inflammation, and apoptosis (a form of programmed cell death) (Jinlian et al., 2007).

Two major apoptosis-associated pathways converge on the effector caspases: the intrinsic and extrinsic cell death pathways. The intrinsic cell death pathway, also known as the mitochondrial apoptotic pathway, is activated by a wide range of signals, including radiation, cytotoxic drugs, cellular stress, and growth factor withdrawal. The intrinsic cell death pathway involves the release of cytochrome c and other proteins from the mitochondrial membrane space (Reed & Pellecchia, 2005) and is regulated by the β-cell lymphoma 2 (Bcl-2) family of proteins. The Bcl-2 family contains both anti-apoptotic proteins [e.g., Bcl-2, myeloid cell factor-1 (Mcl-1), Bcl-xL, and Bcl-w] and pro-apoptotic proteins [e.g., Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), and Bcl-2 homology 3 (BH3) interacting-domain death agonist (Bid)]. Thus, the balance between pro-apoptotic and anti-apoptotic proteins controls cell survival and cell death.

Caspases are proteases that are activated during the early stages of apoptosis by cleavage at specific aspartate residues. Active caspases then go on to cleave additional proteins, including poly-ADP ribose polymerase and other caspases. Eventually, the caspases cleave multiple protein substrates, leading to the irrevocable loss of cellular structure and function, which ultimately results in cell death (Salvesen & Riedl, 2008). In particular, caspases -8, -9, and -3 are implicated in the progression of apoptosis: caspase-9 (an apoptotic initiator) in the mitochondrial intrinsic cell death pathway, caspase-8 in the Fas/cluster of differentiation 95 pathway, and caspase-3 (an apoptotic executor) in multiple downstream signaling pathways.

Mitogen-activated protein kinases (MAPKs) are members of the serine/threonine kinase family and include p38 MAPK, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (Erk1/2). MAPKs are activated by external stress stimuli, such as heat shock, cytokines, and UV radiation, and are involved in cellular proliferation, survival, and apoptosis. Activation of certain MAPKs is closely correlated with the induction of stress-provoked apoptosis (Keshet & Seger, 2010; Kyosseva, 2004; Sumbayev & Yasinska, 2005). For instance, UVB radiation triggers apoptosis in human keratinocytes by activating the p38 MAPK (Nys et al., 2010) and JNK pathways (Cao et al., 2009; Ramaswamy et al., 1998).

Intensive research efforts have been directed toward identifying phytochemical components and extracts that can efficiently protect sun-exposed skin from the deleterious actions of UV radiation. Sargassum muticum (Yendo) Fenshol is an edible brown alga that is widely distributed on the seashores of southern and eastern Korea. Extracts derived from S. muticum demonstrate antioxidant, antimicrobial, and anti-inflammatory properties (Kim et al., 2007; Yoon et al., 2010). Previous work by our group revealed that an ethyl acetate fraction of S. muticum (SME) defended human HaCaT keratinocytes against UVB exposure by enhancing cellular antioxidant systems (Piao et al., 2011). However, the molecular mechanism underlying the action of SME was not elucidated. The present study examined the ability of SME to shield human HaCaT keratinocytes from UVB-induced damage, focusing on its effects on MAPK- and caspase-dependent signaling cascades.

Materials and methods

Preparation of SME

Sargassum muticum specimens were collected from the coast of Daryeodo (Jeju, Republic of Korea). The algae were identified by Dr. Dong Sam Kim (Jeju Biodiversity Research Institute, Jeju, Republic of Korea). A voucher specimen (A06-0000080) has been deposited at the herbarium of Jeju Biodiversity Research Institute. The extraction process of S. muticum was previously reported (Piao et al., 2011); the dried S. muticum was extracted with 80% ethanol at room temperature for 24 h and was then evaporated under reflux. Next, the 80% ethanol extract was suspended in distilled water and successively fractionated with n-hexane, dichloromethane, ethyl acetate, and n-butanol. The ethyl acetate fraction (SME) was used for further study.

Reagents

The primary anti-Mcl-1, -caspase-9, -caspase-3, -PARP, -phospho-p38 (Thr180/Tyr182) (28B10), -phospho-Erk1/2 (Thr202/Tyr204), -Erk1/2, -phospho-stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185), -SAPK/JNK, and -phospho-Akt (Ser473) antibodies were purchased from Cell Signaling Technology (Beverly, MA). The primary anti-Bcl-2 (N-19), -Bax (B-9), and -P38 (C-20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) was purchased from Invitrogen (Carlsbad, CA). The primary anti-actin antibody, Hoechst 33342 dye, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Z-VAD-FMK, a pan-caspase inhibitor, was purchased from Tocris Bioscience (Minneapolis, MN). All other chemicals were of analytical grade.

Cell culture and UVB radiation

The human keratinocyte cell line (HaCaT) was obtained from the Amore Pacific Company (Gyeonggi-do, Republic of Korea) and maintained at 37 °C in a humidified atmosphere of 5% CO₂/90% air. The keratinocytes were routinely cultured in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum, streptomycin (100 μg/ml), and penicillin (100 units/ml).

UV/visible light absorption

To study the UVB absorption spectra of SME, the extract was diluted in dimethyl sulfoxide (DMSO) at a ratio 1:1000 (v/v), and the solution was scanned with UV light (range, 200–500 nm) using a Biochrom Libra S22 UV/visible light spectrophotometer (Biochrom Ltd., Cambridge, UK).

Western blot analysis

Cells were treated with SME (50 μg/ml) and exposed to UVB (30 mJ/cm²) 1 h later. A CL-1000 M UV Crosslinker (UVP, Upland, CA) was used as the UVB source, which delivers a UVB energy spectrum of 280–320 nm. HaCaT keratinocytes were harvested after 24 h and lysed by incubating the cells on ice for 10 min in 100 μl of PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seoul, Republic of Korea). The cell lysates were centrifuged at 13 000 rpm for 5 min and the supernatants were collected. After determining the protein concentrations in the lysates, aliquots (30 μg of protein) were boiled for 5 min and electrophoresed in a 10% sodium dodecyl sulfate-polyacrylamide gel. The proteins were then transferred to nitrocellulose membranes, which were subsequently incubated with the indicated primary antibodies followed by further incubation with the appropriate anti-immunoglobulin-G-horseradish peroxidase secondary antibody conjugates (Pierce, Rockford, IL). Protein bands were detected using an Amersham™ Enhanced Chemiluminescence Plus Western Blotting Detection System (GE Healthcare Life Sciences, Buckinghamshire, UK) according to the manufacturer’s instructions. Actin was used as an internal loading standard.

Determination of mitochondrial membrane potential (Δψ_m)

HaCaT keratinocytes were treated with SME (50 μg/ml) and exposed to UVB radiation 1 h later. Cells were then cultured for an additional 12 h at 37 °C. The Δψ_m was analyzed as follows. Briefly, cells were harvested, washed, and then suspended in phosphate-buffered saline (PBS) containing the mitochondrial membrane-permeate dye, JC-1. Cells were incubated with the dye for 15 min at 37 °C and then analyzed via flow cytometry (Becton Dickinson, Mountain View, CA) (Troiano et al., 2007). In addition, keratinocytes were harvested, and JC-1 was added to each well to stain the cells. Cells were grown on coverslips and then incubated with the dye for 30 min at 37 °C. The stained cells were washed with PBS and the coverslips were mounted onto microscope slides in mounting medium. Slides were examined under a confocal microscope and images were collected using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) (Cossarizza et al., 1993).

DNA fragmentation

Cells were treated with 30 μM Z-VAD-FMK for 1 h and 50 μg/ml SME 1 h later and exposed to UVB radiation. Cells were then incubated for an additional 24 h at 37 °C. Cellular DNA fragmentation was assessed by analyzing the extent of cytoplasmic histone-associated DNA fragmentation with a kit from Roche Diagnostics (Portland, OR) according to the manufacturer’s instructions.

Nuclear staining with Hoechst 33342

Cells were treated with 30 μM Z-VAD-FMK, 50 μg/ml SME, and exposed to UVB radiation 2 h later. Cells were then incubated for an additional 24 h at 37 °C. Hoechst 33342 (10 mg/ml stock; 1.5 μl), a DNA-specific fluorescent dye, was added to each well and the cells were incubated for 10 min at 37 °C. The stained cells were visualized under a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera (Media Cybernetics, Rockville, MD). The degree of nuclear condensation/fragmentation was evaluated and the number of apoptotic cells was quantified. The apoptotic index was calculated as follows: (number of apoptotic cells in treated group/total number of cells in treated group)/(number of apoptotic cells in control group/total number of cells in control group).

Cell viability assay

Seeding the cells onto a 96-well plate at a density of 1 × 10⁵ cells/ml and then treating the cells 16 h later with 30 μM Z-VAD-FMK, 50 μg/ml of SME, was followed 2 h later by 30 mJ/cm² of UVB. After incubating for 24 h at 37 °C, an MTT stock solution (50 μl, 2 mg/ml) was added to each well to yield a total reaction volume of 250 μl. Twenty-four hours later, the plate was centrifuged at 1500 rpm for 5 min and the supernatants were aspirated. The formazan crystals in each well were dissolved in DMSO (150 μl), and the absorbance at 540 nm was read using a scanning multi-well spectrophotometer (Carmichael et al., 1987).

Statistical analysis

All measurements were made in three independent experiments, and all values are expressed as the mean ± the standard error (SE). The results were subjected to an analysis of variance (ANOVA) and to Tukey’s post hoc test to examine differences between conditions. In each case, a p value <0.05 was considered statistically significant.

Results

SME absorbs UVB radiation

We previously showed that UVB exposure increases apoptosis in human HaCaT keratinocytes (Piao et al., 2011). Therefore, the UVB-absorbing capacity of SME was first examined using an UV/visible light spectrophotometry. As shown in Figure 1, SME showed an absorptive capacity for wavelengths in the range of 280–320 nm, corresponding to the UVB portion of the electromagnetic spectrum. Thus, the light-absorbing properties of SME might be closely associated with its anti-apoptotic effects in human skin cells.

Figure 1. SME absorbs UVB radiation. UV/visible light spectroscopic measurements were conducted in the spectral range from 200 to 500 nm. The arrow indicates the position of maximum absorbance (262 nm).

SME modulates the protein expression of mitochondria-associated, pro-, and anti-apoptotic effectors in UVB-irradiated keratinocytes

UVB-induced apoptosis in normal human keratinocytes is, to a large extent, a Bcl-2/Mcl-1-inhibitable process (Assefa et al., 2005; Sitailo et al., 2009; Takahashi et al., 2001). In agreement with these reports, exposure of HaCaT keratinocytes to UVB radiation considerably reduced the expression of the Bcl-2 and Mcl-1 (Figure 2A). SME pretreatment largely prevented this decrease. UVB exposure also increased the expression of the pro-apoptotic protein, Bax (Figure 2A). Conversely, SME reduced Bax expression in UVB-irradiated HaCaT keratinocytes. Therefore, SME modulates the expression of anti- and pro-apoptotic effectors to safeguard HaCaT keratinocytes against UVB radiation.

Figure 2. SME regulates the UVB-stimulated mitochondrial intrinsic cell death pathway. (A) HaCaT cell lysates were electrophoresed in SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with antibodies specific for Bcl-2, Mcl-1, Bax, and actin. The Δψ_m was assessed after JC-1 staining by (B) flow cytometry and (C) confocal microscopy. FI, fluorescence intensity.

To further understand the mechanism underlying SME-mediated protection against UVB-induced apoptosis, we examined changes in mitochondrial membrane potential in the presence of the extract. Mitochondria are the site of oxidative phosphorylation and are intimately involved in the regulation of apoptosis, as well as in the production of reactive oxygen species (ROS). Flow cytometry analysis revealed a loss of Δψ_m in UVB-irradiated cells, confirmed by an increase in the fluorescence of the JC-1 dye (Figure 2B). SME pretreatment, however, blocked the loss of Δψ_m in UVB-irradiated cells.

Aggregates of the JC-1 dye show red fluorescence, whereas the JC-1 monomer shows green fluorescence. Untreated control cells and SME-treated cells showed strong red fluorescence due to the presence of JC-1 aggregates, which indicated mitochondrial polarization (Figure 2C). On one hand, UVB exposure reduced the level of red fluorescence and increased the level of green fluorescence, which is indicative of mitochondrial depolarization. On the other hand, UVB-irradiated cells pretreated with SME showed an increase in red fluorescence and a concomitant decrease in green fluorescence. These observations indicate that SME protects keratinocytes against UVB-induced apoptosis via a mitochondrial mechanism.

SME prevents caspase activation in UVB-irradiated keratinocytes and improves cell viability

To determine the potential involvement of caspases in UVB-induced apoptosis in HaCaT cells, we next explored the ability of SME to alter the expression or activation of key regulatory proteins involved in the caspase pathways. Caspase-9 is activated following mitochondrial membrane disruption (Perkins et al., 2000). Accordingly, the active (cleaved) forms of caspase-9 and its target, caspase-3, were both assessed by western blot analysis. We found that SME inhibited the UVB-induced expression of active caspase-9 (35 and 37 kDa) and active caspase-3 (17 and 19 kDa). These observations were confirmed by the cleavage of poly-ADP ribose polymerase (PARP) (89 kDa), a substrate of active caspases (Figure 3A).

Figure 3. SME prevents UVB-induced apoptosis. (A) HaCaT cell lysates were electrophoresed in SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with antibodies specific for caspase-3, caspase-9, PARP, and actin. (B) Cells were treated with SME (50 μg/ml) and Z-VAD-FMK (30 μM). After 2 h, cells were exposed to UVB radiation and DNA fragmentation was quantified by ELISA assay after further incubation for 24 h. *Significantly different from control cells (p < 0.05), and #significantly different from UVB-irradiated cells (p < 0.05). (C) Cells were treated with SME (50 μg/ml) and Z-VAD-FMK (30 μM). After 2 h, cells were exposed to UVB radiation and after further incubation for 24 h, apoptotic body formation was observed under a fluorescence microscope after Hoechst 33342 staining and quantified. The apoptotic bodies are indicated by arrows. *Significantly different from control cells (p < 0.05), and #significantly different from UVB-irradiated cells (p < 0.05). (D) Cells were treated with SME (50 μg/ml) and Z-VAD-FMK (30 μM). After 2 h, cells were exposed to UVB radiation and cell viability was determined by the MTT assay after further incubation for 24 h. *Significantly different from control cells (p < 0.05), and #significantly different from UVB-irradiated cells (p < 0.05). (C and D are on the following page).

We then examined the ability of the extract to block DNA fragmentation and nuclear condensation/fragmentation, both hallmarks of apoptosis. The level of cytoplasmic histone-associated DNA fragmentation was significantly increased in UVB-irradiated HaCaT keratinocytes compared with that in untreated controls. Conversely, pretreatment with SME or SME plus the pan-caspase inhibitor, Z-VAD-FMK, led to a significant reduction in DNA fragmentation in UVB-irradiated cells (Figure 3B).

Next, the nuclei of HaCaT cells were stained with Hoechst 33342 and visualized by fluorescence microscopy. The resulting images showed intact nuclei in control cells, but significant nuclear fragmentation and apoptotic body formation in UVB-irradiated cells. However, SME or SME + Z-VAD-FMK markedly reduced nuclear fragmentation in UVB-irradiated cells (Figure 3C).

Finally, we investigated whether SME improves the viability of UVB-irradiated cells by assessing the survival of HaCaT keratinocytes after exposure to UVB, SME, UVB + SME, or UVB + SME + Z-VAD-FMK. The cell viability increased significantly, from 53% for UVB-irradiated cells to 71% for SME-pretreated/UVB-irradiated cells, and to 88% for SME/Z-VAD-FMK-pretreated/UVB-irradiated cells (Figure 3D). These results are consistent with the hypothesis that SME protects cells from apoptosis by inhibiting the caspase-dependent apoptotic pathway.

SME regulates UVB-induced MAPK and Akt signaling

Because MAPK signaling plays important roles in modulating apoptosis, we next examined the effects of SME on UVB-stimulated activation of p38 MAPK, JNK, and Erk by measuring the expression of their phosphorylated forms. UVB irradiation led to a marked increase in the levels of phosphorylated p38 MAPK and JNK compared with those in control cells; however, this was reversed by SME. In contrast, UVB irradiation reduced the levels of phosphorylated Erk1/2; again, this was reversed by SME (Figure 4A).

Figure 4. SME prevents the UVB-mediated phosphorylation of p38 MAPK and JNK, as well as the dephosphorylation of Erk1/2 and Akt. (A and B) HaCaT cell lysates were electrophoresed in SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with the indicated primary antibodies.

To further elucidate the mechanism underlying the SME-mediated cell survival observed in Figure 3(D), we examined the activation of Akt in response to UVB and/or SME. Akt, also termed protein kinase B, is a major signaling enzyme involved in cell survival after exposure to oxidative stress. SME increased Akt phosphorylation in HaCaT cells and, in addition, prevented the UVB-induced reduction in phosphorylated Akt levels (Figure 4B). These results suggest that PI3K/Akt is activated by SME and rescues human keratinocytes from UVB-induced apoptosis.

Discussion

The exposure of HaCaT keratinocytes to UVB radiation increases the oxidative state of human HaCaT keratinocytes, thereby sensitizing them to UVB-induced apoptosis (Hyun et al., 2012; Piao et al., 2012). Here, we showed that an ethyl acetate fraction derived from the brown alga S. muticum, SME, protected human HaCaT keratinocytes against UVB-induced apoptotic cell death through a mechanism involving alterations in MAPK- and caspase-dependent signaling cascades.

Apoptosis is mediated by several cellular pathways, including the caspase-mediated pathways, as well as MAPK-regulated signaling pathways (Kohno et al., 2011; Santarpia et al., 2012). The p38 MAPK and JNK pathways are associated with the initiation of UVB-induced apoptosis (Bivik & Ollinger, 2008; Nys et al., 2010), whereas the Erk and/or phosphatidylinositol 3-kinase/Akt signaling pathway play a crucial role in regulating normal cell proliferation, survival, and differentiation (Amaravadi & Thompson, 2005; Cagnol & Chambard, 2010). Notably, the results presented herein showed that SME modulated p38 MAPK, JNK, Erk1/2, and Akt signaling in UVB-irradiated cells to favor cell survival. Specifically, SME reduced the UVB-mediated phosphorylation of p38 MAPK and JNK, and prevented the UVB-mediated dephosphorylation of Erk1/2 and Akt.

The key players that execute the apoptotic cascade are the initiator and effector caspases, caspase-9 and caspase-3, which are activated early in apoptosis by cleavage at particular amino acid residues (Green & Reed, 1998; Spierings et al., 2005). The intrinsic cell death pathway is controlled by various pro- and anti-apoptotic factors, particularly members of the Bcl-2 family of proteins, which in turn regulate the permeability of the mitochondrial membrane (Costantini et al., 2000; Debatin et al., 2002). The anti-apoptotic proteins, Mcl-1, Bcl-2, Bcl-xL, Bcl-w, Bcl-B, and BFL1, all suppress apoptosis induced either by pro-apoptotic Bax or by pro-apoptotic Bak. The anti-apoptotic proteins selectively bind to Bax and/or Bak, block their oligomerization, and induce the closing of mitochondrial membrane pores (Ke et al., 2001; Ruffolo & Shore, 2003; Shangary & Johnson, 2002; Yan et al., 2000). In addition, Bcl-2 directly inhibits members of the caspase family, including caspase-3 and caspase-9 (Deveraux & Reed, 1999; Park et al., 2007). In the current study, SME increased Bcl-2 expression in UVB-irradiated keratinocytes and decreased Bax expression, likely accounting in part for its anti-apoptotic and pro-survival actions.

The function of Mcl-1, another member of the Bcl-2 family, depends on its subcellular localization. Mcl-1 at the mitochondrial outer membrane reduces mitochondrial permeabilization to block apoptosis. However, a cleaved form of Mcl-1 is localized to the mitochondrial matrix, where it controls inner mitochondrial morphology and the oxidative phosphorylation status of cells without directly modulating apoptosis (Andersen & Kornbluth, 2012). Like Bcl-2, the expression of full-length (40 kDa) Mcl-1 was upregulated by SME in the current study. This suggests that the S. muticum extract directed its protective actions toward the anti-apoptotic form of Mcl-1 found at the outer mitochondrial membrane.

Chen et al. (2007) demonstrated that dimerization of caspase-9 results in the loss of mitochondrial membrane potential, in addition to the cleavage (inactivation) of the anti-apoptotic factors, Bcl-2, Bcl-xL, and Mcl-1. Caspases -8, -9, and -3 are situated at pivotal junctions in apoptosis-associated signaling pathways. For instance, the apoptotic executor, caspase-3, is activated by the upstream effectors, caspase-8 and caspase-9; because caspase-3 serves as a convergence-point molecule in a number of different signaling pathways, it is well suited to act as a read-out factor in various apoptosis assays. In the current study, the general caspase inhibitor, Z-VAD-FMK, maintained (and even increased) the protective effect of SME against UVB-induced nuclear fragmentation and cytotoxicity. Moreover, SME inhibited the UVB-mediated disruption of mitochondrial membrane potential and prevented the activation of both caspase-3 and caspase-9.

Marine algae contain many important secondary metabolites, including phenols and polyphenols, resulting from unique linkages known as phenolic coupling (Torres et al., 2008). SME contains an abundance of polyphenolic compounds, as assessed by the Folin–Denis method (Kim et al., 2007; Namvar et al., 2013). Furthermore, Sargassum species in general are characterized by their high polyphenol content relative to Ulva species and Porphyra species (García-Casal et al., 2009). Polyphenols are well-known for their antioxidant properties and protect HaCaT keratinocytes against UVB-induced phototoxic stress and DNA damage (Svobodová et al., 2009; Wu et al., 2009). Recently, it has been reported that apo-9′-fucoxanthinone isolated from S. muticum showed anti-inflammatory effects (Chae et al., 2013; Yang et al., 2013). Apo-9′-fucoxanthinone was a degradative product of fucoxanthin (Mori et al., 2004) and fucoxanthin is known for its antioxidant (Zheng et al., 2013), anti-inflammatory (Heo et al., 2010), anti-obesity (Maeda et al., 2005), antitumor (Kim et al., 2010), and UVB-preventative activities (Urikura et al., 2011).

Taken together with the results presented herein, these observations suggest that SME-derived polyphenols may initiate anti-apoptotic signaling cascades in HaCaT cells.

Conclusions

An ethyl acetate extract of S. muticum, SME, prevented UVB-induced apoptotic cell death in human HaCaT keratinocytes by modulating Bcl-2, Mcl-1, and Bax expression, as well as by regulating MAPK- and mitochondrial signaling pathways. These observations suggest that SME may be a useful tool for use against sun-induced damage in human skin.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-C1ABA001-2011-0021037).