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Ginsenoside F1 Protects Human HaCaT Keratinocytes from Ultraviolet-B-Induced Apoptosis by Maintaining Constant Levels of Bcl-2

2003; Elsevier BV; Volume: 121; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2003.12425.x

1523-1747

Enn Hee Lee, Si Young Cho, Su Jong Kim, Eui Seok Shin, Hui Kyoung Chang, Duck Hee Kim, Myeong Hoon Yeom, Kwang Sik Woe, Jinseon Lee, Young Chul Sim, Tae Ryong Lee,

PARP inhibition in cancer therapy

Ginsenosides, the major active ingredients of ginseng, show a variety of biomedical efficacies such as antiaging and antioxidation. Here, we investigate the protective activity of the ginsenoside F1, an enzymatically modified derivative of ginsenoside Rg1, against ultraviolet-B-induced damage in human HaCaT keratinocytes. Ginsenoside F1 significantly reduced ultraviolet-B-induced cell death and protected HaCaT cells from apoptosis caused by ultraviolet B irradiation. Furthermore, ginsenoside F1 prevented ultraviolet-B-induced cleavage of poly(ADP-ribose) polymerase in HaCaT cells. In search of the molecular mechanism responsible for the antiapoptotic effect of ginsenoside F1, we find that protection from ultraviolet-B-induced apoptosis is tightly correlated with ginsenoside-F1-mediated inhibition of ultraviolet-B-induced downregulation of Bcl-2 and Brn-3a expression. Ginsenosides, the major active ingredients of ginseng, show a variety of biomedical efficacies such as antiaging and antioxidation. Here, we investigate the protective activity of the ginsenoside F1, an enzymatically modified derivative of ginsenoside Rg1, against ultraviolet-B-induced damage in human HaCaT keratinocytes. Ginsenoside F1 significantly reduced ultraviolet-B-induced cell death and protected HaCaT cells from apoptosis caused by ultraviolet B irradiation. Furthermore, ginsenoside F1 prevented ultraviolet-B-induced cleavage of poly(ADP-ribose) polymerase in HaCaT cells. In search of the molecular mechanism responsible for the antiapoptotic effect of ginsenoside F1, we find that protection from ultraviolet-B-induced apoptosis is tightly correlated with ginsenoside-F1-mediated inhibition of ultraviolet-B-induced downregulation of Bcl-2 and Brn-3a expression. poly(ADP-ribose) polymerase Ultraviolet radiation (UV), in particular ultraviolet B (UVB) with a wavelength range between 290 and 320 nm, is one of the most important environmental factors because of its hazardous health effects, which include generation of skin cancer (De Gruijl et al., 1993De Gruijl F.R. Sterenborg H.J. Forbes P.D. et al.Wavelength dependence of skin cancer induction by ultraviolet irradiation of albino hairless mice.Cancer Res. 1993; 53: 53-60PubMed Google Scholar), suppression of the immune system (Beissert and Schwarz, 1999Beissert S. Schwarz T. Mechanisms involved in ultraviolet light-induced immuosuppression.J Invest Dermatol Symp Proc The. 1999; 4: 61-64Abstract Full Text PDF PubMed Scopus (119) Google Scholar), and premature skin aging (Fisher et al., 1997Fisher G.J. Wang Z.Q. Datta S.C. Varani J. Kang S. Voorhees J.J. Pathophysiology of premature skin aging induced by ultraviolet light.N Engl J Med. 1997; 337: 1419-1428Crossref PubMed Scopus (1081) Google Scholar). One of the hallmark events of UV-exposed skin is the formation of apoptotic keratinocytes, which die as sunburn cells within the epidermis. Biochemical analyses reveal that UVB-irradiated keratinocytes show DNA fragmentation accompanied by sequential caspase activation (Takahashi et al., 1997Takahashi H. Kinouchi M. Iizuka H. Interleukin-1β-converting enzyme and CPP 32 are involved in ultraviolet B-induced apoptosis of SV40-transformed human keratinocytes.Biochem Biophys Res Commun. 1997; 236: 194-198Crossref PubMed Scopus (32) Google Scholar). The Bcl-2 protein is constitutively expressed in the human epidermis and regulation of its expression level may have an important role in the generation of sunburn cells (Hockenbery et al., 1991Hockenbery D.M. Zutter M. Hickey W. Nahm M. Korsmeyer S.J. Bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death.Proc Natl Acad Sci USA. 1991; 88 (6956): 6961Crossref PubMed Scopus (1236) Google Scholar;Rodriguez-Villanueva et al., 1995Rodriguez-Villanueva J. Colome M.I. Brisbay S. McDonnell T.J. The expression and localization of bcl-2 protein in normal skin and non-melanoma skin cancers.Pathol Res Pract. 1995; 191: 391-398Crossref PubMed Scopus (53) Google Scholar;Sermadiras et al., 1997Sermadiras S. Dumas M. Joly-Berville R. Bonte F. Meybeck A. Ratinaud M.H. Expression of Bcl-2 and Bax in cultured normal human keratinocytes and melanomacytes: Relationship to differentiation and melanogenesis.Br J Dermatol. 1997; 137: 883-889Crossref PubMed Scopus (23) Google Scholar). Bcl-2 is an antiapoptotic protein that blocks apoptosis probably by inhibiting the channel-forming activity of proapoptotic family members like Bax (Adams and Cory, 1998Adams J.M. Cory S. The bcl-2 protein family: Arbiters of cell survival.Science. 1998; 0: 1322-1326Crossref Scopus (4658) Google Scholar). UVB irradiation has been known to decrease Bcl-2 expression without alteration of Bax expression in human keratinocytes (Gillardon et al., 1994Gillardon F. Eschenfelder C. Uhlmann E. Harschu W. Zimermann M. Differential regulation of c-fos, fos B, c-jun, jun B and bax expression in rat skin following single or chronic ultraviolet irradiation and in vivo modulation by anti-sense oligonucleotide superfusion.Oncogene. 1994; 9: 3219-3225PubMed Google Scholar). Furthermore, Bcl-2-transfected HaCaT cells or Bcl-2-overexpressing transgenic mice were shown to be resistant to UVB-induced apoptosis (Haake and Polakowka, 1995Haake A.R. Polakowka R.R. UV-induced apoptosis in skin equivalents: Inhibition by phorbol ester and Bcl-2 overexpression.Cell Death Diff. 1995; 2: 183-193PubMed Google Scholar;Takahashi et al., 2001Takahashi H. Honma M. Akemi I.Y. et al.In vitro and in vivo transfer of bcl-2 gene into keratinocytes suppresses UVB-induced apoptosis.Photochem Photobiol. 2001; 74: 579-586Crossref PubMed Scopus (27) Google Scholar). Although these findings suggest that Bcl-2 is a key regulator in the apoptotic pathway of UVB-irradiated skin, little is known about the mechanisms governing the downregulation of Bcl-2 expression caused by UVB irradiation. Ginsenosides represent the major active ingredients of ginseng, which show a variety of biomedical efficacies, such as immune modulation, antiaging, anti-inflammation, and antioxidation in the central nervous, cardiovascular, endocrine, and immune systems (Wang et al., 2000Wang Y. Wang B.X. Li T.H. Minami M. Nagata T. Ikejima T. Metabolism of ginsenoside Rg1 and Rh1.Acta Pharmacol Sin. 2000; 21: 792-796PubMed Google Scholar;Yu and Li, 2000Yu S.C. Li X.Y. Effect of ginsenoside on IL-1β and IL-6 mRNA expression in hippopcampal neurons in chronic inflammation model of aged rats.Acta Pharmacol Sin. 2000; 21: 915-918PubMed Google Scholar;Chen et al., 2001Chen X.C. Zhu Y.G. Wang X.Z. Zhu L.A. Huang C. Protective effect of ginsenoside Rg1 on dopamine-induced apoptosis in PC12 cells.Acta Pharmacol Sin. 2001; 22: 673-678PubMed Google Scholar). More than 20 ginsenosides have been isolated and identified to date (Gillis, 1997Gillis C.N. Panax ginseng pharmacolog: A nitric oxide link? Biochem Pharmacol.. 1997; 54: 1-8Google Scholar). Ginsenoside Rg1 prevents rat cortical neurons from apoptosis (Li et al., 1997Li J.Q. Zhang X.G. Zhang J.T. Study on the antiapoptotic mechanism of ginsenoside Rg1 in cultured cortical neurons.Acta Pharmacol Sin. 1997; 32: 406-410Google Scholar), whereas ginsenoside Rh2 inhibits growth in various cancer cells (Ota et al., 1987Ota T. Fujikawa-Yamamoto K. Zong Z.P. et al.Plant glycoside modulation of cell surface related to control of differentiation in cultured B16 melanoma cells.Cancer Res. 1987; 47: 3863-3867PubMed Google Scholar). The action mechanism and potential role of ginsenosides in keratinocytes, however, are poorly understood. In this study we have investigated the role of a ginsenoside in UVB-induced apoptosis of keratinocytes. For the study, the ginsenoside F1 (20-O-β-D-glucopyranosyl-20 (S)-protopanaxatriol), an enzymatically modified molecule of ginsenoside Rg1, is used (Figure 1). We have shown that UVB irradiation downregulates Bcl-2 expression via a downregulation of Brn-3a transcription factor in HaCaT cells, and ginsenoside F1 protects these cells against UVB-induced apoptosis by maintaining constant levels of Brn-3a and the corresponding inhibition of Bcl-2 downregulation. Ten grams of total ginseng extracts (red ginseng, white ginseng, tiny ginseng roots, and ginseng leaves) were dissolved in 2 liters of citrate buffer (pH 4.0) and incubated with 10 g of naringinase (Sigma St. Louis, MO) or 10 g of pectinase (Novozyme Krogshoejvej, Bagsvaerd) for 48 h at 38°C. When the enzymatic hydrolysis reaction was complete, the reaction mixture was extracted with 2 liters of ethyl acetate. After evaporation in vacuo, 2.8 g of the residue was obtained. To obtain pure ginsenoside F1, the resulting products were separated through silica gel column chromatography and eluted with chloroform:methanol (9:1). Further elution with chloroform:methanol (6:1) afforded the pure ginsenoside F1 (0.28 g). The identification was done by FAB-MS, 1H-NMR, and 13C-NMR spectra and the purity was assessed by high performance liquid chromatography and determined to be about 96%. Ginsenoside F1 was dissolved in 100% ethanol to make a stock solution. Ethanol concentration was kept below 0.001% in all the cell cultures and did not exert any detectable change in cell growth or apoptosis.Table ITable IEffect of ginsenoside F1 on the viability and cytotoxicity of HaCaT cellsaHaCaT cells were treated with the indicated concentration of ginsenoside F1 for 24 h.Ginsenoside F1Trypan blue, %±SDbData are mean±SD of triplicate determinations.MTT, OD±SDbData are mean±SD of triplicate determinations.Control92.73±6.301.229±0.031 μm90.07±2.891.204±0.105 μm91.14±2.921.252±0.0910 μm89.08±3.001.210±0.0550 μm56.45±4.560.704±0.10a HaCaT cells were treated with the indicated concentration of ginsenoside F1 for 24 h.b Data are mean±SD of triplicate determinations. Open table in a new tab Human HaCaT keratinocytes were obtained from Dr Fusenig of the German Cancer Research Center (DKFZ) and the cells (1.5–2×105 cells per well) placed in six-well plates were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 24 h. After serum starvation for 24 h, medium was replaced by serum-free medium with various concentrations of ginsenoside F1, and cells were incubated for 24 h prior to irradiation with UVB. Ginsenoside-F1-treated or untreated HaCaT cells were rinsed twice with PBS and exposed to various doses of UVB (0–120 mJ per cm2), the intensity of which was monitored with an IL-1700 radiometer. A UVB radiation source was provided by a bank of Sankyo Denki G15T8E, a fluorescent bulb emitting 270–320 nm wavelength with a peak at 313 nm. Further, a Kodacel cellulose film (Kodacel TA401/407) was used to completely eliminate UVC radiation. After UVB exposure, cells were replenished with serum-free medium including ginsenoside F1 and followed up to 24 h. To determine cell viability, Trypan blue dye exclusion and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assays were performed at 24 h after treatment with different doses of ginsenoside F1 (1, 5, 10, 50 μm) in HaCaT cells as described byFumelli et al., 2000Fumelli C. Marconi A. Salvioli S. et al.Carboxyfullerenes protect human keratinocytes from ultraviolet-B-induced apoptosis.J Invest Dermatol. 2000; 115: 835-841Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar. The results of MTT assays were expressed as optical density units (OD) using an ELISA reader (Thermo Max, Molecular Devices, Sunnyvale, CA) at 540 nm. At 24 h after UVB irradiation (0–120 mJ per cm2), the relative number of living cells was detected by MTT assay. The ratio of viable cells was expressed as a percentage of the OD value obtained without UVB treatment. At 24 h after UVB irradiation, ginsenoside-F1-treated and untreated HaCaT cells were analyzed by TUNEL staining and DNA fragmentation assay. TUNEL staining was performed with the TUNEL Apoptosis Detection Kit (Upstate, New York), as recommended by the manufacturer. The biotin-labeled nuclei cells were detected by fluorescein isothiocyanate (FITC) avidin. Nuclei were counterstained using propidium iodide and cells were analyzed under a fluorescence microscope. For analysis of DNA fragmentation, DNA was isolated from the cells as described byBaba et al., 1998Baba T. Nakano H. Tamai K. Sawamura D. Hanada K. Hashimoto I. Arima Y. Inhibitory effect of β-thujaplicin on ultraviolet B-induced apoptosis in mouse keratinocytes.J Invest Dermatol. 1998; 110: 24-28Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar. Twenty microgram of DNA was subjected to 2% agarose gel electrophoresis. mRNA was extracted from cultures using Oligotex Direct mRNA kits (Qiagen, Hilden, Germany), following the manufacturer's instructions. One microgram of mRNA was used as template for quantitative RT-PCR. cDNA synthesis and PCR amplification were performed using the Platinum Quantitative RT-PCR ThermoScript One-Step System (Invitrogen Grand Island, NY), according to the instructions of the manufacturer. For specific human Bcl-2 or Bax amplification, the oligonucleotides 5′-TACGATAACCGGGAGATAGTGA-3′ (sense, bases 56–77 of human Bcl-2 cDNA) and 5′-CAGGTGCCGGTTCAGGTACT-3′ (antisense, bases 566–586 of human Bcl-2 cDNA) or the oligonucleotides 5′-CAAGAAGCTGAGCGAGTGTCT-3′ (sense, bases 220–240 of human Bax cDNA) and 5′-GGTTCTGATCAGTTCCGGCAC-3′ (antisense, bases 437–457 of human Bax cDNA) were used. For specific human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA amplification, the oligonucleotides 5′-CAACTACATGGTTTACATGTTCC-3′ (sense, bases 174–194 of human GAPDH cDNA) and 5′-GGACTGTGGTCATGAGTCCT-3′ (antisense, bases 570–589 of human GAPDH cDNA) were used (Suschek et al., 1999Suschek C.V. Krischel V. Gerharz D.B. Berendji D. Krutmann J. Kroncke K.D. Bachofen V.K. Nritric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation.J Biol Chem. 1999; 274: 6130-6137Crossref PubMed Scopus (150) Google Scholar). For semiquantitative analysis, agarose gels were scanned by video documentation and the images were analyzed using the ImageMaster 2D Elite software (Amersham Biosciences Buckinghamshire, UK). Relative Bcl-2 and Bax mRNA expression was calculated by normalization to the expression of GAPDH. At 24 h after UVB irradiation (60 mJ per cm2), ginsenoside-F1-treated or untreated cells were washed with PBS and lyzed on ice in RIPA buffer pH 8.5, as described elsewhere (Rodeck et al., 1997Rodeck U. Jost M. DuHadaway J. Kari C. Jensen P.J. Risse B. Ewert D.L. Regulation of Bcl-xL expression in human keratinocytes by cell-substratum adhesion and the epidermal growth factor receptor.Proc Natl Acad Sci USA. 1997; 94: 5067-5072Crossref PubMed Scopus (75) Google Scholar). Forty micrograms of total protein were analyzed under reducing conditions on 8% sodium dodecyl sulfate/polyacrylamide gels and blotted onto nitrocellulose membrane. The blot was incubated with an anti-Bax (0.5 μg per ml, Santa Cruz, Santa Cruz, CA), anti-Bcl-2 (2 μg per ml, Santa Cruz), anti-poly(ADP-ribose) polymerase (anti-PARP) (1 μg per ml, Santa Cruz), anti-Rb (2 μg per ml, Santa Cruz), anti-c-myb (2 mg per ml, Santa Cruz), anti-Brn-3a (2 μg per ml, Santa Cruz), and anti-Hsp 70 (0.5 μg per ml, Santa Cruz) polyclonal antibody. The reaction products were detected by chemiluminescence with the ECL kit (Amersham Biosciences) according to the manufacturer's instructions. Anti-Hsp 70 antibody was used to assess equal loading of the protein. Plasmid constructs were carried out according to the method described bySmith et al., 1998Smith M.D. Ensor E.A. Coffin R.S. Boxer L.M. Latchman D.S. Bcl-2 transcription from the proximal P2 promoter is activated in neuronal cells by the Brn-3a POU family transcription factor.J Biol Chem. 1998; 273: 16715-16722Crossref PubMed Scopus (107) Google Scholar. Briefly, full-length human Brn-3a cDNA was amplified by PCR using primers 5′-GGATCCATGATGTCCATGAACAGCAAGCAGCC-3′ and 5′-GAATTCGTAAGTGGCAGAGAATTTCATCCG-3′ with human brain, whole Marathon-Ready cDNA (Clontech Palo Alto, CA) as template, and subcloned into the BamHI and EcoRI sites of pcDNA4/TO/myc-His A (Invitrogen). The human Bcl-2 P2 promoter region (spanning between –746 and –8 bp relative to the ATG;Smith et al., 1998Smith M.D. Ensor E.A. Coffin R.S. Boxer L.M. Latchman D.S. Bcl-2 transcription from the proximal P2 promoter is activated in neuronal cells by the Brn-3a POU family transcription factor.J Biol Chem. 1998; 273: 16715-16722Crossref PubMed Scopus (107) Google Scholar) were cloned upstream of the luciferase reporter gene in the pGL3 luciferase basic vector (Promega, Madison, WI). Correctness of all constructs was verified by sequencing. Cells (1×105 cells) were transiently transfected with 10 μg of the promoter-reporter plasmid and 10 μg of the Brn-3a expression vector together with 2 mg of pCMV-β-galactosidase control vector using 10 mg of lipofectamine (Invitrogen) reagent, following the directions of the manufacturer. Transfected cells were treated with ginsenoside F1 and irradiated by UVB as described above. The efficiency of transfection of each sample was determined with a chemiluminescence assay for β-galactosidase activity using a commercial kit (Galactolight Plus, Tropics), and the values were subsequently used to equalize the values obtained from luciferase assays. Assays of luciferase activity were carried out using a commercially available kit (Promega, Madison, WI) and a Turner luminometer. In order to understand the effects of ginsenoside F1 on HaCaT cells, we evaluated a possible alteration of vital parameters. Increasing doses of ginsenoside F1 (1–10 μm) did not cause HaCaT cell cytotoxicity as shown by Trypan blue dye exclusion and MTT (Table I). A concentration of 50 μm of ginsenoside F1 displayed a little cytotoxic effect in HaCaT keratinocytes. In order to study the effects of ginsenoside F1 in UVB-induced HaCaT cell apoptosis, we used different doses of ginsenoside F1 in HaCaT cell cultures before the irradiation with different doses of UVB. As expected, UVB radiation drastically reduced viability of HaCaT cells in a dose-dependent manner. On the other hand, we observed a significant increase of viable cells in ginsenoside-F1-treated cells compared with untreated cells (Figure 2a). Indeed, the treatment with 5 mM ginsenoside F1 led to maximal protective effects against UVB-induced cell death. At a UVB dose of 120 mJ per cm2, however, ginsenoside-F1-treated cells showed a higher susceptibility to apoptosis than did untreated cells. UVB irradiation induced distinct morphologic alterations in HaCaT cells, which became round and detached from the plate (Figure 2b, c), whereas the treatment with ginsenoside F1 prevented this alteration, as shown by the morphology of the cells (Figure 2b, d). To further investigate the protective effect of ginsenoside F1 against UVB-induced apoptotic cell death, TUNEL staining and DNA fragmentation assay were performed at 24 h after UVB irradiation using the ginsenoside-F1-treated or untreated cells. As shown in Figure 3, apoptotic nuclei and DNA strand breakages were clearly observed in untreated HaCaT cells at 24 h following 60 mJ per cm2 UVB irradiation. The DNA cleavage, however, was reduced in ginsenoside-F1-treated cells. During the UVB-induced apoptosis, caspase-3 is activated and cleaves several other apoptotic proteins such as PARP. We also examined apoptotic cleavage of the caspase-3 substrate PARP to its 85 kDa form by immunoblot analysis following 60 mJ per cm2 UVB irradiation of HaCaT cells. Cleavage of PARP, as shown by the 85 kDa band, was observed at 24 h post-irradiation in untreated cells (Figure 4). In contrast, ginsenoside F1 markedly diminished the generation of the 85 kDa PARP fragment in HaCaT cells. Taken together, these results demonstrate that ginsenoside F1 exerts an effective protection from UVB-induced apoptosis in HaCaT cells. In search of the molecular mechanism(s) responsible for the protective effects, we examined the mRNA expression and the relative amounts of the two proteins Bax and Bcl-2 in UVB-irradiated HaCaT cells with or without treatment with ginsenoside F1. Figure 5 shows the effects of ginsenoside F1 or UVB irradiation on Bcl-2 and Bax mRNA expression. GAPDH is used as a housekeeper gene to normalize the Bcl-2 and Bax genes. No difference in Bcl-2 mRNA expression was detected between ginsenoside-F1-treated and untreated cells in the absence of UVB irradiation. At 12 h after UVB irradiation, Bcl-2 mRNA expression levels were almost abolished in untreated HaCaT cells (Figure 5a). On the other hand, Bcl-2 levels in ginsenoside-F1-treated cells were not downregulated by UVB irradiation (Figure 5b), compared with those in unirradiated cells. The expression of Bax mRNA was not significantly changed after UVB irradiation with or without treatment with ginsenoside F1 (Figure 5c, d). The pattern of protein expression of Bcl-2 and Bax was similar to that of their mRNA expression (Figure 6). Although it is widely accepted that UVB irradiation decreases the level of Bcl-2 expression, the exact mechanism by which UVB downregulates the expression of Bcl-2 remains unclear. To understand the molecular mechanism for the inhibitory effect of ginsenoside F1 on UVB-induced downregulation of Bcl-2, we examined the expression patterns of various transcription factors that had been shown to regulate transcription of Bcl-2. c-myb has been shown to induce Bcl-2 promoter activity in murine T cells (Taylor et al., 1996Taylor D.P. Badiani P. Weston K. A domain interfering Myb mutant causes apoptosis in T cells.Genes Dev. 1996; 10: 2732-2744Crossref PubMed Scopus (161) Google Scholar) and Rb has been reported to activate transcription of Bcl-2 in epithelial cells by interacting with the activator protein 2 transcription factor (Decary et al., 2002Decary S. Julien T.D. Ogryzko V. Reed J.C. Naguibneva I. Bellan A.H. Cremisi C.E. The retinoblastoma protein binds the promoter of the survival gene bcl-2 and regulates its transcription in epithelial cells through transcription factor AP-2.Mol Cell Biol. 2002; 22: 7877-7888Crossref PubMed Scopus (54) Google Scholar). Brn-3a interacts with the P2 promoter of Bcl-2 gene and activates endogenous Bcl-2 expression in neuronal cells (Smith et al., 1998Smith M.D. Ensor E.A. Coffin R.S. Boxer L.M. Latchman D.S. Bcl-2 transcription from the proximal P2 promoter is activated in neuronal cells by the Brn-3a POU family transcription factor.J Biol Chem. 1998; 273: 16715-16722Crossref PubMed Scopus (107) Google Scholar). At 24 h after UVB radiation, protein extracts from the ginsenoside-F1-treated or untreated HaCaT cells were subjected to immunoblot with antibodies raised to c-myb, Rb, and Brn-3a. No difference in c-myb or Rb protein expression was detected between ginsenoside-F1-treated and untreated cells following UVB irradiation (Figure 7). The level of Brn-3a protein expression was reduced by UVB irradiation in HaCaT cells. Interestingly, the UVB-induced reduction of Brn-3a expression was dramatically restored upon treatment with ginsenoside F1 (Figure 7), suggesting that Brn-3a transcription factor may be involved in the antiapoptotic effect of ginsenoside F1 through the induction of Bcl-2. To establish a correlation between Brn-3a and Bcl-2 expression in UVB-irradiated HaCaT cells, we performed transient transfection assays. Bcl-2 P2 luciferase reporter construct contains the 738 bp of regulatory sequence immediately upstream of the Bcl-2 P2 promoter region (spanning –746 to –8 bp), which has been shown to be responsive to Brn-3a transcription factor (Smith et al., 1998Smith M.D. Ensor E.A. Coffin R.S. Boxer L.M. Latchman D.S. Bcl-2 transcription from the proximal P2 promoter is activated in neuronal cells by the Brn-3a POU family transcription factor.J Biol Chem. 1998; 273: 16715-16722Crossref PubMed Scopus (107) Google Scholar). HaCaT cells were transfected with the Bcl-2 P2 luciferase reporter constructs following the treatment with ginsenoside F1 and UVB irradiation. Whereas UVB irradiation resulted in a 4-fold decrease in luciferase activity in untreated HaCaT cells, no change was observed in luciferase activity in ginsenoside-F1-treated cells, compared with unirradiated cells (Figure 8). To determine if the overexpression of Brn-3a overcomes the repression of luciferase activity under Bcl-2 promoter control caused by UVB irradiation, HaCaT cells were cotransfected with the Bcl-2 P2 luciferase reporter construct and the pBrn-3a expression vector or empty expression vector followed by exposure to UVB. Cotransfection of HaCaT cells with the Brn-3a expression vector restored the decrease of luciferase activity caused by UVB irradiation (Figure 8). These data suggest that UVB irradiation may downregulate Bcl-2 expression via a downregulation of Brn-3a transcription factor in HaCaT cells, and ginsenoside F1 may overcome the repression of Bcl-2 promoter activity via upregulation of Brn-3a expression in UVB-irradiated cells. Ginseng is a highly valued herb in the Far East and has gained popularity in the West during the last decade. The major active components of ginseng are ginsenosides, a diverse group of steroidal saponins, which demonstrate the ability to target a myriad of tissues, producing an array of pharmacologic responses (Gillis, 1997Gillis C.N. Panax ginseng pharmacolog: A nitric oxide link? Biochem Pharmacol.. 1997; 54: 1-8Google Scholar). Many mechanisms of ginsenoside activities still remain unknown, however. In this study we first demonstrate that ginsenoside F1 protects human HaCaT cells from UVB-induced damage in vitro. In particular, ginsenoside F1 effectively reduced cell death and apoptotic DNA cleavage after UVB radiation. In addition, we show that Bcl-2 mRNA expression was not affected by UVB in ginsenoside-F1-treated HaCaT cells, whereas it was markedly downregulated in untreated cells. Thus, it is conceivable that ginsenoside F1 protects human HaCaT cells from UVB-induced apoptosis by maintaining constant levels of Bcl-2. Furthermore, this positive regulation of Bcl-2 might be mediated by Brn-3a transcription factors (Pit-1, Oct-1 and Oct-2, unc-86) binding domain (POU) domain transcription factor in ginsenoside-F1-treated HaCaT cells. Brn-3a, a member of the Brn-3 family of type IV POU domain transcription factors, protects sensory neurons from apoptotic programmed cell death (Smith et al., 1997Smith M.D. Dawson S.J. Latchman D.S. The Brn-3a transcription factor induces neuronal process outgrowth and the coordinate expression of genes encoding synaptic proteins.Mol Cell Biol. 1997; 17: 345-354Crossref PubMed Scopus (62) Google Scholar). The antiapoptotic effect of Brn-3a is mediated via the upregulation of Bcl-2 expression. Brn-3a interacts with the P2 promoter of Bcl-2 gene and activates endogenous Bcl-2 expression in neuronal cells (Smith et al., 1998Smith M.D. Ensor E.A. Coffin R.S. Boxer L.M. Latchman D.S. Bcl-2 transcription from the proximal P2 promoter is activated in neuronal cells by the Brn-3a POU family transcription factor.J Biol Chem. 1998; 273: 16715-16722Crossref PubMed Scopus (107) Google Scholar). Our experiments showed that UVB irradiation caused a decrease in Bcl-2 and Brn-3a expression in HaCaT cells, but not in ginsenoside-F1-treated cells (Figure 6, Figure 7). Furthermore, overexpression of Brn-3a overcame the repression of Bcl-2 promoter activity caused by UVB irradiation (Figure 8). These data suggest that the positive regulation of Bcl-2 gene by Brn-3a provides a possible pathway by which ginsenoside F1 is able to confer HaCaT cells with resistance to UVB-induced apoptosis. Further experiments are needed to precisely define the molecular mechanism by which ginsenoside F1 affects the expression of Bcl-2. We also have to test whether HaCaT cells are resistant to UVB-induced apoptosis when the cells are stably transfected with Brn-3a. UVB radiation activates caspase-3, which cleaves several other apoptotic proteins such as PARP (Mellroy et al., 1999Mellroy D. Sakahira H. Talanian R.V. Nagata S. Involvement of caspase 3 activated Dnase in internucleosomal DNA cleavage induced by diverse apoptotic stimuli.Oncogene. 1999; 18: 4401-4408Crossref PubMed Scopus (105) Google Scholar). During the apoptosis, PARP is cleaved from the 116 kDa intact form into the 85 kDa form. Recently, it has been reported that Bcl-2 can protect cells from apoptosis by acting at a point downstream from release of mitochondrial cytochrome c, thereby preventing a caspase-3-dependent proteolytic cascade (Swanton et al., 1999Swanton E. Savory P. Cosulich S. Clarke P. Woodman P. Bcl-2 regulates a caspase-3/caspase-2 apoptotic cascade in cytosolic extracts.Oncogene. 1999; 18: 1781-1787Crossref PubMed Scopus (106) Google Scholar). In our study, treatment of ginsenoside F1 inhibited UV-induced PARP cleavage in HaCaT cells, suggesting that this molecule may prevent caspase activation through a Bcl-2-mediated antiapoptotic pathway. The death-suppressing activity of Bcl-2 is regulated by Bax, which promotes cell death. The ratio of these two proteins is considered to be important when determining whether the cell undergoes apoptosis after an appropriate stimulus (Yang and Korsmeyer, 1996Yang E. Korsmeyer S.J. Molecular thanatopsis: A discourse on the BCL-2 family and cell death.Blood. 1996; 88: 386-401Crossref PubMed Google Scholar). Previously, it has been shown that Bcl-2 expression is reduced after UVB irradiation in rat and mouse skin (Gillardon et al., 1994Gillardon F. Eschenfelder C. Uhlmann E. Harschu W. Zimermann M. Differential regulation of c-fos, fos B, c-jun, jun B and bax expression in rat skin following single or chronic ultraviolet irradiation and in vivo modulation by anti-sense oligonucleotide superfusion.Oncogene. 1994; 9: 3219-3225PubMed Google Scholar;Li et al., 1996Li G. Mitchell D.L. Ho V.C. Reed J.C. Tron V.A. Decreased DNA repair but normal apoptosis in ultraviolet-irradiated skin of p53-transgenic mice.Am J Pathol. 1996; 148: 1113-1123PubMed Google Scholar), but, in contrast, the expression of Bax is not changed (Gillardon et al., 1994Gillardon F. Eschenfelder C. Uhlmann E. Harschu W. Zimermann M. Differential regulation of c-fos, fos B, c-jun, jun B and bax expression in rat skin following single or chronic ultraviolet irradiation and in vivo modulation by anti-sense oligonucleotide superfusion.Oncogene. 1994; 9: 3219-3225PubMed Google Scholar). Our experiments also showed that neither UVB nor ginsenoside F1 had any influence on Bax mRNA expression. In conclusion, these data demonstrate that ginsenoside F1 protects HaCaT keratinocytes from UVB-induced apoptosis, at least in part through the Brn-3a-mediated transcriptional regulation of Bcl-2. Thus, these results suggest a new rationale for use of ginsenoside F1 to prevent UVB-induced skin damage.