Abstract
Ginseng has been used worldwidely as a traditional medicine of Asian countries for treatment of various diseases including cancer. The purpose of this study was to determine the effect of ginseng saponin mRg2, a mixture of ginsenosides containing 60% Rg2, on the repair and apoptosis of ultraviolet B (UVB)-exposed NIH3T3 cells. When cells were exposed to UVB and then incubated with normal growth medium for 48 h, cell viability, as determined by trypan blue exclusion assay decreased to about 25%. However, when mRg2 was included in the postincubation medium, the UVB-induced loss of cell viability was significantly reduced as compared with that postincubated in normal growth medium. 4,6-diamidino-2-phenylindole (DAPI) staining showed that postincubation of the UVB-exposed cells in medium containing mRg2 significantly reduced the apoptotic nuclear fragmentation. Interestingly, when cells were preincubated with mRg2 for 24 h and then exposed to various doses of UV, the amount of repair synthesis significantly increased as compared with those in cells exposed to UVB alone. Western blot analysis indicated that the mRg2 postincubation after UVB exposure potentiated the level of p53 and p21. The level of Triton nonextractable proliferating cell nuclear antigen (PCNA) also remained elevated by mRg2 postincubation. All these results suggest that mRg2 protects cells against UVB-induced genotoxicity by increasing DNA repair and decreasing apoptosis, in possible association with the modulation of protein levels involved in cell cycle arrest or progression.
Ultraviolet B (UVB) (280 to 320 nm) exposure results in the formation of DNA damages that cause premature skin aging (K-Paiz et al. 2004; Fisher et al. 1997). Most of UVB-induced DNA damages, such as cyclobutane pyrimidine dimers and pyrimidine (6–4) pyrimidine photoproducts, are known to be processed by nucleotide excision repair system mainly consisting of incision/excision of DNA damages by endo/exonucleases, repair synthesis by polymerase with the aid of proliferating cell nuclear antigen (PCNA), and ligation by DNA ligase (Courdavault et al. 2005; Sancar and Sancar 1998). On the other hand, high level of unrepaired DNA damages induced by UVB leads to apoptotic cell death, characterized by the fragmentation of nuclei (D’Errico et al. 2006; Takahashi, Kinouchi, and Iizuka 1997).
DNA damage-induced DNA repair and apoptosis is often accompanied by the p53 response, the stabilization of p53, and transactivation of its downstream effectors including p21, GADD45, mdm2, etc. (Kastan et al. 1991; Fotedar, Bendjennat, and Fotedar 2004). Once activated, p21/waf1/cip1 protein binds to cyclin/CDK (cyclin-dependent protein kinase)complexes and inhibits their kinase activity (El-Deiry 1998; Matsumura and Ananthaswamy 2004). Because CDK activity is required for various cell cycle transitions, p21 is a potent cell cycle inhibitor. p21 also can inhibit the ability of PCNA, which is required for either the initiation of chromosomal DNA replication or DNA repair, to activate DNA polymerase d for viral DNA replication in vitro (Szuts et al. 2005; Waga et al. 1994).
Ginseng (the root of Panax ginseng C. A. Meyer) extracts have been reported to decrease the incidence and proliferation of tumors induced by some carcinogens (Yun and Choi 1990). Human hepatoma SK-HEP-1 cells are known to undergo apoptosis by ginsenosides Rg5, Rs3 and Rs4 via up-regulating p21 and down-regulating cyclin E–dependent protein kinase CDK2 (Lee et al. 1997; Kim et al. 1999). In addition to its antitumor activities, various other effects of ginseng were reported, including in vivo radioprotective effect, protective effect of ginseno-side Rg1 on dopamine-induced apoptosis, protection against free radical–induced hemolysis, and effect on neuronal nicotinic acetylcholine receptors (Lee et al. 2006; Chen et al. 2001; Liu et al. 2002; Sala et al. 2002).
Our previous study elucidated that the ginseng saponin, especially PT saponin, potentiates the alkylating agent–induced p53 responses, which contribute to the apoptosis of NIH3T3 cells (Hwang et al. 2002). On the other hand, our earlier studies suggested that the water extracts of ginseng increase the UV-induced DNA repair synthesis (Kim et al. 1998). We have screened more than 25 ginsenosides by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay and found that Rg2 (95% purity), mRg2, and a mixture of ginsenosides containing Rg2, Rg3, Re, and Rc (60%, 30%, 6%, and 4% [w/w], respectively) effectively protects against the UVB-induced loss of cell viability (data not shown). In the present study, the effect of mRg2 on the UVB-induced DNA repair synthesis and apoptosis in NIH3T3 cells were studied in association with the levels of p53, p21, and PCNA.
MATERIALS AND METHODS
Saponins (>95% purity) and mRg2 were generously provided by Korean Ginseng and Tobacco Research Institute (Taejon, South Korea) and stock solutions of saponins were dissolved in DMSO and stored at –20°C. Trypan blue, NaF, and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (Madrid, Spain). Dulbecco’s modified Eagle’s medium, fetal bovine serum, and trypsin was from Gibco (Paisley, UK). All other agents were either from Sigma-Aldrich or from Merck (Darmstadt, Germany).
Cell Culture and Treatment
NIH3T3 mouse fibroblast cells obtained from the Korean Cell Line Bank were grown in charged Petri dishes in a humidified atmosphere with 5% CO2 at 37°C. The culture medium was Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml). The pH of the medium was adjusted to 7.2 to 7.4 with 10 mM HEPES buffer and 0.37% sodium bicarbonate. The cells were maintained in exponential phase by subculture using 0.025% trypsin-EDTA. Cells were washed twice with phosphate-bufferd saline (PBS; 150 mM NaCl, 3 mM KCl, 12mM Na2HPO4, 2 mM KH2PO4) and exposed to different doses of UVB before incubation for various time periods in medium containing mRg2.
Trypan Blue Exclusion Assay
Cell viability was assessed by trypan blue exclusion assay. Briefly, the cells exposed to 200 J/m2 UVB and postincubated for 24 h in normal medium or medium containing various concentrations of mRg2 were washed with PBS, trypsinized for 1 min, and then neutralized by the addition of fetal bovine serum. After centrifuge (1200 rpm, 6 min), the cells were resuspended in 200 μl PBS and then added with an equal volume of 0.8% trypan blue in PBS. The percentage of blue cells/total cells was counted by scoring 250 cells, thrice per experimental group, using a hemocytometer.
DAPI Staining
The fluorescent dye DAPI was used to evaluate the nuclear fragmentation, a characteristic of apoptotic cells. Cells were exposed to 200 J/m2 UVB and then incubated for 48 h with growth medium or media containing various concentrations of mRg2. At the end of treatment, the cells were fixed with methanol and incubated with 1 μg/ml DAPI (Sigma) PBS for 30 min. Apoptotic cells were determined by evaluating the nuclear morphology using a Zeiss Axioskop 2 Plus fluorescence microscope.
Unscheduled DNA Synthesis (UDS)
DNA repair synthesis was measured by UDS according to Butterworth et al. (1987). Briefly, cells were seeded onto cover slips, and after reaching confluency, the cells were incubated in medium containing 0.1% FBS and various concentrations of mRg2 for 24 h. After washing twice with PBS, the cells were exposed to UVB (20 to 160 J/m2) and then incubated for 2 h in medium containing 0.1% FBS, the appropriate concentrations of mRg2 and 10 μCi [3H]thymidine/ml for labeling during repair synthesis. After fixation of the cells and autoradiography, the grains over the nucleus were counted.
Western Blotting
Cellular lysates were prepared according to the method of Laemmli (1970). Hot Laemmli lysis buffer supplemented with 50 mM NaF was directly added to cells, and after centrifugation, the samples were boiled for 5 min. A 50-μg sample of each lysate was subjected to electrophoresis on 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE). The samples were then electroblotted on PVDF or nitrocellulose membranes. After blocking, blots were incubated with appropriate mouse anti-p53, anti-p21 polyclonal antibody (Santa Cruz), or anti-PCNA antibody (Calbiochem) in PBST (PBS containing 0.05% Tween 20) for 90 min, followed by washing three times (15 min each) with PBST, and then incubated with horseradish peroxidase–conjugated anti-mouse or -rabbit immunoglobulin G (IgG) antibody (Santa Cruz) for 60 min. After washing, the blots were incubated for 3 min with a Western blotting reagent ECL Plus (Amersham), and chemiluminescence was detected by exposure of the filters to enhanced chemiluminescence (ECL)-Western films for 10 s to 10 min.
Statistical Analysis
Results were compared using the Student’s t test to determine whether the groups exposed to UVB and postincubated with medium containing mRg2 were statistically significant when compared with the groups exposed to UVB and postincubated with normal growth medium. For this reason, the statistical significance of the experimental groups versus nontreated control was not shown to avoid the interpretational complexity.
RESULTS
The results of the present study show the effects of mRg2. Because the effects of mRg2 were essentially similar with those of Rg2 (95% purity, data not shown), the design of the present experiments focused to the effect of Rg2 contained in mRg2. In this context, the molar concentration, instead of weight/volumn concentration, of the Rg2 contained in mRg2 was calculated and arbitrarily defined as mRg2 concentration in the results of the present study.
mRg2 Reduces the UVB-Induced Loss of Cell Viability
The trypan blue exclusion assay demonstrated that mRg2 (50 to 200 μM) alone treatment for 48 h did not result in a significant loss of the cell viability as compared to nontreated controls (Figure 1, white bars). However, when the concentration of mRg2 exceeds 400 μM, the cell viability drastically declined. Because the latter data, together with our morphological data (not shown), might represent the detergent-like effect of saponin, we have chosen mRg2 concentrations at below 400 μM for the next series of experiments.
The effect of mRg2 postincubation on the viability of cells exposed to 200 J/m2 UVB was studied (Figure 1, gray bars). When cells were exposed to UVB and then incubated with normal growth medium for 48 h, the cell viability decreased to about 20%. Interestingly, when mRg2 (50 to 200 μM) was included in the postincubation medium, the UVB-induced loss of cell viability was significantly reduced as compared with that postin-cubated in normal growth medium. Essentially similar results were observed by MTT assay (data not shown). As the mRg2 concentrations increase to higher than 400 μM, however, the protective effect of mRg2 against UVB-induced viability loss became less significant.
mRg2 Reduces the UVB-Induced Apoptotic Nuclear Fragmentation
The effect of mRg2 postincubation on the UVB-induced apoptotic nuclear fragmentation was studied by using DAPI staining (Figures 2 and 3). When cells were exposed to UVB (200 J/m2) and then postincubated in growth medium for 48 h, about 90% of the cells showed typical apoptotic nuclear fragmentation (Figures 2g and 3). In contrast, postincubation of the UVB-exposed cells in medium containing mRg2 (50 to 200 μM) for 48 h significantly reduced the apoptotic nuclear fragmentation (Figures 2h –k, and 3). Similar protective effect of mRg2 against UVB-induced apoptosis was also demonstrated in the internucleosomal DNA fragmentation analyses (data not shown). When the concentration of mRg2 increased to 400 μM, however, the protective effect was not demonstrated, possibly due to the increased toxicity in mRg2 alone treated groups (Figure 3, white bars).
mRg2 Increases the UVB-Induced Repair Synthesis
Figure 4A shows that the amount of unscheduled DNA synthesis (UDS) increases in a UVB dose-dependent manner (white bars), whereas the treatment of confluent NIH3T3 cells with 100 μM mRg2 for 24 h did not significantly change the amount of UDS as compared with that in nontreated control. Interestingly, when cells were preincubated with 100 μM mRg2 for 24 h and then exposed to various doses of UV, the amount of UDS (gray bars) significantly increased as compared with those in cells exposed to UVB alone (white bars). Furthermore, as shown in Figure 4B , the mRg2-induced increase of UDS in UVB-exposed cells is dependent on the concentration of mRg2 (gray bars), whereas the amounts of UDS in cells treated with various doses of mRg2 alone (white bars) are all similar to that in nontreated control. These results suggest that mRg2 potentiates the repair synthesis induced by UVB. All the above results (Figures 1 to 4) suggest that mRg2 decreases the UVB-induced apoptosis and enhances the UVB-induced DNA repair. Because the two cellular responses are related to p53 response, we then analyzed the protein levels of p53, p21, and PCNA at various time intervals following UVB exposure.
Effect of mRg2 on the Levels of p53, p21, and PCNA
Western blot analysis indicated that the p53 protein level in cells exposed to UVB and postincubated with normal growth medium gradually increased from about threefold at 1 h postincubation time to about sevenfold at 6 h, as compared with non-treated control, and then declined at 12 h (Figure 5A and B ). Interestingly, however, when the UVB-exposed cells were postincubated with 100 μM mRg2 for 12 h, the level of p53 increased to about more than 10-fold as compared to nontreated control and about 3-fold in comparison with the p53 level in UVB-exposed cells postincubated with normal medium (Figure 5A and B ). These results suggested that the mRg2 postincubation after UVB exposure potentiated both the level and the duration of p53 expression
We also analyzed the effect of mRg2 postincubation on the expression of p21 that is known to be involved in G1 arrest and apoptosis and PCNA that is known to participate in DNA replication and repair (Table 1). Similar to p53 expression, mRg2 postincubation for 3 to 12 h after UVB exposure significantly increased the level of p21 expression as compared with that postincubated in normal medium. PCNA is known to bind to chromatin after UV irradiation (Triton nonextractable), presumably due to participation in DNA repair processes (Celis and Madsen 1986; Bendjennat et al. 2003). Triton-nonextractable PCNA level in cells exposed to UVB gradually increased as a function of normal medium incubation time periods. Interestingly, mRg2 postincubation for 6 to 12 h after UVB exposure significantly increased the PCNA level as compared with that postincubated in normal medium. All these results (Figure 5 and Table 1) suggest that mRg2 potentiates the activation of p53 and p21 and increases the PCNA level subsequent to UVB exposure.
DISCUSSION
Our earlier studies suggested that the water extracts of Panaxia ginseng increase the UV-induced DNA repair synthesis (Kim et al. 1998). After screening of various ginseno-sides, we have selected Rg2, a PT-type ginsenoside, and mRg2, a mixture of ginsenosides containing 60% Rg2, showing effective protection against the UV-induced cell death (data not shown). In the present study, we have demonstrated that mRg2 increases both the DNA repair synthesis and the protein levels of p53, p21, and PCNA following UVB damage and suggested that these increases are associated with the attenuation of the apoptosis.
Our cell viability tests showed that mRg2 postincubation protects against the death of the cells exposed to UVB (Figure 1). The maximal protective effect of mRg2 is shown at concentrations 100 to 200 μM. Similar protective effect of mRg2 is demonstrated in the apoptotic nuclear fragmentation of the cells exposed to UVB (Figures 2 and 3). It is well established that the increase in the viability after DNA damage is due to either the increase in DNA repair or the decrease in the amount of DNA damaging chemicals as reported in the case of antioxidants (Lee, Lee, and Kim 1998).
Our unscheduled DNA synthesis data (Figure 4) showed that mRg2 effectively increases the DNA repair synthesis induced by UVB exposure. The possible detergent-like action of mRg2, which may lead to the overestimation of UDS value, is excluded, because the treatment of mRg2 alone does not increase the amount of UDS significantly when compared with nontreated control (Figure 4B , white bars). The mRg2-mediated increase in the UDS in UVB-exposed cells was shown to be dependent on both the UVB dose and mRg2 concentration. The antigeno-toxic effect of mRg2, shown as decreased nuclear fragmentation and increased UDS in the present study, is in good agreement with those obtained in other lines of investigations. For example, ginseng extracts were shown to reduce mutagenesis and micronuclei formation in mammalian cells after DNA damages (Rhee et al. 1991; Lee et al. 2004).
It is well established that p53 is activated upon various DNA damages such as UVB and transactivates its downstream effectors including p21 (Singh and Agarwal 2005; Islaih et al. 2006; K-Paiz et al. 2004). The present study shows that mRg2 potentiates the p53 and p21 activation after UVB exposure (Figure 5 and Table 1). The present data are in consistence with those of our previous study which demonstrated the similar up-regulation of p53 and p21 by postincubation with ginseng saponins, especially protopanaxatriol, of the cells exposed to methylmethanesulfonate, an alkylating agent (Hwang et al. 2002). The present data are also in agreement with those of Zhang and Wang (2006) demonstrating that another ginsenoside Rg1 blocks the cell cycle in the G1 phase and up-regulates the expression of p53 and p21.
When considering that the mixed components of mRg2 may contribute to the activation of responses other than p53, the mRg2-mediated up-regulation of p53 and p21 seems to be not an exclusive factor underlying the increase in UVB-induced repair (Figure 5). Interestingly, the mRg2 postincubation also increases the level of Triton-nonextractable PCNA as compared with that in cells postincubated in normal medium (Table 1). It has been demonstrated that PCNA is required for DNA excision repair (Shivji, Kenny, and Wood 1992). Because distinct populations of PCNA are known to act separately in DNA replication and DNA excision repair (Szütsl et al. 2005), our present data may suggest that the mRg2 postincubation either increase the expression or decrease the breakdown of PCNA that is involved in DNA repair after UVB damage, or alters the association of PCNA with its multiple partners such as p21 and DNA polymerase (Kelman 1990; Warbrick 2000). The present study and our unpublished data suggest that Rg2 is the active component of mRg2 showing the p53 response and enhanced DNA repair, although the presence of the other components of mRg2, such as Rg3, Re, and Rc does not seem to counteract the action of Rg2. In addition to the latter point, it remains to be clarified whether the possible formation of Rg2–glucocorticoid receptor complex, as reported in the case of Rg1 (Lee et al. 1997), activates p53 response and contributes to the enhanced DNA repair.
Footnotes
Figures and Table
This study was supported by Somang Cosmetics, Seoul, Korea, and in part by KOSEF (Chonbuk 0101). Se Jin Jeong acknowledges support by the Korea Research Foundation grant (KRF-2004-050-C00015).