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Mol Cancer Ther. 2006;5:418-429
© 2006 American Association for Cancer Research

Enhancement of UVB radiation–mediated apoptosis by sanguinarine in HaCaT human immortalized keratinocytes

Shannon Reagan-Shaw1, Jorien Breur1 and Nihal Ahmad1,2,3

1 Department of Dermatology, 2 University of Wisconsin Comprehensive Cancer Center, and 3 Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin

Requests for reprints: Nihal Ahmad, Department of Dermatology, University of Wisconsin, 25B Medical Science Center, 1300 University Avenue, Madison, WI 53706. Phone: 608-263-5359; Fax: 608-263-5223. E-mail: nahmad{at}wisc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this article, we studied the chemopreventive effects of sanguinarine on UVB-mediated responses in human HaCaT immortalized keratinocytes. For our studies, HaCaT cells were treated with a low dose (50 nmol/L) of sanguinarine for 24 hours followed by irradiation with UVB (15 or 30 mJ/cm2). Our data showed that UVB exposure, at both doses, resulted in decreased cell viability and increased apoptosis. Interestingly, pretreatment of the cells with sanguinarine caused a significant enhancement in the antiproliferative response of UVB. These responses on UVB and/or sanguinarine treatments were associated with (a) decrease in Bcl-2 and Bcl-XL and (b) increase in Bax, Bid, and Bak protein levels. Bax knockdown and Bcl-2 overexpression resulted in a rescue of HaCaT cells from sanguinarine-mediated apoptosis. DNA cell cycle analysis revealed that UVB treatment resulted in an accumulation of cells in the G2-M phase of the cell cycle, whereas pretreatment of sanguinarine resulted in a significant shift of cells in the S phase at a low UVB dose and a further accumulation of cells in the G2-M phase at a higher UVB dose. These effects on cell cycle were accompanied with modulations in the protein levels of cyclin (B1, E, and A) and cdc2 and cyclin-dependent kinase 1. Furthermore, sanguinarine treatment was found to result in significant modulations in p53, p66Shc, MsrA, and superoxide dismutase levels. Based on our data, we suggest the sanguinarine may protect skin cells from UVB-mediated damages via apoptotic elimination of damaged cells that escape programmed cell death and therefore possess a potential of clonal expansion. [Mol Cancer Ther 2006;5(2):418–29]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
More than one million new cases of nonmelanoma skin cancers are expected to be newly diagnosed in 2005 in the United States (1). According to the "World Cancer Report," skin cancer constitutes ~30% of all newly diagnosed cancers in the world (2), and solar UV radiation, particularly its UVB component, is an established cause of about 90% of skin cancers (3). UVB radiation causes tumor initiation by DNA damage, and its promoting activity includes transcriptional modulation of genes involved in tumor promotion as well as activation of several signal transduction pathways (4, 5). UVB also indirectly damages DNA by increasing levels of reactive oxygen species (ROS), which facilitate DNA oxidation (6). Whereas low doses of UVB cause DNA mutation leading to tumor initiation, high doses result in irreparable DNA damage causing apoptosis (sunburn) and eventually cell deletion (4, 5). The available options have proven to be inadequate for the management of UV damages, including skin cancers. Therefore, there is an urgent need to develop mechanism-based novel approaches for the prevention or treatment of skin cancer. Chemoprevention by naturally occurring plant-based agents is being investigated as a potential approach for prevention as well as treatment of early treatment of several cancers, including skin cancer (7–12).

The solar UV radiation inflict damages to skin cells that results in the formation of "initiated" cells. The initiated cells may ultimately grow into tumors. The initiated cells generally divide much faster (hyperproliferation) than normal cells and, via the processes of clonal expansion and apoptosis evasion, are transformed into cancerous cells. Therefore, two types of chemopreventive agents could be useful for the management of skin cancer. First, the agents that could inhibit the damages caused by UV may prevent the formation of initiated cells. Second, the agents that could eliminate the initiated cells (with an ability to become cancerous) may reduce the risk of cancer development.

In this study, we evaluated the chemoprotective properties of sanguinarine against UVB exposure–mediated damages in skin cells. Sanguinarine (13-methyl[1,3]benzodioxolo[5,6-c]-1,3-dioxolo[4,5-i]phenanthridinium; Fig. 1A ) is derived from the root of Sanguinaria canadensis and is also found in poppy and Fumaria species (13). Sanguinarine has been shown to have antioxidant properties (14) and to exhibit antimicrobial (15) and anti-inflammatory activities (16). It is used in dental products, such as toothpaste and mouthwashes, to reduce gingival inflammation and supragingival plaque formation (17–19). We have shown previously that sanguinarine induces apoptosis in human epidermoid carcinoma cells but not in normal human epidermal keratinocytes at similar concentrations (20). We also showed that sanguinarine treatment to immortalized human HaCaT keratinocytes resulted in an induction of apoptosis via activation of Bcl-2 family proteins (21).


Figure 1
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  		Figure 1. Sanguinarine treatment significantly enhances UVB-mediated inhibition of cell growth. A, the chemical structure of sanguinarine. B, Following treatment of HaCaT keratinocytes with sanguinarine (Sang) and/or UVB, the effect on cell viability was measured using trypan blue exclusion analysis. Cell viability data is expressed as the percent viable cells out of the total number of cells. Columns, mean of three experiments; bars, SD. *, P < 0.01 for untreated versus treated; ##, P < 0.01 for UVB versus UVB + sanguinarine. C, clonal keratinocyte growth was assessed using the clonogenic assay. Details of the experiments are given in Materials and Methods.

 
In the present study, to assess the photochemopreventive potential of sanguinarine, we evaluated its effects on UVB-mediated damages in HaCaT keratinocytes in vitro. Our data suggested that sanguinarine may protect skin cells from UVB-mediated damages via apoptotic elimination of UV-damaged cells. Our findings may have implications for the management of skin cancer and other hyperproliferative skin conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Sanguinarine chloride (>99.0% pure) was purchased from Sigma Chemical Co. (St. Louis, MO). The following antibodies were used: anti-cyclin B1 and anti–cyclin-dependent kinase 2 (cdk2) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-cdc2, anti-cyclin D1, anti-cyclin D2, anti-cyclin E2, anti–phospho-p53 (Ser15), and anti–phospho-Mdm2 (Ser166) from Cell Signaling Technology (Beverly, MA); anti-cdk4 from BD PharMingen (San Jose, CA); anti-cyclin D3 and anti-Mdm2 from Abcam (Cambridge, MA); anti-cyclin A and anti-cyclin E1 from NeoMarkers (Fremont, CA); anti-Bcl-2, anti-Bax, anti-Bak, anti-Bid, anti–p66-SHC, anti–phospho p66-SHC (Tyr239), anti-Cu/Zn superoxide dismutase (SOD), anti-MnSOD, and anti-MsrA from Upstate Biotechnology (Waltham, MA); anti-Bcl-X, anti–p21-WAF1, and anti-p53 from Biosource International (Camarillo, CA); and anti-ß-actin from Sigma Chemical Co. (St. Louis, MO). The Bax short hairpin RNA and the full-length Bcl-2 plasmids were purchased from Open Biosystems (Huntsville, AL).

Cell Culture and Treatment
HaCaT cells, an immortalized, nontumorigenic human keratinocyte cell line, were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% antibiotics at standard cell culture conditions (37°C, 5% CO2 in a humidified incubator). For our studies, we employed two different experimental protocols. In the first protocol, the cells were pretreated with sanguinarine followed by UVB exposure; whereas in the second protocol, the cells were subjected to UVB irradiation first followed by sanguinarine treatment. For pretreatment studies, the cells (50–60% confluent) were treated with a low concentration of sanguinarine (50 nmol/L) for 24 hours in culture medium. The medium was then removed, and PBS was added, and the cells were exposed to UVB (15 or 30 mJ/cm2). The PBS was aspirated, culture medium was added, and the cells were incubated for another 24 hours. Alternatively, for post-treatment studies, the cells (70% confluent) were treated with UVB (15 or 30 mJ/cm2) in PBS followed immediately by 50 nmol/L sanguinarine treatment for 24 hours.

For UVB irradiation, a "Daavlin Research Irradiators" obtained from Daavlin Co. (Bryan, OH) was used. This equipment consists of a fixture mounted on fixed legs and contains four UVA and four UVB lamps. The exposure system is controlled using two Daavlin Flex Control Integrating Dosimeters. The dose units, in this equipment, could be entered as mJ/cm2 (for UVB) or Joules (for UVA). For accuracy, the machine is periodically calibrated using International Light IL 1400, digital light meter (Daavlin).

Trypan Blue Exclusion Assay
Trypan blue exclusion assay was used to assess the effect of treatments on the growth and viability of HaCaT cells. Briefly, following treatment of cells with sanguinarine and/or UVB in a six-well plate, as described above, the culture medium was collected in a 1.5-mL Eppendorf tube. The cells were trypsinized and collected in the same Eppendorf tube. The cells were pelleted by centrifugation, and the cell pellet was resuspended in 300 µL PBS (10 mmol/L, pH 7.4). Trypan blue (0.4% in PBS, 10 µL) was added to a smaller aliquot (10 µL) of cell suspension, and the number of cells (viable unstained and nonviable blue) were counted using a hemacytometer in duplicate for each sample, with the experiment repeated at least thrice.

Clonogenic Assay
The reproductive potential of treated HaCaT cells was assessed using the clonogenic assay, which is considered to be the optimal assay method for determining survival after radiation in vitro. The colony formation assay for normal keratinocytes treated with UVB has been described previously (22). Cells were treated with 50 nmol/L sanguinarine for 24 hours and were collected by trypsinization. A trypan blue assay was done, and cells were replated in triplicate on a six-well tissue culture plate with 3,000 cells per well. The cells were allowed to adhere overnight and were then exposed to UVB as described earlier. The cells were cultured for 14 days with growth media being replaced every 3 days. The cells were then stained with 0.5% crystal violet (in methanol/H2O, 1:1; Sigma), and the colonies were counted.

Apoptosis and Cell Cycle Analysis by Flow Cytometry
The extent of apoptosis and cell cycle distribution was assessed with the APO-BrdUrd TUNEL Apoptosis Assay kit (Molecular Probes, Eugene, OR) as per the manufacturer's protocol. Cells were treated as described above in a six-well plate. At 24 hours after UVB exposure, the culture medium was collected. The cells were gently trypsinized and added to the culture media and pelleted by centrifugation. The pellet was washed with PBS, the cells were counted and (1 x 106) fixed overnight in ethanol (90%). The cells were washed and labeled with UTP-bromodeoxyuridine (BrdUrd) overnight, washed again with PBS, and incubated with an Alexa 488 anti-BrdUrd antibody followed by counterstaining with propidium iodide. Cells were analyzed using a FACScan benchtop cytometer (BD Biosciences, San Jose, CA) at the Flow Cytometry Facility in the University of Wisconsin Comprehensive Cancer Center. The analyses were done using Cell Quest software (BD Biosciences) for apoptosis and ModFit LT software (Verity Software House, Topsham, ME) for cell cycle analysis, and the data are expressed as the mean of three experiments showing the same trend.

Western Blot Analysis
Following the treatment of the cells with sanguinarine and UVB in 10-cm dishes, as described above, the medium was aspirated, and the cells were washed with ice-cold PBS. Ice-cold radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, 1% NP40] with freshly added 1 mmol/L phenylmethylsulfonyl fluoride and 10 µg/mL protease inhibitors (Protease Inhibitor Cocktail Set III, Pierce, Rockford, IL) was added to the plates. The cells were then scraped, and the cell suspension was transferred into a microfuge tube on ice for 15 minutes with occasional vortexing, ensuring a complete cell lysis. The cell suspension was cleared by centrifugation at 14,000 x g for 15 minutes at 4°C, and the supernatant (total cell lysate) was either used immediately or stored at –70°C. The protein concentration was determined using the Bicinchoninic Acid Protein Assay (Bio-Rad Laboratories, Hercules, CA) as per the manufacturer's protocol.

For immunoblot analysis, 30 µg protein was subjected to SDS-PAGE using 10% to 15% Tris-HCl gel. The protein was transferred onto a nitrocellulose membrane and blocked with TBS-Tween (0.1%) plus 5% dry milk. The membrane was probed with an appropriate primary antibody followed by a secondary horseradish peroxidase–conjugated antibody. The protein levels were detected by freshly prepared chemiluminescent solution [100 mmol/L Tris-HCl (pH 8.5), 0.018% H2O2 (v/v), 1.25 mmol/L Luminol, 225 nmol/L coumaric acid]. The quantification of protein was done by a digital analyses of protein bands (TIFF images) using UN-SCAN-IT software (Silk Scientific, Orem, UT). The data are expressed as the relative density of the protein normalized to ß-actin.

Bivariate Analysis by Flow Cytometry
Following the treatment of the cells with sanguinarine and UVB in six-well plates, as described above, the culture medium was collected. The cells were gently trypsinized and added to the culture media and pelleted by centrifugation. The pellet was washed with PBS and counted, and the cells (1 x 106) were fixed overnight in ethanol (90%). Cells were washed with PBS and pelleted by centrifugation. The pellet was resuspended in 0.25% (v/v) Triton X-100 in PBS on ice and incubated for 5 minutes before washing with PBS and centrifugation. The cells were probed with antibodies diluted in 1% bovine serum albumin (in PBS) for 1 hour at room temperature. The cells were washed with 1% bovine serum albumin (in PBS) and centrifuged. The cell pellet was probed with a FITC-conjugated goat anti-mouse IgG antibody for 30 minutes. The cells were diluted in PBS and were analyzed using a FACScan benchtop cytometer (BD Biosciences) at the Flow Cytometry Facility in the University of Wisconsin Comprehensive Cancer Center. The analyses, repeated at least thrice, were done using Cell Quest software (BD Biosciences).

Transfection of Cells
The Bax short hairpin RNA and full-length Bcl-2 plasmids were grown up in Luria-Bertani broth and were purified by a Perfectprep Plasmid Mini kit (Eppendorf, Hamburg, Germany) according to the manufacturer's instructions. Following pretreatment of the cells with sanguinarine for 24 hours then UVB treatment, the cells were transfected with either of the DNA plasmids. Briefly, each plasmid (0.2 µg per 25 µL media) and the LipofectAMINE 2000 reagent (Invitrogen; 0.5 µL per 25 µL media) were diluted with serum-free media for 5 minutes. Then, the diluted LipofectAMINE was added to the diluted plasmid and incubated at room temperature for 20 minutes. The PBS was aspirated from the cells immediately following UVB treatment, and the plasmid-LipofectAMINE complex was added dropwise to the cells. The cells were incubated at 37°C for 6 hours, at which time fetal bovine serum was added to a 10% concentration, and the cells were incubated further for another 18 hours (for a total incubation of 24 hours after UVB treatment). At this time, the cells were collected and processed for further experiments.

SOD Activity
SOD activity was assessed employing Bioxytech SOD-525 kit (Oxis Research, Portland, OR) as per the manufacturer's protocol. Briefly, following the treatment of the cells with sanguinarine and UVB in six-well plates, as described above, the culture medium was collected. The cells were gently trypsinized and added to the culture media and pelleted by centrifugation. The cells were washed with PBS and repelleted by centrifugation. The cells were resuspended in ice-cold PBS and were sonicated for 30 seconds. The cell suspension was cleared by centrifugation at 14,000 x g for 20 minutes at 4°C, and the supernatant was stored at –70°C. The buffer provided in the kit was warmed, and 40 µL of sample and the solution R2 were added to a microfuge tube and vortexed briefly. The sample was incubated at 37°C for 1 minute, and another solution R1 was added and vortexed briefly. The sample was transferred to a spectrophotometric cuvette, and the absorbance was measured over time. The SOD activity is calculated directly from the rate of absorbance of the sample versus the average rate of the blank control using the provided ratio table, repeated at least thrice for each sample.

Statistical Analysis
The results are expressed as the mean ± SD. Statistical analysis of the data between the untreated versus treatments (*) and UVB-alone treatment versus sanguinarine and UVB treatment (##) were done by Student's t test. P < 0.01 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sanguinarine Enhances UVB-Mediated Inhibition of Viability and Clonogenic Survival of HaCaT Keratinocytes
Human HaCaT keratinocytes were either exposed to sanguinarine alone, UVB alone, or pretreated with sanguinarine followed by UVB exposure. Trypan blue exclusion analysis was used to assess the effect of treatments on the viability of cells. Our data showed that UVB exposure (15 or 30 mJ/cm2) resulted in a significant decrease in the viability of HaCaT cells (Fig. 1B). When cells were pretreated with 50 nmol/L sanguinarine, this loss of cell viability was significantly enhanced at both doses of UVB (Fig. 1B). Sanguinarine alone had no effect on cell viability (Fig. 1B). Interestingly, the after treatment of sanguinarine (following UVB exposure) did not alter the viability of the cells compared with UVB-alone treatments, although it was significantly different from untreated cells (Fig. 1B).

Next, we determined the effects of treatments on the ability of cells to form cellular colonies and thereby assess the reproductive potential and the long-term cell survival after treatments. As shown by the colony formation assay, sanguinarine treatment alone did not have an effect on the clonogenic survival of the cells, whereas UVB exposure resulted in an inhibitory effect on the colony formation ability of HaCaT cells (Fig. 1C). Interestingly, both sanguinarine pretreatment and post-treatment enhanced the antiproliferative effects of UVB with a complete inhibition observed with sanguinarine pretreatment at the 30 mJ/cm2 dose of UVB (Fig. 1C).

Sanguinarine Enhances UVB-Mediated Induction of Apoptosis via Modulations in Bcl-2 Family Proteins in HaCaT Keratinocytes
We determined whether the loss of cell viability observed above was mediated via the apoptotic death of HaCaT cells. For this purpose, we employed a terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) assay kit that used BrdUrd incorporation to measure apoptosis by flow cytometry. This assay is based on the principle that when DNA strands are cleaved (apoptosis), a large number of 3'-hydroxyl ends are exposed, which are detected using an Alexa Fluor 488 dye-labeled anti-BrdUrd monoclonal antibody. This kit also uses propidium iodide staining for determining total cellular DNA content. We found a significant induction of apoptosis in HaCaT cells treated with UVB alone for 24 hours (Fig. 2 ). Similar to the cell viability data, sanguinarine posttreatment did not significantly change the number of apoptotic cells compared with UVB alone (Fig. 2B). Interestingly, pretreatment of cells with sanguinarine appreciably increased the apoptotic cell population from 50% (UVB alone) to 67% at the lower dose of UVB (15 mJ/cm2; Fig. 2). However, at a 30 mJ/cm2 UVB dose, apoptosis was significantly increased from 66% (UVB alone) to 89% (sanguinarine pretreatment plus UVB; Fig. 2). At this point, sanguinarine pretreatment looked more promising and was used for further studies.


Figure 2
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  		Figure 2. Sanguinarine treatment significantly enhances UVB-mediated induction of apoptosis. Following treatment of HaCaT keratinocytes with sanguinarine (Sang) and/or UVB, the extent of apoptosis was assessed with the APO-BrdUrd TUNEL Assay kit. The fragmentation of DNA in apoptotic cells is measured by BrdUrd incorporation, which is visualized by conjugation to an Alexa Fluor 488 dye-labeled anti-BrdUrd antibody. BrdUrd incorporation was analyzed with a flow cytometer (A) followed by a computational analysis (B) of cells staining positive for BrdUrd. Columns, mean of three experiments; bars, SD. *, P < 0.01 for untreated versus treated; #, P < 0.01 for UVB versus UVB + sanguinarine. A, from representative experiment repeated thrice with similar results. Details of the experiments are given in Materials and Methods.

 
UVB radiation has been shown to activate prodeath Bcl-2 family proteins while also reducing levels of prosurvival Bcl-2 family proteins, thereby inducing apoptosis in the skin (23). Studies by others and from our laboratory have shown that sanguinarine modulates Bcl-2 family proteins in favor of apoptosis (21, 24, 25). Immunoblot analysis showed a significant increase in proapoptotic proteins Bax, Bid, and Bak with a concurrent significant decrease in antiapoptotic proteins Bcl-2 and Bcl-XL when the cells were treated with UVB (Fig. 3A and B ). The pretreatment of sanguinarine was found to further enhance the modulatory effects of UVB towards Bcl-2 family proteins in a way that favored apoptosis (Fig. 3A and B). Bcl-2 family proteins can modulate mitochondrial permeability through oxidative phosphorylation during apoptosis. Changes in the Bax/Bcl-2 ratio suggest a corresponding change in mitochondrial permeability to release apoptogenic molecules from the mitochondria to the cytosol. A bivariate analysis by flow cytometry allows reading of multiple protein levels simultaneously in the same population of cells. This analysis showed a significant increase in proapoptotic protein Bax and a significant decrease in antiapoptotic Bcl-2, thereby shifting the Bax/Bcl-2 ratio in favor of apoptosis (Fig. 3C). This shift in the Bax/Bcl-2 ratio toward apoptosis was also seen in the Western Blots, further confirming the UVB-caused apoptosis by sanguinarine (Fig. 3).


Figure 3
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  		Figure 3. Sanguinarine treatment significantly modulates Bcl-2 family proteins in favor of apoptosis. Following treatment of HaCaT keratinocytes with sanguinarine (Sang) and/or UVB, the protein levels of Bcl-2, Bax, Bcl-XL, Bid, and Bak were assessed by Western blot analysis (A) and quantified by densitometric analysis (B) of protein bands. Equal loading was confirmed by stripping the blot and reprobing it for ß-actin. Representative experiment repeated thrice with similar results. The effect of treatment on the Bax/Bcl-2 ratio (C) was determined by both densitometric analysis of the immunoblots relative to ß-actin and by a bivariate analysis by flow cytometry. Columns, mean of three experiments; bars, SD. *, P < 0.01 for untreated versus treated; ##, P < 0.01 for UVB versus UVB + sanguinarine. Following sanguinarine and UVB treatment, the cells were transfected with Bax short hairpin RNA (shRNA), and Bax protein levels were determined by Western blot analysis. The effect of treatments on apoptosis was determined by TUNEL assay (D). Following sanguinarine and UVB treatment, the cells were transfected with Bcl-2, and Bcl-2 protein levels were determined by Western blot analysis. The effect of treatments on apoptosis was determined by TUNEL assay (E). Columns, mean of three experiments; bars, SD. *, P < 0.01 for plasmid alone versus plasmid + treatment; ##, P < 0.01 for plasmid + UVB versus plasmid + UVB + sanguinarine. Details of the experiments are given in Materials and Methods.

 
To determine the cause and effect association between Bax/Bcl-2 and the observed chemopreventive response of sanguinarine, we did gene overexpression and knockdown experiments. Thus, first, we studied the effect of knockdown of Bax using vector-based short hairpin RNA and studied its effect on the observed responses of sanguinarine. Immunoblot analysis showed an efficient knockdown of Bax protein levels in the cells (Fig. 3D). TUNEL analysis confirmed that proapoptotic Bax was a crucial player in sanguinarine pretreatment–mediated apoptosis as cells were found to be rescued from apoptosis on transfection with Bax short hairpin RNA (Fig. 3D).

In the next experiment, we evaluated the effect of a forced overexpression of prosurvival Bcl-2 on the observed proapoptotic response of sanguinarine. As shown by Western Blot analysis (Fig. 3E), the transfection of cells with Bcl-2 resulted in an appreciable increase of Bcl-2 protein levels. Furthermore, our data also showed that Bcl-2 overexpression resulted in a rescue of HaCaT keratinocytes from sanguinarine-mediated apoptosis (Fig. 3E). These results clearly showed that there is a cause and effect relationship between Bax/Bcl-2 and the induction of apoptosis by sanguinarine.

Sanguinarine Enhances UVB-Mediated Cell Cycle Arrest via Modulations in Cell Cycle Regulatory Molecules in HaCaT Keratinocytes
The DNA damage response culminates in activation of cell cycle checkpoints and the appropriate DNA repair pathways or, in certain contexts, initiation of apoptotic programs. The basic purpose of cell cycle regulation is to ensure that DNA is faithfully replicated only once during S phase, and those identical copies of chromosomes are formed and distributed equally to the daughter cells during M phase. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations, leading to cell death or, in multicellular organisms, cancer. A single, high UVB exposure (200 mJ/cm2) to HaCaT keratinocytes is known to induce G2-M phase cell cycle arrest (26). In this study, the DNA cell cycle analysis revealed that UVB treatment resulted in a significant accumulation of cells in the G2-M phase of the cell cycle at 24 hours after exposure (Fig. 4 ). Treatment of sanguinarine (before UVB) resulted in a significant shift of cell accumulation in the S phase at a 15 mJ/cm2 UVB dose and a further accumulation of cells in G2-M phase at a 30 mJ/cm2 UVB dose (Fig. 4).


Figure 4
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  		Figure 4. Sanguinarine treatment significantly enhances a UVB-mediated cell cycle arrest. Following treatment of HaCaT keratinocytes with sanguinarine (Sang) and/or UVB, cell cycle distribution was assessed using the APO-BrdUrd TUNEL Assay kit. The FAC profiles (A), indicating the positions of G0-G1, S, and G2-M are shown along with computational analysis of (B) the cell population in S and G2-M phases. Columns, mean of three experiments; bars, SD. *, P < 0.01 for untreated versus treated; ##, P < 0.01 for UVB versus UVB + sanguinarine. A, from representative experiment repeated thrice with similar results. Details of the experiments are given in Materials and Methods.

 
Progression of normal cell division depends on cyclin interaction with cdks and the degradation of cyclins before chromosomal segregation through ubiquitination. The G2-M transition is regulated by the cyclin B1-cdc2 complex. Binding of cdc2 to cyclin B1 is required for its activity, and repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis. Our data showed that the protein levels of both cyclin B1 and cdc2 were significantly elevated in HaCaT cells treated with 30 mJ/cm2 UVB alone or pretreated with sanguinarine, consistent with the observed G2-M phase arrest in these cells (Fig. 5 ). In addition, protein levels of cyclin B1 and cdc2 were significantly decreased in cells pretreated with sanguinarine followed by a 15 mJ/cm2 UVB dose (Fig. 5). The cyclin A/E-cdk2 complexes play a role in driving replication, regulating the S phase, and in keeping replication to only once per cell cycle. We found that the protein levels of cyclin E1 were elevated in cells treated with 15 mJ/cm2 UVB alone or pretreated with sanguinarine and were significantly decreased in cells pretreated with sanguinarine followed by a 30 mJ/cm2 UVB dose (Fig. 5C and D). On the other hand, cyclin E2 protein levels were found to be significantly down-modulated by UVB treatment and were returned to normal levels with sanguinarine pretreatment (Fig. 5C and D). Our data also showed that the protein levels of cyclin A were significantly decreased in HaCaT cells treated with 30 mJ/cm2 UVB alone or pretreated with sanguinarine (Fig. 5C and D). Furthermore, the protein levels of cdk2 were significantly elevated in cells pretreated with sanguinarine followed by a 15 mJ/cm2 dose of UVB but were found to be significantly decreased in cells pretreated with sanguinarine followed by a 30 mJ/cm2 UVB dose (Fig. 5A and B). Our data also showed a down-regulation of D-type cyclins following UVB treatment, whereas sanguinarine pretreatment resulted in a reversal of levels of both cyclins D2 and D3 (Fig. 5C and D).


Figure 5
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  		Figure 5. Sanguinarine treatment significantly modulates cell cycle regulatory proteins. Following treatment of HaCaT keratinocytes with sanguinarine (Sang) and/or UVB, the protein levels of cdc2, cdk2, and cdk4 were assessed by Western blot analysis (A) and quantified by densitometric analysis (B) of protein bands. The protein levels of cyclins A, B1, D1, D2, D3, E1, and E2 were assessed by Western blot analysis (C) and quantified by densitometric analysis (D) of protein bands. Equal loading was confirmed by stripping the blot and reprobing it for ß-actin. Representative experiment repeated thrice with similar results. Columns, mean of three experiments; bars, SD. *, P < 0.01 for untreated versus treated; ##, P < 0.01 for UVB versus UVB + sanguinarine. Details of the experiments are given in Materials and Methods.

 
Sanguinarine Enhances UVB-Mediated Activation of p53 and p66Shc Proteins in HaCaT Keratinocytes
The p53 protein acts as a sensor of UVB damage and as an initiator of a program of expression of genes involved in growth arrest, repair, or apoptosis. Chouinard et al. showed that UVB irradiation to HaCaT keratinocytes increased expression of phospho-p53 (Ser15) protein but had no appreciable effect on the levels of total p53 protein at up to 10 hours after UVB exposure (27). Our data showed a significant increase in phospho-p53 (Ser15) protein levels at 24 hours after UVB exposure, which was further enhanced significantly when the cells were pretreated with sanguinarine (Fig. 6A and B ). Expression of total p53 protein was slightly down-modulated in response to UVB alone or sanguinarine plus UVB treatment; however, the effect was not significant (Fig. 6A and B). It is known that phosphorylation of p53 on Ser15 impairs the binding of Mdm2, which correlates with p53 accumulation following genotoxic stress (28, 29). Mdm2 protein expression was found to be significantly decreased, whereas phospho-Mdm2 levels were significantly increased with sanguinarine pretreatment (Fig. 6A and B) in HaCaT keratinocytes.


Figure 6
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  		Figure 6. Sanguinarine treatment significantly enhanced UVB-mediated activation of p53 and p66Shc and modulates an oxidative stress response. Following treatment of HaCaT keratinocytes with sanguinarine (Sang) and/or UVB, the protein levels of p53, phospho-p53, Mdm2, phospho-Mdm2, p66Shc, and phospho-p66Shc were assessed by Western blot analysis (A) and quantitated by densitometric analysis (B) of protein bands. Following treatment of HaCaT keratinocytes with sanguinarine and/or UVB, SOD activity was assessed with the Bioxytech SOD-525 kit (C). Following treatment of HaCaT keratinocytes with sanguinarine and/or UVB, the protein levels of CuZnSOD, MnSOD, and MsrA were assessed by Western blot analysis (D) and quantified by densitometric analysis (E) of protein bands. Equal loading was confirmed by stripping the blot and reprobing it for ß-actin. Columns, mean of three experiments; bars, SD. *, P < 0.01 for untreated versus treated; ##, P < 0.01 for UVB versus UVB + sanguinarine. Details of the experiments are given in Materials and Methods.

 
The p66Shc protein regulates intracellular levels of ROS and mediates ROS up-regulation during p53-induced apoptosis (30). p53 induces p66Shc protein up-regulation by increasing its stability (30). Shc proteins are phosphorylated by all receptor tyrosine kinases and, upon phosphorylation, Shc proteins form stable complexes with cellular tyrosine-phosphorylated polypeptides (31–33). A model has been proposed whereby the p53-p66Shc pathway is a sensor of the levels of intracellular oxidative signals and regulates intracellular levels of oxidants and of oxidative damage (30). Western blot analysis showed a small, but significant decrease in p66Shc protein levels after UVB treatment alone or with sanguinarine pretreatment (Fig. 6A and B). Expression of phospho-p66Shc (Tyr239) was significantly increased at 24 hours after UVB treatment, and this effect was found to be significantly enhanced with sanguinarine pretreatment (Fig. 6A and B).

Sanguinarine Treatment Modulates UVB-Mediated Oxidative Stress in HaCaT Keratinocytes
The most important enzymatic antioxidant to protect cells from UVB damage is SOD (34). Two types of SOD, copper-zinc SOD (Cu/Zn SOD) and manganese-SOD (MnSOD), have been identified in mammalian cells, and keratinocytes have been reported to contain both enzymes of SOD (35, 36). Examination of total SOD activity showed a significant dose-dependent increase in total SOD activity at 24 hours after UVB exposure, and to our surprise, sanguinarine was found to significantly decrease SOD activity only at the 30 mJ/cm2 UVB dose (Fig. 6C). Earlier studies have shown that a single UVB exposure to human keratinocytes decreased MnSOD protein levels and increased Cu/Zn SOD protein levels at 24 after UVB (37). Our data showed that UVB exposure resulted in a significant dose-dependent decrease in MnSOD and increase in CuZnSOD protein levels at 24 hours after UVB exposure in HaCaT cells (Fig. 6D and E). Interestingly and surprisingly, pretreatment with sanguinarine significantly enhanced these effects (Fig. 6D and E).

Protein-bound methionine residues are among the most susceptible to oxidation by ROS, resulting in formation of methionine sulfoxide residues. However, this modification can be repaired by methionine sulfoxide reductase (MsrA; refs. 38, 39). MsrA then has an important function in cellular metabolism as an antioxidant enzyme that scavenges ROS by facilitating the cyclic interconversion of methionine/protein-methionine residues between oxidized and reduced forms (38, 40, 41). In this study, we found that UVB exposure to HaCaT cells resulted in a significant decrease in MsrA protein levels at 24 hours after UVB, and pretreatment with sanguinarine resulted in a significant enhancement in this inhibitory effect of UVB (Fig. 6D and E).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Skin cancer is of great concern as its rates have been steadily increasing in recent years accounting for >1.3 million new cases each year; and the same trend is expected to continue in the future (42). UVB radiation from the sun is an established cause for >90% of skin cancers. Therefore, mechanism-based strategies aimed at protection from UVB exposure–mediated cellular damages, which may lead to the development of neoplasms, could be useful in the management of skin cancer. Chemoprevention seems to be a plausible strategy for protection of UVB radiation-induced damage to the skin. UVB radiation is regarded as a complete carcinogen leading to cancer initiation and promotion (4–6). Plant-based agents, often antioxidants, have been shown to inhibit many UVB-induced signal transduction pathways responsible of cancer development (43, 44). In this study, we determined the protective effect of sanguinarine, a plant alkaloid with documented anti-inflammatory and anticancer effects, against the UVB exposure–mediated damages to human HaCaT keratinocytes.

UVB radiation imparts damage to skin cells, and the cells respond to this damage in three ways: by tolerating the damage, by repairing the damage via delays in the cell cycle, or by undergoing apoptosis (a programmed cell death). The latter two responses represent defense mechanisms of the system to either repair the damage or eliminate the defective cells (containing damages). However, the unrepaired cells (with genetic abnormalities) may undergo a clonal expansion to acquire a hyperproliferative phenotype that could ultimately result in a neoplastic condition.

In this study, we found that sanguinarine enhances the ability of HaCaT cells to undergo cell cycle arrest and apoptosis as a result of UVB-caused damages. This is clearly an important observation because apoptosis is a mechanism of defense and acts by opposing the creation of a damaged (preneoplastic) cell and expansion of this cell into a clone. Once mutations arise, apoptosis also removes damaged (preneoplastic) cells that are aberrantly proliferating due to genetic defects.

Similarly, cell cycle arrest increases the time available for DNA repair before DNA replication and mutation fixation. The checks and balances governing the cell cycle prevent inappropriate DNA replication, and a breakdown in these checkpoints can lead to genomic instability and cancer. It is known that UVB exposure causes a G2-M phase cell cycle arrest in the keratinocytes, and our data verified this finding. However, we showed that sanguinarine pretreatment had a differential effect on the distribution of cells in different phases of cell cycle. Thus, sanguinarine pretreatment resulted in a significant arrest of cells in the S phase when exposed to 15 mJ/cm2 dose of UVB and in G2-M phase when exposed to 30 mJ/cm2 dose of UVB. The reason for this differential effect, however, remains unknown at present.

Our data also showed the involvement of p53, Bcl-2 family proteins, and cell cycle regulatory proteins during the chemoprotective effects of sanguinarine. The mechanism of G2-M arrest in mammalian cells is controlled by the cyclin B1/cdc2 kinase. Activation of this kinase is suppressed by DNA damage, and this may result from the imposition of inhibitory phosphorylation on the cdc2 kinase as well as down-regulation of cyclin B1 levels. p53 represses the cdc2 gene, to help ensure that cells do not escape the initial block. In our study, the cells that were arrested in G2-M phase showed an accumulation in both cdc2 and cyclin B1 protein levels (Figs. 4 and 5). On the other hand, formation and activation of the pre-replication complex requires coordinate actions of cyclin E-cdk2 and cyclin A-cdk2 complexes. And our data showed that the cells that S-phase arrest as a result of sanguinarine pretreatment was associated with an accumulation of cyclin E, cdk2, and/or cyclin A proteins (Figs. 4 and 5).

DNA damage elicited by UVB is thought to be an important trigger for p53 accumulation and transcriptional activation, leading to cell cycle arrest and allowing more time for DNA repair or elimination of damaged cells through apoptosis. Most nonmelanoma skin cancers have mutations in p53 (45–47). The HaCaT cell line bears mutations in both alleles of the p53 gene, rendering a nonfunctional, transcriptionally inactive protein with an increased half-life (48). The mutations in p53 present in HaCaT cells are characteristic of the UV signature (48). We found a significant increase in phospho-p53 (Ser15) protein levels that was significantly enhanced with sanguinarine pretreatment along with no appreciable changes in total p53 protein levels in HaCaT cells (Fig. 6). p53 protects mammals from neoplasia by inducing apoptosis, DNA repair, and cell cycle arrest in response to a variety of stresses. The regulation of tumor suppressor p53 depends not only on the level of expression of this molecule but also on its activation that regulates its stability and capacity to bind to DNA and trigger transcription (49–51). It is known that phosphorylation of p53 on Ser15 impairs the binding of Mdm2, which correlates with p53 accumulation following genotoxic stress (28, 29). The phosphorylation of p53 is affected by its conformation, which, in turn, is modified by mutation of the protein (52).

Another interesting finding of our study was the observed significant accumulation of phospho-p66Shc (Tyr239) in HaCaT cells exposed to UVB. This response of UVB was found to be further enhanced by sanguinarine pretreatment (Fig. 6). P66Shc is a splice variant of p52Shc/p46Shc, a cytoplasmic signal transducer involved in the transmission of mitogenic signals from tyrosine kinases to Ras (53). P66Shc is not involved in Ras regulation but rather functions in the intracellular pathway that converts oxidative signals into apoptosis (54, 55). P66Shc is serine phosphorylated in cells treated with UV or other inducers of oxidative stress, and p66Shc–/– fibroblasts are resistant to UV-induced apoptosis, a finding mirrored by the increased UV sensitivity conferred by overexpression of p66Shc (54). p53 induces p66Shc protein up-regulation by increasing its stability (30). It has been shown that p53 and p66Shc regulate steady-state levels of intracellular ROS, and the p66Shc gene increases intracellular ROS, thereby affecting the rate of oxidative damage to the nucleic acids (30). The p53/p66Shc pathway could be a sensor for the levels of intracellular oxidative signals and regulate intracellular levels of oxidants and of oxidative damage (30). High-intensity oxidative signals would result in high-level activation of the p53/p66Shc pathway and apoptosis (30). Low-intensity oxidative signals could result in chronic, low-level activation of the p53/p66Shc signaling pathway, thus allowing moderate ROS increases and accumulation of the oxidative damage (30).

Thus, because of the documented association of the p53/p66Shc pathway with the oxidative stress, our data implicated that the observed effects of sanguinarine might be mediated via modulations in the oxidative stress within the cells. To further confirm the involvement of oxidative stress, we determined the effect of treatments on the levels of antioxidant enzyme SOD that is known to be modulated by the ROS and oxidative stress in the cells.

It is important to mention here that ROS, at normal physiologic levels, play a role in regulating signaling pathways and gene expression and are also involved in cancer development (56). It is likely that in skin cancers, a diminished antioxidant defense caused by chronic UV exposure may lead, indirectly, to a clonal expansion of initiated, promotable cells that are resistant to excessive oxidative damage. SOD has been shown to protect human keratinocytes against UVB-induced injury (37, 57). In human skin, single exposures of solar-simulated UV resulted in a transient reduction of SOD activity (58); however, chronic UV exposure induced epidermal SOD activity (59). An immunohistochemical investigation of SCC and BCC revealed a decreased CuZnSOD and MnSOD expression within the tumors, indicating a UV-dependent impairment of the antioxidant defense (35). A single dose of UVB irradiation dose-dependently regulated expression of MnSOD in HeLa cells, although it had no effect of its enzymatic activity (60). In contrast, UVB irradiation reduced both the enzymatic activity and the expression of CuZnSOD in HeLa cells (60). Overexpression of MnSOD has been shown to block or delay apoptosis (34). We found a significant increase in SOD activity at 24 hours after UVB exposure along with a significant increase in CuZnSOD and decrease in MnSOD protein levels, and pretreatment with sanguinarine significantly enhanced these effects in HaCaT cells (Fig. 6).

We also determined effect of treatments on the protein levels of MsrA, which plays an important role in cellular metabolism as an antioxidant enzyme that scavenges ROS by facilitating the cyclic interconversion of methionine/protein-methionine residues between oxidized and reduced forms (38, 40, 41). Mice lacking the MsrA gene exhibit heightened sensitivity to oxidative stress (61). Our data showed that UVB exposure to HaCaT cells resulted in a significant decrease in MsrA protein levels, which was further down-regulated with sanguinarine pretreatment (Fig. 6). Thus, our data showed that sanguinarine, often referred as an antioxidant, in fact, enhances the oxidative stress in the cells that are damaged by UVB. Interestingly, sanguinarine did not induce oxidative stress in the cells that were not exposed to UVB.

Taken together, based on our data, we suggest that sanguinarine may protect skin cells from UVB-mediated damages via apoptotic elimination of damaged cells that escape programmed cell death and therefore possess a potential of clonal expansion.


   Acknowledgments
 
We thank undergraduate student Stefanie Jones for her technical assistance.


   Footnotes
 
Grant support: NIH grants CA099076 and CA098368 and Department of Defense grant W81XWH-04-1-0220.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/18/05; revised 11/ 3/05; accepted 12/ 8/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10–30.[Abstract/Free Full Text]
  2. Stewart BW, Kleihues P, editors. World cancer report. Washington (DC): IARC Press; 2003.
  3. Gailani MR, Leffell DJ, Ziegler A, et al. Relationship between sunlight exposure and a key genetic alteration in basal cell carcinoma. J Natl Cancer Inst 1996;20:349–54.
  4. Cleaver JE, Crowley E. UV damage, DNA repair and skin carcinogenesis. Front Biosci 2002;7:1024–43.
  5. Sarasin A. The molecular pathways of ultraviolet-induced carcinogenesis. Mutat Res 1999;428:5–10.[Medline]
  6. de Gruijl FR. Photocarcinogenesis: UVA vs. UVB radiation. Skin Pharmacol Appl Skin Physiol 2002;15:316–20.[CrossRef][Medline]
  7. Hawk ET, Umar A, Viner JL. Colorectal cancer chemoprevention-an overview of the science. Gastroenterology 2004;126:1423–47.[CrossRef][Medline]
  8. Klein EA, Thompson IM. Update on chemoprevention of prostate cancer. Curr Opin Urol 2004;14:143–9.[CrossRef][Medline]
  9. Mehta RG, Pezzuto JM. Discovery of cancer preventive agents from natural products: from plants to prevention. Curr Oncol Rep 2002;4:478–86.[Medline]
  10. Park EJ, Pezzuto JM. Botanicals in cancer chemoprevention. Cancer Metastasis Rev 2002;21:231–55.[CrossRef][Medline]
  11. Scheer M, Kuebler AC, Zoller JE. Chemoprevention of oral squamous cell carcinomas. Onkologie 2004;27:187–93.[CrossRef][Medline]
  12. Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003;3:768–80.[CrossRef][Medline]
  13. Shamma M, Guinaudeau H. Aporphinoid alkaloids. Nat Prod Rep 1986;3:345–51.[CrossRef][Medline]
  14. Vavreckova C, Ulrichova J, Hajduch M, et al. Effect of quaternary benzo[c]phenanthridine alkaloids sanguinarine, chelerythrine and fagaronine on some mammalian cells. Acta Univ Palacki Olomuc Fac Med 1994;138:7–10.[Medline]
  15. Mitscher LA, Park YH, Clark D, et al. Antimicrobial agents from higher plants. An investigation of Hunnemannia fumariaefolia pseudoalcoholates of sanguinarine and chelerythrine. Lloydia 1978;41:145–50.[Medline]
  16. Lenfeld J, Kroutil M, Marsalek E, et al. Antiinflammatory activity of quaternary benzophenanthridine alkaloids from Chelidonium majus. Planta Med 1981;43:161–5.[Medline]
  17. Mandel ID. Chemotherapeutic agents for controlling plaque and gingivitis. J Clin Periodontol 1988;15:488–98.[CrossRef][Medline]
  18. Mandel ID. Antimicrobial mouthrinses: overview and update. J Am Dent Assoc 1994;125 Suppl 2:2–10S.
  19. Walterova D, Ulrichova J, Valka I, et al. Benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine: biological activities and dental care applications. Acta Univ Palacki Olomuc Fac Med 1995;139:7–16.[Medline]
  20. Ahmad N, Gupta S, Husain MM, Heiskanen KM, Mukhtar H. Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells. Clin Cancer Res 2000;6:1524–8.[Abstract/Free Full Text]
  21. Adhami VM, Aziz MH, Mukhtar H, Ahmad N. Activation of prodeath Bcl-2 family proteins and mitochondrial apoptosis pathway by sanguinarine in immortalized human HaCaT keratinocytes. Clin Cancer Res 2003;9:3176–82.[Abstract/Free Full Text]
  22. Gniadecki R, Hansen M, Wulf HC. Two pathways for induction of apoptosis by ultraviolet radiation in cultured human keratinocytes. J Invest Dermatol 1997;109:163–9.[CrossRef][Medline]
  23. Lee JK, Kim JH, Nam KT, Lee SH. Molecular events associated with apoptosis and proliferation induced by ultraviolet-B radiation in the skin of hairless mice. J Dermatol Sci 2003;32:171–9.[CrossRef][Medline]
  24. Weerasinghe P, Hallock S, Tang SC, Liepins A. Role of Bcl-2 family proteins and caspase-3 in sanguinarine-induced bimodal cell death. Cell Biol Toxicol 2001;17:371–81.[CrossRef][Medline]
  25. Weerasinghe P, Hallock S, Liepins A. Bax, Bcl-2, and NF-kappaB expression in sanguinarine induced bimodal cell death. Exp Mol Pathol 2001;71:89–98.[CrossRef][Medline]
  26. Athar M, Kim AL, Ahmad N, et al. Mechanism of ultraviolet B-induced cell cycle arrest in G2/M phase in immortalized skin keratinocytes with defective p53. Biochem Biophys Res Commun 2000;277:107–11.[CrossRef][Medline]
  27. Chouinard N, Valerie K, Rouabhia M, Huot J. UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53. Biochem J 2002;365:133–45.[CrossRef][Medline]
  28. Bean LJ, Stark GR. Phosphorylation of serines 15 and 37 is necessary for efficient accumulation of p53 following irradiation with UV. Oncogene 2001;20:1076–84.[CrossRef][Medline]
  29. Shieh SY, Ikeda M, Taya Y, Prives C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 1997;91:325–34.[CrossRef][Medline]
  30. Trinei M, Giorgio M, Cicalese A, et al. A p53–66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis. Oncogene 2002;21:3872–8.[CrossRef][Medline]
  31. Songyang Z, Margolis B, Chaudhuri M, Shoelson SE, Cantley LC. The phosphotyrosine interaction domain of SHC recognizes tyrosine-phosphorylated NPXY motif. J Biol Chem 1995;270:14863–6.[Abstract/Free Full Text]
  32. van der GP, Wiley S, Lai VK, et al. A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides. Curr Biol 1995;5:404–12.[CrossRef][Medline]
  33. McGlade J, Cheng A, Pelicci G, Pelicci PG, Pawson T. Shc proteins are phosphorylated and regulated by the v-Src and v-Fps protein-tyrosine kinases. Proc Natl Acad Sci U S A 1992;89:8869–73.[Abstract/Free Full Text]
  34. Shindo Y, Hashimoto T. Ultraviolet B-induced cell death in four cutaneous cell lines exhibiting different enzymatic antioxidant defences: involvement of apoptosis. J Dermatol Sci 1998;17:140–50.[CrossRef][Medline]
  35. Kobayashi T, Matsumoto M, Iizuka H, Suzuki K, Taniguchi N. Superoxide dismutase in psoriasis, squamous cell carcinoma and basal cell epithelioma: an immunohistochemical study. Br J Dermatol 1991;124:555–9.[CrossRef][Medline]
  36. Kobayashi T, Saito N, Takemori N, et al. Ultrastructural localization of superoxide dismutase in human skin. Acta Derm Venereol 1993;73:41–5.[Medline]
  37. Sasaki H, Akamatsu H, Horio T. Effects of a single exposure to UVB radiation on the activities and protein levels of copper-zinc and manganese superoxide dismutase in cultured human keratinocytes. Photochem Photobiol 1997;65:707–13.[Medline]
  38. Moskovitz J, Weissbach H, Brot N. Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins. Proc Natl Acad Sci U S A 1996;93:2095–9.[Abstract/Free Full Text]
  39. Moskovitz J, Berlett BS, Poston JM, Stadtman ER. The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo. Proc Natl Acad Sci U S A 1997;94:9585–9.[Abstract/Free Full Text]
  40. Stadtman ER, Moskovitz J, Berlett BS, Levine RL. Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism. Mol Cell Biochem 2002;234–5:3–9.
  41. Levine RL, Berlett BS, Moskovitz J, Mosoni L, Stadtman ER. Methionine residues may protect proteins from critical oxidative damage. Mech Ageing Dev 1999;107:323–32.[CrossRef][Medline]
  42. Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004. CA Cancer J Clin 2004;54:8–29.[Abstract/Free Full Text]
  43. Afaq F, Mukhtar H. Photochemoprevention by botanical antioxidants. Skin Pharmacol Appl Skin Physiol 2002;15:297–306.[CrossRef][Medline]
  44. Sander CS, Chang H, Hamm F, Elsner P, Thiele JJ. Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis. Int J Dermatol 2004;43:326–35.[CrossRef][Medline]
  45. Kanjilal S, Strom SS, Clayman GL, et al. p53 mutations in nonmelanoma skin cancer of the head and neck: molecular evidence for field cancerization. Cancer Res 1995;55:3604–9.[Abstract/Free Full Text]
  46. Oram Y, Orengo I, Baer SC, Ocal T. p53 Protein expression in squamous cell carcinomas from sun-exposed and non-sun-exposed sites. J Am Acad Dermatol 1994;31:417–22.[Medline]
  47. Ziegler A, Leffell DJ, Kunala S, et al. Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers. Proc Natl Acad Sci U S A 1993;90:4216–20.[Abstract/Free Full Text]
  48. Lehman TA, Modali R, Boukamp P, et al. p53 mutations in human immortalized epithelial cell lines. Carcinogenesis 1993;14:833–9.[Abstract/Free Full Text]
  49. Chehab NH, Malikzay A, Stavridi ES, Halazonetis TD. Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage. Proc Natl Acad Sci U S A 1999;96:13777–82.[Abstract/Free Full Text]
  50. Giaccia AJ, Kastan MB. The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev 1998;12:2973–83.[Free Full Text]
  51. Steegenga WT, van der Eb AJ, Jochemsen AG. How phosphorylation regulates the activity of p53. J Mol Biol 1996;263:103–13.[CrossRef][Medline]
  52. Adler V, Pincus MR, Minamoto T, et al. Conformation-dependent phosphorylation of p53. Proc Natl Acad Sci U S A 1997;94:1686–91.[Abstract/Free Full Text]
  53. Pelicci G, Lanfrancone L, Grignani F, et al. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell 1992;70:93–104.[CrossRef][Medline]
  54. Migliaccio E, Giorgio M, Mele S, et al. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 1999;402:309–13.[CrossRef][Medline]
  55. Migliaccio E, Mele S, Salcini AE, et al. Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway. EMBO J 1997;16:706–16.[CrossRef][Medline]
  56. Durocher D, Jackson SP. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol 2001;13:225–31.[CrossRef][Medline]
  57. Sasaki H, Akamatsu H, Horio T. Protective role of copper, zinc superoxide dismutase against UVB-induced injury of the human keratinocyte cell line HaCaT. J Invest Dermatol 2000;114:502–7.[CrossRef][Medline]
  58. Punnonen K, Autio P, Kiistala U, Ahotupa M. In-vivo effects of solar-simulated ultraviolet irradiation on antioxidant enzymes and lipid peroxidation in human epidermis. Br J Dermatol 1991;125:18–20.[CrossRef][Medline]
  59. Punnonen K, Lehtola K, Autio P, Kiistala U, Ahotupa M. Chronic UVB irradiation induces superoxide dismutase activity in human epidermis in vivo. J Photochem Photobiol B 1995;30:43–8.[Medline]
  60. Isoherranen K, Peltola V, Laurikainen L, et al. Regulation of copper/zinc and manganese superoxide dismutase by UVB irradiation, oxidative stress and cytokines. J Photochem Photobiol B 1997;40:288–93.[CrossRef][Medline]
  61. Moskovitz J, Bar-Noy S, Williams WM, et al. Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc Natl Acad Sci U S A 2001;98:12920–5.[Abstract/Free Full Text]



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