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Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells Shogo Abe a, Keizo Yuasa a, b, * a b
Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan Department of Bioscience and Bioindustry, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 August 2019 Received in revised form 26 August 2019 Accepted 4 September 2019 Available online xxx
Although we recently reported that sudachitin (5,7,40 -trihydroxy-6,8,30 -trimethoxyflavone), a polymethoxyflavone isolated from the peel of Citrus sudachi, can induce apoptosis in human keratinocyte HaCaT cells, the mechanism underlying its action remains unclear. In this study, we explored the mechanisms underlying sudachitin-induced apoptosis in HaCaT cells. Sudachitin activated p38MAPK and inhibited ERK1/2, whereas another polymethoxyflavone, nobiletin (5,6,7,8,30 ,40 -hexamethoxyflavone), activated ERK1/2. The p38MAPK inhibitor SB203580 significantly attenuated sudachitin-induced heat shock protein 27 phosphorylation, downstream of p38MAPK, and subsequent apoptosis, indicating that sudachitin induces apoptosis via the p38MAPK pathway. Additionally, sudachitin inhibited serum- and EGF-stimulated Raf-1-ERK1/2 activation, and blocked EGF-induced cell migration and proliferation in HaCaT cells. These results suggest that small structural differences in polymethoxyflavones can induce different cellular responses by altering the regulation of MAPK activities and that sudachitin may be a potential candidate for developing new drugs for skin diseases such as psoriasis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Polymethoxyflavone Keratinocyte Apoptosis MAPK Cell migration and proliferation
1. Introduction The skin is the largest and outermost organ of the body and functions as a primary barrier protecting the body from harmful compounds, pathogenic microbes, and ultraviolet (UV) radiation. The skin comprises three layers: the epidermis, dermis, and subcutaneous layer. The outermost epidermis is composed of a stratified epithelium with distinct layers of keratinocytes [1]. Exposure to UV radiation results in DNA damage, which causes mutations; this is considered as an event initiating skin carcinogenesis. The elimination of UV radiation-damaged keratinocytes via apoptosis is an essential mechanism for protecting the skin from sunlight. However, the increased proliferative and reduced apoptotic abilities of keratinocytes causes abnormal proliferation, leading to
Abbreviations: UV, ultraviolet; PMF, polymethoxyflavone; EGF, epidermal growth factor; PARP, poly (ADP-ribose) polymerase; HSP, heat-shock protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Bid, BH3-interacting-domain death agonist; MKK, MAP kinase kinase. * Corresponding author. Department of Bioscience and Bioindustry, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan. E-mail address: [email protected] (K. Yuasa).
conditions such as skin cancer, psoriasis, keratosis, verrucae, and lichen simplex chronicus [2]. A precise balance of apoptosis and cell proliferation in keratinocytes is important for the treatment and prevention of such skin diseases. The MAPK family, which includes ERK1/2, p38MAPK, and JNK, is involved in the regulation of various cellular responses, such as cell proliferation, survival, apoptosis, and inflammation [2,3]. Particularly, in keratinocytes, p38MAPK activation promotes UVB-induced apoptosis [2]. Therefore, the identification and characterization of compounds that can modulate MAPK signaling in keratinocytes may help in developing novel drugs for treating skin diseases. Citrus fruits are widely produced and consumed as fresh fruits and processed products. Citrus peels, considered as a waste byproduct of citrus-processing industries (fruit slices and citrus juice), contain a high content of polyphenolic compounds. Thus, these are considered as a potentially valuable resource. Polymethoxyflavones (PMFs), which are found in citrus peels, belong to a specific flavonoid subclass, in which all or most hydroxyl groups are methylated. They have been reported to have chemopreventive effects, such as anti-cancer, anti-inflammation, and antiatherosclerosis as well as neuroprotective effects [4]. Sudachitin (5,7,40 -trihydroxy-6,8,30 -trimethoxyflavone) is extracted from the
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Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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peel of Citrus sudachi (a well-known fruit in Tokushima Prefecture, Japan) and is a PMF. It has been reported to exhibit several biological activities, such as anti-inflammatory, immune-enhancing, and anti-metabolic syndrome effects [5e8]. A previous study demonstrated that sudachitin could exert its biological activity via the regulation of the MAPK pathways [6]. Sudachitin suppresses inflammatory bone destruction by inhibiting RANKL-induced ERK1/2 and JNK activations. However, a study reported contradictory results that sudachitin did not affect TNFa-induced activation of MAPK family members [7]. Thus, the effect of sudachitin on MAPK signaling pathways remains controversial and unclear. We recently reported that in human keratinocyte HaCaT cells, sudachitin and nobiletin (5,6,7,8,30 ,40 -hexamethoxyflavone) can induce apoptosis and autophagy, respectively, indicating that the two PMFs induce distinct cellular responses despite small structural differences [9]. Other studies suggested that hydroxylated PMFs, in which some of the methoxy groups are replaced by a hydroxyl group, show a higher ability to induce apoptosis than nonhydroxylated PMFs [10,11]. These PMFs include 3,5-dihydroxy6,7,30 40 -tetramethoxyflavone and 5-demethyltangeretin (5hydroxy-6,7,8,40 -tetramethoxyflavone). Although the methoxy groups at the C-30 and C-8 positions may be involved in the induction of apoptosis [9,12,13], the structureeactivity relationship and molecular mechanism underlying PMF-induced apoptosis are not yet fully elucidated. Understanding the mechanisms of distinct cellular responses caused by small structural differences in PMFs may lead to the discovery of more effective apoptotic inducers and identify different biological activities of PMFs. In this study, we investigated the mechanism underlying sudachitin-induced apoptosis via the MAPK pathways. We demonstrated that sudachitin induced caspase-dependent apoptosis through the activation of the p38MAPK pathway. Furthermore, we demonstrated that sudachitin inhibited cell migration and proliferation by suppressing epidermal growth factor (EGF)-induced ERK1/2 activation. Together, these results demonstrate that small structural differences in PMFs can alter their MAPK regulation activities, and suggest that sudachitin may be a potential therapeutic drug for skin disease. 2. Materials and methods 2.1. Materials and cell culture Sudachitin and nobiletin (Wako Pure Chemical Industries) were dissolved in 100% DMSO, and cells were exposed to a final concentration of 0.1% DMSO. EGF (Peptide Institute) and mitomycin C were dissolved in sterile water, while Z-VAD-FMK (Peptide Institute) and SB203580 (Calbiochem) were dissolved with DMSO. Human keratinocyte HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 C in a humidified incubator with 5% CO2.
intensities were quantified using the ImageJ software (NIH). 2.3. Luciferase reporter assay HaCaT cells were plated at a density of 3 104 cells in a 24-well plate, and were transfected with pFR-Luc, pFA2-Elk1, and pCMV-bgal using FuGENE HD (Roche) according to the manufacturer's instructions. After serum-starvation for 2 h and subsequent treatment with 30 mM sudachitin for 1 h, cells were stimulated with 1 nM EGF for an additional 6 h. After harvest, luciferase and bgalactosidase activities were performed as previously described [14]. Luciferase activity was normalized to b-galactosidase activity. 2.4. In vitro wound healing assay In vitro wound healing assay was performed as previously described [15]. In brief, HaCaT cells were seeded on a 24-well plate. After cells were grown to confluence, the culture medium was replaced with serum-free medium for 2 h. Then, cells were pretreated with DMSO or 30 mM sudachitin in the presence of 10 mg/ml mitomycin C. After 1 h, the confluent monolayers were scratched with a yellow pipette tip. The detached cells were removed by washing with PBS three times. Subsequently, cells were treated with DMSO or 30 mM sudachitin in the presence or absence of 1 nM EGF for 24 h. Images were captured in the same position at 0 and 24 h after the scratch using an IN Cell Analyzer 6000 system (GE Healthcare), and the wound areas were quantified using ImageJ software. The migration area was calculated as the difference between the initial and final wound area. 2.5. Proliferation assay Cell proliferation was measured by 5-bromo-20 -deoxyuridine (BrdU) incorporation assay using a CycLex Cellular BrdU ELISA Kit Ver.2 (Medical & Biological Laboratories) according to the manufacturer's instructions. Briefly, HaCaT cells were plated on 96-well plates and were serum-starved for 24 h. After pretreatment with 10 mM sudachitin for 1 h, cells were treated with 1 nM EGF. After 24 h, the cells were incubated with BrdU for 2 h. Following treatment with anti-BrdU antibody and substrate, the absorbance at 450 nm with 540 nm as reference was measured using the Infinite M200 plate reader (TECAN Japan). 2.6. Statistical analysis All experiments were performed multiple times to confirm their reproducibility. One representative set of data was shown in the figures, and data were expressed as the mean ± standard error. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's or Bonferroni's multiple comparison tests using GraphPad Prism (GraphPad Software). 3. Results
2.2. Immunoblot analysis Immunoblot analysis was performed as previously described [9]. Cells were treated with sudachitin (30, 100 mM), nobiletin (100 mM), or DMSO for different time periods. Total cell lysates were prepared and subjected to immunoblot analysis using antibodies against Bid, cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), total/phospho-ERK1/2, total/phospho-p38MAPK, total/ phospho-JNK, total/phospho-MKK3/6, phospho-HSP27, phosphoRaf-1 (Ser-338) (Cell Signaling Technology), Raf-1 (BD Transduction Laboratories), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Wako Pure Chemical Industries). Immunoblot band
3.1. Sudachitin induces caspase-dependent apoptosis through the regulation of MAPK pathways We recently reported that sudachitin and nobiletin induce apoptosis and autophagy in HaCaT cells, respectively [9]. However, the mechanisms underlying apoptosis are not yet fully understood. In this study, we aimed to characterize the molecular pathways underlying sudachitin-induced apoptosis. To confirm the apoptotic effect of sudachitin, we measured caspase activation in HaCaT cells using immunoblot analysis. Consistent with previous results [9], sudachitin induced PARP cleavage, which is dependent on caspase-
Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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3 activation and is an apoptotic hallmark, in a concentrationdependent manner (Fig. 1A). Sudachitin also caused a dosedependent proteolytic cleavage and the activation of caspase-3. BH3-interacting-domain death agonist (Bid) is a pro-apoptotic Bcl-2 family protein activated by caspase-8. Sudachitin significantly decreased the level of full-length Bid, accompanied by an increased amount of its cleaved product, truncated Bid (tBid). Furthermore, HaCaT cells were pretreated with the pan-caspase inhibitor Z-VAD-FMK prior to being exposed to sudachitin. As shown in Fig. 1B, Z-VAD-FMK significantly reduced the sudachitininduced cleavage of PARP, further reinforcing that sudachitin induces apoptosis via the caspase pathway. Previous studies have demonstrated that some polyphenolic compounds induce apoptosis by modulating the MAPK pathways (e.g., ERK1/2, p38MAPK, and JNK) [16,17]. Therefore, we assessed whether sudachitin-induced apoptosis is also associated with the ERK1/2, p38MAPK, and JNK pathways. Sudachitin increased p38MAPK and JNK phosphorylation, but significantly decreased ERK1/2 phosphorylation (Fig. 1C). In contrast, nobiletin significantly increased ERK1/2 phosphorylation. These results suggest that sudachitin induces apoptosis through different signaling pathways from nobiletin.
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3.2. Sudachitin induces apoptosis through the activation of the p38MAPK pathway A previous study has revealed that p38MAPK plays an important role in chemotherapeutic agent-induced apoptosis [18]. As sudachitin, not nobiletin, induced p38MAPK phosphorylation and activation, we next examined the dose-dependence of the effect of sudachitin on p38MAPK phosphorylation. Sudachitin promoted p38MAPK phosphorylation, whereas it decreased ERK1/2 phosphorylation in a concentration-dependent manner (Fig. 2A). Furthermore, we examined the course of p38MAPK phosphorylation over time in response to sudachitin. We observed that p38MAPK phosphorylation was significantly increased at 4 h after stimulation by 100 mM sudachitin, and that this increase was sustained for up to 24 h (Fig. 2B). As p38MAPK is phosphorylated and activated by MAP kinase kinase 3/6 (MKK3/6), we subsequently examined whether the MKK3/6-p38MAPK cascade is activated by sudachitin. MKK3/6 phosphorylation was significantly increased following sudachitin treatment, but not following nobiletin treatment (Fig. 2C). To confirm the involvement of p38MAPK in sudachitin-induced apoptosis, a specific p38MAPK inhibitor, SB203580, was used. As shown in Fig. 2D, SB203580 significantly reduced the sudachitin-induced phosphorylation of heat shock
Fig. 1. Sudachitin induces caspase-mediated apoptosis and regulates MAPK pathways in HaCaT cells. (A) HaCaT cells were stimulated with sudachitin (30 and 100 mM) or DMSO for 24 h. (B) After pretreatment with 50 mM Z-VAD-FMK for 1 h, HaCaT cells were treated with 30 mM sudachitin (Sud) for 24 h. (C) HaCaT cells were stimulated with 100 mM nobiletin (N), 100 mM sudachitin (S), or DMSO (D) for 4 h. The cell lysates were subjected to immunoblot analysis with antibodies specific for the indicated proteins, and the detected bands were quantitated by ImageJ software. The levels of full-length Bid and cleaved caspase-3 were normalized to those of GAPDH. The levels of cleaved PARP and phosphorylated MAPKs were normalized to those of full-length PARP and total MAPKs, respectively. The data were expressed as the mean ± standard error derived from at least three independent experiments, and statistical analysis was performed by ANOVA with either Tukey's (A) or Bonferroni's (B and C) multiple comparison. ***p < 0.001, *p < 0.05.
Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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Fig. 2. Sudachitin induces apoptosis through the activation of the p38MAPK pathway. (A) HaCaT cells were treated with sudachitin (30 and 100 mM) or DMSO for 4 h. (B) HaCaT cells were stimulated with 100 mM sudachitin for indicated times. (C) HaCaT cells were stimulated with 30 mM nobiletin (N), 30 mM sudachitin (S), or DMSO (D) for 4 h. (D) HaCaT cells were treated with 30 mM sudachitin (Sud) in the presence of 10 mM SB203580 (SB) or DMSO (D) for 4 or 24 h. The cell lysates were analyzed by immunoblot analysis. The levels of phosphorylated p38MAPK, MKK3/6, HSP27, and cleaved PARP were normalized to those of total p38MAPK, MKK3/6, GAPDH, and full-length PARP, respectively. The data were expressed as the mean ± standard error derived from at least three independent experiments, and statistical analysis was performed by ANOVA with either Tukey's (A) or Bonferroni's (BeD) multiple comparison. ***p < 0.001, **p < 0.01, *p < 0.05.
protein (HSP) 27, a protein that functions downstream of p38MAPK. Moreover, sudachitin-induced PARP cleavage was significantly inhibited by SB203580, indicating that sudachitin-induced apoptosis occurs through the activation of the p38MAPK pathway. 3.3. Sudachitin suppresses EGF-induced ERK1/2 activation Sudachitin, in contrast to nobiletin, induced a decrease in ERK1/ 2 phosphorylation (Fig. 1C). The activation of the ERK1/2 pathway is typically triggered by the interaction of the cell surface receptors with mitogens such as EGF, followed by the activation of Ras, Raf, MEK, and ERK. In keratinocytes, EGF provides protection against UV-induced apoptosis, and the inhibitors of the EGF receptor enhance UVB-induced apoptosis [19], suggesting that the EGF
signaling pathway may function as a cell survival pathway. As the ERK1/2 pathway has recently attracted some attention as a potential target for the treatment of psoriasis and in anti-cancer therapy [20], we decided to further study the inhibitory effect of sudachitin on the ERK1/2 pathway. ERK1/2 phosphorylation was significantly decreased at 4 and 8 h after sudachitin treatment, but returned to pretreatment levels at 24 h after treatment (Fig. 3A). During in vitro cell culture, cells are typically cultured in FBSsupplemented media containing several growth factors such as EGF. To determine the precise effect of sudachitin on seruminduced ERK1/2 phosphorylation, serum-starved cells were stimulated with 5% FBS in the presence or absence of sudachitin. Raf-1 phosphorylation on Ser-338, required for Raf activation, and ERK1/ 2 phosphorylation were observed in serum-depleted cells;
Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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Fig. 3. Sudachitin suppresses EGF-induced ERK1/2 activation. (A) HaCaT cells were stimulated with 100 mM sudachitin for indicated times. (B and C) HaCaT cells were serum-starved for 2 h and pretreated with 30 mM sudachitin for 1 h. Then, cells were treated with (B) 5% FBS or (C) 1 nM EGF for 4 h. The cell lysates were subjected to the immunoblot analysis with the indicated antibodies. The levels of phosphorylated Raf-1 and ERK1/2 were normalized to those of total Raf-1 and ERK1/2, respectively. (D) HaCaT cells were transfected with a GAL4-responsive luciferase reporter plasmid and a GAL4-Elk-1 expression plasmid together with pCMV-b-gal. After serum-starvation for 2 h and subsequent treatment with 30 mM sudachitin for 1 h, cells were stimulated with 1 nM EGF for another 6 h. After harvest, luciferase and b-galactosidase activities were measured, and the luciferase activity was normalized to the b-galactosidase activity. All data are presented as the mean ± standard error derived from at least three independent experiments, and statistical analysis was performed by one-way ANOVA with Bonferroni's multiple comparison. ***p < 0.001, **p < 0.01, *p < 0.05.
however, these were significantly increased in the presence of serum (Fig. 3B). Pretreatment with sudachitin significantly inhibited the serum-stimulated activation of Raf-1 and ERK1/2. A similar result was obtained when serum-starved cells were stimulated with EGF (Fig. 3C). We also analyzed the effect of sudachitin on EGFstimulated ERK1/2 activation using the GAL4/Elk-1 reporter system [14]. The Elk-1 transcription factor is a downstream target of the ERK pathway. EGF stimulated an increase in the Elk-1-dependent luciferase activity, and this increase was significantly suppressed by sudachitin treatment (Fig. 3D). These results indicate that sudachitin can inhibit EGF-stimulated ERK1/2 signaling.
3.4. Sudachitin suppresses EGF-induced migration and proliferation of HaCaT cells The ERK1/2 pathway plays important roles in many cellular functions, including cell proliferation, differentiation, migration, senescence, and apoptosis. In HaCaT cells, EGF-induced ERK1/2 activation has been shown to be associated with cell migration and proliferation [21,22]. Therefore, we tested whether sudachitin could inhibit EGF-induced migration using the wound healing assay. The assay revealed that sudachitin significantly reduced the EGF-stimulated cell migration of HaCaT cells (Fig. 4A). Furthermore, the effect of sudachitin on HaCaT cell proliferation was evaluated using BrdU incorporation assay. EGF stimulation caused a
significant increase in HaCaT cell proliferation, and this was significantly inhibited by pretreatment with sudachitin (Fig. 4B). These results suggest that sudachitin can inhibit HaCaT cell migration and proliferation through the inhibition of the ERK1/2 pathway.
4. Discussion The MAPK pathways are involved in many cellular responses, such as apoptosis, cell proliferation, differentiation, migration, and inflammation, and are regulated by various compounds, including flavonoids. The present study demonstrated that sudachitin can activate the p38MAPK pathway and inhibit the ERK1/2 pathway in HaCaT cells. In contrast, nobiletin, which has methoxy groups instead of hydroxyl groups at the C-5, C-7, and C-40 positions in sudachitin, activated ERK1/2. A previous study has reported that nobiletin activates ERK1/2 in rat pheochromocytoma PC12D cells [23]. Contrary to these, it has been shown that in human osteosarcoma U2OS and HOS cells, nobiletin suppressed both ERK1/2 and JNK activities [24]. These data suggest that the regulation of MAPK pathways by PMFs may be cell type-specific. A study on the structureeactivity relationships of nobiletin and its analogs showed that nobiletin most potently activated ERK1/2, and that other compounds, such as tangeretin (5,6,7,8,40 -pentamethoxyflavone) and 6-demethoxynobiletin (6-hydroxy-5,7,8,30 ,40 -
Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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Fig. 4. Sudachitin suppresses EGF-induced migration and proliferation of HaCaT cells. (A) Serum-starved HaCaT cells were pretreated with 30 mM sudachitin in the presence of 10 mg/ml mitomycin C for 1 h, and then subjected to the in vitro wound healing assay in the presence or absence of 1 nM EGF. Images were obtained using an IN Cell Analyzer 6000 system, and wound area was quantified using ImageJ software. The migration area was calculated as the area covered by the cells after incubation for 24 h. (B) Serum-starved HaCaT cells were pretreated with 10 mM sudachitin for 1 h, and then treated with 1 nM EGF. After 24 h, cells were subjected to the BrdU incorporation assay. The data were expressed as the mean ± standard error derived from at least three independent experiments, and statistical analysis was performed by ANOVA with Bonferroni's multiple comparison. ***p < 0.001, **p < 0.01.
hexamethoxyflavone), were also able to activate ERK1/2 [23]. However, 5-demethylnobiletin (5-hydroxy-6,7,8,30 ,40 -hexamethoxyflavone), sinensetin (5,6,7,30 ,40 -pentamethoxyflavone), and 6demethoxytangeretin (6-hydroxy-5,7,8,40 -pentamethoxyflavone) had no effect at a dose of 100 mM, suggesting that the methoxy substituents at the C-5, C-6, C-8, and C-30 positions are critical for activating the ERK1/2 pathway. Based on the findings of the present study, we suggest that introducing a methoxy group at the C-5 position is important for ERK1/2 activation, and that hydroxyl groups at the C-7 and C-40 positions lead to the inhibition of ERK1/2 activity. Further investigation regarding the structureeactivity relationship of other PMFs is necessary to further clarify the regulation of MAPK pathways by PMFs. We demonstrated that sudachitin induced apoptosis and inhibited EGF-induced HaCaT cell migration and proliferation. HaCaT cells are immortalized keratinocytes that retain their differentiation ability, and are widely used as an in vitro model system to study not only keratinocyte proliferation and differentiation but also skin diseases such as psoriasis [25]. Psoriasis is a chronic inflammatory skin disease that leads to pain, itching, and bleeding in patients; it is caused by excessive cell proliferation, angiogenesis, inflammation, altered apoptotic ability, and the promotion of migration in keratinocytes [26,27]. The induction of apoptosis and inhibition of cell migration and proliferation in keratinocytes may be ideal mechanisms to target for the treatment of psoriasis. Recently, treatment for psoriasis based on the anti-proliferative and apoptotic activities of natural compounds have gained some attention [28]. A recent study showed that the EGF receptor and its ligands (transforming growth factor-a, amphiregulin, and heparinbinding-EGF) are overexpressed in the epidermis of active psoriatic lesions [29]. Furthermore, it has been reported that serum EGF levels are elevated in patients with psoriasis and that these correlate with disease severity [30]. As the induction of apoptosis and
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Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells Shogo Abe a, Keizo Yuasa a, b, * a b
Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan Department of Bioscience and Bioindustry, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 August 2019 Received in revised form 26 August 2019 Accepted 4 September 2019 Available online xxx
Although we recently reported that sudachitin (5,7,40 -trihydroxy-6,8,30 -trimethoxyflavone), a polymethoxyflavone isolated from the peel of Citrus sudachi, can induce apoptosis in human keratinocyte HaCaT cells, the mechanism underlying its action remains unclear. In this study, we explored the mechanisms underlying sudachitin-induced apoptosis in HaCaT cells. Sudachitin activated p38MAPK and inhibited ERK1/2, whereas another polymethoxyflavone, nobiletin (5,6,7,8,30 ,40 -hexamethoxyflavone), activated ERK1/2. The p38MAPK inhibitor SB203580 significantly attenuated sudachitin-induced heat shock protein 27 phosphorylation, downstream of p38MAPK, and subsequent apoptosis, indicating that sudachitin induces apoptosis via the p38MAPK pathway. Additionally, sudachitin inhibited serum- and EGF-stimulated Raf-1-ERK1/2 activation, and blocked EGF-induced cell migration and proliferation in HaCaT cells. These results suggest that small structural differences in polymethoxyflavones can induce different cellular responses by altering the regulation of MAPK activities and that sudachitin may be a potential candidate for developing new drugs for skin diseases such as psoriasis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Polymethoxyflavone Keratinocyte Apoptosis MAPK Cell migration and proliferation
1. Introduction The skin is the largest and outermost organ of the body and functions as a primary barrier protecting the body from harmful compounds, pathogenic microbes, and ultraviolet (UV) radiation. The skin comprises three layers: the epidermis, dermis, and subcutaneous layer. The outermost epidermis is composed of a stratified epithelium with distinct layers of keratinocytes [1]. Exposure to UV radiation results in DNA damage, which causes mutations; this is considered as an event initiating skin carcinogenesis. The elimination of UV radiation-damaged keratinocytes via apoptosis is an essential mechanism for protecting the skin from sunlight. However, the increased proliferative and reduced apoptotic abilities of keratinocytes causes abnormal proliferation, leading to
Abbreviations: UV, ultraviolet; PMF, polymethoxyflavone; EGF, epidermal growth factor; PARP, poly (ADP-ribose) polymerase; HSP, heat-shock protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Bid, BH3-interacting-domain death agonist; MKK, MAP kinase kinase. * Corresponding author. Department of Bioscience and Bioindustry, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan. E-mail address: [email protected] (K. Yuasa).
conditions such as skin cancer, psoriasis, keratosis, verrucae, and lichen simplex chronicus [2]. A precise balance of apoptosis and cell proliferation in keratinocytes is important for the treatment and prevention of such skin diseases. The MAPK family, which includes ERK1/2, p38MAPK, and JNK, is involved in the regulation of various cellular responses, such as cell proliferation, survival, apoptosis, and inflammation [2,3]. Particularly, in keratinocytes, p38MAPK activation promotes UVB-induced apoptosis [2]. Therefore, the identification and characterization of compounds that can modulate MAPK signaling in keratinocytes may help in developing novel drugs for treating skin diseases. Citrus fruits are widely produced and consumed as fresh fruits and processed products. Citrus peels, considered as a waste byproduct of citrus-processing industries (fruit slices and citrus juice), contain a high content of polyphenolic compounds. Thus, these are considered as a potentially valuable resource. Polymethoxyflavones (PMFs), which are found in citrus peels, belong to a specific flavonoid subclass, in which all or most hydroxyl groups are methylated. They have been reported to have chemopreventive effects, such as anti-cancer, anti-inflammation, and antiatherosclerosis as well as neuroprotective effects [4]. Sudachitin (5,7,40 -trihydroxy-6,8,30 -trimethoxyflavone) is extracted from the
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Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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peel of Citrus sudachi (a well-known fruit in Tokushima Prefecture, Japan) and is a PMF. It has been reported to exhibit several biological activities, such as anti-inflammatory, immune-enhancing, and anti-metabolic syndrome effects [5e8]. A previous study demonstrated that sudachitin could exert its biological activity via the regulation of the MAPK pathways [6]. Sudachitin suppresses inflammatory bone destruction by inhibiting RANKL-induced ERK1/2 and JNK activations. However, a study reported contradictory results that sudachitin did not affect TNFa-induced activation of MAPK family members [7]. Thus, the effect of sudachitin on MAPK signaling pathways remains controversial and unclear. We recently reported that in human keratinocyte HaCaT cells, sudachitin and nobiletin (5,6,7,8,30 ,40 -hexamethoxyflavone) can induce apoptosis and autophagy, respectively, indicating that the two PMFs induce distinct cellular responses despite small structural differences [9]. Other studies suggested that hydroxylated PMFs, in which some of the methoxy groups are replaced by a hydroxyl group, show a higher ability to induce apoptosis than nonhydroxylated PMFs [10,11]. These PMFs include 3,5-dihydroxy6,7,30 40 -tetramethoxyflavone and 5-demethyltangeretin (5hydroxy-6,7,8,40 -tetramethoxyflavone). Although the methoxy groups at the C-30 and C-8 positions may be involved in the induction of apoptosis [9,12,13], the structureeactivity relationship and molecular mechanism underlying PMF-induced apoptosis are not yet fully elucidated. Understanding the mechanisms of distinct cellular responses caused by small structural differences in PMFs may lead to the discovery of more effective apoptotic inducers and identify different biological activities of PMFs. In this study, we investigated the mechanism underlying sudachitin-induced apoptosis via the MAPK pathways. We demonstrated that sudachitin induced caspase-dependent apoptosis through the activation of the p38MAPK pathway. Furthermore, we demonstrated that sudachitin inhibited cell migration and proliferation by suppressing epidermal growth factor (EGF)-induced ERK1/2 activation. Together, these results demonstrate that small structural differences in PMFs can alter their MAPK regulation activities, and suggest that sudachitin may be a potential therapeutic drug for skin disease. 2. Materials and methods 2.1. Materials and cell culture Sudachitin and nobiletin (Wako Pure Chemical Industries) were dissolved in 100% DMSO, and cells were exposed to a final concentration of 0.1% DMSO. EGF (Peptide Institute) and mitomycin C were dissolved in sterile water, while Z-VAD-FMK (Peptide Institute) and SB203580 (Calbiochem) were dissolved with DMSO. Human keratinocyte HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 C in a humidified incubator with 5% CO2.
intensities were quantified using the ImageJ software (NIH). 2.3. Luciferase reporter assay HaCaT cells were plated at a density of 3 104 cells in a 24-well plate, and were transfected with pFR-Luc, pFA2-Elk1, and pCMV-bgal using FuGENE HD (Roche) according to the manufacturer's instructions. After serum-starvation for 2 h and subsequent treatment with 30 mM sudachitin for 1 h, cells were stimulated with 1 nM EGF for an additional 6 h. After harvest, luciferase and bgalactosidase activities were performed as previously described [14]. Luciferase activity was normalized to b-galactosidase activity. 2.4. In vitro wound healing assay In vitro wound healing assay was performed as previously described [15]. In brief, HaCaT cells were seeded on a 24-well plate. After cells were grown to confluence, the culture medium was replaced with serum-free medium for 2 h. Then, cells were pretreated with DMSO or 30 mM sudachitin in the presence of 10 mg/ml mitomycin C. After 1 h, the confluent monolayers were scratched with a yellow pipette tip. The detached cells were removed by washing with PBS three times. Subsequently, cells were treated with DMSO or 30 mM sudachitin in the presence or absence of 1 nM EGF for 24 h. Images were captured in the same position at 0 and 24 h after the scratch using an IN Cell Analyzer 6000 system (GE Healthcare), and the wound areas were quantified using ImageJ software. The migration area was calculated as the difference between the initial and final wound area. 2.5. Proliferation assay Cell proliferation was measured by 5-bromo-20 -deoxyuridine (BrdU) incorporation assay using a CycLex Cellular BrdU ELISA Kit Ver.2 (Medical & Biological Laboratories) according to the manufacturer's instructions. Briefly, HaCaT cells were plated on 96-well plates and were serum-starved for 24 h. After pretreatment with 10 mM sudachitin for 1 h, cells were treated with 1 nM EGF. After 24 h, the cells were incubated with BrdU for 2 h. Following treatment with anti-BrdU antibody and substrate, the absorbance at 450 nm with 540 nm as reference was measured using the Infinite M200 plate reader (TECAN Japan). 2.6. Statistical analysis All experiments were performed multiple times to confirm their reproducibility. One representative set of data was shown in the figures, and data were expressed as the mean ± standard error. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's or Bonferroni's multiple comparison tests using GraphPad Prism (GraphPad Software). 3. Results
2.2. Immunoblot analysis Immunoblot analysis was performed as previously described [9]. Cells were treated with sudachitin (30, 100 mM), nobiletin (100 mM), or DMSO for different time periods. Total cell lysates were prepared and subjected to immunoblot analysis using antibodies against Bid, cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), total/phospho-ERK1/2, total/phospho-p38MAPK, total/ phospho-JNK, total/phospho-MKK3/6, phospho-HSP27, phosphoRaf-1 (Ser-338) (Cell Signaling Technology), Raf-1 (BD Transduction Laboratories), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Wako Pure Chemical Industries). Immunoblot band
3.1. Sudachitin induces caspase-dependent apoptosis through the regulation of MAPK pathways We recently reported that sudachitin and nobiletin induce apoptosis and autophagy in HaCaT cells, respectively [9]. However, the mechanisms underlying apoptosis are not yet fully understood. In this study, we aimed to characterize the molecular pathways underlying sudachitin-induced apoptosis. To confirm the apoptotic effect of sudachitin, we measured caspase activation in HaCaT cells using immunoblot analysis. Consistent with previous results [9], sudachitin induced PARP cleavage, which is dependent on caspase-
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3 activation and is an apoptotic hallmark, in a concentrationdependent manner (Fig. 1A). Sudachitin also caused a dosedependent proteolytic cleavage and the activation of caspase-3. BH3-interacting-domain death agonist (Bid) is a pro-apoptotic Bcl-2 family protein activated by caspase-8. Sudachitin significantly decreased the level of full-length Bid, accompanied by an increased amount of its cleaved product, truncated Bid (tBid). Furthermore, HaCaT cells were pretreated with the pan-caspase inhibitor Z-VAD-FMK prior to being exposed to sudachitin. As shown in Fig. 1B, Z-VAD-FMK significantly reduced the sudachitininduced cleavage of PARP, further reinforcing that sudachitin induces apoptosis via the caspase pathway. Previous studies have demonstrated that some polyphenolic compounds induce apoptosis by modulating the MAPK pathways (e.g., ERK1/2, p38MAPK, and JNK) [16,17]. Therefore, we assessed whether sudachitin-induced apoptosis is also associated with the ERK1/2, p38MAPK, and JNK pathways. Sudachitin increased p38MAPK and JNK phosphorylation, but significantly decreased ERK1/2 phosphorylation (Fig. 1C). In contrast, nobiletin significantly increased ERK1/2 phosphorylation. These results suggest that sudachitin induces apoptosis through different signaling pathways from nobiletin.
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3.2. Sudachitin induces apoptosis through the activation of the p38MAPK pathway A previous study has revealed that p38MAPK plays an important role in chemotherapeutic agent-induced apoptosis [18]. As sudachitin, not nobiletin, induced p38MAPK phosphorylation and activation, we next examined the dose-dependence of the effect of sudachitin on p38MAPK phosphorylation. Sudachitin promoted p38MAPK phosphorylation, whereas it decreased ERK1/2 phosphorylation in a concentration-dependent manner (Fig. 2A). Furthermore, we examined the course of p38MAPK phosphorylation over time in response to sudachitin. We observed that p38MAPK phosphorylation was significantly increased at 4 h after stimulation by 100 mM sudachitin, and that this increase was sustained for up to 24 h (Fig. 2B). As p38MAPK is phosphorylated and activated by MAP kinase kinase 3/6 (MKK3/6), we subsequently examined whether the MKK3/6-p38MAPK cascade is activated by sudachitin. MKK3/6 phosphorylation was significantly increased following sudachitin treatment, but not following nobiletin treatment (Fig. 2C). To confirm the involvement of p38MAPK in sudachitin-induced apoptosis, a specific p38MAPK inhibitor, SB203580, was used. As shown in Fig. 2D, SB203580 significantly reduced the sudachitin-induced phosphorylation of heat shock
Fig. 1. Sudachitin induces caspase-mediated apoptosis and regulates MAPK pathways in HaCaT cells. (A) HaCaT cells were stimulated with sudachitin (30 and 100 mM) or DMSO for 24 h. (B) After pretreatment with 50 mM Z-VAD-FMK for 1 h, HaCaT cells were treated with 30 mM sudachitin (Sud) for 24 h. (C) HaCaT cells were stimulated with 100 mM nobiletin (N), 100 mM sudachitin (S), or DMSO (D) for 4 h. The cell lysates were subjected to immunoblot analysis with antibodies specific for the indicated proteins, and the detected bands were quantitated by ImageJ software. The levels of full-length Bid and cleaved caspase-3 were normalized to those of GAPDH. The levels of cleaved PARP and phosphorylated MAPKs were normalized to those of full-length PARP and total MAPKs, respectively. The data were expressed as the mean ± standard error derived from at least three independent experiments, and statistical analysis was performed by ANOVA with either Tukey's (A) or Bonferroni's (B and C) multiple comparison. ***p < 0.001, *p < 0.05.
Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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Fig. 2. Sudachitin induces apoptosis through the activation of the p38MAPK pathway. (A) HaCaT cells were treated with sudachitin (30 and 100 mM) or DMSO for 4 h. (B) HaCaT cells were stimulated with 100 mM sudachitin for indicated times. (C) HaCaT cells were stimulated with 30 mM nobiletin (N), 30 mM sudachitin (S), or DMSO (D) for 4 h. (D) HaCaT cells were treated with 30 mM sudachitin (Sud) in the presence of 10 mM SB203580 (SB) or DMSO (D) for 4 or 24 h. The cell lysates were analyzed by immunoblot analysis. The levels of phosphorylated p38MAPK, MKK3/6, HSP27, and cleaved PARP were normalized to those of total p38MAPK, MKK3/6, GAPDH, and full-length PARP, respectively. The data were expressed as the mean ± standard error derived from at least three independent experiments, and statistical analysis was performed by ANOVA with either Tukey's (A) or Bonferroni's (BeD) multiple comparison. ***p < 0.001, **p < 0.01, *p < 0.05.
protein (HSP) 27, a protein that functions downstream of p38MAPK. Moreover, sudachitin-induced PARP cleavage was significantly inhibited by SB203580, indicating that sudachitin-induced apoptosis occurs through the activation of the p38MAPK pathway. 3.3. Sudachitin suppresses EGF-induced ERK1/2 activation Sudachitin, in contrast to nobiletin, induced a decrease in ERK1/ 2 phosphorylation (Fig. 1C). The activation of the ERK1/2 pathway is typically triggered by the interaction of the cell surface receptors with mitogens such as EGF, followed by the activation of Ras, Raf, MEK, and ERK. In keratinocytes, EGF provides protection against UV-induced apoptosis, and the inhibitors of the EGF receptor enhance UVB-induced apoptosis [19], suggesting that the EGF
signaling pathway may function as a cell survival pathway. As the ERK1/2 pathway has recently attracted some attention as a potential target for the treatment of psoriasis and in anti-cancer therapy [20], we decided to further study the inhibitory effect of sudachitin on the ERK1/2 pathway. ERK1/2 phosphorylation was significantly decreased at 4 and 8 h after sudachitin treatment, but returned to pretreatment levels at 24 h after treatment (Fig. 3A). During in vitro cell culture, cells are typically cultured in FBSsupplemented media containing several growth factors such as EGF. To determine the precise effect of sudachitin on seruminduced ERK1/2 phosphorylation, serum-starved cells were stimulated with 5% FBS in the presence or absence of sudachitin. Raf-1 phosphorylation on Ser-338, required for Raf activation, and ERK1/ 2 phosphorylation were observed in serum-depleted cells;
Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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Fig. 3. Sudachitin suppresses EGF-induced ERK1/2 activation. (A) HaCaT cells were stimulated with 100 mM sudachitin for indicated times. (B and C) HaCaT cells were serum-starved for 2 h and pretreated with 30 mM sudachitin for 1 h. Then, cells were treated with (B) 5% FBS or (C) 1 nM EGF for 4 h. The cell lysates were subjected to the immunoblot analysis with the indicated antibodies. The levels of phosphorylated Raf-1 and ERK1/2 were normalized to those of total Raf-1 and ERK1/2, respectively. (D) HaCaT cells were transfected with a GAL4-responsive luciferase reporter plasmid and a GAL4-Elk-1 expression plasmid together with pCMV-b-gal. After serum-starvation for 2 h and subsequent treatment with 30 mM sudachitin for 1 h, cells were stimulated with 1 nM EGF for another 6 h. After harvest, luciferase and b-galactosidase activities were measured, and the luciferase activity was normalized to the b-galactosidase activity. All data are presented as the mean ± standard error derived from at least three independent experiments, and statistical analysis was performed by one-way ANOVA with Bonferroni's multiple comparison. ***p < 0.001, **p < 0.01, *p < 0.05.
however, these were significantly increased in the presence of serum (Fig. 3B). Pretreatment with sudachitin significantly inhibited the serum-stimulated activation of Raf-1 and ERK1/2. A similar result was obtained when serum-starved cells were stimulated with EGF (Fig. 3C). We also analyzed the effect of sudachitin on EGFstimulated ERK1/2 activation using the GAL4/Elk-1 reporter system [14]. The Elk-1 transcription factor is a downstream target of the ERK pathway. EGF stimulated an increase in the Elk-1-dependent luciferase activity, and this increase was significantly suppressed by sudachitin treatment (Fig. 3D). These results indicate that sudachitin can inhibit EGF-stimulated ERK1/2 signaling.
3.4. Sudachitin suppresses EGF-induced migration and proliferation of HaCaT cells The ERK1/2 pathway plays important roles in many cellular functions, including cell proliferation, differentiation, migration, senescence, and apoptosis. In HaCaT cells, EGF-induced ERK1/2 activation has been shown to be associated with cell migration and proliferation [21,22]. Therefore, we tested whether sudachitin could inhibit EGF-induced migration using the wound healing assay. The assay revealed that sudachitin significantly reduced the EGF-stimulated cell migration of HaCaT cells (Fig. 4A). Furthermore, the effect of sudachitin on HaCaT cell proliferation was evaluated using BrdU incorporation assay. EGF stimulation caused a
significant increase in HaCaT cell proliferation, and this was significantly inhibited by pretreatment with sudachitin (Fig. 4B). These results suggest that sudachitin can inhibit HaCaT cell migration and proliferation through the inhibition of the ERK1/2 pathway.
4. Discussion The MAPK pathways are involved in many cellular responses, such as apoptosis, cell proliferation, differentiation, migration, and inflammation, and are regulated by various compounds, including flavonoids. The present study demonstrated that sudachitin can activate the p38MAPK pathway and inhibit the ERK1/2 pathway in HaCaT cells. In contrast, nobiletin, which has methoxy groups instead of hydroxyl groups at the C-5, C-7, and C-40 positions in sudachitin, activated ERK1/2. A previous study has reported that nobiletin activates ERK1/2 in rat pheochromocytoma PC12D cells [23]. Contrary to these, it has been shown that in human osteosarcoma U2OS and HOS cells, nobiletin suppressed both ERK1/2 and JNK activities [24]. These data suggest that the regulation of MAPK pathways by PMFs may be cell type-specific. A study on the structureeactivity relationships of nobiletin and its analogs showed that nobiletin most potently activated ERK1/2, and that other compounds, such as tangeretin (5,6,7,8,40 -pentamethoxyflavone) and 6-demethoxynobiletin (6-hydroxy-5,7,8,30 ,40 -
Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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Fig. 4. Sudachitin suppresses EGF-induced migration and proliferation of HaCaT cells. (A) Serum-starved HaCaT cells were pretreated with 30 mM sudachitin in the presence of 10 mg/ml mitomycin C for 1 h, and then subjected to the in vitro wound healing assay in the presence or absence of 1 nM EGF. Images were obtained using an IN Cell Analyzer 6000 system, and wound area was quantified using ImageJ software. The migration area was calculated as the area covered by the cells after incubation for 24 h. (B) Serum-starved HaCaT cells were pretreated with 10 mM sudachitin for 1 h, and then treated with 1 nM EGF. After 24 h, cells were subjected to the BrdU incorporation assay. The data were expressed as the mean ± standard error derived from at least three independent experiments, and statistical analysis was performed by ANOVA with Bonferroni's multiple comparison. ***p < 0.001, **p < 0.01.
hexamethoxyflavone), were also able to activate ERK1/2 [23]. However, 5-demethylnobiletin (5-hydroxy-6,7,8,30 ,40 -hexamethoxyflavone), sinensetin (5,6,7,30 ,40 -pentamethoxyflavone), and 6demethoxytangeretin (6-hydroxy-5,7,8,40 -pentamethoxyflavone) had no effect at a dose of 100 mM, suggesting that the methoxy substituents at the C-5, C-6, C-8, and C-30 positions are critical for activating the ERK1/2 pathway. Based on the findings of the present study, we suggest that introducing a methoxy group at the C-5 position is important for ERK1/2 activation, and that hydroxyl groups at the C-7 and C-40 positions lead to the inhibition of ERK1/2 activity. Further investigation regarding the structureeactivity relationship of other PMFs is necessary to further clarify the regulation of MAPK pathways by PMFs. We demonstrated that sudachitin induced apoptosis and inhibited EGF-induced HaCaT cell migration and proliferation. HaCaT cells are immortalized keratinocytes that retain their differentiation ability, and are widely used as an in vitro model system to study not only keratinocyte proliferation and differentiation but also skin diseases such as psoriasis [25]. Psoriasis is a chronic inflammatory skin disease that leads to pain, itching, and bleeding in patients; it is caused by excessive cell proliferation, angiogenesis, inflammation, altered apoptotic ability, and the promotion of migration in keratinocytes [26,27]. The induction of apoptosis and inhibition of cell migration and proliferation in keratinocytes may be ideal mechanisms to target for the treatment of psoriasis. Recently, treatment for psoriasis based on the anti-proliferative and apoptotic activities of natural compounds have gained some attention [28]. A recent study showed that the EGF receptor and its ligands (transforming growth factor-a, amphiregulin, and heparinbinding-EGF) are overexpressed in the epidermis of active psoriatic lesions [29]. Furthermore, it has been reported that serum EGF levels are elevated in patients with psoriasis and that these correlate with disease severity [30]. As the induction of apoptosis and
the suppression of EGF-induced cell migration and proliferation of keratinocytes are important for the treatment of psoriasis, sudachitin may be a potential choice to search for more effective treatments for psoriasis and other skin diseases. Acknowledgements The authors would like to thank Enago (www.enago.jp) for the English language review. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.010. References [1] M. Gaur, M. Dobke, V.V. Lunyak, Mesenchymal stem cells from adipose tissue in clinical applications for dermatological indications and skin aging, Int. J. Mol. Sci. 18 (2017) 208. [2] D. Raj, D.E. Brash, D. Grossman, Keratinocyte apoptosis in epidermal development and disease, J. Investig. Dermatol. 126 (2006) 243e257. [3] J. Cursons, J. Gao, D.G. Hurley, C.G. Print, P.R. Dunbar, M.D. Jacobs, E.J. Crampin, Regulation of ERK-MAPK signaling in human epidermis, BMC Syst. Biol. 9 (2015) 41. [4] C.S. Lai, J.C. Wu, C.T. Ho, M.H. Pan, Disease chemopreventive effects and molecular mechanisms of hydroxylated polymethoxyflavones, Biofactors 41 (2015) 301e313. [5] K. Yuasa, K. Tada, G. Harita, T. Fujimoto, M. Tsukayama, A. Tsuji, Sudachitin, a polymethoxyflavone from Citrus sudachi, suppresses lipopolysaccharideinduced inflammatory responses in mouse macrophage-like RAW264 cells, Biosci. Biotechnol. Biochem. 76 (2012) 598e600. [6] Y. Ohyama, J. Ito, V.J. Kitano, J. Shimada, Y. Hakeda, The polymethoxy flavonoid sudachitin suppresses inflammatory bone destruction by directly inhibiting osteoclastogenesis due to reduced ROS production and MAPK activation in osteoclast precursors, PLoS One 13 (2018), e0191192. [7] Y. Hosokawa, I. Hosokawa, K. Ozaki, T. Matsuo, Sudachitin inhibits matrix metalloproteinase-1 and -3 production in tumor necrosis factor-a-stimulated human periodontal ligament cells, Inflammation 42 (2019) 1456e1462.
Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010
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Please cite this article as: S. Abe, K. Yuasa, Sudachitin, a polymethoxyflavone from Citrus sudachi, induces apoptosis via the regulation of MAPK pathways in human keratinocyte HaCaT cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.09.010