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Glycyrrhizic acid attenuates stem cell-like phenotypes of human dermal papilla cells
Phytomedicine 22 (2015) 1269–1278
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Glycyrrhizic acid attenuates stem cell-like phenotypes of human dermal papilla cells Chayanin Kiratipaiboon a, Parkpoom Tengamnuay b, Pithi Chanvorachote c,d,∗ a
Pharmaceutical Technology (International) Program, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand c Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand d Cell-Based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand b
a r t i c l e
i n f o
Article history: Received 28 April 2015 Revised 5 November 2015 Accepted 5 November 2015
Keywords: Glycyrrhizic acid Dermal papilla cell Stem cell Epithelial-mesenchymal transition β -catenin Glycogen synthase kinase3β
a b s t r a c t Background: Although the growth of unwanted hair or hirsutism is a harmless condition, many people find it bothersome and embarrassing. Maintaining stem cell features of dermal papilla cells is a critical biological process that keeps the high rate of hair growth. Glycyrrhizic acid has been reported to impair hair growth in some studies; however, its underlying mechanism has not yet been investigated. Purpose: This study aimed to explore the effect and underlying mechanism of glycyrrhizic acid on stemness of human dermal papilla cells. Study design/Methods: The stem cell molecular markers, epithelial to mesenchymal markers and Wnt/β catenin-associated proteins of human dermal papilla cell line and primary human dermal papilla cells were analysed by western blot analysis and immunocytochemistry. Results: The present study demonstrated that glycyrrhizic acid significantly depressed the stemness of dermal papilla cells in dose- and time-dependent manners. Clonogenicity and stem cell markers in the glycyrrhizic acid-treated cells were found to gradually decrease in the culture in a time-dependent manner. Our results demonstrated that glycyrrhizic acid exerted the stem cell suppressing effects through the interruption of ATP-dependent tyrosine kinase/glycogen synthase kinase3β -dependent mechanism which in turn down-regulated the β -catenin signalling pathway, coupled with decreased its down-stream epithelialmesenchymal transition and self-renewal transcription factors, namely, Oct-4, Nanog, Sox2, ZEB1 and Snail. The effect of glycyrrhizic acid on the reduction of stem cell features was also observed in the primary dermal papilla cells directly obtained from human hair follicles. Conclusion: These results revealed a novel molecular mechanism of glycyrrhizic acid in regulation of dermal papilla cells and provided the evidence supporting the use of this compound in suppressing the growth of unwanted hair. © 2015 Elsevier GmbH. All rights reserved.
Introduction The dermal papilla cells (DPCs) are recognized as the signalling centre in hair follicle morphogenesis and cycling (Stenn and Paus, 2001). The DPCs release various cytokines to induce proliferation and differentiation of keratinocytes resulting in hair shaft outgrowth. Several lines of evidence indicated that the cytokine level and the
Abbreviations: DPCs, Dermal papilla cells; GSK3β , Glycogen synthase kinase3β ; Akt, ATP-dependent tyrosine kinase; EMT, Epithelial-mesenchymal transition; FGF, Fibroblast growth factor; IGF, Insulin-like growth factor; GA, Glycyrrhizic acid; TCF, T-cell factor; LEF, Lymphoid enhancing factor. ∗ Corresponding author at: Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Phatumwan, Bangkok 10330, Thailand. Tel.: +662 218 8344; fax: +662 2188340. E-mail address: [email protected], [email protected] (P. Chanvorachote). http://dx.doi.org/10.1016/j.phymed.2015.11.002 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
amount of DPCs lying underneath hair follicles determine the rate of hair growth as well as duration of anagen phase (Elliott et al., 1999; Paus and Foitzik, 2004). As the DPCs are multipotent stem cells, their functions in regulation of the hair follicle as described were shown to link with their stem cell phenotypes (Ito et al., 2007; Driskell et al., 2009; Clavel et al., 2012; Driskell et al., 2012). This concept was supported by the fact that Sox2, one of the major transcription factors that maintain pluripotency of stem cells, in DPCs plays an important role on hair growth in transgenic animals (Driskell et al. 2009; Clavel et al. 2012). Also, recent study demonstrated that Wnt pathway and fibroblast growth factor genes that maintain hair inductive ability of dermal papilla cells were more prominent in the Sox2-positive DPCs (Driskell et al., 2009). According to the comprehensive knowledge of the stem cell biology, evidence suggested that the expression of CD133, a protein marker of human stem cells, is associated with the hair inductive
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cultured in Prigrow III medium (Applied Biological Materials Inc, Richmond, BC) supplemented with 10% fetal bovine serum (FBS) and 100 units/ml of penicillin/streptomycin (Life technologies, MD, USA) at 37 °C in a 5% CO2 atmosphere. For primary human DPCs, they were purchased from PromoCell (Heidelberg, Germany). The cells were maintained in medium containing bovine pituitary extract 4 μl/ml, fetal calf serum 0.05 ml/ml, basic fibroblast growth factor 1 ng/ml, recombinant human insulin 5 μg/ml and phenol red 0.62 ng/ml from PromoCell (Heidelberg, Germany), and 100 units/ml of penicillin/ streptomycin at 37 °C in a 5% CO2 atmosphere. Plant material Glycyrrhizic acid (GA; batch number: BCBL 1535 V) having high purity (≥95%; see supplementary materials for certificate of analysis) was bought from Sigma-Aldrich (St. Louis, MO). Structural formula, nomenclature, chemical formula and molecular weight of GA are given in Fig. 1. GA was prepared by dissolving it in dimethyl sulfoxide (DMSO; Lab-Scan, Dublin, Ireland) and stock sample was further diluted in culture medium resulting in final test concentrations, with final concentration of 0.1% DMSO. The non-treated control cells were treated with an equivalent volume of DMSO (0.1% final concentration). Cell viability assay
Fig. 1. Structural formula, nomenclature, chemical formula and molecular weight (MW) of glycyrrhizic acid (GA).
property of DPCs in transgenic mice (Ito et al., 2007; Driskell et al., 2009). In addition, an ablation of stem cell-related transcription factors including Sox2 in DPCs leads to the impairment of the hair shaft outgrowth (Clavel et al., 2012). In addition, the Wnt/β -catenin signalling appears to lend strong support to hair follicle morphogenesis and regeneration (Kishimoto et al., 2000; Shimizu and Morgan, 2004; Enshell-Seijffers et al., 2010). Indeed, β -catenin was shown to regulate crucial signalling pathways in hair follicle formation in response to several stimuli, including fibroblast growth factor (FGF) and insulin-like growth factor (IGF) (Enshell-Seijffers et al., 2010). In the transgenic mice model, the suppression of β -catenin in the DPCs resulted in the inhibition of hair follicle formation (Enshell-Seijffers et al., 2010). Therefore, the loss of stem cell-like phenotypes or the suppression of such stem cell-related signals in DPCs may shorten the anagen phase as well as reduce the rate of hair growth and benefit the treatment of hirsutism or managing unwanted hair. Glycyrrhizic acid (GA) (Fig. 1) is a triterpenoid saponin isolated from the roots of Glycyrrhiza glabra L., colloquially known as licorice (Alam, 2014). Recent studies demonstrated that GA was effective for the impairment of hair growth in rat without any signs of skin irritation even after long-term treatment (Ivosevic-Zaper et al., 2014); however, the underlying mechanisms as well as the effect of GA in regulation of DPCs function and stemness have not been clarified. Therefore, the present study aimed to investigate the molecular mechanism of the compound on the cellular signals regulating stem cell phenotypes in DPCs. This study reveals for the first time that GA exhibits strong activity in suppression of stemness in DPCs and that the information may facilitate the use of this relatively safe compound in hair removal products. Materials and methods Cells culture Immortalized dermal papilla cells (DPCs) were obtained from Applied Biological Materials Inc (Richmond, BC). The cells were
Cell viability was determined utilising the MTT assay. Cells were seeded at a density of 1 × 104 cells/well onto each 96-well plate and incubated overnight for cell attachment. The cells were then treated with different concentrations of GA (0–200 μM) for 24 h. After indicated treatments, the cells were incubated with 5 mg/ml of MTT (Invitrogen, Carlsbad, CA) for 4 h at 37 °C. Subsequently, the supernatant was removed and replaced with 100 μl of DMSO to dissolve the formazan crystal. The intensity of MTT product was measured at 570 nm using a microplate reader (Anthos, Durham, NC). Cell viability was calculated as percentage relative to non-treated value using the following formula
Cell viability (%) =
A570 of treatment × 100 A570 of control
Nuclear staining assay Apoptotic and necrosis cell deaths were detected by Hoechst 33342 and PI co-staining. Cells were seeded at a density of 1 × 104 cells/well onto each 96-well plate and incubated overnight for cell attachment. Subsequently, the cells were treated with various concentrations of GA (0–200 μM) for 24 h. After indicated treatments, the cells were stained with 10 μM of Hoechst and 5 μg/ml of PI (Molecular Probes, Eugene, OR) for 30 min at 37 °C and visualized by a fluorescence microscope (Olympus IX 51 with DP70; Olympus America Inc., Center valley, PA). Cell cycle analysis Cells were seeded at a density of 3 × 104 cells/well onto each 6well plate and incubated overnight for cell attachment. The cells were cultured in the presence or absence of GA (200 μM) for 72 h. After indicated treatment, the cells were incubated in the absence of growth factors for 24 h. The cells were then incubated with complete media for 12 h, trypsinized and fixed with 70% absolute ethanol at – 20 °C overnight. The cells were washed with cold PBS and incubated in PI solution containing 0.1% Triton-X, 1 μg/ml RNase, and 1 mg/ml propidium iodide at 37 °C for 30 min. The cells at the early passages (passages 2–3) without serum-starvation were used as a non-treated control at 0 h. DNA in whole cells were stained with PI, and cell cycle profile was analysed using flow cytometry (FACSort, Becton Dickinson, Rutherford, NJ, USA).
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Cell morphology and aggregation behaviour evaluation Cells were seeded at a density of 6 × 103 cells/well onto each 24-well plate and incubated overnight for cell attachment. The cells were then treated with various concentration of GA (0–200 μM) for 72 h. After indicated treatment, morphology of cells was observed at 0, 24, 48 and 72 h and aggregation behaviour of cells was determined at 72 h. Morphology and aggregation behaviour of cells were photographed by a phase-contrast microscope (Olympus IX51 with DP70; Olympus America Inc., Center valley, PA). Immunofluorescence Cells were seeded at a density of 3 × 104 cells/well onto each coverslip in 6-well plate and incubated overnight for cell attachment. The cells were cultured in the presence or absence of GA (200 μM) for 72 h. The cells at the early passages (passages 2–3) were used as a non-treated control at 0 h. The coverslips were fixed with 4% paraformaldehyde for 20 min and permeabilised with 0.1% Triton-X for 10 min at room temperature. Thereafter, the coverslips were incubated with 3% bovine serum albumin (BSA) for 30 min at room temperature to prevent nonspecific binding. The coverslips were washed and incubated with CD133 rabbit monoclonal antibodies (Cell Applications, San Diego, CA) at 1:100 dilution overnight at 4 °C. After primary antibody incubation, the coverslips were washed with PBS and subsequently incubated with Alexa Fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, CA) for 1 h at room temperature. Samples were examined with Confocal Laser Scanning Microscopy (Zeiss LSM 510) to analyse the expression of CD133. Western blot analysis Cells were seeded at a density of 3 × 104 cell/well onto each 6well plate overnight and cultured in various concentrations of GA (25–200 μM) for 72 h. Afterward, the cells were incubated in lysis buffer containing 20 mM TrisHCl (pH 7.5), 0.5% Triton X, 150 mM sodium chloride, 10% glycerol, 1 mM sodium orthovanadade, 50 mM sodium fluoride, 100 mM phenylmethylsulfonyl fluoride and commercial protease inhibitor cocktail (Roche Molecular Biochemicals) for 30 min on ice. Cell lysates were then collected and determined for protein concentration using the Bradford method (Bio-Rad, Hercules, CA). Equal amount of proteins of each sample (40 μg) were heated in Laemmli loading buffer at 95 °C for 5 min and subsequently loaded on 10% SDS-polyacrylamide electrophoresis. After separation, proteins were transferred onto 0.45 μm nitrocellulose membranes (Bio-Rad). Subsequently, the membranes were blocked with 5% non-fat milk in TBST [25 mM TrisHCl (pH 7.5), 125 mM NaCl, 0.05% Tween-20] at room temperature for 1 h, membranes were incubated with specific primary antibodies against CD133 (Cell Applications, San Diego, CA), aldehyde dehydrogenase 1A1 (ALDH1A1; Santa Cruz Biotechnology, Dallas, Texas), integrinβ 1, phosphorylated ATP-dependent tyrosine kinase (Akt; Ser 473), Akt, phosphorylated glycogen synthase kinase3β (GSK3β ; Ser 9), GSK3β , ZEB1, Oct-4, Nanog, Sox2, Slug, Snail and β -actin (Cell Signaling, Danvers, MA) at 4 °C overnight. Membranes were washed twice with TBST for 10 min and incubated with horseradish peroxidase-coupled secondary antibodies for 1 h at room temperature. The immune complexes were detected with Chemiluminescence substrate (super signal West Pico; Pierrce, Rockford, IL) and quantified using analyst/PC densitometry software (BioRad, Hercules, California). Statistical analysis Data were obtained from at least three independent experiments and presented as means ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA with Dunnett’s test at a
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significance level (α ) of 0.05. These analyses were performed using SPSS Version 19 (SPSS Inc., Chicago, IL). Results Effect of GA on cell viability and death of DPCs To study the role of GA on the stem cell property of DPCs, we first determined cell viability and cell death response to GA treatment using cell viability MTT and apoptosis/necrosis detection Hoechst 33342/propidium iodide (PI) co-staining assays. Fig. 2a shows that treatment of the DCPs with GA at the concentrations of 0–200 μM for 24 h caused no significant change in cell viability compared with the non-treated control. Consistently, the Hoechst/PI apoptosis assay indicated that the treatment of the compound at such concentrations caused neither apoptosis nor necrosis assessed by Hoechst and PI, respectively (Fig. 2b). The effect of GA on cell cycle distribution of DPCs Further, we determined the effect of GA on the cell cycle distribution of DPCs. The DPCs were cultured in the presence or absence of GA for 72 h and subjected to cell cycle evaluation. The cells were incubated in the absence of growth factors for 24 h. Then, the cells were incubated with complete media for 12 h and the percentage of cells in G1, S and G2/M phases of cell cycle was analysed by flowcytometer using PI. Also, the DPCs at the early passages without serumstarvation were used as a control. Fig. 2c d shows that at 12 h after the cells receive growth factors, GA-treated cells showed no significant difference in terms of cell proliferation in comparison to that of the non-treated control cells. These results indicated that GA treatment caused no significant effect on the cell cycle. Therefore, these results may be used to clarify that the following effects of GA on DPCs were not a consequence of cytotoxic effect or cell stress. GA alters morphology and aggregative pattern of DPCs DPCs have been characterized as multipotent stem cells and the stemness of DPCs was shown to associate with their ability to induce hair follicles and control hair growth (Ito et al. 2007; Driskell et al. 2009; Driskell et al. 2012; Clavel et al. 2012). In order to test whether GA affects the stem cell-like characteristics in these DPCs, the cells were treated with GA at the concentrations of 0–200 μM for 0–72 h, and morphology of the cells as well as aggregative pattern were determined. These results indicated that the shape and appearance of most GA-treated cells at the concentrations of 25–200 μM are dramatically altered toward fibroblast-like morphology at 48 and 72 h (Fig. 3a). Meanwhile, the morphology of most non-treated control and GA-treated cells at the concentration of 10 μM remained unaltered (Fig. 3a). Because the hair follicle inductive property of the DPCs is associated with their aggregate behaviours (Osada et al. 2007), we further investigated the effect of GA treatments on the aggregative growth pattern of the cells. The DPCs at early passages (passages 2–3) were cultured in the presence or absence of GA for 72 h and the aggregate size and number were determined. Fig. 3b–d demonstrates that GA at the concentrations of 100–200 μM significantly decreased the size as well as the number of cell aggregations in comparison to those of non-treated control at 72 h. GA reduces expression level of CD133 in DPCs Having shown that culture of the DPCs in the presence of GA caused the decline of stem cell-like phenotypes, we next investigated the presence of the stem cell marker in these cells by immunocytochemistry analysis. Because CD133 has been widely accepted as a marker of several stem cells including DPCs (Ito et al. 2007; Li 2013),
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Fig. 2. Cytotoxicity and proliferative effect of GA on DPCs. (a) Cells were treated with GA (0–200 μM) for 24 h. Cytotoxicity was determined by MTT assay. (b) After indicated treatment for 24 h, mode of cell death was examined by Hoechst 33342/PI co-staining assay. Scale bar is 100 μm. The data represent the means of three independent samples ± SD. (c–d) Cells were cultured in the presence or absence of GA (200 μM) for 72 h and serum-starved for 24 h. After serum-starvation, cells were incubated with complete media for 12 h. The cells at the early passages (passages 2–3) without serum-starvation were also used as a non-treated control at 0 h. Cell distributions in G1, S and G2/M phases of cell cycle were determined by PI staining and flow cytometry.
we analysed the expression of such a protein in the DPCs treated with GA at the concentrations of 200 μM for 72 h and the control cells. Fig. 4a shows that the expression of CD133 was suppressed in the GAtreated cells after being cultivated for 72 h in comparison to that of control cells at 0 and 72 h. These results suggested that GA decreased the stem cell-like phenotypes of DPCs during culture.
GA down-regulates stem cell markers in DPCs In order to provide further information of GA on the stemness of DPCs, the expression levels of stem cell markers including CD133, integrin β 1 and ALDH1A1 in response to GA treatment in time- and concentration-dependent manners were determined by western blot
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Fig. 3. Effects of GA on stem cell-like characteristics in DPCs. (a) Cells were treated with GA (0–200 μM) for various times (0–72 h). After indicated treatment, morphology of DPCs was observed. Scale bar is 100 μm. (b) Aggregation behavior of cells was determined after indicated treatment for 72 h. Scale bar is 100 μm. (c–d) Aggregation size and aggregation number were determined by image analyser. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control.
analysis. For time-dependent experiments, the cells were incubated in the presence or absence of GA 200 μM and protein expression was determined at various times (0–72 h). Fig. 4b shows that all stem cellrelated proteins including CD133, Integrin β 1 and ALDH1A1 were gradually decreased in GA treated cells in a time-dependent fashion compared with non-treated control cells. The effect of GA on stemness of DPCs was confirmed by dose-dependent study. The cells were incubated in the presence or absence of GA (25–200 μM) and protein expression was determined at 72 h. The results confirmed the suppressive effect of GA on the stem cell markers in these cells (Fig. 4c). GA attenuates Wnt/β -catenin signalling and epithelial-mesenchymal transition (EMT) in DPCs The Wnt/β -catenin signal has been accepted as an important feature to maintain stemness in human stem cells (Merrill 2012). In addition, the hair follicle inductive effect of cultured DPCs could be prolonged by exposing the cells to Wnt/β -catenin activator (Kishimoto et al. 2000; Shimizu and Morgan 2004). To determine the effect of GA
on Wnt/β -catenin pathway, the signalling proteins related to Wnt/β catenin including activated Akt (phosphorylated Akt at Ser 473), total Akt, inactivated glycogen synthase kinase3β (phosphorylated GSK3β at Ser 9), parental GSK3β and β -catenin were analysed by western blot analysis. The activated Akt is shown to phosphorylate its downstream target GSK3β . As a negative regulator of β -catenin, such phosphorylation will inhibit the function of GSK3β , which in turn leaves the β -catenin to function as a co-transcription factor. Our results showed that treatment of the cells with GA at the concentrations of 100–200 μM significantly decreased the level of activated Akt (Fig. 5a). Consequently, the phosphorylation of GSK3β was strongly decreased in the GA-treated DPCs (Fig. 5a). As GSK3β was shown to play the major role in the degradation process of β -catenin, GAtreated DPCs demonstrated corresponding results indicating that the decrease of phosphorylated GSK3β leads to a dramatic reduction of cellular β -catenin (Fig. 5a). These data suggested that GA decreased the stemness of DPCs at least in part by the reduction of cellular β catenin via Akt/GSK3β -dependent pathway. Recently, increasing evidence indicated that the process of the cell transition from epithelial-mesenchymal phenotypes has a
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Fig. 4. Effects of GA on stem cell markers in DPCs. (a) Cells were cultured in the presence or absence of GA (200 μM) for 72 h. The cells at the early passages (passages 2–3) were used as a non-treated control at 0 h. Expression of CD133 was analysed by immunofluorescence staining. Scale bar is 50 μm. (b) Time-dependent effects of GA treatment on the expression of stem cell markers were determined. Cells were cultured in the presence or absence of GA (200 μM) for 0–72 h. The level of CD133, integrin β 1 and ALDH1A1 were determined by western blot analysis. Blots were reprobed with β -actin to confirm equal loading. The immunoblot signals were quantified by densitometry and the mean data from independent experiments were normalized to the results. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control at 0 h. (c) Cells were treated with GA (0–200 μM) for 72 h. After indicated treatment, level of CD133, integrin β 1 and ALDH1A1 were analysed by western blot. β -actin was served as the loading control. The immunoblot signals were quantified by densitometry and the mean data from independent experiments were normalized to the results. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control.
significant impact on cell biology as it is related with the stem cell like properties in various cells (Mani et al. 2008; Shimono et al. 2009). Furthermore, the transcription factors up-regulated during EMT like Snail were shown to maintain the stem cell-like phenotypes in many cells (Mani et al. 2008; Shimono et al. 2009). In order to clarify whether GA plays a part in the regression of stem cell phenotypes through this pathway, the EMT-activating transcription factors including ZEB1, Slug, and Snail were determined. After incubation with GA for 72 h, the cellular levels of ZEB1 and Snail were significantly down-regulated. However, our results found only a slight change in the case of Slug (Fig. 5b). Taken together, our results suggested that GA suppresses the stem cell-like phenotypes in the DPCs by negatively regulating β -catenin and EMT.
GA decreases the self-renewal transcription factors in DPCs Because the self-renewal is an important signature of stem cells (He et al. 2009), we next investigated whether the treatment of the cells with GA could affect the self-renewal-related proteins. Transcription factors that have been shown to maintain pluripotency and self-renewal in human stem cells (He et al. 2009), including Oct-4, Nanog, and Sox2 were determined in the GA-treated and non-treated DPCs. The results indicated that Oct-4 strongly decreased in response to GA treatment at the concentrations of 50–200 μM (Fig. 5b). Consistently, the expression levels of Nanog and Sox2 were found to significantly reduce after treatment with GA at 100–200 μM (Fig. 5b). These results suggested that the treatment of GA was able to decrease the
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Fig. 5. Effects of GA on Wnt/β -catenin signalling, EMT and self-renewal transcription factors in DPCs. (a) Cells were treated with GA (0–200 μM) for 72 h. After indicated treatments, the level of Wnt/β -catenin signalling (Akt, p-Akt (Ser 473), GSK3β , p-GSK3β (Ser 9) and β -catenin) were analysed by western blot analysis. The immunoblot signals were quantified by densitometry and mean data from independent experiments were normalized to the results. (b) EMT and self-renewal transcription factors (ZEB1, Oct-4, Nanog, Sox2, Slug and Snail) were determined by western blot analysis. β -actin was used as the loading control. The western blot signals were quantified by densitometry and mean data from independent experiments were normalized to the results. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control.
self-renewal machinery in these cells by suppressing such transcription factors.
mary DPCs (Fig. 6e). Taken together, these data supported our findings that GA decreases the stemness of DPCs.
GA mediates the down-regulation of the stem cell-like phenotypes in primary human DPCs
Discussion
Having shown that the treatment of GA represses the stemness of DPC line, we next confirmed such an effect of the compound in primary human DPCs. The isolated human DPCs were cultured in primary DPC medium in the presence of 0-200 μM GA. The stem celllike characteristics were evaluated as described. Fig. 6a and b indicates that treatment of the cells with 0–200 μM GA caused no direct cytotoxicity in these cells. In response to GA treatment for 72 h, western blot analysis revealed that levels of stem cell markers including CD133, integrin β 1 and ALDH1A1 significantly decreased in a dose-dependent manner (Fig. 6c). Furthermore, treatment of the cells with GA significantly decreased the levels of proliferation and stemness signals like active Akt, inactive GSK3β , and β -catenin (Fig. 6d). The EMT-related transcription factors ZEB1 and Snail were also found to be down-regulated in the GA-treated pri-
DPCs have been shown to exhibit stem cell-like plasticity by their ability to differentiate to different cell types (Richardson et al. 2005). Interestingly, multipotency as well as stemness of DPCs is accepted to be an important factor determining an ability to induce hair follicle formation (Ito et al. 2007; Driskell et al. 2009; Driskell et al. 2012; Clavel et al. 2012). The functions of active DPCs are not only limited to the feature of hair follicle generation, but also their promoting ability on hair growth as well as the maintenance or prolongation of anagen phase have been widely accepted (Elliott et al. 1999; Paus and Foitzik 2004). Therefore, it is possible that diminishing the stemness in these specialized DPCs may reduce the rate of hair growth and benefit the treatment of hirsutism or related approaches. In an attempt to evaluate the mechanism of GA in negatively regulating hair growth, we have discovered for the first time that GA could mediate the loss of stemness in DPCs. The primitive DPCs usually appearing
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Fig. 6. Effects of GA on stem cell-like phenotypes in primary human DPCs. (a) Cells were treated with GA (0–200 μM) for 24 h. Cytotoxicity was determined by MTT assay. The data represent the means of three independent triplicate samples ± SD. (b) After indicated treatment, mode of cell death was examined by Hoechst 33342/PI co-staining assay. Scale bar is 100 μm. (c–e) Cells were treated with GA (0–200 μM) for 72 h. After indicated treatment, the level of stem cell markers (CD133, integrin β 1 and ALDH1A1), Wnt/β -catenin signalling (Akt, p-Akt, GSK3β , p-GSK3β and β -catenin) and EMT transcription factors (ZEB1, Slug and Snail) were determined by western blot analysis, respectively. β -actin was served as the loading control. The immunoblot signals were quantified by densitometry and mean data from independent experiments were normalized to the results. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control.
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Fig. 7. Schematic diagram summarizes the effects of GA on the stemness of human DPCs. (a) Wnt activation leads to the inhibition of GSK3β function. The GSK3β is also inactivated by the phosphorylation through the function of activated Akt. The function of GSK3β is to phosphorylate β -catenin leading to β -catenin degradation. Therefore, in the presence of Wnt and activated Akt, β -catenin accumulates in the cells and activates transcription of genes associating with EMT and self-renewal. (b) Addition of GA resulted in the decrease of activated Akt leading to the increase level of active GSK3β . As a consequence, β -catenin is phosphorylated and degraded. Hence, the decrease of β -catenin resulted in the reduction of transcription of target genes, and the decrease of stem cell phenotypes in human DPCs.
as spindle-shaped cells changed to flat multipolar cells with elongated shapes (Fig. 3a). Treatment of the DPCs with non-toxic concentrations of the compound caused the spontaneous alteration of cell morphology toward fibroblast-like cells (Fig. 3a). Also, our immunecytochemistry as well as protein analysis revealed that treatment of DPCs with GA led to the significant decrease of stem cell markers (Fig. 4a–c). Regarding stem cell research, CD133, a transmembrane glycoprotein, has been widely used as a standard biomarker of human stem cells (Li 2013). It was shown that DPCs at the early passages exhibited a high level of CD133. Together with other stem cell markers, GA was shown to induce the loss of stem cell features in DPCs as indicated by the significant decrease of CD133, ALDH1A1, and integrin β 1 (Fig. 4b and c). In terms of molecular mechanism in regulating stemness, considerable progress has been obtained in elucidating stem cell signalling pathways, and in particular Wnt/β -catenin has been shown to be a critical factor in maintaining stem cell characteristics as well as functions (Merrill 2012). Previous study reported that an ablation of β -catenin in DPCs causes the dramatic suppression of both hair growth and regeneration (Enshell-Seijffers et al. 2010). The β catenin functions as a co-trancription factor of T-cell factor/lymphoid enhancing factor (TCF/LEF) and consequently increases the expression of proteins facilitating stem cell functions (Fukumoto et al. 2001). Indeed, the presence of cellular β -catenin is tightly controlled by the function of GSK3β . The phosphorylation of β -catenin by active GSK3β induces ubiquitin-mediated proteasomal degradation of β catenin. This regulation of GSK3β on β -catenin was shown to be regulated by the very well known proliferative and survival signals Akt. The active Akt is shown to inhibit the function of GSK3β by phospho-
rylating GSK3β at serine 9 (Fukumoto et al. 2001). Herein, we showed that GA treatment decreased the level of activated Akt resulting in the decrease of β -catenin (Figs. 5a and 6d), suggesting that the decline of stemness in GA-treated cells is due to the interruption of Akt/β catenin pathway. Recent evidence have suggested that β -catenin interacts with many signalling pathways involving in pluripotency and EMT (Kim et al. 2002; Miki et al. 2011; Li et al. 2012; Merrill 2012; Su et al. 2014). Wnt/β -catenin signalling activation was shown to increase the expression of EMT proteins and pluripotent activating transcription factors (Van Raay et al. 2005; Pereira and Merrill 2006; Cole et al. 2008; Ten Berge et al. 2008; Lambertini et al. 2010; Wu et al. 2012). Consistent with previous reports that the transcription factor Snail and ZEB1 plays an important role in EMT process (Mani et al. 2008; Shimono et al. 2009), our results showed the significant decrease of such proteins in the DPCs treated with GA (Figs. 5b and 6e), and the decrease of EMT markers was found to be corresponding to the stem cell-like morphology and aggregative behaviour. Our findings also lend strong support to the view that Akt/GSK3β -dependent β -catenin activation is important for the DPCs to maintain their stemness. These results unveiled that the transcription factors which are down-stream targets of Wnt/β -catenin, namely Oct-4, Nanog and Sox2 were downregulated in the GA treated cells. In conclusion, we systematically evaluated the role of GA treatment on the reduction of stemness in DPCs. Together, these results revealed a novel stemness inhibitory mechanism of GA in DPCs that is through the interruption of Akt/GSK3β -dependent β -catenin regulation resulting in a down-regulation of transcription factors associated with EMT and self-renewal (Fig. 7a and b). This study
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provides the knowledge facilitating further investigations that may benefit the therapeutic as well as cosmeceutic approaches of this compound. Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This research has been supported by the Thailand Research Fund through a Research and Researchers for industries program and the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (CU-57-003-HR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.11.002. References Alam, P., Alajmi, M.F., Siddiqui, N.A., Al-Rehaily, A., Basudan, O.A., 2014. Determination of bioactive marker glycyrrhizin in Glycyrrhiza glabra root and commercial formulation by validated PHTLC-densitometric method. J. Coast. Life Med. 2, 882– 887. Clavel, C., Grisanti, L., Zemla, R., Rezza, A., Barros, R., Sennett, R., Mazloom, A.R., Chung, C.-Y., Cai, X., Cai, C.-L., 2012. Sox2 in the dermal papilla niche controls hair growth by fine-tuning BMP signaling in differentiating hair shaft progenitors. Dev. Cell 23, 981–994. Cole, M.F., Johnstone, S.E., Newman, J.J., Kagey, M.H., Young, R.A., 2008. Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells. Genes Dev 22, 746–755. Driskell, R.R., Giangreco, A., Jensen, K.B., Mulder, K.W., Watt, F.M., 2009. Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis. Development 136, 2815–2823. Driskell, R.R., Juneja, V.R., Connelly, J.T., Kretzschmar, K., Tan, D.W.-M., Watt, F.M., 2012. Clonal growth of dermal papilla cells in hydrogels reveals intrinsic differences between Sox2-positive and-negative cells in vitro and in vivo. J. Invest. Dermatol 132, 1084–1093. Elliott, K., Stephenson, T.J., Messenger, A.G., 1999. Differences in hair follicle dermal papilla volume are due to extracellular matrix volume and cell number: implications for the control of hair follicle size and androgen responses. J. Invest. Dermatol 113, 873–877. Enshell-Seijffers, D., Lindon, C., Kashiwagi, M., Morgan, B.A., 2010. β -catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair. Dev. Cell 18, 633–642.
Fukumoto, S., Hsieh, C.-M., Maemura, K., Layne, M.D., Yet, S.-F., Lee, K.-H., Matsui, T., Rosenzweig, A., Taylor, W.G., Rubin, J.S., 2001. Akt participation in the Wnt signaling pathway through Dishevelled. J. Biol. Chem 276, 17479–17483. He, S., Nakada, D., Morrison, S.J., 2009. Mechanisms of stem cell self-renewal. Annu. Rev. Cell Dev. Biol 25, 377–406. Ito, Y., Hamazaki, T.S., Ohnuma, K., Tamaki, K., Asashima, M., Okochi, H., 2007. Isolation of murine hair-inducing cells using the cell surface marker prominin-1/CD133. J. Invest. Dermatol 127, 1052–1060. Ivosevic-Zaper, J., Hofmann, M., Kakadjanova, A., Valesky, E., Meissner, M., BereiterHahn, J., Kaufmann, R., Bernd, A., Kippenberger, S., 2014. Topically applied glycyrrhizic acid causes hair removal in rats. Pharm. Biol 52, 1362–1365. Kim, K., Lu, Z., Hay, E.D., 2002. Direct evidence for a role of beta-cetenin/LEF-1 signaling pathway in induction of EMT. Cell Biol. Int 26, 463–476. Kishimoto, J., Burgeson, R.E., Morgan, B.A., 2000. Wnt signaling maintains the hairinducing activity of the dermal papilla. Genes Dev 14, 1181–1185. Lambertini, E., Franceschetti, T., Torreggiani, E., Penolazzi, L., Pastore, A., Pelucchi, S., Gambari, R., Piva, R., 2010. Slug: a new target of lymphoid enhancer factor-1 in human osteoblasts. BMC Mol. Biol 11, 13. Li, J., Li, J., Chen, B., 2012. Oct4 was a novel target of Wnt signaling pathway. Mol. Cell Biochem 362, 233–240. Li, Z., 2013. CD133: a stem cell biomarker and beyond. Exp. Hematol. Oncol 2, 17. Mani, S.A., Guo, W., Liao, M.-J., Eaton, E.N., Ayyanan, A., Zhou, A.Y., Brooks, M., Reinhard, F., Zhang, C.C., Shipitsin, M., 2008. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704–715. Merrill, B.J., 2012. Wnt pathway regulation of embryonic stem cell self-renewal. Cold Spring Harb. Perspect. Biol 4, a007971. Miki, T., Yasuda, S.-Y., Kahn, M., 2011. Wnt/β -catenin signaling in embryonic stem cell self-renewal and somatic cell reprogramming. Stem Cell Rev 7, 836–846. Osada, A., Iwabuchi, T., Kishimoto, J., Hamazaki, T.S., Okochi, H., 2007. Long-term culture of mouse vibrissal dermal papilla cells and de novo hair follicle induction. Tissue Eng 13, 975–982. Paus, R., Foitzik, K., 2004. In search of the “hair cycle clock”: a guided tour. Differentiation 72, 489–511. Pereira, L., Merrill, B.J., 2006. Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal. Mol. Cell. Biol 26, 7479–7491. Richardson, G.D., Arnott, E.C., Whitehouse, C.J., Lawrence, C.M., Reynolds, A.J., Hole, N., Jahoda, C.A., 2005. Plasticity of rodent and human hair follicle dermal cells: implications for cell therapy and tissue engineering. J. Investig. Dermatol. Symp. Proc 10, 180–183. Shimizu, H., Morgan, B.A., 2004. Wnt signaling through the β -catenin pathway is sufficient to maintain, but not restore, anagen-phase characteristics of dermal papilla cells. J. Investig. Dermatol. 122, 239–245. Shimono, Y., Zabala, M., Cho, R.W., Lobo, N., Dalerba, P., Qian, D., Diehn, M., Liu, H., Panula, S.P., Chiao, E., 2009. Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell 138, 592–603. Stenn, K.S., Paus, R., 2001. Controls of hair follicle cycling. Physiol. Rev 81, 450–481. Su, T., Dan, S., Wang, Y., 2014. Akt–Oct4 regulatory circuit in pluripotent stem cells. Chin. Sci. Bull 59, 936–943. Ten Berge, D., Koole, W., Fuerer, C., Fish, M., Eroglu, E., Nusse, R., 2008. Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3, 508–518. Van Raay, T.J., Moore, K.B., Iordanova, I., Steele, M., Jamrich, M., Harris, W.A., Vetter, M.L., 2005. Frizzled 5 signaling governs the neural potential of progenitors in the developing Xenopus retina. Neuron 46, 23–36. Wu, K., Fan, J., Zhang, L., Ning, Z., Zeng, J., Zhou, J., Li, L., Chen, Y., Zhang, T., Wang, X., 2012. PI3K/Akt to GSK3β /β -catenin signaling cascade coordinates cell colonization for bladder cancer bone metastasis through regulating ZEB1 transcription. Cell Signal 24, 2273–2282.
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Glycyrrhizic acid attenuates stem cell-like phenotypes of human dermal papilla cells Chayanin Kiratipaiboon a, Parkpoom Tengamnuay b, Pithi Chanvorachote c,d,∗ a
Pharmaceutical Technology (International) Program, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand c Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand d Cell-Based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand b
a r t i c l e
i n f o
Article history: Received 28 April 2015 Revised 5 November 2015 Accepted 5 November 2015
Keywords: Glycyrrhizic acid Dermal papilla cell Stem cell Epithelial-mesenchymal transition β -catenin Glycogen synthase kinase3β
a b s t r a c t Background: Although the growth of unwanted hair or hirsutism is a harmless condition, many people find it bothersome and embarrassing. Maintaining stem cell features of dermal papilla cells is a critical biological process that keeps the high rate of hair growth. Glycyrrhizic acid has been reported to impair hair growth in some studies; however, its underlying mechanism has not yet been investigated. Purpose: This study aimed to explore the effect and underlying mechanism of glycyrrhizic acid on stemness of human dermal papilla cells. Study design/Methods: The stem cell molecular markers, epithelial to mesenchymal markers and Wnt/β catenin-associated proteins of human dermal papilla cell line and primary human dermal papilla cells were analysed by western blot analysis and immunocytochemistry. Results: The present study demonstrated that glycyrrhizic acid significantly depressed the stemness of dermal papilla cells in dose- and time-dependent manners. Clonogenicity and stem cell markers in the glycyrrhizic acid-treated cells were found to gradually decrease in the culture in a time-dependent manner. Our results demonstrated that glycyrrhizic acid exerted the stem cell suppressing effects through the interruption of ATP-dependent tyrosine kinase/glycogen synthase kinase3β -dependent mechanism which in turn down-regulated the β -catenin signalling pathway, coupled with decreased its down-stream epithelialmesenchymal transition and self-renewal transcription factors, namely, Oct-4, Nanog, Sox2, ZEB1 and Snail. The effect of glycyrrhizic acid on the reduction of stem cell features was also observed in the primary dermal papilla cells directly obtained from human hair follicles. Conclusion: These results revealed a novel molecular mechanism of glycyrrhizic acid in regulation of dermal papilla cells and provided the evidence supporting the use of this compound in suppressing the growth of unwanted hair. © 2015 Elsevier GmbH. All rights reserved.
Introduction The dermal papilla cells (DPCs) are recognized as the signalling centre in hair follicle morphogenesis and cycling (Stenn and Paus, 2001). The DPCs release various cytokines to induce proliferation and differentiation of keratinocytes resulting in hair shaft outgrowth. Several lines of evidence indicated that the cytokine level and the
Abbreviations: DPCs, Dermal papilla cells; GSK3β , Glycogen synthase kinase3β ; Akt, ATP-dependent tyrosine kinase; EMT, Epithelial-mesenchymal transition; FGF, Fibroblast growth factor; IGF, Insulin-like growth factor; GA, Glycyrrhizic acid; TCF, T-cell factor; LEF, Lymphoid enhancing factor. ∗ Corresponding author at: Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Phatumwan, Bangkok 10330, Thailand. Tel.: +662 218 8344; fax: +662 2188340. E-mail address: [email protected], [email protected] (P. Chanvorachote). http://dx.doi.org/10.1016/j.phymed.2015.11.002 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
amount of DPCs lying underneath hair follicles determine the rate of hair growth as well as duration of anagen phase (Elliott et al., 1999; Paus and Foitzik, 2004). As the DPCs are multipotent stem cells, their functions in regulation of the hair follicle as described were shown to link with their stem cell phenotypes (Ito et al., 2007; Driskell et al., 2009; Clavel et al., 2012; Driskell et al., 2012). This concept was supported by the fact that Sox2, one of the major transcription factors that maintain pluripotency of stem cells, in DPCs plays an important role on hair growth in transgenic animals (Driskell et al. 2009; Clavel et al. 2012). Also, recent study demonstrated that Wnt pathway and fibroblast growth factor genes that maintain hair inductive ability of dermal papilla cells were more prominent in the Sox2-positive DPCs (Driskell et al., 2009). According to the comprehensive knowledge of the stem cell biology, evidence suggested that the expression of CD133, a protein marker of human stem cells, is associated with the hair inductive
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cultured in Prigrow III medium (Applied Biological Materials Inc, Richmond, BC) supplemented with 10% fetal bovine serum (FBS) and 100 units/ml of penicillin/streptomycin (Life technologies, MD, USA) at 37 °C in a 5% CO2 atmosphere. For primary human DPCs, they were purchased from PromoCell (Heidelberg, Germany). The cells were maintained in medium containing bovine pituitary extract 4 μl/ml, fetal calf serum 0.05 ml/ml, basic fibroblast growth factor 1 ng/ml, recombinant human insulin 5 μg/ml and phenol red 0.62 ng/ml from PromoCell (Heidelberg, Germany), and 100 units/ml of penicillin/ streptomycin at 37 °C in a 5% CO2 atmosphere. Plant material Glycyrrhizic acid (GA; batch number: BCBL 1535 V) having high purity (≥95%; see supplementary materials for certificate of analysis) was bought from Sigma-Aldrich (St. Louis, MO). Structural formula, nomenclature, chemical formula and molecular weight of GA are given in Fig. 1. GA was prepared by dissolving it in dimethyl sulfoxide (DMSO; Lab-Scan, Dublin, Ireland) and stock sample was further diluted in culture medium resulting in final test concentrations, with final concentration of 0.1% DMSO. The non-treated control cells were treated with an equivalent volume of DMSO (0.1% final concentration). Cell viability assay
Fig. 1. Structural formula, nomenclature, chemical formula and molecular weight (MW) of glycyrrhizic acid (GA).
property of DPCs in transgenic mice (Ito et al., 2007; Driskell et al., 2009). In addition, an ablation of stem cell-related transcription factors including Sox2 in DPCs leads to the impairment of the hair shaft outgrowth (Clavel et al., 2012). In addition, the Wnt/β -catenin signalling appears to lend strong support to hair follicle morphogenesis and regeneration (Kishimoto et al., 2000; Shimizu and Morgan, 2004; Enshell-Seijffers et al., 2010). Indeed, β -catenin was shown to regulate crucial signalling pathways in hair follicle formation in response to several stimuli, including fibroblast growth factor (FGF) and insulin-like growth factor (IGF) (Enshell-Seijffers et al., 2010). In the transgenic mice model, the suppression of β -catenin in the DPCs resulted in the inhibition of hair follicle formation (Enshell-Seijffers et al., 2010). Therefore, the loss of stem cell-like phenotypes or the suppression of such stem cell-related signals in DPCs may shorten the anagen phase as well as reduce the rate of hair growth and benefit the treatment of hirsutism or managing unwanted hair. Glycyrrhizic acid (GA) (Fig. 1) is a triterpenoid saponin isolated from the roots of Glycyrrhiza glabra L., colloquially known as licorice (Alam, 2014). Recent studies demonstrated that GA was effective for the impairment of hair growth in rat without any signs of skin irritation even after long-term treatment (Ivosevic-Zaper et al., 2014); however, the underlying mechanisms as well as the effect of GA in regulation of DPCs function and stemness have not been clarified. Therefore, the present study aimed to investigate the molecular mechanism of the compound on the cellular signals regulating stem cell phenotypes in DPCs. This study reveals for the first time that GA exhibits strong activity in suppression of stemness in DPCs and that the information may facilitate the use of this relatively safe compound in hair removal products. Materials and methods Cells culture Immortalized dermal papilla cells (DPCs) were obtained from Applied Biological Materials Inc (Richmond, BC). The cells were
Cell viability was determined utilising the MTT assay. Cells were seeded at a density of 1 × 104 cells/well onto each 96-well plate and incubated overnight for cell attachment. The cells were then treated with different concentrations of GA (0–200 μM) for 24 h. After indicated treatments, the cells were incubated with 5 mg/ml of MTT (Invitrogen, Carlsbad, CA) for 4 h at 37 °C. Subsequently, the supernatant was removed and replaced with 100 μl of DMSO to dissolve the formazan crystal. The intensity of MTT product was measured at 570 nm using a microplate reader (Anthos, Durham, NC). Cell viability was calculated as percentage relative to non-treated value using the following formula
Cell viability (%) =
A570 of treatment × 100 A570 of control
Nuclear staining assay Apoptotic and necrosis cell deaths were detected by Hoechst 33342 and PI co-staining. Cells were seeded at a density of 1 × 104 cells/well onto each 96-well plate and incubated overnight for cell attachment. Subsequently, the cells were treated with various concentrations of GA (0–200 μM) for 24 h. After indicated treatments, the cells were stained with 10 μM of Hoechst and 5 μg/ml of PI (Molecular Probes, Eugene, OR) for 30 min at 37 °C and visualized by a fluorescence microscope (Olympus IX 51 with DP70; Olympus America Inc., Center valley, PA). Cell cycle analysis Cells were seeded at a density of 3 × 104 cells/well onto each 6well plate and incubated overnight for cell attachment. The cells were cultured in the presence or absence of GA (200 μM) for 72 h. After indicated treatment, the cells were incubated in the absence of growth factors for 24 h. The cells were then incubated with complete media for 12 h, trypsinized and fixed with 70% absolute ethanol at – 20 °C overnight. The cells were washed with cold PBS and incubated in PI solution containing 0.1% Triton-X, 1 μg/ml RNase, and 1 mg/ml propidium iodide at 37 °C for 30 min. The cells at the early passages (passages 2–3) without serum-starvation were used as a non-treated control at 0 h. DNA in whole cells were stained with PI, and cell cycle profile was analysed using flow cytometry (FACSort, Becton Dickinson, Rutherford, NJ, USA).
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Cell morphology and aggregation behaviour evaluation Cells were seeded at a density of 6 × 103 cells/well onto each 24-well plate and incubated overnight for cell attachment. The cells were then treated with various concentration of GA (0–200 μM) for 72 h. After indicated treatment, morphology of cells was observed at 0, 24, 48 and 72 h and aggregation behaviour of cells was determined at 72 h. Morphology and aggregation behaviour of cells were photographed by a phase-contrast microscope (Olympus IX51 with DP70; Olympus America Inc., Center valley, PA). Immunofluorescence Cells were seeded at a density of 3 × 104 cells/well onto each coverslip in 6-well plate and incubated overnight for cell attachment. The cells were cultured in the presence or absence of GA (200 μM) for 72 h. The cells at the early passages (passages 2–3) were used as a non-treated control at 0 h. The coverslips were fixed with 4% paraformaldehyde for 20 min and permeabilised with 0.1% Triton-X for 10 min at room temperature. Thereafter, the coverslips were incubated with 3% bovine serum albumin (BSA) for 30 min at room temperature to prevent nonspecific binding. The coverslips were washed and incubated with CD133 rabbit monoclonal antibodies (Cell Applications, San Diego, CA) at 1:100 dilution overnight at 4 °C. After primary antibody incubation, the coverslips were washed with PBS and subsequently incubated with Alexa Fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, CA) for 1 h at room temperature. Samples were examined with Confocal Laser Scanning Microscopy (Zeiss LSM 510) to analyse the expression of CD133. Western blot analysis Cells were seeded at a density of 3 × 104 cell/well onto each 6well plate overnight and cultured in various concentrations of GA (25–200 μM) for 72 h. Afterward, the cells were incubated in lysis buffer containing 20 mM TrisHCl (pH 7.5), 0.5% Triton X, 150 mM sodium chloride, 10% glycerol, 1 mM sodium orthovanadade, 50 mM sodium fluoride, 100 mM phenylmethylsulfonyl fluoride and commercial protease inhibitor cocktail (Roche Molecular Biochemicals) for 30 min on ice. Cell lysates were then collected and determined for protein concentration using the Bradford method (Bio-Rad, Hercules, CA). Equal amount of proteins of each sample (40 μg) were heated in Laemmli loading buffer at 95 °C for 5 min and subsequently loaded on 10% SDS-polyacrylamide electrophoresis. After separation, proteins were transferred onto 0.45 μm nitrocellulose membranes (Bio-Rad). Subsequently, the membranes were blocked with 5% non-fat milk in TBST [25 mM TrisHCl (pH 7.5), 125 mM NaCl, 0.05% Tween-20] at room temperature for 1 h, membranes were incubated with specific primary antibodies against CD133 (Cell Applications, San Diego, CA), aldehyde dehydrogenase 1A1 (ALDH1A1; Santa Cruz Biotechnology, Dallas, Texas), integrinβ 1, phosphorylated ATP-dependent tyrosine kinase (Akt; Ser 473), Akt, phosphorylated glycogen synthase kinase3β (GSK3β ; Ser 9), GSK3β , ZEB1, Oct-4, Nanog, Sox2, Slug, Snail and β -actin (Cell Signaling, Danvers, MA) at 4 °C overnight. Membranes were washed twice with TBST for 10 min and incubated with horseradish peroxidase-coupled secondary antibodies for 1 h at room temperature. The immune complexes were detected with Chemiluminescence substrate (super signal West Pico; Pierrce, Rockford, IL) and quantified using analyst/PC densitometry software (BioRad, Hercules, California). Statistical analysis Data were obtained from at least three independent experiments and presented as means ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA with Dunnett’s test at a
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significance level (α ) of 0.05. These analyses were performed using SPSS Version 19 (SPSS Inc., Chicago, IL). Results Effect of GA on cell viability and death of DPCs To study the role of GA on the stem cell property of DPCs, we first determined cell viability and cell death response to GA treatment using cell viability MTT and apoptosis/necrosis detection Hoechst 33342/propidium iodide (PI) co-staining assays. Fig. 2a shows that treatment of the DCPs with GA at the concentrations of 0–200 μM for 24 h caused no significant change in cell viability compared with the non-treated control. Consistently, the Hoechst/PI apoptosis assay indicated that the treatment of the compound at such concentrations caused neither apoptosis nor necrosis assessed by Hoechst and PI, respectively (Fig. 2b). The effect of GA on cell cycle distribution of DPCs Further, we determined the effect of GA on the cell cycle distribution of DPCs. The DPCs were cultured in the presence or absence of GA for 72 h and subjected to cell cycle evaluation. The cells were incubated in the absence of growth factors for 24 h. Then, the cells were incubated with complete media for 12 h and the percentage of cells in G1, S and G2/M phases of cell cycle was analysed by flowcytometer using PI. Also, the DPCs at the early passages without serumstarvation were used as a control. Fig. 2c d shows that at 12 h after the cells receive growth factors, GA-treated cells showed no significant difference in terms of cell proliferation in comparison to that of the non-treated control cells. These results indicated that GA treatment caused no significant effect on the cell cycle. Therefore, these results may be used to clarify that the following effects of GA on DPCs were not a consequence of cytotoxic effect or cell stress. GA alters morphology and aggregative pattern of DPCs DPCs have been characterized as multipotent stem cells and the stemness of DPCs was shown to associate with their ability to induce hair follicles and control hair growth (Ito et al. 2007; Driskell et al. 2009; Driskell et al. 2012; Clavel et al. 2012). In order to test whether GA affects the stem cell-like characteristics in these DPCs, the cells were treated with GA at the concentrations of 0–200 μM for 0–72 h, and morphology of the cells as well as aggregative pattern were determined. These results indicated that the shape and appearance of most GA-treated cells at the concentrations of 25–200 μM are dramatically altered toward fibroblast-like morphology at 48 and 72 h (Fig. 3a). Meanwhile, the morphology of most non-treated control and GA-treated cells at the concentration of 10 μM remained unaltered (Fig. 3a). Because the hair follicle inductive property of the DPCs is associated with their aggregate behaviours (Osada et al. 2007), we further investigated the effect of GA treatments on the aggregative growth pattern of the cells. The DPCs at early passages (passages 2–3) were cultured in the presence or absence of GA for 72 h and the aggregate size and number were determined. Fig. 3b–d demonstrates that GA at the concentrations of 100–200 μM significantly decreased the size as well as the number of cell aggregations in comparison to those of non-treated control at 72 h. GA reduces expression level of CD133 in DPCs Having shown that culture of the DPCs in the presence of GA caused the decline of stem cell-like phenotypes, we next investigated the presence of the stem cell marker in these cells by immunocytochemistry analysis. Because CD133 has been widely accepted as a marker of several stem cells including DPCs (Ito et al. 2007; Li 2013),
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Fig. 2. Cytotoxicity and proliferative effect of GA on DPCs. (a) Cells were treated with GA (0–200 μM) for 24 h. Cytotoxicity was determined by MTT assay. (b) After indicated treatment for 24 h, mode of cell death was examined by Hoechst 33342/PI co-staining assay. Scale bar is 100 μm. The data represent the means of three independent samples ± SD. (c–d) Cells were cultured in the presence or absence of GA (200 μM) for 72 h and serum-starved for 24 h. After serum-starvation, cells were incubated with complete media for 12 h. The cells at the early passages (passages 2–3) without serum-starvation were also used as a non-treated control at 0 h. Cell distributions in G1, S and G2/M phases of cell cycle were determined by PI staining and flow cytometry.
we analysed the expression of such a protein in the DPCs treated with GA at the concentrations of 200 μM for 72 h and the control cells. Fig. 4a shows that the expression of CD133 was suppressed in the GAtreated cells after being cultivated for 72 h in comparison to that of control cells at 0 and 72 h. These results suggested that GA decreased the stem cell-like phenotypes of DPCs during culture.
GA down-regulates stem cell markers in DPCs In order to provide further information of GA on the stemness of DPCs, the expression levels of stem cell markers including CD133, integrin β 1 and ALDH1A1 in response to GA treatment in time- and concentration-dependent manners were determined by western blot
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Fig. 3. Effects of GA on stem cell-like characteristics in DPCs. (a) Cells were treated with GA (0–200 μM) for various times (0–72 h). After indicated treatment, morphology of DPCs was observed. Scale bar is 100 μm. (b) Aggregation behavior of cells was determined after indicated treatment for 72 h. Scale bar is 100 μm. (c–d) Aggregation size and aggregation number were determined by image analyser. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control.
analysis. For time-dependent experiments, the cells were incubated in the presence or absence of GA 200 μM and protein expression was determined at various times (0–72 h). Fig. 4b shows that all stem cellrelated proteins including CD133, Integrin β 1 and ALDH1A1 were gradually decreased in GA treated cells in a time-dependent fashion compared with non-treated control cells. The effect of GA on stemness of DPCs was confirmed by dose-dependent study. The cells were incubated in the presence or absence of GA (25–200 μM) and protein expression was determined at 72 h. The results confirmed the suppressive effect of GA on the stem cell markers in these cells (Fig. 4c). GA attenuates Wnt/β -catenin signalling and epithelial-mesenchymal transition (EMT) in DPCs The Wnt/β -catenin signal has been accepted as an important feature to maintain stemness in human stem cells (Merrill 2012). In addition, the hair follicle inductive effect of cultured DPCs could be prolonged by exposing the cells to Wnt/β -catenin activator (Kishimoto et al. 2000; Shimizu and Morgan 2004). To determine the effect of GA
on Wnt/β -catenin pathway, the signalling proteins related to Wnt/β catenin including activated Akt (phosphorylated Akt at Ser 473), total Akt, inactivated glycogen synthase kinase3β (phosphorylated GSK3β at Ser 9), parental GSK3β and β -catenin were analysed by western blot analysis. The activated Akt is shown to phosphorylate its downstream target GSK3β . As a negative regulator of β -catenin, such phosphorylation will inhibit the function of GSK3β , which in turn leaves the β -catenin to function as a co-transcription factor. Our results showed that treatment of the cells with GA at the concentrations of 100–200 μM significantly decreased the level of activated Akt (Fig. 5a). Consequently, the phosphorylation of GSK3β was strongly decreased in the GA-treated DPCs (Fig. 5a). As GSK3β was shown to play the major role in the degradation process of β -catenin, GAtreated DPCs demonstrated corresponding results indicating that the decrease of phosphorylated GSK3β leads to a dramatic reduction of cellular β -catenin (Fig. 5a). These data suggested that GA decreased the stemness of DPCs at least in part by the reduction of cellular β catenin via Akt/GSK3β -dependent pathway. Recently, increasing evidence indicated that the process of the cell transition from epithelial-mesenchymal phenotypes has a
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Fig. 4. Effects of GA on stem cell markers in DPCs. (a) Cells were cultured in the presence or absence of GA (200 μM) for 72 h. The cells at the early passages (passages 2–3) were used as a non-treated control at 0 h. Expression of CD133 was analysed by immunofluorescence staining. Scale bar is 50 μm. (b) Time-dependent effects of GA treatment on the expression of stem cell markers were determined. Cells were cultured in the presence or absence of GA (200 μM) for 0–72 h. The level of CD133, integrin β 1 and ALDH1A1 were determined by western blot analysis. Blots were reprobed with β -actin to confirm equal loading. The immunoblot signals were quantified by densitometry and the mean data from independent experiments were normalized to the results. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control at 0 h. (c) Cells were treated with GA (0–200 μM) for 72 h. After indicated treatment, level of CD133, integrin β 1 and ALDH1A1 were analysed by western blot. β -actin was served as the loading control. The immunoblot signals were quantified by densitometry and the mean data from independent experiments were normalized to the results. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control.
significant impact on cell biology as it is related with the stem cell like properties in various cells (Mani et al. 2008; Shimono et al. 2009). Furthermore, the transcription factors up-regulated during EMT like Snail were shown to maintain the stem cell-like phenotypes in many cells (Mani et al. 2008; Shimono et al. 2009). In order to clarify whether GA plays a part in the regression of stem cell phenotypes through this pathway, the EMT-activating transcription factors including ZEB1, Slug, and Snail were determined. After incubation with GA for 72 h, the cellular levels of ZEB1 and Snail were significantly down-regulated. However, our results found only a slight change in the case of Slug (Fig. 5b). Taken together, our results suggested that GA suppresses the stem cell-like phenotypes in the DPCs by negatively regulating β -catenin and EMT.
GA decreases the self-renewal transcription factors in DPCs Because the self-renewal is an important signature of stem cells (He et al. 2009), we next investigated whether the treatment of the cells with GA could affect the self-renewal-related proteins. Transcription factors that have been shown to maintain pluripotency and self-renewal in human stem cells (He et al. 2009), including Oct-4, Nanog, and Sox2 were determined in the GA-treated and non-treated DPCs. The results indicated that Oct-4 strongly decreased in response to GA treatment at the concentrations of 50–200 μM (Fig. 5b). Consistently, the expression levels of Nanog and Sox2 were found to significantly reduce after treatment with GA at 100–200 μM (Fig. 5b). These results suggested that the treatment of GA was able to decrease the
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Fig. 5. Effects of GA on Wnt/β -catenin signalling, EMT and self-renewal transcription factors in DPCs. (a) Cells were treated with GA (0–200 μM) for 72 h. After indicated treatments, the level of Wnt/β -catenin signalling (Akt, p-Akt (Ser 473), GSK3β , p-GSK3β (Ser 9) and β -catenin) were analysed by western blot analysis. The immunoblot signals were quantified by densitometry and mean data from independent experiments were normalized to the results. (b) EMT and self-renewal transcription factors (ZEB1, Oct-4, Nanog, Sox2, Slug and Snail) were determined by western blot analysis. β -actin was used as the loading control. The western blot signals were quantified by densitometry and mean data from independent experiments were normalized to the results. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control.
self-renewal machinery in these cells by suppressing such transcription factors.
mary DPCs (Fig. 6e). Taken together, these data supported our findings that GA decreases the stemness of DPCs.
GA mediates the down-regulation of the stem cell-like phenotypes in primary human DPCs
Discussion
Having shown that the treatment of GA represses the stemness of DPC line, we next confirmed such an effect of the compound in primary human DPCs. The isolated human DPCs were cultured in primary DPC medium in the presence of 0-200 μM GA. The stem celllike characteristics were evaluated as described. Fig. 6a and b indicates that treatment of the cells with 0–200 μM GA caused no direct cytotoxicity in these cells. In response to GA treatment for 72 h, western blot analysis revealed that levels of stem cell markers including CD133, integrin β 1 and ALDH1A1 significantly decreased in a dose-dependent manner (Fig. 6c). Furthermore, treatment of the cells with GA significantly decreased the levels of proliferation and stemness signals like active Akt, inactive GSK3β , and β -catenin (Fig. 6d). The EMT-related transcription factors ZEB1 and Snail were also found to be down-regulated in the GA-treated pri-
DPCs have been shown to exhibit stem cell-like plasticity by their ability to differentiate to different cell types (Richardson et al. 2005). Interestingly, multipotency as well as stemness of DPCs is accepted to be an important factor determining an ability to induce hair follicle formation (Ito et al. 2007; Driskell et al. 2009; Driskell et al. 2012; Clavel et al. 2012). The functions of active DPCs are not only limited to the feature of hair follicle generation, but also their promoting ability on hair growth as well as the maintenance or prolongation of anagen phase have been widely accepted (Elliott et al. 1999; Paus and Foitzik 2004). Therefore, it is possible that diminishing the stemness in these specialized DPCs may reduce the rate of hair growth and benefit the treatment of hirsutism or related approaches. In an attempt to evaluate the mechanism of GA in negatively regulating hair growth, we have discovered for the first time that GA could mediate the loss of stemness in DPCs. The primitive DPCs usually appearing
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Fig. 6. Effects of GA on stem cell-like phenotypes in primary human DPCs. (a) Cells were treated with GA (0–200 μM) for 24 h. Cytotoxicity was determined by MTT assay. The data represent the means of three independent triplicate samples ± SD. (b) After indicated treatment, mode of cell death was examined by Hoechst 33342/PI co-staining assay. Scale bar is 100 μm. (c–e) Cells were treated with GA (0–200 μM) for 72 h. After indicated treatment, the level of stem cell markers (CD133, integrin β 1 and ALDH1A1), Wnt/β -catenin signalling (Akt, p-Akt, GSK3β , p-GSK3β and β -catenin) and EMT transcription factors (ZEB1, Slug and Snail) were determined by western blot analysis, respectively. β -actin was served as the loading control. The immunoblot signals were quantified by densitometry and mean data from independent experiments were normalized to the results. The data represent the means of three independent samples ± SD. ∗P < 0.05 versus non-treated control.
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Fig. 7. Schematic diagram summarizes the effects of GA on the stemness of human DPCs. (a) Wnt activation leads to the inhibition of GSK3β function. The GSK3β is also inactivated by the phosphorylation through the function of activated Akt. The function of GSK3β is to phosphorylate β -catenin leading to β -catenin degradation. Therefore, in the presence of Wnt and activated Akt, β -catenin accumulates in the cells and activates transcription of genes associating with EMT and self-renewal. (b) Addition of GA resulted in the decrease of activated Akt leading to the increase level of active GSK3β . As a consequence, β -catenin is phosphorylated and degraded. Hence, the decrease of β -catenin resulted in the reduction of transcription of target genes, and the decrease of stem cell phenotypes in human DPCs.
as spindle-shaped cells changed to flat multipolar cells with elongated shapes (Fig. 3a). Treatment of the DPCs with non-toxic concentrations of the compound caused the spontaneous alteration of cell morphology toward fibroblast-like cells (Fig. 3a). Also, our immunecytochemistry as well as protein analysis revealed that treatment of DPCs with GA led to the significant decrease of stem cell markers (Fig. 4a–c). Regarding stem cell research, CD133, a transmembrane glycoprotein, has been widely used as a standard biomarker of human stem cells (Li 2013). It was shown that DPCs at the early passages exhibited a high level of CD133. Together with other stem cell markers, GA was shown to induce the loss of stem cell features in DPCs as indicated by the significant decrease of CD133, ALDH1A1, and integrin β 1 (Fig. 4b and c). In terms of molecular mechanism in regulating stemness, considerable progress has been obtained in elucidating stem cell signalling pathways, and in particular Wnt/β -catenin has been shown to be a critical factor in maintaining stem cell characteristics as well as functions (Merrill 2012). Previous study reported that an ablation of β -catenin in DPCs causes the dramatic suppression of both hair growth and regeneration (Enshell-Seijffers et al. 2010). The β catenin functions as a co-trancription factor of T-cell factor/lymphoid enhancing factor (TCF/LEF) and consequently increases the expression of proteins facilitating stem cell functions (Fukumoto et al. 2001). Indeed, the presence of cellular β -catenin is tightly controlled by the function of GSK3β . The phosphorylation of β -catenin by active GSK3β induces ubiquitin-mediated proteasomal degradation of β catenin. This regulation of GSK3β on β -catenin was shown to be regulated by the very well known proliferative and survival signals Akt. The active Akt is shown to inhibit the function of GSK3β by phospho-
rylating GSK3β at serine 9 (Fukumoto et al. 2001). Herein, we showed that GA treatment decreased the level of activated Akt resulting in the decrease of β -catenin (Figs. 5a and 6d), suggesting that the decline of stemness in GA-treated cells is due to the interruption of Akt/β catenin pathway. Recent evidence have suggested that β -catenin interacts with many signalling pathways involving in pluripotency and EMT (Kim et al. 2002; Miki et al. 2011; Li et al. 2012; Merrill 2012; Su et al. 2014). Wnt/β -catenin signalling activation was shown to increase the expression of EMT proteins and pluripotent activating transcription factors (Van Raay et al. 2005; Pereira and Merrill 2006; Cole et al. 2008; Ten Berge et al. 2008; Lambertini et al. 2010; Wu et al. 2012). Consistent with previous reports that the transcription factor Snail and ZEB1 plays an important role in EMT process (Mani et al. 2008; Shimono et al. 2009), our results showed the significant decrease of such proteins in the DPCs treated with GA (Figs. 5b and 6e), and the decrease of EMT markers was found to be corresponding to the stem cell-like morphology and aggregative behaviour. Our findings also lend strong support to the view that Akt/GSK3β -dependent β -catenin activation is important for the DPCs to maintain their stemness. These results unveiled that the transcription factors which are down-stream targets of Wnt/β -catenin, namely Oct-4, Nanog and Sox2 were downregulated in the GA treated cells. In conclusion, we systematically evaluated the role of GA treatment on the reduction of stemness in DPCs. Together, these results revealed a novel stemness inhibitory mechanism of GA in DPCs that is through the interruption of Akt/GSK3β -dependent β -catenin regulation resulting in a down-regulation of transcription factors associated with EMT and self-renewal (Fig. 7a and b). This study
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