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DOI: 10.1055/s-2005-916176
© Georg Thieme Verlag KG Stuttgart · New York
Suppression of Fas-Mediated Apoptosis of Keratinocyte Cells by Chikusetsusaponins Isolated from the Roots of Panax japonicus
Prof. Dr. Haruki Yamada
Kitasato Institute for Life Sciences & Graduate School of Infection Control Sciences
Kitasato University
5-9-1 Shirokane
Minato-ku
Tokyo 108-8641
Japan
Email: yamada@lisci.kitasato-u.ac.jp
Publication History
Received: March 18, 2005
Accepted: July 25, 2005
Publication Date:
05 December 2005 (online)
Abstract
Fas-mediated apoptotic cell death of a human keratinocyte cell line, HaCaT cells, and of a murine keratinocyte cell line, Pam212 cells, was suppressed by pre-treatment with a methanol extract from the roots of Panax japonicus C.A. Meyer. Activity-guided fractionation led to the isolation of chikusetsusaponins IV, IVa, V and polysciasaponin P5 as the active ingredients. Of these compounds, chikusetsusaponin IV, was most active when applied at a concentration of 12.5 μg/mL. The intracellular hallmark events of apoptosis such as DNA fragmentation and chromatin condensation were significantly reduced by the treatment with chikusetsusaponin IV. The apoptotic cell death of Jurkat cells was also suppressed by treatment with the active saponins. These results suggest that the use of chikusetsusaponins IV, IVa, V, polysciasaponin P5, or a crude extract of P. japonicus containing these saponins is expected to relieve cutaneous symptoms caused by excessive apoptotic cell death in the skin through the Fas/FasL pathway.
#Introduction
Fas (CD95/Apo-1) is a 45 kDa cell-surface receptor belonging to the tumor necrosis factor/nerve growth factor receptor family, which is highly expressed in activated lymphocytes and a variety of cells of lymphoid or non-lymphoid origin as well as tissues and tumor cells [1]. Fas ligand (FasL/CD95L) is a membrane protein belonging to the tumor necrosis factor family. Although the expression of FasL was originally thought to be restricted to activated T cells and natural killer cells, it is now known that FasL is widely expressed and functional in many tissues. When FasL binds to Fas on FasL-sensitive target cells, the target cells die because of apoptosis. The Fas/FasL interaction is recognized as a major pathway of apoptosis and has been determined to have important functions in relation to homeostasis and biological defense mechanisms. Its deregulation is involved in the occurrence of many diseases.
Fas-mediated apoptosis of keratinocytes has multiple functions [2] and has been implicated in various conditions such as allergic contact dermatitis [3], lupus erythematosus [4], toxic epidermal necrolysis [5] and the formation of sun burnt cells by UV radiation [6], [7]. Recent studies demonstrated Fas expression associated with apoptotic keratinocytes in aging of human epidermis [8], [9]. Similarly, Haake et al. [10] showed occasional premature apoptotic cells in the spinous and basal layers, pointing to possible age-related changes in the rate of intrinsic apoptosis. Therefore, inhibitory substances of excessive apoptotic cell death in skin are expected to relieve such symptoms.
In the course of our screening for anti-apoptotic activity using a human keratinocyte cell line, HaCaT cells, it was found that the extract from the roots of Panax japonicus C. A. Meyer (Araliaceae) potently inhibted Fas-mediated apoptotic cell death. P. japonicus has been used to promote the function of the stomach and as expectorant, antitussive, tonic, and anti-inflammatory agent [11], [12]. Recently, a methanol extract of P. japonicus has been found to exert a significant enhancement of the outgrowth activity of cultured human neuroblastoma cells [13]. However, there is no available literature concerning the anti-apoptotic activity of this plant. In the present paper, we report the protective effects of saponins isolated from P. japonicus against Fas-mediated apoptotic cell death. To our knowledge, this is the first report on anti-apoptotic substances from P. japonicus.
#Materials and Methods
#Plant material
The roots of P. japonicus were purchased from Uchida Wakan-yaku Co. Ltd. (Tokyo, Japan), and a voucher (No. 402 418) was deposited at the Herbarium of Oriental Medicine Research Center, the Kitasato Institute (Tokyo, Japan). The aerial parts of P. japonicus were kindly provided by Drs. K. Kawaguchi and T. Watanabe (Medicinal Plant Garden, Kitasato University).
#Chemicals
The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), was obtained from MBL (Nagoya, Japan). Recombinant human soluble Fas Ligand (FasL) and enhancer™ protein were from Alexis Biochemicals (San Diego, CA, USA). Recombinant human interferon-γ (rhIFN-γ) was from Strathmann Biotec AG (Hamburg, Germany). Recombinant murine interferon-γ (rmIFN-γ) was from PeproTech EC (London, UK). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Dojindo (Kumamoto, Japan). Alamar BlueTM was from Alamar Bio-Sciences (Sacramento, CA, USA). 4′,6-Diamidino-2-phenylindole (DAPI) and actinomycin D were from Sigma (St. Lous, MO, USA). Alexa Fluor 594 phalloidin was from Molecular Probes (Eugene, OR, USA). The DeadEndTM Fluorometric TUNEL System was from Promega (Madison, WI, USA)
#Activity-guided purification of the active substances from the extract of P. japonicus
The roots of P. japonicus (480 g) were extracted with MeOH (1.5 L × 3) at room temperature. Evaporation of the solvent gave a crude methanol extract (91.8 g), and the anti-apoptotic activity of this crude extract at a concentration of 25 μg/mL was 28.8 ± 4.6 % against FasL-induced cell death of HaCaT cells. The crude extract was re-dissolved in MeOH (500 mL) and partitioned with n-hexane (500 mL × 4) for removal of the waxy material. The MeOH layer was evaporated and re-suspended in H2O (200 mL), and then the solution was acidified to pH 3 with trifluoroacetic acid (TFA). The resulting aqueous layer was partitioned with AcOEt (500 mL × 4). The aqueous layer was successively partitioned with n-BuOH (500 mL × 4). Each fraction was evaporated to give an n-hexane fr. (2.4 g), an AcOEt fr. (3.6 g), an n-BuOH fr. (37.4 g) and a H2O fr. (45.2 g). The anti-apoptotic activity of these fractions against FasL-induced cell death of HaCaT cells was less than 5 %, 11.5 ± 1.9 %, 41.7 ± 5.6 %, and 24.6 ± 1.2 %, respectively. Since the n-BuOH fraction showed the most potent activity, the n-BuOH fraction (500 mg) was subjected to HPLC (PEGASIL 5 μm ODS, 20 × 250 mm) eluted with a gradient solvent system of 0.01 % TFA-CH3CN (20 - 60 %; linear for 45 min; flow rate, 9.0 mL/min), to afford compound 1 (133.6 mg, tR = 25.5 min), 2 (136.4 mg, tR = 27.5 min), 3 (14.3 mg, tR = 28.5 min) and 4 (6.8 mg, tR = 37.0 min). The purity of obtained each compound was confirmed by 3D-HPLC.
#FasL-induced cell death of HaCaT cells and Pam212 cells
A human keratinocyte cell line, HaCaT cells [14], and a murine keratinocyte cell line, Pam212 cells [15], were kindly provided by Prof. Dr. N. E. Fusenig (German Cancer Research Center, Heidelberg, FRG) and Prof. Dr. S. Kobayashi (Kyoritsu University of Pharmacy, Tokyo, Japan), respectively.
HaCaT cells were grown in D-MEM supplemented with 8 % FBS, penicillin (100 IU/mL) and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air. HaCaT cells were seeded at a density of 2.2 × 104 cells/well in 96-well tissue culture plates (Falcon 3072, Becton-Dickinson, Franklin Lakes, NJ, USA) and cultured in the presence of 10 ng/mL rhIFN-γ for 48 h to up-regulate the expression of Fas molecules. After changing to the fresh culture medium containing 10 ng/mL rhIFN-γ, the cells were pre-cultured with the test compound for 1 h followed by stimulation with 50 ng/mL FasL and 500 ng/mL enhancer™ protein for 24 h. The cell viability was determined with the MTT assay [16], and absorbance at 570 nm (A570) was measured with a microplate reader (Bio-Rad, Model 250). For induction of Fas-mediated apoptotic cell death in Pam212 cells, the same procedure was used as described above except for use of rhIFN-γ. For stimulation of Pam212 cells, rmIFN-γ was used. Anti-apoptotic activity was calculated as follows:
Inhibition (%) = {(sample + FasL) A570 - FasL A570}/(control A570 - FasL A570) × 100.
In this assay, some variations of the value of anti-apoptotic activity were observed from assay to assay. The cells were cultured with IFN-γ for 48 h and then cultured further with FasL for 24 h to induce apoptotic cell death. The variation might be partially due to two stages stimulation and due to long periods of incubation time.
#FasL-induced cell death of Jurkat cells
A human lymphoma cell line, Jurkat cells, was cultured in RPMI 1640 medium supplemented with 8 % FBS, penicillin (100 IU/mL) and streptomycin (100 μg/mL) in a humidified atmosphere of 5 % CO2 and 95 % air. The cell suspension was aliquoted (160 μL; 5 × 105 cells/mL) into a 96-well culture plate, and the cells were pre-treated with the test compound for 1 h followed by stimulation with 5 ng/mL FasL and culturing for 18 h. Cell viability was measured using a fluorometric assay, Alamar BlueTM reduction assay [17]. The fluorescence intensity was measured with Fluoroskan II (Labsystems, Helsinki, Finland) at an excitation wavelength 544 nm and an emission wavelength 590 nm (F590). The DeltaSoft 3 for Mac (Ver. 1.45 FL, Biometallics Inc., Princeton, NJ, USA) was used for data management. Anti-apoptotic activity was calculated as follows:
Inhibition (%) = {(sample + FasL) F590 - FasL F590}/(control F590 - FasL F590)} × 100.
#Actinomycin D-induced cell death of Jurkat cells
The cell suspension was aliquoted (160 μL; 5 × 105 cells/mL) into 96 well culture plates, and the cells were pre-treated with the tested compound for 1 h followed by stimulation with 2 μg/mL actinomycin D and culturing for 18 h. Cell viability was measured with the Alamar BlueTM reduction assay, and anti-apoptotic activity was calculated as described above.
#Detection of DNA fragmentation and chromatin condensation
HaCaT cells were fixed with 4 % paraformaldehyde and permeabilized for 5 min in PBS containing 0.2 % Triton X-100 at room temperature, and then DNA fragmentation and chromatin condensation were evaluated by TUNEL (TdT-mediated dUTP nick-end labeling) assay and DAPI staining, respectively. The actin cytoskeleton was visualized with Alexa Fluor 594 phalloidin as a counter staining. Images were recorded on a fluorescence microscope.
#Statistical analysis
Data were expressed as mean ± SD, and differences between groups were analyzed by ANOVA followed by post hoc analysis using Scheffe’s test using a personal computer with the StatView-J program for Macintosh (SAS Institute Inc., Cary, NC, USA).
#Results and Discussion
In the screening for inhibitors of Fas-mediated apoptotic cell death of HaCaT cells, a potent inhibitory activity was found in the methanol extract of P. japonicus, which inhibited Fas-mediated apoptotic cell death of HaCaT cells in a dose-dependent manner (Table [1]). The methanol extract of P. japonicus also inhibited Fas-mediated apoptotic cell death of a murine keratinocyte cell line, Pam212 cells, in a dose-dependent manner (Table [2]).
To identify the active principles of P. japonicus, fractionation was accomplished. Activity-guided fractionation led to the isolation of compounds 1 (chikusetsusaponin V), 2 (chikusetsusaponin IV), 3 (chikusetsusaponin IVa) and 4 (polysciasaponin P5). Although these saponins were obtained as purified active compounds, the anti-apoptotic activities were weaker than expected because these saponins are the main ingredients of the methanol extract and because the sum of the yield of each active saponin was 23.7 %. These compounds are classified as oleanane-type triterpenoidal saponins, and the chemical structures are shown in Fig. [1]. Structural identification was carried out by analysis of their NMR and mass spectroscopic data and by comparison with the previously reported spectral values [18], [19], [20]. Each carbohydrate moiety was also analyzed by high performance anion-exchange chromatography equipped using a pulsed amperometric detector after acid hydrolysis or glycosidase digestion. Copies of the original spectra are obtainable from the author of correspondence. The anti-apoptotic activity was also observed in the methanol extract from the aerial parts of P. japonicus, and the extract contained the same saponins (data not shown).
The saponins 1 - 4 inhibited Fas-mediated apoptosis of HaCaT cells and Pam212 cells in a concentration-dependent manner (Tables [1] and [2]). The positive control, Z-VAD-FMK, also inhibited Fas-mediated apoptosis of both cell types. Of these compounds, chikusetsusaponin IV (2) was most active when applied at a concentration of 12.5 μg/mL (Tables [1] and [2]). Therefore, the following experiments were performed using chikusetsusaponin IV (2). DNA fragmentation and chromatin condensation are typical intracellular events of apoptosis. To confirm that chikusetsusaponin IV (2) has a potent anti-apoptotic effect against Fas-mediated apoptotic cell death of HaCaT cells, the effect of chikusetsusaponin IV (2) on DNA fragmentation and chromatin condensation was evaluated by TUNEL and DAPI staining, respectively. Only a small number of TUNEL positive cells were observed in untreated HaCaT cells. When HaCaT cells were treated with FasL, a marked increase in TUNEL-positive cells was observed, and this increase in TUNEL positive cells was significantly reduced in the presence of 50 μg/mL (54 μM) chikusetsusaponin IV (2) (Figs. [2] A and B). When HaCaT cells were treated with FasL, marked chromatin condensation was also observed. This increase in chromatin condensation was significantly inhibited by treatment of the cells with 50 μg/mL (54 μM) chikusetsusaponin IV (2) (Figs. [2] C and D). Inhibition of DNA fragmentation and chromatin condensation were also observed upon treatment of FasL-stimulated Pam212 cells with 50 μg/mL methanol extract of P. japonicus (data not shown).
It is widely known that Jurkat cells constitutively express the Fas molecule and are therefore sensitive to killing by FasL without any other stimuli such as IFN-γ. The effects of the methanol extract of P. japonicus and the saponins 1 - 4 were examined using Jurkat cells. The methanol extract of P. japonicus and the saponins 1 - 4 each inhibited Fas-mediated apoptotic cell death of Jurkat cells in a concentration-dependent manner (Table [3]), and the IC50 values of the saponins 1 - 4 were 47.5, 15.6, 48.9, and 44.6 μM, respectively. To know whether the anti-apoptotic activity of the saponins were due to specific inhibitory activity of the Fas/FasL pathway, the effects of the saponins 1 - 4 on actinomycin D-induced apoptotic cell death was examined. The methanol extract of P. japonicus and the saponins 1 - 4 each inhibited actinomycin D-induced apoptotic cell death of Jurkat cells (Table [4]). These results suggest that the methanol extract of P. japonicus and the saponins 1 - 4 may inhibit the intracellular signal transduction pathways involved in apoptosis, but that they do not inhibit the Fas/FasL interaction on the cell surface. It is assumed that there are several target molecules in the cells since the potency of the different compounds varies with the cell type investigated. However, the precise mechanism of the anti-apoptotic activity is not known at the present. Details of the molecular events of the anti-apoptotic activity are being studied and will be described in a following publication.
Programmed cell death through apoptosis plays a fundamental role in a variety of regulated physiological processes, and its deregulation is involved in many diseases. Deregulation of apoptosis in skin is involved in cutaneous disorders and symptoms such as allergic contact dermatitis, sunburn dermatitis, eczema, and others. In this study, the saponins 1 - 4 and the methanol extract of P. japonicus showed anti-apoptotic activity against Fas-mediated apoptotic cell death of keratinocyte cell lines, HaCaT cells and Pam212 cells. These results suggest that these saponins 1 - 4 and the crude extract of P. japonicus containing these saponins are expected to relieve cutaneous diseases involved in excessive apoptotic cell death in skin through Fas/FasL pathway.
Fig. 1 Chemical structures of compounds 1 - 4.
Fig. 2 Effect of chikusetsusaponin IV (2) on DNA fragmentation and chromatin condensation of FasL stimulated HaCaT cells. HaCaT cells were treated with 50 μg/mL (54 μM) chikusetsusaponin IV (2) or 2 μM Z-VAD-FMK, as a positive control, for 1 h and then stimulated with 50 ng/mL FasL and 500 ng/mL enhancer™ protein for 18 h. DNA fragmentation was evaluated using the TUNEL assay (A and B), and chromatin condensation was evaluated by DAPI staining (C and D). The TUNEL positive cells and apoptotic cells exhibiting morphological features of chromatin condensation were counted in 6 randomly selected fields. Data are expressed as mean ± SD. The experiment was repeated twice and similar results were obtained. Images were recorded on a fluorescence microscope.
| Tested sample | Inhibition of cell death (%) | ||
| 50 μg/mL | 25 μg/mL | 12.5 μg/mL | |
| Exp. 1 | |||
| Methanol extract of P. japonicus | 29.0 ± 6.5 | 21.8 ± 8.1 | n. d. |
| Z-VAD-FMK (2 μM) | 41.8 ± 2.4 | ||
| Exp. 2 | |||
| 1 (chikusetsusaponin V) | 43.2 ± 1.2 | 17.7 ± 9.0 | < 5 |
| 2 (chikusetsusaponin IV) | 37.4 ± 4.1 | 23.5 ± 6.6 | 25.5 ± 6.9 |
| 3 (chikusetsusaponin IVa) | 24.2 ± 5.2 | 18.9 ± 7.2 | 8.4 ± 8.0 |
| 4 (polysciasaponin P5) | 19.3 ± 8.3 | 13.2 ± 7.7 | 11.7 ± 8.7 |
| Z-VAD-FMK (2 μM) | 72.3 ± 1.4 | ||
| Anti-apoptotic activity was calculated as described in Materials and Methods. Z-VAD-FMK was used as a positive control at a concentration of 2 μM. n. d. = not determined. It is not possible to compare the anti-apoptotic activity measured in experiments 1 and 2 because of the differing cell passage number. No measurable cytotoxicity was observed upon use of the tested samples alone (data not shown). Data are expressed as mean ± SD (n = 4). The experiment was repeated twice and similar results were obtained. | |||
| Tested sample | Inhibition of cell death (%) | ||
| 50 μg/mL | 25 μg/mL | 12.5 μg/mL | |
| Methanol extract of P. japonicus | 57.2 ± 9.6 | 27.2 ± 5.0 | 27.1 ± 9.2 |
| 1 (chikusetsusaponin V) | 53.7 ± 9.7 | 37.3 ± 6.0 | 29.0 ± 8.8 |
| 2 (chikusetsusaponin IV) | 87.5 ± 5.3 | 58.1 ± 9.0 | 52.3 ± 6.2 |
| 3 (chikusetsusaponin IVa) | 43.6 ± 5.3 | 32.5 ± 12.1 | 26.5 ± 10.9 |
| 4 (polysciasaponin P5) | 31.9 ± 7.6 | 34.3 ± 3.7 | 41.5 ± 10.9 |
| Z-VAD-FMK (2 μM) | 44.5 ± 3.7 | ||
| Anti-apoptotic activity was calculated as described in Materials and Methods. Z-VAD-FMK was used as a positive control at a concentration of 2 μM. No measurable cytotoxicity was observed upon use of the tested samples alone (data not shown). Data are expressed as mean ± SD (n = 4). The experiment was repeated twice and similar results were obtained. | |||
| Tested sample | Inhibition of cell death (%) | IC50 (μM) | ||
| 50 μg/mL | 25 μg/mL | 12.5 μg/mL | ||
| Methanol extract of P. japonicus | 40.7 ± 4.8 | 21.9 ± 4.4 | 11.5 ± 2.9 | |
| 1 (chikusetsusaponin V) | 53.2 ± 2.8 | 34.5 ± 5.3 | 21.4 ± 5.8 | 47.5 |
| 2 (chikusetsusaponin IV) | 71.1 ± 9.4 | 60.7 ± 2.7 | 47.1 ± 1.9 | 15.6 |
| 3 (chikusetsusaponin IVa) | 59.0 ± 6.9 | 40.5 ± 3.0 | 21.7 ± 2.0 | 48.9 |
| 4 (polysciasaponin P5) | 53.6 ± 2.5 | 49.2 ± 2.7 | 39.9 ± 3.0 | 44.6 |
| Z-VAD-FMK (2 μM) | 60.1 ± 4.3 | |||
| Anti-apoptotic activity was calculated as described in Materials and Methods. Z-VAD-FMK was used as a positive control at a concentration of 2 μM. No measurable cytotoxicity or antiproliferative effect was observed upon use of the tested samples alone (data not shown). Data are expressed as mean ± SD (n = 4). The experiment was repeated twice and similar results were obtained. | ||||
| Tested sample | Inhibition of cell death (%) |
| 25 μg/mL | |
| Methanol extract of P. japonicus | 47.0 ± 3.4 |
| 1 (chikusetsusaponin V) | 41.8 ± 2.9 |
| 2 (chikusetsusaponin IV) | 64.5 ± 5.9 |
| 3 (chikusetsusaponin IVa) | 53.9 ± 3.2 |
| 4 (polysciasaponin P5) | 59.5 ± 5.5 |
| Anti-apoptotic activity was calculated as described in Materials and Methods. No measurable cytotoxicity or antiproliferative effect was observed upon use of the tested samples alone (data not shown). Data are expressed as mean ± SD (n = 4). The experiment was repeated twice and similar results were obtained. | |
Acknowledgements
The authors gratefully acknowledge the excellent technical assistance of Ms. M. Sato for NMR measurements. The authors also thank to Ms. C. Sakabe and A. Nakagawa for measurement of mass spectra. A part of this work was supported by a grant of the 21st Century COE Program from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan.
#References
- 1 Walczak H, Krammer P H. The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems. Exp Cell Res. 2000; 256 58-66
- 2 Wehrli P, Viard I, Bullani R, Tschopp J, French L E. Death receptors in cutaneous biology and disease. J Invest Dermatol. 2000; 115 141-8
- 3 Trautmann A, Akdis M, Kleemann D, Altznauer F, Simon H U, Graeve T. et al . T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis. J Clin Invest. 2000; 106 25-35
- 4 Baima B, Sticherling M. Apoptosis in different cutaneous manifestations of lupus erythematosus. Br J Dermatol. 2001; 144 958-66
- 5 Viard I, Wehrli P, Bullani R, Schneider P, Holler N, Salomon D. et al . Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. Science. 1998; 282 490-3
- 6 Leverkus M, Yaar M, Gilchrest B A. Fas/Fas ligand interaction contributes to UV-induced apoptosis in human keratinocytes. Exp Cell Res. 1997; 232 255-62
- 7 Murphy G, Young A R, Wulf H C, Kulms D, Schwarz T. The molecular determinants of sunburn cell formation. Exp Dermatol. 2001; 10 155-60
- 8 Gilhar A, Ullmann Y, Karry R, Shalaginov R, Assy B, Serafimovich S, Kalish R S. Aging of human epidermis: reversal of aging changes correlates with reversal of keratinocyte Fas expression and apoptosis. J Gerontol A Biol Sci Med Sci. 2004; 59 411-5
- 9 Wang X, Bregegere F, Soroka Y, Kayat A, Redziniak G, Milner Y. Enhancement of Fas-mediated apoptosis in ageing human keratinocytes. Mech Ageing Dev. 2004; 125 237-49
- 10 Haake A R, Roublevskaia I, Cooklis M. Apoptosis: a role in skin aging?. J Investig Dermatol Symp Proc. 1998; 3 28-35
- 11 Saito H, Lee Y M, Takagi K, Shibata S, Shoji J, Kondo N. Pharmacological studies of Panacis japonici rhizoma. I. Chem Pharm Bull. 1977; 25 1017-25
- 12 Lee Y, Saito H, Takagi K, Shibata S, Shoji J. Pharmacological studies of Panacis japonici rhizoma. II. Chem Pharm Bull. 1977; 25 1391-8
- 13 Zou K, Zhu S, Meselhy M R, Tohda C, Cai S, Komatsu K. Dammarane-type saponins from Panax japonicus and their neurite outgrowth activity in SK-N-SH cells. J Nat Prod. 2002; 65 1288-92
- 14 Boukamp P, Petrussevska R T, Breitkreutz D, Hornung J, Markham A, Fusenig N E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988; 106 761-71
- 15 Yuspa S H, Hawley-Nelson P, Koehler B, Stanley J R. A survey of transformation markers in differentiating epidermal cell lines in culture. Cancer Res. 1980; 40 4694-703
- 16 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65 55-63
- 17 Page B, Page M, Noel C. A new fluorometric assay for cytotoxicity measurements in vitro . Int J Oncol. 1993; 3 473-6
- 18 Nie R L, Morita T, Kasai R, Zhou J, Wu C Y, Tanaka O. Saponins from Chinese medicinal plants. (I). Isolation and structures of hemslosides. Planta Med. 1984; 50 322-7
- 19 Kinjo J, Suyama K, Nohara T. Triterpenoidal saponins from Dumasia truncata . Phytochemistry. 1995; 40 1765-7
- 20 Marouf A, Desbene S, Khanh T C, Wagner H, Correia M, Chauffert B, Lacaille-Dubois M A. Triterpene saponins from the roots of Achyranthes bidentata . Pharm Biol. 2001; 39 263-7
Prof. Dr. Haruki Yamada
Kitasato Institute for Life Sciences & Graduate School of Infection Control Sciences
Kitasato University
5-9-1 Shirokane
Minato-ku
Tokyo 108-8641
Japan
Email: yamada@lisci.kitasato-u.ac.jp
References
- 1 Walczak H, Krammer P H. The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems. Exp Cell Res. 2000; 256 58-66
- 2 Wehrli P, Viard I, Bullani R, Tschopp J, French L E. Death receptors in cutaneous biology and disease. J Invest Dermatol. 2000; 115 141-8
- 3 Trautmann A, Akdis M, Kleemann D, Altznauer F, Simon H U, Graeve T. et al . T cell-mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis. J Clin Invest. 2000; 106 25-35
- 4 Baima B, Sticherling M. Apoptosis in different cutaneous manifestations of lupus erythematosus. Br J Dermatol. 2001; 144 958-66
- 5 Viard I, Wehrli P, Bullani R, Schneider P, Holler N, Salomon D. et al . Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. Science. 1998; 282 490-3
- 6 Leverkus M, Yaar M, Gilchrest B A. Fas/Fas ligand interaction contributes to UV-induced apoptosis in human keratinocytes. Exp Cell Res. 1997; 232 255-62
- 7 Murphy G, Young A R, Wulf H C, Kulms D, Schwarz T. The molecular determinants of sunburn cell formation. Exp Dermatol. 2001; 10 155-60
- 8 Gilhar A, Ullmann Y, Karry R, Shalaginov R, Assy B, Serafimovich S, Kalish R S. Aging of human epidermis: reversal of aging changes correlates with reversal of keratinocyte Fas expression and apoptosis. J Gerontol A Biol Sci Med Sci. 2004; 59 411-5
- 9 Wang X, Bregegere F, Soroka Y, Kayat A, Redziniak G, Milner Y. Enhancement of Fas-mediated apoptosis in ageing human keratinocytes. Mech Ageing Dev. 2004; 125 237-49
- 10 Haake A R, Roublevskaia I, Cooklis M. Apoptosis: a role in skin aging?. J Investig Dermatol Symp Proc. 1998; 3 28-35
- 11 Saito H, Lee Y M, Takagi K, Shibata S, Shoji J, Kondo N. Pharmacological studies of Panacis japonici rhizoma. I. Chem Pharm Bull. 1977; 25 1017-25
- 12 Lee Y, Saito H, Takagi K, Shibata S, Shoji J. Pharmacological studies of Panacis japonici rhizoma. II. Chem Pharm Bull. 1977; 25 1391-8
- 13 Zou K, Zhu S, Meselhy M R, Tohda C, Cai S, Komatsu K. Dammarane-type saponins from Panax japonicus and their neurite outgrowth activity in SK-N-SH cells. J Nat Prod. 2002; 65 1288-92
- 14 Boukamp P, Petrussevska R T, Breitkreutz D, Hornung J, Markham A, Fusenig N E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988; 106 761-71
- 15 Yuspa S H, Hawley-Nelson P, Koehler B, Stanley J R. A survey of transformation markers in differentiating epidermal cell lines in culture. Cancer Res. 1980; 40 4694-703
- 16 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65 55-63
- 17 Page B, Page M, Noel C. A new fluorometric assay for cytotoxicity measurements in vitro . Int J Oncol. 1993; 3 473-6
- 18 Nie R L, Morita T, Kasai R, Zhou J, Wu C Y, Tanaka O. Saponins from Chinese medicinal plants. (I). Isolation and structures of hemslosides. Planta Med. 1984; 50 322-7
- 19 Kinjo J, Suyama K, Nohara T. Triterpenoidal saponins from Dumasia truncata . Phytochemistry. 1995; 40 1765-7
- 20 Marouf A, Desbene S, Khanh T C, Wagner H, Correia M, Chauffert B, Lacaille-Dubois M A. Triterpene saponins from the roots of Achyranthes bidentata . Pharm Biol. 2001; 39 263-7
Prof. Dr. Haruki Yamada
Kitasato Institute for Life Sciences & Graduate School of Infection Control Sciences
Kitasato University
5-9-1 Shirokane
Minato-ku
Tokyo 108-8641
Japan
Email: yamada@lisci.kitasato-u.ac.jp
Fig. 1 Chemical structures of compounds 1 - 4.
Fig. 2 Effect of chikusetsusaponin IV (2) on DNA fragmentation and chromatin condensation of FasL stimulated HaCaT cells. HaCaT cells were treated with 50 μg/mL (54 μM) chikusetsusaponin IV (2) or 2 μM Z-VAD-FMK, as a positive control, for 1 h and then stimulated with 50 ng/mL FasL and 500 ng/mL enhancer™ protein for 18 h. DNA fragmentation was evaluated using the TUNEL assay (A and B), and chromatin condensation was evaluated by DAPI staining (C and D). The TUNEL positive cells and apoptotic cells exhibiting morphological features of chromatin condensation were counted in 6 randomly selected fields. Data are expressed as mean ± SD. The experiment was repeated twice and similar results were obtained. Images were recorded on a fluorescence microscope.