Food and Chemical Toxicology 63 (2014) 30–37

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Vanillin protects human keratinocyte stem cells against Ultraviolet B irradiation Jienny Lee a, Jae Youl Cho c, Sang Yeol Lee d, Kyung-Woo Lee a,⇑, Jongsung Lee b,e,⇑, Jae-Young Song a a

Viral Disease Division, Animal and Plant Quarantine Agency, 175 Anyang-Ro, Manan-Gu, Anyang-Si, 430-757 Gyeonggi-Do, Republic of Korea Department of Dermatological Health Management, College of Health Science, Eulji University, Seongnam-Si, 461-713 Gyeonggi-Do, Republic of Korea c Department of Genetic Engineering, Sungkyunkwan University, Suwon-Si, 440-746 Gyeonggi-Do, Republic of Korea d Department of Life Science, Gachon University, San 65, Bokjeong-Dong, Sujeong-Gu, Seongnam-Si, 461-701 Gyeonggi-Do, Republic of Korea e Department of Convergence Biomedical Science & Engineering, Eulji University, Seongna-Si, 461-713 Gyeonggi-Do, Republic of Korea b

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Article history: Received 6 August 2013 Accepted 22 October 2013 Available online 30 October 2013 Keywords: Ultraviolet B Keratinocyte stem cells p53 MDM2 Vanillin

a b s t r a c t Ultraviolet-B (UVB) irradiation is one of major factors which induce cellular damages in the epidermis. We investigated protective effects and mechanisms of vanillin, a main constituent of vanilla beans, against UVB-induced cellular damages in keratinocyte stem cells (KSC). Here, vanillin significantly attenuated UVB irradiation-induced cytotoxicity. The vanillin effects were also demonstrated by the results of the senescence-associated b-galactosidase and alkaline comet assays. In addition, vanillin induced production of pro-inflammatory cytokines. Attempts to elucidate a possible mechanism underlying the vanillin-mediated effects revealed that vanillin significantly reduced UVB-induced phosphorylation of ataxia telangiectasia mutated (ATM), serine threonine kinase checkpoint kinase 2 (Chk2), tumor suppressor protein 53 (p53), p38/mitogen-activated protein kinase (p38), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), S6 ribosomal protein (S6RP), and histone 2A family member X (H2A.X). UVB-induced activation of p53 luciferase reporter was also significantly inhibited by vanillin. In addition, while ATM inhibitor had no effect on the vanillin effects, mouse double minute 2 homolog (MDM2) inhibitor significantly attenuated suppressive effects of vanillin on UVB-induced activation of p53 reporter in KSC. Taken together, these findings suggest that vanillin protects KSC from UVB irradiation and its effects may occur through the suppression of downstream step of MDM2 in UVB irradiation-induced p53 activation. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Increased ultraviolet (UV) irradiation at the Earth’s surface due to the depletion of the stratospheric ozone layer have enhanced interest in the mechanisms of various effects it might have on organisms. Especially, DNA is one of the main targets for UV-induced damage in a variety of organisms ranging from bacteria to humans (Sinha and Häder, 2002; Häder and Sinha, 2005). UV Irradiation induces the formation of several types of mutagenic DNA lesions. Cellular senescence is also an irreversible cell cycle arrest in response to UVB-induced DNA damage (Harley et al., 1990; Serrano et al., 1997; te Poele et al., 2002). UV has three different UV wavelength components, including UVA (320–400 nm), UVB (280–320 nm), and UVC (200–280 nm), which have distinct mutagenic properties. Among three UV wavelength components, UVB irradiation is a main causer of DNA damage in the epidermis and is the most ⇑ Corresponding authors. Tel.: +82 31 463 1864; fax: +82 31 463 4565 (K.-W. Lee), tel.: +82 31 740 7209; fax: +82 31 740 7358 (J. Lee). E-mail addresses: [email protected] (K.-W. Lee), [email protected] (J. Lee). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.10.031

important environmental mutagen and carcinogen of epidermal cells. Epidermal keratinocytes absorb the bulk of cutaneous UV exposure. As a result of carcinogenic UVB exposure, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage (Zhuang et al., 1999). The human epidermis is a stratified epithelium that maintains its integrity through a process of constant regeneration, driven by a population of keratinocyte stem cells (KSC) in the basal layer (Green, 1977). The search to identify human KSC has focused on the principle of adhesion to the basement membrane. In vitro, it has been shown that the keratinocytes that rapidly adhere to cell culture plates form tightly packed colonies, termed holoclones, which have the greatest longterm growth potential and so are likely to contain stem cells (Barrandon and Green, 1987; Jones and Watt, 1993). In the epidermis, KSC are promising clinical candidates for the treatment of photodamage, chronic wounds, and ulcers (Kim et al., 2004). Several natural plant-derived compounds, including vanillin, cinnamaldehyde, coumarin, umbelliferone and tannic acid, have moderate anti-mutagenic properties. They also sensitise cells to the lethal effects of DNA-damaging agents (Ohta, 1993). Among them, vanillin (4-hydroxy-3-methoxybenzaldehyde), an

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anticlastogen, has been demonstrated to inhibit gene mutations in both bacterial and mammalian cells (Watanabe et al., 1990; Keshava et al., 1998; Gustafson et al., 2000; Akagi et al., 1995). However, the mechanisms underlying its effect against UVB radiation-induced cellular damage remain undefined. Therefore, this study was conducted to investigate the effects of vanillin on cellular damages induced by UVB irradiation, and the possible mechanism underlying this protective effect.

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2.6. b-Gal staining Cells were washed twice with PBS and fixed with 2% formaldehyde/0.2% glutaraldehyde at room temperature (RT) for 10 min. After two additional washes with PBS, 2 mL of staining solution (150 mM sodium chloride, 25.2 mM sodium phosphate dibasic, 7.36 mM citric acid, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM magnesium chloride, and 1 ng/ml 5-bromo-4-chloro-3-indolyl-b-d-galactoside, pH 6.0) were added to the cells, and they were incubated at 37 °C overnight (Dimri et al., 1995). After the incubation, the cells were washed with PBS and examined microscopically at 20 magnification (Nikon Eclipse Ti, Nikon, Tokyo, Japan).

2. Materials and methods 2.7. Enzyme-linked immunosorbent assay (ELISA) 2.1. Materials Vanillin was purchased from ChromaDex (ChromaDex, Irvine, CA, USA). Mouse double minute 2 homolog (MDM2) and ataxia telangiectasia mutated (ATM) inhibitors were obtained from EMD millipore (EMD millipore, Billerica, MA, USA). Antibodies in this study were used according to the manufacturers’ instructions and purchased as follows: CD29, CD34, CD49f, and CD71-antibodies from BD Pharmingen (BD Biosciences, San Jose, CA, USA), b-catenin-antibody from eBioscience (eBioscience, San Diego, CA, USA), p63-antibody from AbD serotec (AbD serotec, Raleigh, NC, USA), keratin 19 (K19) and ATM-antibodies from abcam (abcam, Cambridge, MA, USA), serine threonine kinase checkpoint kinase 2 (Chk2), tumor suppressor protein 53 (p53), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/ SAPK), p38/mitogen-activated protein kinase (p38/MAPK), ribosomal protein S6 (S6RP), histone 2A family member X (H2A.X)-anibodies, and Hoechst 33342 from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA).

2.2. Cell isolation and culture Cells were isolated from human child’s foreskin. Skin specimens were processed according to the method of Rheinwald and Green (Rheinwald and Green, 1975), as modified in our laboratory using method of Kim (Kim et al., 2004). In brief, the primary epidermal cells from foreskin were plated onto the collagen-coated dishes which had been prepared by incubating 100 mm dishes with type IV collagen (20 lg/mL) at 4 °C overnight. In this study, we used collagen ‘‘type IV’’ which is the ligand of integrin-b1 (CD29). It was reported that human interfollicular epidermal stem cells express high levels of CD29. Thus, collagen type IV is considered a potential candidate for the selection of epidermal stem cells (Jones and Watt, 1993). Then, a portion of these cells plated was selected according to their ability to adhere to the dishes within 1 h-incubation at 37 °C, and non-adhering cells was discarded. Only rapidly adhering (RA) cells were cultured in EpiLife™ keratinocyte medium (Invitrogen, Carlsbad, CA, USA) at 5% CO2 and 37 °C under sterile conditions. The medium was changed every 3 days and the cells at 80% confluence were passaged. Before each experiment, the RA cells were trypsinized, counted, washed twice with phosphate-buffered saline (PBS) and resuspended in PBS (Invitrogen). The harvested cells highly expressed KSC-specific markers such as CD29 and integrin-a6 (CD49f), but did not express CD71.

2.3. Characterization of KSC To immunophenotypically characterize KSC, the cells were stained using monoclonal antibodies against the KSC specific markers (CD29, CD34, CD49f, CD71, bcatenin, p63 and keratin19) according to the manufacturer’s instructions. The cells were then analyzed on BD FACSCalibur™ flow cytometer (BD, Franklin Lakes, NJ, USA) with BD FACS software.

2.4. UVB radiation To understand the biological consequences in skin by UVB irradiation, an in vitro model system was used to probe the response of KSC to UVB exposure at the intensity of 30 mJ/cm2 (Luzchem Research Inc., Ottawa, Canada). For UVB exposure, when the cells were 70% confluent, the medium was removed, and the cells were washed with PBS and gently overlaid with PBS. The cells were then irradiated for 20 s at 30 mJ/cm2. At the indicated times after UVB irradiation, the cells were harvested and assayed for senescence-related specific experiments.

2.5. MTT assay Cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were exposed to MTT (0.1 mg/mL) for 3 h at 37 °C under 5% CO2 incubator. The medium was then removed, and the cells were solubilized with dimethyl sulfoxide (1 mL). After complete solubilization, the presence of blue formazan was evaluated spectrophotometrically by measuring the absorbance at a wavelength of 570 nm. Intact cells were also employed and used as a positive control.

Supernatants of the KSC cultured in different conditions were analyzed for various cytokines using commercially available ELISA kits (R&D systems, Minneapolis, Minn, USA) according to the manufacturer’s protocols. The supernatants were assayed for epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), transforming growth factor-beta1 (TGF-b1), tumor necrosis factor-alpha (TNF-a), interleukin-1beta (IL-1b), and interleukin-6 (IL-6). The standard curve was linearized and subjected to regression analysis. The EGF, FGF-2, TGF-b1, TNF-a, IL-1b, and IL-6 concentration of the unknown samples was calculated using the standard curve. The intact KSC were also employed as a positive control. All samples and standards were measured in duplicate.

2.8. Immunoblotting Cells were harvested, lysed with lysis buffer and a protease inhibitor cocktail kit (Roche, Mannheim, Germany), and centrifuged at 12,000 rpm for 10 min to obtain the supernatants. Proteins in the supernatants were then separated by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Uppsala, Sweden) at 250 mA in transfer buffer (20 mM Tris base, 150 mM glycine, 20% MeOH in 1 L distilled water). The membranes were then washed and incubated with primary phospho-p53, phospho-Chk2, phospho-JNK/SAPK, phospho-p38/ MAPK, phospho-S6 ribosomal protein (S6RP), phospho-H2A.X, and b-actin antibodies (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, washed and incubated with horseradish peroxidase-conjugated IgG secondary antibody (Cell Signaling Technology, Danvers, MA, USA). Immediately after washing, the immunoreactive proteins were detected by chemiluminescence (ECL kit, GE Healthcare, Uppsala, Sweden). The detected proteins were normalized to b-actin as appropriate.

2.9. Luciferase reporter gene assay Cells were transiently transfected with 2 lg of the firefly luciferase reporter gene under the control of p53 responsible elements (Stratagene, La Jolla, CA, USA) and 0.2 lg of Renilla luciferase expression vector driven by thymidine kinase promoter (Promega, Madison, WI, USA) by superfect reagent (Invitrogen, Carlsbad, CA, USA). Cells (2  105 cells/mL) prepared in a 12 well culture plate were transfected transiently with p53-Luc. After 24 h, the cells were irradiated by UVB (30 mJ/ cm2). The non-irradiated intact cells were used as a control. After 12 h, the cells were harvested and a luciferase assay was performed using the dual-luciferase reporter assay system (Promega, Madison, WI, USA), as described previously (Tian et al., 2011). Firefly and Renilla luciferase activities were measured using a LB953 luminometer (Berthold Technologies, Bad Wildbad, Germany). Firefly luciferase activity was normalized to Renilla luciferase activity for each sample.

2.10. Comet assay To detect cellular DNA damage by single-strand breaks, alkaline micro-gel electrophoresis was performed essentially as described by Singh et al (Singh et al., 1988). The pelleted cells (2.5  104) were washed and resuspended in 85 lL PBS. The cell suspension was then mixed with 85 lL of 1% low gelling temperature agarose dissolved in PBS. Next, 75 lL of the cell-agarose mixture was transferred onto a frosted microscope slide that had been pre-coated with 85 lL of 1% normal gelling temperature agarose. A third layer of 0.5% low gelling temperature agarose (75 lL) was applied over the second layer containing the cells. In establishing each of the three layers of agarose, glass coverslips were applied on top of the liquid agarose to spread the agarose across the surface of the slides. Each layer of agarose was congealed by placing the slides on a metal tray which was positioned in crushed ice. After removing coverslips, the slides were immersed in ice-cold alkaline cell lysis solution and allowed to incubate for 1 h. Slides were then placed carefully in a horizontal electrophoresis unit and left undisturbed at for 20 min. Next, electrophoresis was performed at 300 mA for 20 min. Afterwards slides were gently immersed in neutralization buffer for 5 min, and this step repeated. After applying 60 lL ethidium bromide solution (20 lg/mL) on the top of the agarose and covering with coverslips, the slides were viewed with a fluorescence microscope equipped with camera (Nikon Eclipse Ti, Nikon). The percentage of the total fluorescence in the tail and the tail length of the 50 cells per slide were recorded.

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2.11. Immunofluorescence 4

Cells (2  10 cells/100 lL) were prepared in a 96 well culture plate for 24 h before treatment. After 24 h, cells were irradiated by UVB (30 mJ/cm2). The non-irradiated cells were used as a positive control. The fixation and staining for UVB-induced phosphorylation of p53 and H2A.X were performed previously described by al Rashid et al. (2005). Cells were imaged on the GE IN Cell Analyzer 1000 (GE Healthcare Lifescience, Uppsala, Sweden) at 20 objective magnification. The cells were then analyzed on IN Cell Analyzer 1000 with high-content image analysis software, IN Cell Investigator. 2.12. Statistics All experiments were conducted at least three times, and data were expressed as the mean ± SD. Data was analyzed by Student’s t-test. A value of P < 0.05 was considered statistically significant.

vealed that vanillin significantly recovered the reduced cell viability of KSC induced by UVB irradiation (Fig. 2B). Specifically, vanillin induced a 40% increase in the cell viability of KSC when compared to the UVB-irradiated group. In addition, we investigated cytotoxicity of vanillin itself using the MTT assay. We found that vanillin did not exert any cytotoxic effects at the treated concentrations of vanillin (data not shown). These effects of vanillin were further demonstrated by the results of the senescenceassociated b-galactosidase (SA-b-gal) assay. As shown in Fig. 2C, vanillin significantly inhibited the UVB-induced accumulation of SA-b-gal in KSC. Also, we observed the UVB irradiation-induced cellular DNA damage of KSC was attenuated by vanillin in an alkaline comet assay (Fig. 2D). These findings suggest that vanillin could effectively contribute to the protection of KSC against UVB irradiation.

3. Results 3.1. Characterization of keratinocyte stem cells Firstly, characteristics of the cells isolated from human child’s foreskin was verified using FACS analysis. As shown in Fig. 1, while KSC-specific markers such as integrin b-1 (CD29), integrin a-6 (CD49f), b-catenin, p63, and keratin19 were positively expressed, CD34 and CD71 were negatively expressed in the cells. These results indicate that KSC was isolated successfully from human foreskin. 3.2. Protective effects of vanillin against UVB irradiation-induced cytotoxicity We investigated protective effects of vanillin on UVB irradiation-induced damage of KSC using an MTT assay. The results re-

3.3. Inhibitory effects of vanillin against UVB-induced production of pro-inflammatory cytokines As a next step, we examined effects of vanillin against UVB-induced production of pro-inflammatory cytokines. We measured the levels of epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), interleukin-1beta (IL-1b), interleukin-6 (IL-6), transforming growth factor-beta1 (TGF-b1), and tumor necrosis factor-alpha (TNF-a) in KSC irradiated with UVB and found that the production of pro-inflammatory cytokines such as TNF-a, IL-1b, and IL-6 was significantly up-regulated in response to UVB irradiation when compared to the untreated control in KSC. Moreover, vanillin led to a significant decrease in the production of TNF-a, IL-1b, and IL-6 in KSC (Fig. 2E). However, vanillin was found to exert any significant effects on the UVB irradiation-induced

Fig. 1. Phenotypic characterization of KSC. Immunofluorescence staining was conducted to examine surface makers of KSC which was isolated from human child’s foreskin. KSC-specific markers (integrin b-1 (CD29), integrin a-6 (CD49f), b-catenin, p63, and keratin19) were positively expressed and the KSC-negative markers (CD34 and CD71) were not detected. Data are representative of at least three independent experiments.

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Fig. 2. Effects of vanillin against UVB irradiation on KSC. (A) Chemical structure of vanillin. (B) Using an MTT assay, we examined the protective effects of vanillin on the cell damage of KSC under UVB irradiation condition. (C) Also, protective effects of vanillin were confirmed by the senescence-associated beta-galactosidase (SA-b-gal) assay. (D) To detect cellular DNA damage as single-strand breaks, alkaline micro-gel electrophoresis was performed. (E) Next, supernatants of the KSC cultured in different conditions were analyzed using a commercially available ELISA kit according to the manufacturer’s protocol. All experiments were performed 24 h after UVB irradiation (vanillin W/ WO). Data are representative of at least three independent experiments. Results are mean ± standard deviation (SD). P < 0.05 vs. UVB-irradiated control.

production of EGF, FGF-2, and TGF-b1 which was known to have anti-inflammatory activity. 3.4. Protective effects of vanillin against ATM-related signaling pathway We investigated phosphorylation of ATM under UVB irradiation in KSC using FACS analysis. As shown in Fig. 3A, flow cytometric result showed that fluorescence intensity of ATM phosphorylation

stained by ATM1981 dye was [M2: 56.1%] value in vanillin-treated, and UVB-irradiated KSC, compared with [M2: 74.5%] value of fluorescence intensity in the UVB-irradiated KSC (Fig. 3A). Also, we observed that while red/green fluorescence intensity of phosphorylated p53/H2AX detected using a confocal microscope was enhanced in the UVB-irradiated KSC; vanillin significantly reduced red/ green fluorescence intensity in the UVB-irradiated KSC (Fig. 3B). The above data imply that vanillin could contribute to the interaction between ATM and p53 under UVB irradiation condition.

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Fig. 3. Protective effects of vanillin against ATM-related signaling pathway. (A) UVB-irradiated cells were harvested to examine phosphorylation level of ATM in KSC using FACS analysis. Flow cytometric result showed that fluorescence intensity of phosphorylated ATM stained by ATM1981 dye was [M2: 56.1%] value in vanillin-treated irradiated KSC compared with [M2: 74.5%] value of fluorescence intensity in irradiated KSC. (B) Cells (2  104 cells/100 lL) were prepared in a 96 well culture plate and fixed/ stained to measure levels of phosphorylated p53 and H2A.X. Cells were imaged on the GE IN Cell Analyzer 1000 at 20 objective magnification. (C) To investigate the protective molecular mechanisms of vanillin effects, we conducted immunoblot analysis using phospho-Chk2, phospho-p53, phospho-JNK, phospho-p38, phospho-S6RP, and phospho-H2A.X antibody in KSC extracts at the indicated times after UVB irradiation. Only ATM phosphorylation assay was performed using FACS analysis. (D) Densitometric analysis of Fig. 3C. Data are representative of at least three independent experiments. Results are mean ± standard deviation (SD). P < 0.05 vs. UVB-irradiated control. ( ): no UVB irradiation, (+): UVB irradiation, V/20: vanillin (20 lM), V/100: vanillin (100 lM).

Therefore, to investigate the protective molecular mechanisms of vanillin, we conducted immunoblot analysis using phosphoChk2, phospho-p53, phospho-JNK/SAPK, phospho-p38/MAPK, phospho-S6RP, and phospho-H2A.X antibodies in KSC extracts at the indicated times after UVB irradiation. As shown in Fig. 3C, ATM-related signaling pathways (ATM, Chk2, p53, and H2A.X),

MAPKs (JNK and p38), and S6RP were rapidly phosphorylated in response to UVB irradiation in KSC. Overall, vanillin significantly inhibited the UVB-induced phosphorylation of ATM, Chk2, p53, JNK, p38, and H2A.X in KSC (Fig. 3C). These results indicate that ATM/p53/MAPK pathway is involved in the protection of KSC by vanillin against UVB irradiation.

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Fig. 3 (continued)

3.5. Protective effects of vanillin through p53-MDM interaction We found that ATM/p53 pathway was involved in the vanillininduced protective mechanisms of KSC under UVB irradiation condition. Therefore, the correlation between them was investigated. It is well known that MDM2 is an important negative regulator of the p53 tumor suppressor. MDM2 has been identified as a p53 interacting protein that represses p53 transcriptional activity (Zhang and Chen, 2008). As shown in Fig. 4A, we observed that while UVB irradiation induced p53 reporter activation in KSC, vanillin significantly reduced the UVB-irradiated effects. In addition, while ATM inhibitor had no effect on the vanillin effects, MDM2 inhibitor significantly attenuated suppressive effects of vanillin on UVB-induced activation of p53 reporter in KSC. These results suggest that vanillin operate downstream of MDM2 in UVB irradiation-induced p53 activation.

4. Discussion The results of this study provide evidence of the effects of vanillin on UVB-induced cellular damages, as well as its inhibitory mechanisms in KSC. Specifically, the findings presented here demonstrated that vanillin suppresses cytotoxicity, cellular senescence, production of pro-inflammatory cytokines which are induced by UVB irradiation. Ataxia telangiectasia mutated (ATM)-related

signaling plays a central role in the UVB-induced phenomenon and is linked to the characteristic changes that occur during UVB irradiation. Here, we demonstrated that vanillin protects KSC from UVB irradiation by suppressing downstream step of MDM2 molecule. UVB induces production of various pro-inflammatory cytokines in keratinocytes (Konnikov et al., 1989; Kirnbauer et al., 1991; Grandjean-Laquerriere et al., 2002) which mediates cutaneous inflammatory responses (Kondo, 1999). Among the cytokines, TNF-a plays an important role in photodamage of epidermal cells. TNF-a release after UVB exposure induces nearby endothelial cells and keratinocytes to display cell adhesion molecules, thereby recruiting inflammatory cells that secrete elastases and collagenases, leading to damage and aging of the skin (Strickland et al., 1997; Rijken et al., 2006; Bashir et al., 2009). In this study, the production of TNF-a, IL-1b, and IL-6 induced by UVB irradiation was found to be significantly reduced by vanillin molecule. These findings suggest that vanillin could effectively contribute to the attenuation of inflammatory function of UVB irradiation. UVB irradiation induces cellular DNA damage and cytotoxicity. In addition, cellular senescence is one of UVB irradiation-induced celluar damages. Senescent cells have several major characteristics. They include enlarged cell size, flattened morphology, inability to synthesize DNA, and expression of the biomarkers, senescenceassociated b-galactosidase (SA-b-gal) (Blackburn, 2005; Kim et al., 2002). In this study, vanillin improved cell viability of UVB-irradi-

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Senescent cells express activated forms of ATM and its downstream target, Chk2. Also, phosphorylation of p53 by ATM and Chk2 inhibits p53 degradation by MDM2, a RING finger E3 ligase which ubiquitinates and degrades p53 through ubiquitin-mediated proteolysis. MDM2 achieves this repression by binding to and blocking the N-terminal trans-activation domain of p53. MDM2 is a p53 responsive gene-that is, its transcription can be activated by p53. Thus when p53 is stabilized, the transcription of MDM2 is also induced, resulting in higher MDM2 protein levels. Various stresses inhibit MDM2 activity and induce post-translational modifications of p53, which stabilize and activate p53 (Zhang and Chen, 2008). In this study, we found that ATM/p53 pathway is involved in the vanillin-induced protective mechanisms of KSC under UVB irradiation conditions. Therefore, the correlation between them was investigated. Vanillin was found to inhibit UVB-irradiated p53 reporter activation in KSC. Also, ATM inhibitor did not affect the vanillin effects in UVB irradiation-induced p53 activation. However, vanillin repressed p53 striking activation against MDM2 inhibition in KSC. Overall, these findings suggest that vanillin suppresses the UVB-induced cellular damages and its effects may be mediated through the inhibition of downstream step of MDM2 in UVB irradiation-induced p53 activation.

Fig. 4. Protective effects of vanillin through p53-MDM interaction. Cells were transfected with p53 reporter according to the protocol described in Materials and Methods. After 24 h of transfection, the medium was changed to complete medium. Next, at the 12 h after UVB irradiation (vanillin W/WO, ATM inhibitor W/WO, and MDM2 inhibitor W/WO), cells were harvested and assayed for p53 luciferase activaton. The p53 reporter activity values were expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates. Data are representative of at least three independent experiments. Results are mean ± standard deviation (SD). P < 0.05 vs. UVB-unirradiated control. P < 0.05 vs. UVB-irradiated control. TF: transfection, ATM (In): ATM inhibitor, MDM2 (In): MDM2 inhibitor.

ated KSC and attenuated the accumulation of SA-b-gal and cellular DNA damage induced by UVB. These findings suggest that vanillin is involved in the protection of KSC from UVB irradiation. In addition, we did not observe UV-absorbing properties of vanillin (data not shown). Therefore, these results suggest that protective effects of vanillin are attributed to its cellular activities, not its UVB-filtering activity. Ataxia telangiectasia mutated (ATM) plays a critical role in the cellular response to UVB-induced DNA damage. Upon activation, ATM phosphorylates a number of substrates including targets that initiate cell cycle arrest, DNA repair, and apoptosis (So et al., 2009). ATM is one of the major upstream regulators of the p53 response to ionizing radiation-induced damage. p53 protein also has many modulating functions in cells playing a role in gene transcription, cell cycle control, DNA repair, senescence and apoptosis (Amundson et al., 1998; Fridman and Lowe, 2003; Schmitt et al., 2002; Komarova and Gudkov, 2001). Under normal conditions of cell growth, p53 protein has a relatively short half-life, being rapidly targeted for ubiquitination and degradation. Following cellular stress, p53 is phosphorylated on a number of sites, increasing its half-life and transactivation activity (Meek, 1994). Many different kinase families phosphorylate p53, including DNA-PK, the casein kinase family, MAP kinases, SAP kinases and CDKs (Meek, 1998). Also, these kinases phosphorylate numerous constituents of the repair machinery and checkpoint proteins Chk2, Chk1, and histone H2A.X (Kitagawa and Kastan, 2005; Shiloh, 2006). We observed that ATM-related signaling pathways (ATM, Chk2, p53, and H2A.X), MAPKs (JNK/SAPK and p38/MAPK), and S6RP were rapidly phosphorylated in response to UVB irradiation in KSC. Also, vanillin significantly inhibited UVB-induced phosphorylation of ATM, Chk2, p53, JNK, p38, S6RP, and H2A.X in KSC.

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