Dual efficacy of silibinin in protecting or enhancing ultraviolet B radiation-caused apoptosis in HaCaT human immortalized keratinocytes
Sivanandhan Dhanalakshmi1,
G. U. Mallikarjuna1,
Rana P. Singh1 and
Rajesh Agarwal1,2,3
1 Department of Pharmaceutical Sciences, School of Pharmacy and 2 University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, CO 80262, USA
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Abstract
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An increasing incidence of human skin cancer and other adverse effects of solar ultraviolet (UV) radiation enhance the need for novel chemoprevention strategies. Here, we have studied the effect of silibinin on UVB-induced apoptosis in HaCaT cells. Silibinin strongly prevented lower doses (15 and 30 mJ/cm2) of UVB-induced apoptosis, as observed by a reversal in UVB-caused poly(ADP-ribose) polymerase (PARP) cleavage, caspase 9 activation and an increase in apoptotic cells. UVB-induced PARP cleavage was also abolished by all caspase inhibitor, suggesting that it is a caspase-dependent effect. In other studies, silibinin restored UVB-caused depletion of a protein inhibitor of apoptosis, survivin, concomitant with up-regulation of transcription factor nuclear factor
B DNA binding activity, without any noticeable effect on UVB-caused activated protein-1 activation. Further, silibinin treatment up-regulated UVB-induced extracellular signal regulated kinase 1/2 phosphorylation, suggesting a possible role as a survival event in the protective effect of silibinin. In other studies, silibinin caused a moderate increase in phospho-Bcl-2, without any noticeable changes in total Bcl-2 levels, and down-regulated bax levels moderately. Silibinin also caused a strong decrease in Bad heterodimerization with Bclx(L), which was consistent with an increased translocation of Bclx(L) to the mitochondria from the cytosol. Consistent with its protective effect on UVB-caused apoptosis, silibinin also increased S phase arrest, possibly providing a prolonged time for efficient DNA repair. Interestingly, the protective effects of silibinin in HaCaT cells were lost at a higher dose of UVB (120 mJ/cm2) and instead it further enhanced UVB-caused apoptosis together with a strong decrease in UVB-caused activated protein-1 activation. Together, these results clearly demonstrate the dual efficacy of silibinin in protecting or enhancing UVB-caused apoptosis in the same cellular system and suggest that silibinin possibly works as a UVB damage sensor to exert its biological action.
Abbreviations: AP-1, activated protein-1; DMSO, dimethyl sulfoxide; EMSA, electrophoretic mobility shift assay; Erk1/2, extracellular signal regulated kinase 1/2; NF-
B, nuclear factor
B; NMSC, non-melanoma skin cancer; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PI, propidium iodide; UV, ultraviolet
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Introduction
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Chronic exposure to solar ultraviolet (UV) radiation is the major cause of non-melanoma skin cancer (NMSC), which is the most frequently diagnosed malignancy in Caucasians around the world (1,2). The UV radiation reaching the Earth's surface comprises UVA (320400 nm) and UVB (280320 nm), the latter being crucial in inducing chronic skin damage and cutaneous malignancy and suppressing the immune system (36). UVB also activates various signal transduction pathways and induces the expression of several specific genes (79). UVB causes tumor initiation by inducing chromosomal alterations and DNA damage and its promoting activity includes transcriptional modulation of genes involved in tumor promotion (1014). One of the characteristics of UVB-caused DNA damage is the formation of cyclobutane pyrimidine dimers and (64) photoproducts. Further indirect DNA damage is also caused by increases in the level of reactive oxygen species that facilitate DNA oxidation (15). UVB causes photodamage in cells that can be subdivided into acute and chronic photodamage (16). Whereas lower doses of UVB cause DNA mutation leading to tumor initiation, high doses result in irreparable DNA damage causing apoptosis (sunburn) and eventually cell deletion; formation of sunburnt cells is linked to the severity of UVB-induced DNA damage (17).
The above summarized adverse effects of UVB clearly highlight the need for the development and implementation of novel prevention approaches. Chemoprevention of UVB-caused skin damage and skin cancer is one such approach that involves the use of natural or synthetic agents (1822). Such agents that can prevent UVB-induced DNA damage and/or other biological events occurring following UVB exposure of the skin could be of potential importance in preventing UVB-induced skin damage as well as NMSC (2326). Several recent studies by others and us have shown that silymarin and silibinin (the major active compound in silymarin), derived from milk thistle (Silybum marianum) extract, are highly effective against photo-induced as well as chemical carcinogenesis and are also effective against other epithelial cancers (27). The antioxidant properties of these agents, such as scavenging free radicals and up-regulation of the antioxidant defense system, are also well documented (28,29).
Although the role of nuclear factor
B (NF-
B) in cell survival and the tumorigenic response has been studied in great detail, NF-
B has also been shown to be a major player in the regulation of epidermal homeostasis and in the regulation of various normal physiological processes (30). Similar to NF-
B, the activated protein-1 (AP-1) family of transcription factors are also involved in normal development of keratinocytes (31). Hydroxyeicosatrienoic acid, an anti-proliferative compound, has been shown to inhibit skin hyperplasia by up-regulation of AP-1 (32). Further, up-regulation of AP-1 and extracellular signal regulated kinase 1/2 (Erk1/2) activities has been reported in normal human skin as part of the wound healing response (33). Further to these molecular events, Bcl-2 family proteins are known to play a crucial role in the modulation of apoptosis in response to different death stimuli (34). Cell cycle regulation is another event that is required for cell proliferation and in the maintenance of homeostasis in response to cellular stress (35).
Taken together, these anti-apoptotic and mitogenic signaling events, as well as effects on cell cycle progression, are potential mechanisms of UVB-induced photodamage and apoptosis, suggesting that they could also be potential molecular targets for chemoprevention. Accordingly, in the present study, employing HaCaT human immortalized keratinocyte cells, we have assessed the effect of silibinin on UVB-caused apoptosis and various crucial survival responses. HaCaT cells have non-functional p53 (36) and defective NF-
B signaling (37), which make them more sensitive to UVB-induced apoptosis. Here we report the dual efficacy of silibinin in protecting or enhancing UVB-caused apoptosis in HaCaT cells, which could be of great significance in (i) protecting normal human skin against UVB-caused cell damage and (ii) preventing carcinogenesis by simultaneously deleting initiated cells.
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Materials and methods
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Reagents
Dulbecco's modified Eagle's medium, penicillin/streptomycin and other culture materials were from Gibco BRL Life Technologies (Grand Island, NY). Fetal bovine serum was from Hyclone Laboratories (Logan, UT). Silibinin (Sigma Chemical Co., St Louis, MO) was dissolved in dimethyl sulfoxide (DMSO). Unless specified otherwise, the final concentration of DMSO in the culture medium during different treatments did not exceed 0.1% (v/v). Anti-caspases, anti-phospho-Bcl-2 (Ser70) and anti-Bad primary antibodies and peroxidase-conjugated anti-rabbit secondary antibody were from Cell Signaling Technology (Beverly, MA). Anti-Bclx(L) and poly(ADP-ribose) polymerase (PARP) antibodies were from BD Pharmingen (San Diego, CA). Anti-Bcl-2 antibody was from Upstate Biotechnologies (Lake Placid, NY). Consensus NF-
B and AP-1 specific oligonucleotides and the gel shift assay system were from Promega (Madison, WI). Caspase inhibitor was from Enzyme Systems Products (Livermore, CA). The ECL detection system and peroxidase-conjugated anti-mouse secondary antibody were from Amersham Corp. (Arlington Heights, IL). Other chemicals were obtained in their highest commercially available purity grade.
UVB irradiation
HaCaT cells were grown to near confluency. Before UVB irradiation, the medium was removed from the culture plates and the cells were washed with phosphate-buffered saline (PBS) twice and then covered with a thin layer of PBS. This was followed by UVB irradiation. Control cultures were identically processed but not irradiated. The UVB light source was a bank of four FS24T12-UVB-HO sunlamps equipped with a UVB Spectra 305 Dosimeter (Daavlin Co., Bryan, OH), which emitted
80% radiation in the range 280340 nm with a peak emission at 314 nm, as monitored with a SEL 240 photodetector, 103 filter and 1008 diffuser attached to an IL1400A Research Radiometer (International Light, Newburyport, MA). The UVB irradiation doses were also calibrated using an IL1400A radiometer.
Immunoblotting and immunoprecipitation
Following the desired treatments, cell lysates were prepared in non-denaturing lysis buffer (10 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.3 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 0.5% NP-40, 5 U/ml aprotinin) and protein concentration in the lysates was determined using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). For immunoblot analyses, 40100 µg of protein lysates per sample were denatured in 2x SDSPAGE sample buffer and subjected to SDSPAGE on 8% (for PARP), 12% (for Erk1/2 and Bcl-2 family proteins) or 16% (for caspase 9) Trisglycine gels. The separated proteins were transferred to nitrocellulose membrane followed by blocking with 5% (w/v) non-fat milk powder in TBS (10 mM Tris, 100 mM NaCl, 0.1% Tween 20) for 1 h at room temperature or overnight at 4°C. Membranes were then probed with specific primary antibodies followed by peroxidase-conjugated secondary antibody and visualized with an ECL detection system.
For immunoprecipitation studies, 200 µg of protein was immunoprecipitated overnight with anti-Bad primary antibody and A/G beads. Immunoprecipitates thus obtained were washed three times with lysis buffer and samples were boiled in 2x sample buffer for 5 min followed by centrifugation. The resulting clear supernatants were subjected to SDSPAGE on a 12% gel. The separated proteins were electrophoretically transferred to nitrocellulose membrane followed by blocking with 5% (w/v) non-fat milk powder in Tris-buffered saline for 1 h at room temperature. The membrane was then probed and visualized for Bclx(L) and Bad as detailed above.
Apoptotic cell death assay by annexin V and propidium iodide (PI) staining
For analysis of apoptotic cells, HaCaT cells were plated in 60 mm dishes and 24 h later either exposed to the desired doses of UVB or treated with silibinin (100 µM) immediately after UVB exposure. Cells were collected after 6 h UVB exposure, stained with annexin V and PI (Molecular Probes) following the manufacturer's protocol and apoptotic cells were then analyzed immediately by flow cytometry using the FACS Analysis Core Facility of the University of Colorado Cancer Center.
Preparation of nuclear and cytosolic extracts
Following the desired treatments, cells were scraped from plates with ice-cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride and 10% NP-40) and left on ice for 20 min. After vigorous vortexing for 10 s, homogenates were centrifuged at 4000 r.p.m. for 30 s. The pellet was then resuspended in buffer C (20 mM HEPES, pH 7.9, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), vigorously rocked at 4°C for 15 min, then centrifuged for 5 min at 14 000 r.p.m. The supernatant thus obtained was used to assay the DNA binding activity of NF-
B and AP-1.
Electrophoretic mobility shift assay (EMSA)
For EMSA, NF-
B or AP-1 specific oligonucleotides (3.5 pmol) were end-labeled with [
-32P]ATP (3000 Ci/mmol at 10 mCi/ml) using T4 polynucleotide kinase in 10x kinase buffer as per the manufacturer's protocol (Promega, Madison, WI). Labeled double-stranded oligo probe was separated from free [
-32P]ATP using a G-25 Sephadex column. The consensus sequences of the oligonucleotides used were: 5'-AGT TGA GGG GAC TTT CCC AGG C-3' and 3'-TCA ACT CCC CTG AAA GGG TCC G-5' for NF-
B; 5'-CGC TTG ATG AGT CAG CCG GAA-3' and 3'-GCG AAC TAC TCA GTC GGC CTT-5' for AP-1. For EMSAs, 4 or 8 µg protein (for AP-1 and NF-
B, respectively) from nuclear extracts was first incubated with 5x gel shift binding buffer [20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 50 mM TrisHCl and 0.25 mg/ml poly(dI-dC)·poly(dI-dC)] and then with 32P-end-labeled consensus oligonucleotide for 20 min at 37°C. DNAprotein complexes thus formed were resolved on 6% DNA retardation gels (Invitrogen, Gaithersburg, MD). The gels were dried and bands were visualized by autoradiography.
Preparation of mitochondrial and cytosolic fractions
After the desired treatments, mitochondrial and cytosolic lysates were prepared as described (38). Briefly, cells were lysed in S-100 buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.9 mM MgCl2, 1 mM EGTA, 1 mM EDTA and a mixture of protease inhibitors) and left on ice for 20 min. They were then homogenized using a Dounce pestle homogenizer for
4045 strokes. After a short centrifugation at 1000 g for 5 min, the supernatant was again centrifuged at 14 000 g for 30 min and the cytosolic supernatant and mitochondrial pellet were collected. The pellet was washed once with extraction buffer and then finally suspended in lysis buffer (150 mM NaCl, 50 mM TrisHCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA and protease inhibitors).
Cell cycle progression assay
For cell cycle progression studies, HaCaT cells were plated in 60 mm dishes and 24 h later either exposed to UVB (30 mJ/cm2) alone or treated with silibinin (100 µM) immediately after UVB exposure. Cells were collected after 6 h UVB exposure and
0.5 x 106 cells in 0.5 ml of saponin/PI solution (0.3% w/v saponin, 25 µg/ml PI, 0.1 mM EDTA and 10 µg/ml RNase in PBS) were incubated overnight at 4°C in the dark. Cell cycle distribution was then analyzed by flow cytometry using the FACS Analysis Core Services of the University of Colorado Cancer Center.
Statistical analysis
All experiments were repeated at least twice. The data were analyzed using the Jandel Scientific SigmaStat 2.03 softwere. For all measurements, as needed, Student's t-test was employed to assess the statistical significance of difference between control and treated groups. A statistically significant difference was considered to be present at P < 0.05.
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Results
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Effect of silibinin on UVB-induced PARP cleavage, caspase activation and apoptosis in HaCaT cells
In order to assess the effect of silibinin on UVB-induced apoptosis, HaCaT cells were either exposed to UVB alone, pre-treated with different doses of silibinin followed by UVB exposure or exposed to UVB and immediately thereafter treated with different doses of silibinin, followed by PARP cleavage analysis, a hallmark of apoptosis, by western blotting. As shown in Figure 1A, treatment of cells with 100 or 200 µM doses of silibinin immediately after UVB exposure resulted in a strong protection against UVB-induced PARP cleavage; lower doses of silibinin (1050 µM), however, were not effective in this treatment protocol. Similarly, treatment with silibinin prior to UVB exposure also showed some protection against UVB-induced apoptosis in HaCaT cells, however, the maximum effect was evident when silibinin was added immediately after UVB irradiation (data not shown). Based on these observations, we selected a dose of 100 µM silibinin immediately after UVB exposure and assessed its effect on different doses of UVB-caused PARP cleavage and other associated events. Accordingly, HaCaT cells were post-treated with silibinin (100 µM) immediately after exposure to 15, 30 and 60 mJ/cm2 doses of UVB and cells were harvested 6 h after UVB exposure. As shown in Figure 1B, UVB irradiation resulted in dose-dependent PARP cleavage, observed as disappearance of the PARP band at 116 kDa concomitant with an increasing band for cleaved PARP at
89 kDa. In the studies assessing silibinin efficacy, post-treatment protected against UVB-induced PARP cleavage, however, the maximum protective effect was evident against the lowest dose of UVB studied (15 mJ/cm2), with moderate protection against 30 mJ/cm2 UVB-caused PARP cleavage, but no effect against the higher UVB dose of 60 mJ/cm2 (Figure 1B). However, it should also be noticed that the PARP level is about twice as high and the cleaved PARP level less than half when compared with silibinin treatment alone. Therefore, it could be reasoned that silibinin provides a more than 4-fold protection against apoptosis even at 30 mJ/cm2 UVB.

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Fig. 1. Protective effect of silibinin post-treatment on UVB-induced, caspase-dependent apoptosis. HaCaT cells grown to near confluency were either exposed to the indicated dose(s) of silibinin alone or immediately after UVB (15 mJ/cm2). Cell lysates were prepared 6 h later and western blotting was carried out for PARP cleavage (A). Cells were exposed to the indicated doses of UVB alone or post-treated with silibinin (100 µM) after UVB exposure or treated only with silibinin and harvested after 6 h. Cell lysates were prepared and western blotting was carried out for PARP cleavage (B) and cleaved caspase 9 (C). To confirm the involvement of caspases, cells were exposed to UVB (30 mJ/cm2) with or without silibinin and caspase inhibitor (50 µM) pre-treatment for 2 h. Cells were harvested 6 h after UVB exposure and PARP cleavage was analyzed by western blotting (D). To quantify apoptosis, cells were stained with annexin V and PI for flow cytometric analysis of apoptotic cells (E), as described in Materials and methods. Sb, silibinin; CI, caspase inhibitor.
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In general, caspases are known to be involved in apoptosis induced by different stimuli and PARP cleavage is an ultimate effect of caspase activation. Accordingly, we next assessed the levels of cleaved caspase 9 under identical experimental conditions. Consistent with the PARP cleavage results, the level of cleaved caspase 9 increased following UVB exposure in a dose-dependent manner and a silibinin post-treatment produced a similar reversal in cleaved caspase 9 levels as observed for PARP cleavage (Figure 1C). To confirm the role of caspase activation in UVB-induced apoptosis, we next used all caspase inhibitor and assessed the level of PARP cleavage (Figure 1D). Pre-treatment of cells with caspase inhibitor completely abolished UVB (30 mJ/cm2)-induced PARP cleavage with or without silibinin post-treatment (Figure 1D). These results suggest that caspase activation plays a major role in UVB-induced apoptosis and that silibinin protects HaCaT cells from UVB-induced apoptosis at least in part via inhibition of caspase activation.
To further substantiate the results shown in Figure 1B and C, we next performed a quantitative apoptosis assay in which HaCaT cells were either sham irradiated or irradiated with 15 and 30 mJ/cm2 doses of UVB followed immediately by vehicle or an identical dose of silibinin treatment. Six hours after UVB exposure, cells were harvested, stained with annexin V and PI and analyzed by flow cytometry. As shown by the data in Figure 1E, compared with the sham control, a 15 mJ/cm2 dose of UVB resulted in
28% (P < 0.001) apoptotic cells, however, a silibinin post-treatment resulted in only
14% apoptotic cells, accounting for a significant protection (
50%, P < 0.05) against UVB-caused apoptosis. In the other studies, 30 mJ/cm2 UVB resulted in
30% apoptotic cells and a silibinin post-treatment showed
23% apoptotic cells, accounting for 23% protection (data not shown), which is again consistent with the caspase 9 and PARP cleavages results. Silibinin alone did not show any cytotoxic or apoptotic effects in these studies in HaCaT cells (Figure 1E).
Effect of UVB and silibinin on survivin, activation of NF-
B and AP-1 and Erk1/2 phosphorylation in HaCaT cells
Since silibinin inhibited UVB-induced apoptosis in HaCaT cells, we next assessed the involvement of different anti-apoptotic and mitogenic signaling molecules as plausible targets for the protective effect of silibinin. First we analyzed survivin level, which is known to inhibit apoptosis by directly binding to caspases (39). Whereas a 15 mJ/cm2 dose of UVB did not show any change, the 30 mJ/cm2 dose of UVB resulted in a modest decrease in survivin protein level. This was not only restored following silibinin post-treatment, but there was a marked increase in survivin levels compared with both sham control and UVB-exposed samples (Figure 2A). The same membrane was re-probed with an anti-ß-actin antibody as a loading control (Figure 2B).

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Fig. 2. Effect of UVB + silibinin treatment on survival and mitogenic signaling in HaCaT cells. Cells were either mock irradiated or irradiated with the indicated doses of UVB or treated with silibinin (100 µM) immediately after UVB exposure or treated with silibinin (100 µM) only. Six hours after exposure either cell lysates were prepared to analyze protein levels of survivin (A), ß-actin (B) and levels of phosphorylated and total Erk1/2 (E and F) or nuclear extracts were prepared to analyze DNA binding activity of NF- B (C) and AP-1 (D) transcription factors, as described in Materials and methods. Sb, silibinin.
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Next we assessed NF-
B activation, which is known to play a crucial role in protecting cells from various death stimuli, including DNA damage (30). As shown in Figure 2C, UVB irradiation resulted in only a modest increase, if any, in NF-
B activation, however, higher doses of UVB irradiation followed immediately by silibinin treatment caused a significant up-regulation of NF-
B activation. The AP-1 family of transcription factors is also known to play an important role in the survival response of cells (31). As shown in Figure 2D, UVB exposure resulted in a dose-dependent increase in the DNA binding activity of AP-1, which was not affected by silibinin post-treatment. MAPK/Erk1/2 is another important signaling molecule that is up-regulated in response to a variety of stresses and is also known to be essential for cell survival (33). We, therefore, also assessed Erk1/2 activation under similar UVB and silibinin treatment conditions. Irradiation of cells with 15 and 30 mJ/cm2 UVB resulted in a moderate and strong increase in Erk1/2 phosphorylation, however, silibinin post-treatment caused a further increase in the phosphorylation of Erk1/2 (Figure 2E), which was stronger with the lower dose of UV (Figure 2E). No noticeable changes were observed in total Erk1/2 protein (Figure 2F). Together, these results are consistent with the protective effect of silibinin against UVB-caused apoptosis, whereby silibinin increases the levels of an inhibitor of the apoptosis protein survivin and at the same time keeps both the NF-
B and Erk1/2AP-1 survival pathways active to protect the cells from apoptotic death by UVB exposure.
Silibinin modulates levels, interaction and translocation of Bcl-2 family members in affording protection against UVB-induced apoptosis
Bcl-2 family proteins are one of the key regulators of apoptosis; Bcl-2 is known to be anti-apoptotic and overexpression of Bcl-2 has been shown to protect cells from apoptosis (40). Phosphorylation of Bcl-2 at Ser70 leads to its dimerization with other anti-apoptotic proteins of the Bcl-2 family, which subsequently blocks the mitochondrial release of cytochrome c. Several other interactions between Bcl-2 family members also determine a pro- or anti-apoptotic cellular response. Accordingly, we next assessed whether silibinin-mediated protection against UVB-caused apoptosis also involves an alteration in the levels, interaction and/or translocation of Bcl-2 family members. We observed a moderate increase in Ser70 phosphorylation of Bcl-2 following silibinin post-treatment of UVB-irradiated cells (Figure 3A), without any noticeable changes in total Bcl-2 levels in any of the treatment groups (Figure 3B). In other studies, whereas UVB irradiation did not affect the protein level of Bax, post-treatment with silibinin down-regulated Bax protein levels moderately (Figure 3C). Membranes were re-probed with anti-ß-actin to ensure equal loading (Figure 3D). Further, as observed by binding studies, UVB exposure (30 mJ/cm2) did not alter the heterodimerization of Bad with Bclx(L) as compared with sham irradiated controls, whereas silibinin post-treatment resulted in a significant decrease (Figure 3E); immunoblotting for total Bad protein confirmed that the observed decrease in Bclx(L) interaction with Bad was not due to an overall decrease in Bad protein levels (Figure 3F). Based on these observations, we next assessed whether an alteration in heterodimerization of Bad and Bclx(L) also affects the translocation of Bclx(L) to mitochondria (Figure 3G and H). Consistent with the binding studies results, analysis of mitochondrial lysates revealed that the level of Bclx(L) was much lower in UVB-irradiated cells and increased strongly following silibinin post-treatment, as well as with silibinin alone (Figure 3H). These observations in the mitochondrial fraction correlated with cytosolic levels of Bclx(L), where an increased level of Bclx(L) was observed in unirradiated and UVB-irradiated cells, but silibinin alone or silibinin post-treatment significantly reduced this level (Figure 3G). Together, these results suggest that at least part of the protective effect of silibinin against UVB-caused apoptosis is via modulation of Bcl-2 family members.

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Fig. 3. Effect of UVB + silibinin treatment on BadBclx(L) interaction and Bclx(L) translocation. HaCaT cells were either unirradiated or exposed to 30 mJ/cm2 UVB or post-treated with silibinin (100 µM) or treated with silibinin only. Cell lysates were prepared and equal amounts of protein was resolved on a Trisglycine gel to determine phospho-Bcl-2 (A), Bcl-2 (B), Bax (C) or ß-actin (D) or protein was immunoprecipitated with anti-Bad antibody and immunoblotted for Bclx(L) (E) and equal loading was verified by immunoblotting with Bad (F). To analyze translocation of Bclx(L) from cytosol to mitochondria, after the above mentioned treatments, cytosolic (G) and mitochondrial (H) fractions were prepared and western blotting was carried out as described in Materials and methods.
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Effect of silibinin on UVB-caused cell cycle modulation
Cell cycle progression is important for maintaining homeostasis, especially when there is an insult to DNA (41,42). Since UVB radiation is known to damage DNA directly, we next assessed the effect of UVB radiation and silibinin post-treatment on cell cycle progression. HaCaT cells were exposed to 30 mJ/cm2 UVB and immediately after being treated with silibinin (100 µM). Cells were harvested after 6 h, stained with saponin and PI and analyzed for DNA content by flow cytometry. UVB exposure caused a G1 arrest (51 versus 37% in control), largely at the expense of a decrease in S phase cells (30 versus 39% in control) (Figure 4AC). Post-treatment with silibinin, however, reversed the UVB-caused G1 arrest, resulting in an increase in S phase cells (37% in UVB + silibinin versus 30% in UVB alone) (Figure 4AC). UVB exposure resulted in a slight decrease in the G2/M phase of the cell cycle as compared with the control (19 versus 23% in control) and silibinin post-treatment did not cause any further changes as compared with UVB alone. In general, an arrest in S phase of the cell cycle allows cells more time to repair damaged DNA. Since silibinin treatment resulted in an accumulation of cells in S phase, part of the protective effect of silibinin against UVB-caused apoptosis might be due to its effect on cell cycle distribution.

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Fig. 4. Effect of UVB + silibinin treatment on cell cycle distribution of HaCaT cells. Cells were grown to 5060% confluency and then either unirradiated or exposed to 30 mJ/cm2 UVB or post-treated with silibinin (100 µM) or treated with silibinin only. Six hours later, cells were harvested, stained with saponin and PI and analyzed for DNA content by flow cytometry. Numbers of cells present in the G1 (A), S (B) and G2/M (C) phases are represented as percentages. *, Significantly different from the control (P < 0.001); #, significantly different from the UVB-irradiated cells (P < 0.05). Sb, silibinin.
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Silibinin enhances apoptosis caused by higher dose UVB in HaCaT cells
Whereas the results shown in Figure 1 clearly demonstrate that silibinin strongly protects against apoptosis induced by lower doses of UVB (15 and 30 mJ/cm2), additional studies were needed to assess the effect of silibinin on apoptosis caused by higher doses of UVB. In the next set of experiments, when cells were exposed to 120 mJ/cm2 UVB, post-treatment with silibinin failed to protect the cells from UVB-induced apoptosis, but rather moderately enhanced apoptosis, as observed by PARP cleavage (Figure 5A) and quantitation of apoptotic cells (42% in UVB alone versus 51% after silibinin post-treatment) (Figure 5B). Furthermore, in contrast to the lack of an effect of silibinin on AP-1 activation caused by lower doses of UVB as a survival response (shown in Figure 2C), the AP-1 activation caused by the higher dose of UVB (120 mJ/cm2) was strongly inhibited following silibinin post-treatment (Figure 5C). Together, these results clearly demonstrate the dual efficacy of silibinin in protecting or enhancing UVB-caused apoptosis in the same cellular system and suggest that silibinin possibly works as a UVB damage sensor to exert its biological action.

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Fig. 5. Apoptotic effect of UVB + silibinin exposure on HaCaT cells with higher dose UVB. Cells were exposed to 120 mJ/cm2 UVB with or without silibinin post-treatment and then either (A) cell lysates were prepared for western blot analysis of PARP cleavage, (B) cells were stained with annexin V and PI for flow cytometric analysis of apoptotic cells or (C) nuclear lysates were prepared to analyze AP-1 DNA binding activity by EMSA as detailed in Materials and methods. Sb, silibinin.
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Discussion
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The central finding of the present study is a dual efficacy of silibinin in protecting or enhancing UVB-caused apoptotic death of HaCaT cells. These cells are non-malignant when injected into nude mice and are essentially used as normal keratinocytes. HaCaT cells harbor mutant p53 alleles, which accounts for their sensitivity to UVB-induced apoptosis (36), as stabilization of p53 is essential for up-regulation of DNA damage repair proteins or those that can arrest cell proliferation (43). Consistent with these reports, in the present study neither UVB exposure nor silibinin treatment resulted in any change in p53 protein level (data not shown), suggesting that in HaCaT cells silibinin acts in a p53-independent way in affording protection against UVB-caused damage. In this regard, UVB-caused apoptosis in these cells is caspase-dependent and expression of dominant negative caspase 9 has been shown to block UVB-induced apoptosis (44). Again, consistent with this report, we observed caspase 9 activation following UVB exposure of HaCaT cells, silibinin treatment resulted in a strong inhibition of UVB-caused caspase 9 activation, suggesting that this event is one of the major mechanisms behind silibinin efficacy.
Other important anti-apoptotic and survival factors include NF-
B and AP-1 family members. Consistent with the previous reports (37), UVB exposure failed to activate NF-
B in HaCaT cells at the doses used in the present study, however, silibinin post-treatment after UVB exposure resulted in a significant up-regulation of NF-
B DNA binding activity. In keratinocytes, NF-
B is known to activate p21, which is involved in growth arrest (30). L-Ascorbic acid, a known scavenger of free radicals, has been shown to activate NF-
B synergistically in HaCaT cells as a photoprotective response to UVA (31). It has been reported that IKK is mutated in these cells (37) and in some cases MAPK family members have been shown to have a role in activating NF-
B (45). In our study, following exposure to UVB, AP-1 DNA binding activity was significantly elevated. Treatment with silibinin significantly up-regulated NF-
B DNA binding activity but produced no change in UVB-induced AP-1 DNA binding activity. Furthermore, silibinin treatment further enhanced UVB-caused MAPK/Erk1/2 phosphorylation. Together, these observations strengthen a possible role of Erk1/2AP-1 in NF-
B activation in UVB + silibinin treated cells as compared with UVB alone. Since up-regulation of NF-
B DNA binding activity is one of the essential events for protection of cells under various kinds of stress, the results of the present study suggest that such an event is also important in the protective effect of silibinin against UVB-induced cell damage. Survivin is another anti-apoptotic molecule that acts by binding to caspases and inhibiting their activation, and overexpression of survivin has been shown to counteract UVB-induced apoptosis even after loss of the p53 allele (46). Since silibinin up-regulated UVB-depleted survivin levels and since survivin is a NF-
B-responsive gene, there is a possibility that the observed increase in survivin is regulated by activation of NF-
B. More detailed studies, however, are needed in future to support this possibility. It is interesting to mention here that recent studies by others and us have shown that silibinin inhibits NF-
B DNA binding activity in DU145 human malignant prostate carcinoma cells and sensitized them to TNF
-induced apoptosis (47), suggesting a selective and cell type-specific effect of silibinin.
The Bcl-2 family of proteins is another important factor that decides the fate of the cell during cellular stress (34). The Bcl-2 family consists of both apoptotic and anti-apoptotic proteins and the balance between these proteins turns the cellular apoptotic machinery on and off (34). Whereas the precise mechanisms by which Bcl-2 family members act remain unclear, it has been established that they play a key role in the mitochondrial apoptotic pathway (48). Bcl-2 is an anti-apoptotic protein and Ser70 phosphorylation of Bcl-2 helps it to dimerize with other anti-apoptoic proteins of the Bcl-2 family. These dimers are known to play a role in blocking cytochrome c release from mitochondria, thereby preventing the initiation of caspase activation. Erk1/2, among other MAPK family members, is involved in Bcl-2 phosphorylation, which has been shown to be required for its anti-apoptotic function (48). Further, epidermal cell proliferation has been shown to occur in an Erk1/2-dependent pathway (49). In our study, we observed that UVB-induced Erk1/2 levels were up-regulated by silibinin treatment. Also, homo- and/or heterodimerization of anti- and pro-apoptotic proteins of the Bcl-2 family can decide the fate of the cell; whether it will follow the apoptotic or survival pathway (34). Since silibinin treatment resulted in the release of anti-apoptotic Bclx(L) from pro-apoptotic Bad, which further led to translocation of Bclx(L) to the mitochondria, this event possibly contributes to protection against mitochondrial membrane damage and subsequent cytochrome c release, followed by the observed inhibition of caspase 9 activation by silibinin as its mechanism of action against UVB-caused apoptosis. However, how silibinin treatment alone affects translocation of Bclx(L) and Bad and its significance with regards to HaCaT cell survival is not clear at present and needs further study.
Another important mechanism by which cellular homeostasis is maintained is via regulation of the cell cycle (41,42). When exposed to physiological stress or when there is an insult to DNA, cells are arrested in different stages of the cell cycle (41,42). An arrest in S phase of the cell cycle allows more time for the cells to repair DNA damage prior to proceeding through the mitotic phase (50). It is suggested that activation of the p53 pathway is essential in causing an arrest in cell proliferation in response to DNA damage (51,52). Since HaCaT cells have a non-functional p53 allele, it is possible that they are unable to undergo an S phase arrest, although a marked G1 arrest was observed. Vitamin E has been shown to protect HaCaT cells from UVB-induced growth abnormalities and to increase cell proliferation (53). Silibinin treatment caused a significant increase in the number of S phase cells even after 3 h UVB exposure (data not shown), suggesting that it allows more time for the damaged cells to repair, however, detailed studies are needed in future to further delineate the molecular mechanism involved in this action of silibinin.
In the present study, silibinin afforded strong protection against UVB-induced apoptosis at lower doses, which was completely lost at a higher dose of UVB and, in fact, an increase in apoptosis together with strong down-regulation of AP-1 DNA binding activity were observed. These findings suggest that silibinin could protect normal human skin keratinocytes from sunburn or apoptosis when the damage is moderate but that silibinin acts differently, causing apoptotic cell death, when the UVB damage is severe. This dual efficacy of silibinin might be of significance in deleting DNA damaged cells from cell cycle progression and could be of considerable importance to humans that are exposed to sunlight in their daily life, especially since the dose(s) that we used in this study falls within the physiological range of UVB exposure (54). In this regard, it is also important to mention here that other than cancer induction, skin aging and induction of erythema are other hazardous effects of UVB irradiation that occur at physiological doses of UVB exposure and which could be selectively counteracted by silibinin.
Another important issue in the present study is the fact that silibinin post-treatment was used throughout the study, which totally rules out the possibility of any sunscreen effect of silibinin and suggests that the observed molecular/signaling effects are associated with the protective effects of silibinin against UVB-caused damage. Together, these findings encourage further mechanistic and in vivo studies to develop silibinin as a chemopreventive and/or chemotherapeutic agent against UVB-caused skin damage and cancer in humans.
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Notes
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3 To whom correspondence should be addressed Email: rajesh.agarwal{at}uchsc.edu 
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Acknowledgments
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This work was supported by USPHS grant CA64514 from the National Cancer Institute, NIH.
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Received August 11, 2003;
revised September 16, 2003;
accepted September 25, 2003.