Effects and Interactions of Low Doses of Arsenic and UVB on

Although arsenic and ultraviolet light B (UVB) are both causes for skin cancers, lesions of arsenic-induced Bowen's disease are often confined to sun-...
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Chem. Res. Toxicol. 2004, 17, 1199-1205

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Effects and Interactions of Low Doses of Arsenic and UVB on Keratinocyte Apoptosis Chih-Hung Lee,† Chia-Li Yu,‡ Wei-Ting Liao,§ Ying-Hsien Kao,† Chee-Yin Chai,| Gwo-Shing Chen,† and Hsin-Su Yu*,†,⊥ Departments of Dermatology, Biochemistry, and Pathology, Kaohsiung Medical University, Kaohsiung, Taiwan, and Departments of Medicine and Dermatology and Institute of Molecular Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan Received February 21, 2004

Although arsenic and ultraviolet light B (UVB) are both causes for skin cancers, lesions of arsenic-induced Bowen’s disease are often confined to sun-protected skin. UVB may play a modulatory role in skin carcinogenesis by arsenic. The purpose of this study was to evaluate the effects and interactions of arsenic and UVB on cell cycle progression and apoptosis. Cultured human keratinocytes were treated with sodium arsenite (1 µM) and/or UVB (50 mJ/cm2) irradiation in different combinations: (i) arsenic alone, (ii) UVB alone, (iii) arsenic followed by UVB (As-UVB), and (iv) UVB followed by arsenic (UVB-As) treatments. Cell cycle analysis and BrdU pulsing revealed S phase arrest in all treatment groups and growth arrest in AsUVB and UVB-As groups. The terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling assay showed a higher apoptosis rate in the UVB-As group as compared to that of the As-UVB and UVB groups. UVB irradiation significantly decreased Bcl-2 expression. In either the As-UVB or the UVB-As group, the expression of Bcl-2 was further suppressed as compared to the UVB group. The caspase-3, -8, and -9 relative activities were all increased in the UVB group; however, arsenic significantly enhanced caspase-8 and -3 relative activities in UVB-irradiated keratinocytes (the UVB-As group). Pretreatment with the caspase inhibitor(s) rescued the keratinocytes viability to different degrees with the least in the UVB-As group. Our findings revealed that arsenic enhances UVB-induced keratinocyte apoptosis via suppression of Bcl-2 expression and stimulation of caspase-8 activity. Combined UVB and arsenic treatment resulted in the antiproliferative and proapoptotic effects in keratinocytes. Our results provide the explanation for the rare occurrences of arsenical cancers in the sun-exposed skin and the potential therapeutic role of UVB in arsenic-induced Bowen’s disease.

Introduction Epidemiological studies revealed that long-term ingestion of artesian well water contaminated with high concentrations of arsenic (As) is the causal factor for endemic arsenical cancers on the southwestern coast of Taiwan (1-3). Clinically, As-induced skin cancer lesions are usually multiple and on nonsun-exposed areas (1, 4). Ultraviolet light B (UVB) irradiation has been used in treating many hyperproliferative dermatoses including psoriasis (5) and cutaneous T-cell lymphoma (6). Our previous study revealed that UVB irradiation reduced mutant p53 and Ki-67 expression in As-induced Bowen’s disease lesions and resulted in an inhibitory effect on its proliferation (7, 8). UVB may exert a negative effect on the arsenical carcinogenesis in skin. The proapoptotic and antiproliferative effects of UVB on As-induced Bowen’s disease may explain the rare occurrence of arsenical cancers in the sun-exposed skin and provide a basis for * To whom correspondence should be addressed. Tel: 886-2-23562140. Fax: 886-2-2393-4177. E-mail: [email protected]. † Department of Dermatology, Kaohsiung Medical University. ‡ Department of Medicine and Institute of Molecular Medicine, College of Medicine, National Taiwan University. § Department of Biochemistry, Kaohsiung Medical University. | Department of Pathology, Kaohsiung Medical University. ⊥ Department of Dermatology, National Taiwan University Hospital, College of Medicine, National Taiwan University.

the potential therapeutic role of UVB on As-induced Bowen’s disease. As, by itself, is not mutagenic in bacterial or mammalian cells but reinforces mutations induced by various mutagens including UVB. Previous studies investigating the interaction of UVB and As focused on the DNA excision repair and replication. The inhibition of pyrimidine dimers excision (9) and postreplication repair (10, 11) by As is responsible for the cytotoxicity and mutagenesis of UV in Chinese hamster ovary cells. As treatment enhances cytotoxicity, mutagenicity, and clastogenicity of UV light in Chinese hamster ovary cells (9, 12). However, these studies utilized ultraviolet light within the spectrum of UVC instead of UVB. UVC (220-280 nm) from the sun is largely blocked by the ozone layer in the atmosphere, and thus, UVB (280-315 nm) comprises the major part of ultraviolet rays hitting the earth surface. In addition, it is well-known that UVB is the major cause of photoaging and carcinogenesis of human skin. As a result, we believe that UVB serves as a better model in studying the interaction of UV and As in keratinocytes. There are two mechanisms for cells to correct DNA damage: direct repair of the DNA damage or induction of apoptosis. There are evidences to suggest that p53 is involved in both DNA repair and apoptosis. G1 arrest is induced when p53 transcriptionally upregulates p21WAF1/CIP1 (13). It is assumed that p53 induces cell cycle

10.1021/tx049938m CCC: $27.50 © 2004 American Chemical Society Published on Web 08/25/2004

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arrest to provide extra time for the cell to repair DNA damage before the replication. In apoptosis, p53 regulates the expression of Bcl-2/Bax (14). The Bcl-2 protein is thought to prevent most types of apoptotic cell death, while its heterodimerization with Bax protein promotes apoptosis (15, 16). p53 is found to be a direct transcriptional activator of the human Bax gene (17). However, low Bax expression is not exclusively caused by p53 mutations (18). UV-induced DNA damage leads to p53mediated apoptosis (19, 20). Upon severe DNA damage, p53 upregulates Bax that binds to the mitochondrial membrane and induces cytochrome c release, which subsequently activates caspase-9 and caspase-3 leading to downstream apoptotic responses (21). The other pathway of UV-induced apoptosis is through activation of the membrane death receptor Fas (CD95) (22, 23), which initiates apoptosis by activation of caspase-8 followed by activation of caspase-3 (24). However, caspase-9 has been described as the main caspase involved in UVB-induced apoptosis in human keratinocytes (25). As causes apoptosis of human keratinocytes through the Fas-FasL pathway with enhancement of AP-1 activity. Downstream signals of the Fas-FasL pathway, including FADD, caspase-8, caspase-3, and PARP cleavage, are activated (26). Therefore, activation of a different primary caspase is involved in apoptosis induced by As as compared to UVB-induced apoptosis. The purpose of this study is to elucidate the molecular mechanisms responsible for keratinocyte apoptosis by As and UVB interaction through the assessment of the cell cycle, the apoptotic markers, and the caspases in vitro.

Materials and Methods Keratinocyte Culture and Treatments. The normal human keratinocytes were obtained from adult foreskins through routine circumcision. The method for keratinocyte cultivation was described in our previous report (26). Briefly, skin specimens were washed with phosphate-buffered saline (PBS; pH 7.2), cut into small pieces, and incubated in medium containing 0.25% trypsin (Gibco, Grand Island, NY) overnight at 4 °C. The epidermal sheet was lifted from the dermis using a fine forcep. The epidermal cells were pelleted by centrifugation (500g, 10 min) and dispersed into individual cells by repeated aspiration with a 2 mL pipet. The keratinocytes were gently resuspended in approximately 5 mL of keratinocyte-SFM medium (Gibco), which contained 25 µg/mL bovine pituitary extract (BPE) and 5 ng/mL recombinant human epidermal growth factor (rhEGF). The medium was changed every 2 days. Keratinocytes at the third passage were then grown in a keratinocyte-SFM medium without BPE and rhEGF for 24 h before experimentation. The following experiments were all carried out in three repeated experiments. The cells were treated with various regimens of sodium arsenite (1 µM) and/or UVB (50 mJ/cm2) as follows: (i) nontreated (control), (ii) incubated with As for 48 h (As treatment), (iii) irradiated with UVB (50, 100, and 200 mJ/cm2) after a 48 h incubation (UVB treatment), (iv) incubated with As for 24 h and then irradiated with UVB, followed by a 24 h incubation in a culture medium (As-UVB treatment), and (v) irradiated with UVB and then incubated for 24 h in a culture medium, followed by a 24 h As exposure (UVB-As treatment). The irradiation UVB came from SVL (311 nm, 1 × 15 W, intensity 1.28 mW/cm2, France) and was measured by a UVX digital radiometer (UVR-305/365-D detector, Tokyo Optical Co., LTD.) to match 1 mW/cm2 at 15 cm height. Cell Cycle Analysis. A cell cycle profile was studied by two different methods including flow cytometry and the bromodeoxyuridine test. In the first method, keratinocytes treated were harvested by trypsinization and centrifugation. After they were

Lee et al. washed with PBS, the cells were fixed with ice cold 70% ethanol for 30 min, washed with PBS, and then treated with 1 mL of 1 mg/mL of RNase A solution (containing 0.112 mg/mL of trisodium citrate) at 37 °C for 30 min. The cells were further harvested by centrifugation at 400g for 5 min and further stained with 250 µL of DNA staining solution (10 mg of propidium iodide, 0.1 mg of trisodium citrate, and 0.03 mL of Triton X-100 were dissolved in 100 mL of H2O) at room temperature for 30 min in the dark. After 750 µL of PBS was loaded, we collected the DNA contents of 10 000 events. FACScan (Becton Dickinson, San Jose, CA) and the cell cycle profile were analyzed with CellFit software. The percentage of cell subpopulation in each phase of the cell cycle was calculated with the RFIT analytic mode from the DNA content histograms. The second method allowed an assessment of the proportion of cells undergoing replicative DNA synthesis. The keratinocytes were pulse-labeled with 10 µM bromo-deoxyuridine (BrdU) for 2 h before harvest. The cells were then fixed with 70% ethanol, treated with 0.1 N HCl, and heated for 10 min at 97 °C to expose the labeled double-stranded DNA. The cells were then stained with anti-BrdU-conjugated FITC (Immunotech, France) and counterstained with propidium iodide. The cell cycle analysis was carried out on the FACScan, using Lysis II software. The percentage in the S phase was calculated directly from the BrdU/PI dot plots. Detection of Apoptosis. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling) assay was conducted for the detection of apoptosis in keratinocytes. The commercially available apoptosis detection system (Promega, Madison, WI) was used. Keratinocytes were harvested by trypsinization and centrifugation. After they were washed with PBS, the cells were fixed in 4% phosphate-buffered paraformaldehyde on ice for 20 min. The cells were then washed with PBS, followed by permeabilization with 0.2% Triton X-100 solution for 5 min on ice. After equilibration with terminal transferase buffer (TT buffer, 25 mM Tris-HCl, pH 6.6, 0.2 mM DTT, 200 mM potassium cacodylate, 2.5 mM cobalt chloride, and 0.25 mg/mL BSA) at room temperature for 5 min, the cells were then exposed to 0.5 U/µL terminal transferase and 5 µM fluoresceinated dUTP in TT buffer for 60 min at 37 °C in a moist chamber. The reaction was terminated by adding 1 mL of 20 mM EDTA and washed with phosphate buffer. Ten thousand keratinocytes in total were assessed by FACScan (Beckman Coulter, Brea, CA), and the positive staining rate was analyzed with Windows Multiple Document Interface (WinMDI) software for flow cytometry. The specificity of staining was ascertained with a negative control omitting terminal transferase enzyme. Protein Extraction and Western Blotting. The total cell extracts from cultured human keratinocytes were obtained by lysing the cells in cold RIPA buffer (50 mM Tris-HCL, pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1% nonidet P-40, and 150 mM NaCl) in the presence of protease inhibitors (Roche, Molecular Biochemicals, Mannheim, Germany). After the mixtures were centrifuged to remove cell debris, we collected supernatants to run SDS-PAGE, which was made up by 12 or 15% acrylamide gels under reducing conditions (in the presence of 250 mM β-mercaptoethanol). The proteins were subsequently electrotransferred onto a nitrocellulose membrane (Sartorius AG, Go¨ttingen, Germany) following conventional protocols. The blots were blocked in 5% skimmed milk/ PBS with 0.1% Tween-20 (PBS-T) at 4 °C. For p53, p21, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), blocking was done for 1 h, followed by 1 h of incubation with primary antibodies at room temperature. The working concentrations of primary antibodies in PBS-T with 5% skimmed milk were as follows: 1 µg/mL for Bcl-2 (Santa Cruz Biotech, Santa Cruz, CA) at 1:500 dilution, 1 µg/mL for Bax (Santa Cruz Biotech) at 1:500 dilution, 2 µg/mL for p53 (Chemicon International, Temecula, CA), 1 µg/mL p21WAF1/CIP1 (Upstate Biotech), and 1 µg/mL for GAPDH (Chemicon), respectively. After five washes in PBS-T, the blots were incubated with secondary antibodies (horseradish peroxidase coupled with anti-mouse or anti-rabbit

UVB and Arsenic in Keratinocyte Apoptosis immunoglobulin G) at a 1:5000 dilution. The enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) detection system and BioMax film (Kodak, Rochester, NY) were used to visualize the presence of specific proteins on the blots according to the manufacturer’s instructions. The exposure times for each protein ranging from 1 min to 1 h were calibrated for documenting linearity and permitting semiquantitative analysis of density. The visualized films were recorded on a digital imaging system (Alpha Imager 2000, Alpha Innotech Corp., San Leandro, CA) and analyzed in a densitometrical analysis system (Alpha Ease ver. 3.23, Alpha Innotech Corp.). The inductive ratio of p53 and p21WAF1/CIP1 was expressed as the ratio of density of the protein to that of GAPDH in the same specimen, and the control group was taken as 1.0. The experiments were repeated three times to determine the final value. Caspase Activity Assay. The commercially available ApoAlert caspase-3 colorimetric assay kit, ApoAlert caspase-8 colorimetric assay kit, and ApoAlert caspase-9/6 fluorescent assay kit (BD Biosciences Clontech, Palo Alto, CA) were used to detect the caspase activity. Keratinocytes (2 × 106 cells) were washed with PBS, and the cell lysates were obtained according to the manufacturer’s instructions. We added 50 µL of Reaction Buffer/ DTT Mix (provided by the kits) and caspase substrates (50 µM DEVD-pNA for caspase-3; 200 µM IETD-pNA for caspase-8; and 250 µM LEHD-AMC for caspase-9/6) to 50 µL of cell lysate. These mixtures were incubated at 37 °C for 1 h in a water bath. After reaction, the samples for the caspase-3 or caspase-8 activity test were read at 405 nm in a spectrophotometer. For the caspase-9/6 activity test, the samples were read in a fluorometer with a 380 nm excitation filter and a 460 nm emission filter. The experiments were repeated three times to determine the final value. The values were normalized with a control group and shown as relative caspase activity. Caspase Inhibition Tests. For caspase inhibitions, the third passage of keratinocytes (2 × 105 cells/mL) was seeded into 96 well plates and incubated with caspase-3 inhibitor, caspase-8 inhibitor, caspase-9 inhibitor, or pancaspase inhibitor for 30 min. The working concentrations of the caspase inhibitors (Enzyme Systems Products, Livermore, CA) were as follows: (i) 200 µM zVAD-fmk for pancaspase inhibition, (ii) 100 µM DEVD-cho for caspase-3 inhibition, (iii) 100 µM IETD-cho for caspase-8 inhibition, and (iv) 100 µM LEHD-cmk for caspase-9/6 inhibition. The cells were then treated with As 1 µM and/or UVB 50 mJ/cm2 without removing the caspase inhibitors. The cell viability was then measured by the XTT assay. The commercially available kit for cell proliferation (XTT assay kit, Roche, Mannheim, Germany) was used according to the manufacturer’s instruction. After treatment, XTT reagents were added into each well and incubated at 37 °C for 4 h to generate colorimetric formazan products. The absorbance of the formazan product was measured at a wavelength of 450 nm with a reference wavelength of 630 nm. The experiments were repeated three times to determine the final value. The results were expressed as relative cell viability. Statistical Analysis. All results were expressed as mean values ( standard deviation (SD) and analyzed by using the statistical analysis system (SPSS, SPSS Inc.). Differences among groups were analyzed by one way analysis of variance. Scheffe’s postcomparison method was employed to further demonstrate the difference sources between each group. A difference at p < 0.05 was considered statistically significant.

Results Interaction of As and UVB Treatments Exhibited an Inhibitory Effect on Cell Cycle Progression in Cultured Keratinocytes. The effects of As and/or UVB interaction on cell cycle progression were examined, and the results are listed in Table 1. Both the As-treated and UVB-irradiated groups showed a decrease at the G1 phase and an increase at the S and G2/M phases as compared to the control group (p < 0.05). In parallel, the

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Figure 1. Effect of As and UVB on apoptotic induction. The TUNEL reaction was used for the detection of apoptosis. The percentage (%) of the positive cells and mean fluorescence intensity (MFI#) of cell distribution were analyzed and noted on the figure of each group: (A) nontreated control group, (B) As group, (C) UVB group, (D) As-UVB group, and (E) UVBAs group. The left margin of marker (M1) set at channel 67.5 was selected to determine the positive rate of cells. As treatment did not increase the apoptotic index significantly (TUNEL positive rate 3.93%). The apoptotic index was increased in the UVB (12.48%) and the As-UVB groups (15.2%), and a further enhancement of apoptosis was noticed in the UVB-As group (38.58%). Table 1. Effect of As and UVB on Cell Cycle Progressiona group

% G1 phase

% S phase

control As UVB As-UVB UVB-As

74.5 ( 2.7 64.7 ( 3.7* 62.3 ( 5.0* 55.0 ( 11.8* 67.2 ( 5.7

11.4 ( 2.2 19.1 ( 1.0* 18.6 ( 2.4* 34.6 ( 13.7* 14.8 ( 1.7*

% G2/M phase % BrdU (+) 14.1 ( 0.6 16.2 ( 2.8 19.1 ( 0.6* 10.4 ( 1.9* 18.0 ( 1.0*

4.4 ( 0.3 6.0 ( 0.5* 6.6 ( 0.7* 2.0 ( 0.3* 3.7 ( 0.3*

a Cell cycle distributions of cultured keratinocytes with As and/ or UVB treatments were measured by flow cytometric method and BrdU incorporation. The data were expressed as means ( SD (n ) 3). An asterisk denotes p < 0.05 when compared with the control.

positive rates of BrdU (6.0 ( 0.5 and 6.6 ( 0.7% for the As group and UVB group, respectively) in both groups were significantly higher than those of the control group (4.4 ( 0.3%), suggesting proliferation in either 1 µM Astreated or 50 mJ/cm2 UVB-irradiated groups. In the AsUVB group, the cell population at the S phase (34.6 ( 13.7%) was about three times higher than that in the control group (11.4 ( 2.2%). On the other hand, G1distributed cells were significantly decreased to 55.0 ( 11.8% as compared to 74.5 ( 2.7% in the control group (p < 0.05). In the UVB-As group, the cell populations at the S phase (14.8 ( 1.7%) and G2/M phase (18.0 ( 1.0%) were higher than those in the control groups (11.4 ( 2.2 and 14.1 ( 0.6%, respectively). The BrdU labeling rates in the As-UVB (2.0 ( 0.3%) and UVB-As (3.7 (

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Figure 2. Effects of As and UVB on the p53 and p21WAF1/CIP1 protein expression. (A) Expression of p53 (53 kDa) and p21WAF1/CIP1 (21 kDa) by Western blotting after As treatment and/or UVB irradiation. (B) The expression levels of proteins in each experimental group were expressed as relative ratios of integrated density value of p53 or p21WAF1/CIP1 to GAPDH (36 kDa) with normalization to that of the control group. An induction of p53 was demonstrated only in the As-UVB group; however, an induction of p21WAF1/CIP1 was demonstrated in the As, UVB, As-UVB, and UVB-As groups. An asterisk denotes p < 0.05 when compared with the control (n ) 3).

0.3%) groups were lower than those in the control group (4.4 ( 0.3%), indicating that cells at the S phase failed to progress into G2/M and were arrested with a DNA content between 2N and 4N. These results suggest that the interaction of As and UVB exhibits an inhibitory effect on cell cycle progression in cultured keratinocytes, especially in the group pretreated with As. Apoptosis Was Significantly Increased in the UVB-As Group. The effects of As and UVB on apoptotic induction in keratinocytes are shown in Figure 1. TUNEL positive keratinocytes (12.48 ( 2.13%) were significantly increased by UVB irradiation at 50 mJ/cm2 (Figure 1C). UVB in 100 and 200 mJ/cm2 resulted in a significant increase in keratinocyte apoptosis (TUNEL positive rates of 16.73 ( 1.72 and 68.13 ( 13.35%, respectively). As treatment (Figure 1B) showed no significant induction of apoptosis (3.93 ( 0.77%) as compared to the control group (4.75 ( 0.66%) (Figure 1A). The apoptosis rate (38.58 ( 4.56%) in the UVB-As group (Figure 1E) was significantly increased as compared to those of other groups. In contrast, the TUNEL positive rate (15.20 ( 3.15%) in the As-UVB group (Figure 1D) was not different from those in the UVB group. Upregulation of p53 and p21WAF1/CIP1 Expression in the As-UVB Group. Levels of p53 and p21WAF1/CIP1 proteins were measured by Western blotting after treatment with As and/or UVB (Figure 2). The inductive ratio of p53 (ratio of p53/GAPDH normalized with that in the

Lee et al.

Figure 3. Effects of As and UVB on the Bcl-2 and Baxprotein expression. Cultured human keratinocytes (KCs) were treated with As and/or UVB. (A) The proteins were probed with monoclonal antibodies against human Bcl-2 (26 kDa) and Bax (21 kDa) molecules, respectively. (B) The expression level of the protein in each experimental group was expressed as relative ratios of integrated density value of Bcl-2 or Bax to GAPDH. The Bax expression was similar in all treatment groups. However, a decrease in the expression of Bcl-2 was noticed in the UVB, As-UVB, and UVB-As groups. An asterisk denotes p < 0.05 when compared with the control (n ) 3).

control group) was 1, 0.8 ( 0.4, 1.1 ( 0.3, 2.1 ( 0.6, and 1.1 ( 0.4 in the control, As, UVB, As-UVB, and UVBAs groups, respectively. An upregulation of p53 was demonstrated in the As-UVB group (Figure 2B). In addition, the inductive ratio of p21WAF1/CIP1 (p21WAF1/CIP1/ GAPDH normalized with that in the control group) was 1.0, 1.8 ( 0.3, 6.2 ( 0.5, 7.2 ( 0.4, and 6.5 ( 0.2 in the control, As, UVB, As-UVB, and UVB-As groups, respectively. A marked induction of p21WAF1/CIP1 was noticed in the As, UVB, UVB-As, and As-UVB groups (Figure 2B). Our results revealed that a significantly inductive effect on p53 and p21WAF1/CIP1 was exhibited in the AsUVB group (p < 0.05). Downregulation of Bcl-2 and Bax in the As-UVB and UVB-As Groups. Figure 3 shows the Bcl-2 and Bax expression by Western blotting. The relative densities of Bax to GAPDH calculated by integrated density values of these two proteins were similar in all treated groups. The relative density of Bax to GAPDH was 0.67 ( 0.14, 0.71 ( 0.13, 0.69 ( 0.15, 0.67 ( 0.17, and 0.67 ( 0.15 in the control, As, UVB, As-UVB, and UVB-As groups, respectively. On the other hand, the relative density of Bcl-2 to GAPDH was 0.53 ( 0.11, 0.45 ( 0.14, 0.25 ( 0.13, 0.13 ( 0.07, and 0.14 ( 0.10 in the control, As, UVB, As-UVB, and UVB-As groups, respectively. As treatment did not significantly alter the Bcl-2 level (0.45 ( 0.14) as compared to the nontreated control (0.53 ( 0.11) (p > 0.05). However, 50 mJ/cm2 UVB irradiation significantly decreased Bcl-2 expression. In either the As-UVB or the UVB-As group, the expression of Bcl-2 was further suppressed as compared to the UVB group. As Enhanced UVB-Induced Caspase-8 and Caspase-3 Activities. The caspase-8 relative activity was elevated in the following decreasing order: the

UVB and Arsenic in Keratinocyte Apoptosis

Figure 4. Effects of As and UVB on caspase-8, caspase-9, and caspase-3 activities. The caspase-8 relative activity was increased in the UV and As-UV groups, and a significant enhancement of caspase-8 relative activity was demonstrated in the UV-As group. In parallel to the result of caspase-8, the caspase-3 relative activity displayed the same sequential activity in these groups. The caspase-9 relative activity was increased similarly in the UV, As-UV, and UV-As groups. An asterisk denotes the significant restoration of cell viability with caspase inhibitor (p < 0.05, n ) 3).

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fact that caspase-3 is a downstream target of both caspase-8 and caspase-9 in the apoptotic caspase cascade. Pancaspase Inhibitor, but not Caspase-8, -9, and -3 Inhibitors, Significantly Enhanced Cell Viability in the UVB-As Group. The cell viability did not decrease in the As group (Figure 5). However, it was significantly decreased in the UVB group (72.6 ( 5.7%), the As-UVB group (76.3 ( 6.8%), and the UVB-As group (51.2 ( 4.6%). The sequence of the cell viability, i.e., the control group/the As group, the UVB group/the As-UVB group, the UVB-As group, is just reverse as that of apoptosis among the five experimental groups. All tested caspase inhibitors revealed no significant effects on cell viability in the As group. In the UVB group, the cell viability (72.6 ( 5.7%) was partially restored after blocking with the caspase inhibitors (87.2 ( 4.5, 85.5 ( 3.6, and 80.2 ( 5.8% in the pancaspase, caspase-9, and caspase-3 inhibitors, respectively) except for the casapse-8 inhibitor. The cell viability was partially restored after blocking with the pancaspase inhibitor (84.9 ( 6.3%) and caspase-3 inhibitor (81.6 ( 5.3%) in the As-UVB group (76.3 ( 6.8%). Caspase-8 and caspase-9 inhibitors showed no significant effect on cell viability in this group. In contrast, only pancaspase inhibitor significantly enhanced cell viability (76.8 ( 3.3%) in the UVB-As group (51.2 ( 4.6%).

Discussion

Figure 5. Results of caspase blocking test in cell viability. The cell viability was significantly decreased in the UVB group (72.6 ( 5.7%), the As-UVB group (76.3 ( 6.8%), and the UVB-As group (51.2 ( 4.6%) but not in the As group. In the UVB group, all caspase inhibitors except the caspase-8 inhibitor partially restored the cell viability. In the As-UVB group, only the pancaspase and caspase-3 inhibitors partially rescued the keratinocytes. In the UVB-As group, only the pancaspase inhibitor was able to rescue the keratinocytes. An asterisk denotes significant differences in indicating groups. # denotes the significant recovery of cell viability after blocking with individual caspase inhibitors in each group (p < 0.05, n ) 3).

UVB-As group (1040.8 ( 104.2%), the As-UVB group (467.2 ( 43.2%)/the UVB group (509.7 ( 68.4%), the As group (302.4 ( 43.2%), and the control group (100%) (Figure 4). As significantly enhanced caspase-8 relative activity in UVB-irradiated keratinocytes. In contrast, the caspase-9 relative activity was elevated in the decreasing sequence of the UVB-As group (1273.2 ( 149.3%)/the As-UVB group (1320.7 ( 140.5%)/the UVB group (1254.9 ( 125.4%), the As group (129.3 ( 16.8%)/the control group (100%). A significant increase in the relative activity of caspase-9 was noticed in UVB-irradiated keratinocytes. However, As did not further change caspase-9 activity in keratinocytes by UVB. The caspase-3 relative activity was decreased in the following descending order: UVB-As group (1607.0 ( 178.4%), the AsUVB group (1155.3 ( 139.9%) or the UVB group (1089.4 ( 110.6%), and the As group (85.1 ( 9.8%) or the control group (100%). Our findings are consistent with the known

An increase in β-adrenergic receptor and cAMP levels was reported to result in inhibition of cell growth in keratinocytes (27, 28). Our previous studies revealed that the β-adrenergic receptor density on keratinocytes was reduced in either As (1 µM) treatment or UVB (50 mJ/ cm2) irradiation. However, the β-adrenergic receptor density on keratinocytes was unchanged when keratinocytes were irradiated with UVB followed by As treatment (29, 30). In this study, As at 1 µM exhibited no obvious apoptosis-inducing effect on keratinocytes by the TUNEL assay. In contrast, analysis of cell cycle progression under As treatment showed that cells were arrested in the S phase and had increased BrdU uptake. This result demonstrates that cell proliferation, but not apoptosis, was induced by 1 µM As treatment. A low apoptotic index by the TUNEL assay further confirmed this effect. In this study, an obvious UVB-induced cytotoxicity by XTT test was not noticed until the dosage reached to 100 mJ/cm2 (data not shown). UVB at 50 mJ/ cm2 had a dual effect on cell proliferation and apoptosis. After UVB irradiation, the proliferation tendency was displayed by the cell cycle analysis and the BrdU uptake; however, the apoptotic index was increased by the TUNEL assay. UVB causes DNA damage by inducing cyclobutane pyrimidine dimers, which activate p53 and result in p53-dependent G1 phase arrest regulated by p21WAF1/CIP1 (31). p21WAF1/CIP1 expression is associated with nucleotide excision repair, and Bax expression is associated with apoptosis in human cells irradiated with UVB (32). Our results of an increase in p21WAF1/CIP1 expression, decrease in Bcl-2 expression, and increase in Bax expression demonstrated the apoptotic effect at UVB 50 mJ/ cm2. The modest proliferative effect at 1 µM As and apoptotic effect at 50 mJ/cm2 UVB in keratinocytes provided reasonable dosages for studying As and UVB interaction. In the As-UVB group, a significant increase in cell population at the S phase and a decrease in BrdU uptake

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Figure 6. Schematic picture demonstrates the enhancement of As in UVB-induced apoptosis in keratinocytes. UVB induces apoptosis of keratinocytes mainly through DNA damage, p53 activation, and decreased Bcl-2, caspase-9, and caspase-3 activation (gray arrows). On the other hand, As induces apoptosis of keratinocytes mainly through the death receptor(s) and caspase-9 and caspase-3 activation. As, which does not induce keratinocytes apoptosis at 1 µM, enhances the UVB-induced keratinocyte apoptosis through the activation of caspase-8 and caspase-3 as well as a decrease in Bcl-2 (black arrows).

were noticed (Table 1). These results implied that keratinocytes were arrested at the S phase but could not progress to the G2/M phase, suggesting that active proliferation was inhibited. UVB revealed an inhibitory effect on proliferation in As-pretreated keratinocytes. However, As pretreatment did not significantly alter the apoptosis index induced by UVB. As induced modest stimulatory effects on p21WAF1/CIP1 and caspase-8 activity in keratinocytes. However, in the As-UVB group, a significant increase in p53, p21WAF1/CIP1, caspase-9, caspase3, and caspase-8 was noticed. These findings are similar to those of the UVB group except that UVB did not have increased p53 expression. The increase of p53 expression is parallel to the S phase arrest in the As-UVB group. These results provide an explanation for lack of difference in apoptotic index between the UVB group and the AsUVB group in our findings (Figure 1). In the As-UVB group, there was a significant decrease in cell viability as compared to the As group. Pancapase and caspase-3 inhibitors, but not caspase-8 and caspase-9 inhibitors, were able to partially restore keratinocyte viability. In contrast, the caspase-9 inhibitor could significantly enhance keratinocytes viability in the UVB group, which suggests that caspase-9 may play an important role in UVB-induced keratinocyte (28) but not in the As-UVB group. As treatment could potentiate the DNA damaging effect induced by other agents, including UV (11, 12, 33, 34). In this study, we demonstrated the As enhanced UVB-induced apoptosis in cultured keratinocytes. In Figure 1, a significant increase was seen in the apoptotic index of keratinocytes in the UVB-As group as compared to other groups. UVB-induced apoptosis is mainly through the p53 activation, the decrease in Bcl-2/Bax ratio, followed by caspase-9 and caspase-3 activation (35). In contrast, Fas-FADD activation followed by caspase-9 and caspase-3 activation is able to induce apoptosis in Aspretreated keratinocytes (26). In both UVB-As and AsUVB groups, a significant increase in p21WAF1/CIP1 and a decrease in Bcl-2 expression were seen (Figures 2 and

Lee et al.

3). However, caspase-8 and caspase-3 activities were significantly higher in the UVB-As group as compared to those of the As-UVB group. As may enhance UVBinduced keratinocytes apoptosis through activation of caspase-8 and caspase-3. Furthermore, the pancaspase inhibitor was the only inhibitor, which was able to partially rescue keratinocyte apoptosis in the UVB-As group. Our results suggest that other pathways may be involved in the keratinocyte apoptosis in this group (Figure 6). In this study, we found new evidences for understanding molecular mechanisms of keratinocyte apoptosis in As and UVB interaction. Our results provide the theoretical basis for the rare occurrence of arsenical skin cancers in the sun-exposed skin and the potential therapeutic role of UVB treatment for As-induced Bowen’s disease.

Acknowledgment. This study was supported by the grant from the National Health Research Institute (NHRI-EX90-8929SL), Taiwan, Republic of China.

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