Liposome-AE on Human Nonmelanoma Skin Cancer Cells and

Nov 24, 2009 - R1-associated death domain and Fas-associated death domain, and ... death in A431 and SCC25 cells than only the use of AE or 5-Fu, like...
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Chem. Res. Toxicol. 2009, 22, 2017–2028

2017

The Molecular Effects of Aloe-Emodin (AE)/Liposome-AE on Human Nonmelanoma Skin Cancer Cells and Skin Permeation Tzung-Han Chou and Chia-Hua Liang* Department of Cosmetic Science, Chia Nan UniVersity of Pharmacy and Science, 60 Erh-Jen Road, Section 1, Pao-An, Jen-Te Hsiang, Tainan 717, Taiwan ReceiVed September 4, 2009

In this study, aloe-emodin (AE) was less cytotoxic to human noncancerous skin cells (premalignant keratinocytic HaCaT and fibroblast Hs68) than to nonmelanoma cancer cells (epidermoid carcinoma A431 and head and neck squamous cell carcinoma SCC25). Notably, AE induced apoptosis by upregulating tumor necrosis factor-R and Fas ligand and their cognate receptors, downstream adaptor TNFR1-associated death domain and Fas-associated death domain, and activated caspase-8 in A431 and SCC25 cells. Moreover, AE up-regulated p53, increased intracellular reactive oxygen species levels, depleted intracellular-reduced GSH, up-regulated cytochrome c and Bax, down-regulated Bcl-2, and activated caspase-9 and -3. The combinatory use of AE and 5-fluorouracil (5-Fu) achieved significantly more cell death in A431 and SCC25 cells than only the use of AE or 5-Fu, likely via regulation of caspase-8, -9, and -3 expressions. Incorporating AE into the liposomal formulation accelerated cell death of A431 and SCC25 cells within a short time. Furthermore, skin permeation profiles of drug suggest that the liposomal formulation enhances transdermal delivery of AE. Experimental data demonstrate the feasibility of applying liposome to deliver AE in clinical therapy. Introduction Nonmelanoma skin cancer is the most common malignancy worldwide among Caucasians, accounting for over 90% of all incidences of skin cancer (1). Cutaneous squamous cell carcinoma (SCC) is one of the most common human nonmelanoma skin malignancies. Its incidence varies considerably and is reportedly increasingly worldwide (2). Thus, developing novel antiskin cancer drugs characterized by selective targeting and low toxicity for dividing normal host tissues is very important. Natural ingredients that have been used traditionally to treat many diseases for hundreds of years are considered as a good choice for carcinoma therapy (3). Aloe-emodin (AE), a hydroxyanthraquinone naturally occurring in Aloe Vera leaves, reportedly alters the expression of a number of proteins involved in oxidative stress, antimetastasis, cell cycle arrest, and apoptosis in several cancer cell lines, including human hepatoma, leukemia, glioma, and lung carcinoma cells (4). Furthermore, experimental findings indicate that AE has selective activity against neuroectodermic tumors, particularly against neuroblastoma, which is a tumor exhibiting low susceptibility to available drugs, and has caused clinical interest (5). On the basis of its unique in vitro antitumor activity, selective toxicity, and cellular pharmacokinetics, AE can be considered a novel anticancer agent that may contribute to the development of targeted nontoxic drugs (6). However, the underlying interactive mechanisms of AE on cancer cells have not been fully elucidated, especially those on skin cancer, and currently, there is a shortage of new clinical drugs for antiskin cancers. Thus, determining the molecular effects of AE on human skin cancers is very worthy. The selectivity of in vitro killing of skin cancer cells and the antiskin cancer activity of AE are investigated for the first time in this study. * To whom correspondence should be addressed. Tel: (886)-6-26649112441. Fax: (886)-6-2667324. E-mail: [email protected].

Apoptosis is a potential mechanism that contributes to the antiproliferative and antineoplastic effects of AE. Two major apoptotic pathways, intrinsic and extrinsic apoptosis signaling, in cells responsive to apoptotic stimuli have been identified (7, 8). The extrinsic apoptotic pathway is mediated by such death receptors as receptors for tumor necrosis factor (TNFRs) and Fas ligand (FasL); caspase-8 is a major initiator caspase in this pathway (7). Conversely, the intrinsic apoptotic pathway is a mitochondria-involved signaling cascade in which caspase-9 is the predominant initiator caspase. Alteration of the balance between the Bcl-2 family proteins causes the release of cytochrome c and induces caspase-9 activation and eventually apoptosis (8). The generation of reactive oxygen species (ROS) may contribute to mitochondrial damage, reduction of mitochondrial transmembrane potential, release of cytochrome c, and subsequent caspase activation and apoptosis (9). AE can induce DNA damage through excessive production of ROS in human lung carcinoma (10) and hepatoma cells (4). This study determines whether AE induces apoptosis of skin cancer cells via signaling from death receptors and downstream mitochondria-mediated oxidative stress pathways. Identifying tumors that are likely responsive or resistant to first-line chemotherapy with 5-fluorouracil (5-Fu) before treatment is extremely challenging (11). Notably, although topical 5-Fu cream is an effective therapy, successful treatment is inevitably accompanied by pain, pruritus, burning, erythema, erosion, and scarring (12). Patients with an unfavorable clinical or genetic makeup are candidates for alternative treatment modalities using novel agents with novel action mechanisms. Because drug combinations such as the traditional Chinese medicines have been applied for treating diseases and reducing suffering, this study assesses the efficacy of 5-Fu combined with AE for skin cancer cells. In addition, AE, a hydrophobic drug, was found to crystallize in water (image not shown). Even though this low solubility

10.1021/tx900318a  2009 American Chemical Society Published on Web 11/24/2009

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may be a major hindrance to clinical application of AE, an appropriate drug delivery system is needed. Liposome, a spherical self-closed lipid vesicle, has been widely utilized in many drug/agent delivery systems due to its good biocompatibility, biodegradability, low toxicity, and ability to encapsulate hydrophilic and hydrophobic drugs simultaneously (13). Liposomal encapsulation of anticancer drugs reportedly improves pharmacokinetics and biodistribution significantly by reducing drug release (14). Therefore, liposome has been chosen in this study as the AE carrier for the first time, and the ability of AE to inhibit cancer cell proliferation has been explored. In this study, AE (5 mol %) is incorporated into a liposomal formulation (liposome-AE) prepared using the thin film hydration method. This study demonstrates that liposome-AE elevates the efficacy of inhibition on skin cancer cells, suggesting that such a formulation can be applied in future clinical trials of human skin cancer treatments. Additionally, the transdermal absorption route has garnered considerable attention as a drug delivery system due to such advantages as good patient acceptability (noninvasiveness), avoidance of gastrointestinal disturbances, and application ease. Liposomes are regarded as a potential vehicle for transdermal drug delivery. In this study, the physicochemical characteristics and in vitro skin penetration behavior of liposome-AE are investigated to preliminarily assess the clinical feasibility of transdermal administration of AE.

Experimental Procedures Materials. AE and 5-Fu (Sigma, St. Louis, MO) were dissolved at a concentration of 200 mM in 100% dimethyl sulfoxide (DMSO) as a stock solution. Hydrogenated soybean phosphatidylcholine (HSPC) (>98% PC purity) was a gift from Nippon Oil and Fat Corp. (Tokyo, Japan). Dihexadecyldimethylammonium bromide (DHDAB) (purity >97%) was obtained from Fluka (Buchs SG, Switzerland). Cholesterol (Chol) was purchased from Sigma Aldrich Inc. (St. Louis, MO). Methanol and chloroform of HPLC analytical grade were purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). All materials and solvents were utilized as received without further purification. The pure water with a resistivity of 18.2 MΩ cm used in all experiments was obtained from a Milli-Q plus purification system (Millipore, United States). A 5 mM concentration of liposome was prepared with ddH2O as a stock solution. Cell Culture. Human epidermoid carcinoma A431, human head and neck SCC SCC25, human skin melanoma A375, and nontransformed human skin fibroblasts Hs68 cells were purchased from the American Type Culture Collection (Rockville, MD). Human premalignant keratinocytic HaCaT cells were kindly provided by Prof. Hamm-Ming Sheu (National Cheng Kung University Medical College, Tainan, Taiwan). Cells were cultured in medium supplemented with 10% fetal bovine serum (Hazelton Product, Denver, PA) and 1% penicillin-streptomycin at 37 °C in 5% CO2; specifically, A431, A375, HaCaT, and Hs68 cells in Dulbecco’s modified Eagle medium (DMEM) and SCC25 cells in DMEM/ F12 medium supplemented with 0.4 µg/mL hydrocortisone (GIBCO, Grand Island, NY). Assessment of Cell Viability and Morphology Change. Cells (1.5 × 104 cells/well) were each seeded in 100 µL 96-well multidishes for at least 24 h prior to use. The cells were separately treated with serial concentrations of 5-Fu, AE, and 5-Fu plus AE for indicated times. The control groups were treated with DMSO, and the final DMSO concentration did not exceed 0.1%. For cell viability of the liposome-AE assay, mixed phosphatidylcholine/ DHDAB vesicles at a concentration of 5 mM were diluted to various concentrations (0.001, 0.01, 0.1, and 1 mM) by adding the appropriate volume of culture medium and then were treated with the cells for 24 and 48 h. The control groups were, respectively, treated with 1, 1, 2, and 20 µL of 1× phosphate-buffered saline (PBS), which were the same volumes as those of the vesicles in 100 µL of the medium for 24 and 48 h. After the new medium

Chou and Liang was replaced, the effects on cell growth were examined by MTS [3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium] assay according to the manufacturer’s procedures (CellTiter 96 AQ, Promega, Madison, WI). The absorbance at 490 nm (A490) was measured with an automated plate reader (Synergy2, BioTek Instruments, Inc., United States). Values are expressed as the percentage of mean cell viability relative to the control group cultures. After treatment drugs for 72 h, the IC20, IC50, and IC80 values were calculated from the drug concentration that induced 20, 50, and 80% of the cell survival rate. For morphological analysis, after incubation of the IC80 concentration of AE for serial times, the A431 and SCC25 cells in each well were washed once with 1× PBS, and analysis was performed using a phase contrast inverted light microscope (Nikon, TE2000U, Japan). Assessment of Cell Cycle Distribution and Apoptotic Cells. A431 and SCC25 cells (1.5 × 105 cells/mL) were seeded in 24well plates and incubated with or without AE (IC50 and IC80 concentrations) for 24, 48, and 72 h. Cells were resuspended in 1 mL of 1× PBS and 4 mL of 70% ice cold ethanol. The cells were incubated overnight at 4 °C and then washed with 1× PBS. The cells were then resuspended in 250 µL of propidium iodide solution (50 µg/mL PI in 1× PBS) containing 1 µL of 20 µg/mL RNase A. The cells were incubated in the dark setting for 15 min at room temperature and then analyzed by FACScan flow cytometer (Becton-Dickinson, San Jose, CA). A minimum of 10000 cells were collected, and the cell distribution in each phase (sub-G1, G0/G1, S, and G2/M phases) of the cell cycle was determined using Windows Multiple Document Interface software (WinMDI), including subG1-peak of apoptotic cells. Fluorescent Immunocytochemistry Analysis. A431 and SCC25 cells (1.5 × 105 cells/mL) were seeded in 24-well multidishes and treated with or without AE (IC50 concentration) for 72 h, and adherent cells were fixed by incubation with 100% ice-cold methanol for 30 min at -20 °C and exposed to 3% (v/v) H2O2 to inactivate endogenous peroxidase. Cells were then washed twice with ice-cold 1× PBS and stained with mouse antihuman TNFR1, TNF-R2, Fas, TNF-R1-associated death domain (TRADD), Fasassociated death domain (FADD), p53, Bax, Bcl-2 (Santa Cruz, CA), cytochrome c (BD Transduction Laboratories), and cleaved caspase-3, -8, and -9 (Cell Signaling Technology) monoclonal antibodies (10 µg/mL) or human recombinant TNF-R and FasL proteins (50 ng/100 µL) (Deisenhofen, Germany) in 1× PBS containing 0.5% bovine serum albumin (BSA) and 0.1% sodium azide (Sigma-Aldrich) for 1 h at 4 °C. Cells were then washed twice with cold 1× PBS and incubated with FITC-conjugated antimouse IgG (1:500) (Santa Cruz, CA) (Ex, 485 nm; Em, 528 nm) at 4 °C for 30 min. The cell nuclei were stained with 0.1 µg/ mL of Hoechst 33342 (Promega) (Ex, 360 nm; Em, 460 nm) and photographed by fluorescent microscope (Nikon, TE2000-U). For quantitative analysis of fluorescent staining, cells (1.5 × 105 cells/ mL) were seeded in 96-well plates, and these experiments were repeated on cell treatments with AE (IC50 and IC80 concentrations) for 72 h, fixed in 4% para-formaldehyde, and permeabilized in saponin (0.1% v/v in PBS-BSA) (pH 7.4) at 4 °C for 30 min and detected with antihuman TNF-R1, TNF-R2, Fas, TRADD, FADD, p53, Bax, Bcl-2, cytochrome c, and cleaved caspase-3, -8, and -9 mouse IgG and antimouse IgG-FITC. Cell nuclei were stained with 0.1 µg/mL of Hoechst 33342 (Promega) for 5 min. The protein expressions (Ex, 495 nm; Em, 525 nm) and the cell nuclei (Ex, 346 nm; Em, 460 nm) were measured from three independent experiments by Multi-Detection Microplate Reader (Synergy2, BioTek Instruments, Inc.). The fluorescence percentage is defined in terms of FITC intensity/Hoechst 33342 intensity when the cells were treated with the designated drug concentration. For flow cytometry analysis, cells (1.5 × 105 cells/mL) were seeded in 6-well plates and treatment with AE (IC50 and IC80 concentrations) for 72 h. For the 5-Fu combined AE experiments, the cells were separately treated with IC50 concentrations of 5-Fu, AE, and 5-Fu plus AE for 72 h. Then, the cell suspension was centrifuged at 300g for 5 min. The supernatant was removed, and

AE/Liposome-AE Interacted with Nonmelanoma Skin Cancer Cells cells were fixed by incubation with ice-cold 4% para-formaldehyde in 1× PBS (pH 7.4) for 30 min at 4 °C. After centrifugation at 300g for 5 min at 4 °C, cells were resuspended in 200 µL of 1× PBS. Cells were then stained with antihuman TNF-R1, TNF-R2, Fas, TRADD, FADD, p53, Bax, Bcl-2, cytochrome c, and cleaved caspase-3, -8, and -9 monoclonal antibodies in 1× PBS containing 0.5% BSA (PBS-BSA) and 0.1% sodium azide (Sigma-Aldrich) as the primary antibodies for 1 h at 4 °C. The cells were washed twice with ice-cold 1× PBS and incubated with FITC-conjugated human IgG (1:500) (Santa Cruz, CA) as the secondary antibodies at 4 °C for 1 h. The negative controls, the primary antibodies, were replaced with 1× PBS under the same conditions. Two additional washing steps with ice-cold 1× PBS were performed before the cells were analyzed by a FACScan flow cytometer (BectonDickinson), and a minimum of 10000 events was acquired for analysis by WinMDI software. Determination of Intracellular ROS and GSH Levels. To evaluate intracellular ROS levels, a fluorometric assay using intracellular oxidation of 2′,7′-dichlorofluorescein diacetate (DCFHDA) was performed. The nonpolar DCFH-DA is converted to the polar derivative DCFH by esterases when it is taken up by the cell. DCFH is nonfluorescent but is rapidly oxidized to the highly fluorescent dichlorofluoroscein (DCF) by intracellular H2O2 or nitric oxide. The GSH content was determined, measuring the formation of a fluorescent complex of o-phthalaldehyde (OPA) with reduced GSH. To investigate the relationship between the increased ROS and the level of antioxidant materials in cells, the intracellular GSH level was determined. Briefly, 1.5 × 105 cells/well was exposed to AE with various concentrations and different incubation times. To measure ROS generation, after the medium was replaced, cells were incubation with DCFH-DA (10 µM) in the dark at 37 °C for 1 h. To evaluate GSH content, cells were washed with 1× PBS, and then, added was 50 µg/mL of OPA in 0.1 M Na2HPO4-5 mM EDTA buffer in the dark at 25 °C for 1 h. Cell nuclei were stained with 0.1 µg/mL of Hoechst 33342 solution for 5 min. After two additional washing steps with 1× PBS, the DCF fluorescence (Ex, 504 nm; Em, 524 nm), GSH fluorescence (Ex, 355 nm; Em, 460 nm), and the cell nuclei (Ex, 346 nm; Em, 460 nm) were measured from three independent experiments by a Multi-Detection Microplate Reader (Synergy2, BioTek Instruments, Inc.) and photographed by fluorescent microscope (Nikon, TE2000-U). The fluorescence percentage is defined in terms of DCF intensity/ Hoechst 33342 intensity and GSH intensity/Hoechst 33342 intensity when the cells were treated with the designated drug concentration. Immunoblot Analysis. Cells were separately treated with IC50 concentrations of 5-Fu, AE, and 5-Fu plus AE for 72 h, and the protein expressions of cleaved caspase-3, -8, and -9 were determined by immunoblot analysis. Cells (1 × 106) were lysed at 4 °C in 400 µL of lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 1% Nonidet P-40, 100 mM sodium fluoride, and 5 mM EDTA) and protease inhibitor mix Complete (Boehringer Mannheim). The lyses were centrifuged to remove cellular debris. The protein concentration of the extracts was determined using an ESL protein assay (Boehringer Mannheim) with BSA as the standard. Cell lysates (40 µg) were fractionated on a 12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA). After blocking with 5% skim milk, the membranes were probed with mouse anticleaved caspase-3, -8, and -9 monoclonal antibodies (10 µg/mL) (Cell Signaling Technology) and β-actin antibodies (1 µg/ mL) (Santa Cruz, CA), respectively. The membranes were incubated with the appropriate secondary antibodies conjugated horseradish peroxidase (Bio-Rad, Hercules, CA) in 1:3000 dilutions for 1 h and subjected to ECL detection (Amersham, Piscataway, NJ) and autoradiography by standard procedures. Preparation of Liposome-AE. The liposomal formulation encapsulated AE was prepared by the thin film hydration method as described elsewhere (15) The liposome system investigated here was composed of HSPC, Chol, and DHDAB in the designed molar ratio of 7/3/1 with 5 mol % (drug/total lipid) of AE (liposomeAE). All constituents of liposomal membrane were weighted and dissolved in the chloroform/methanol (1/1, v/v) cosolvent. Then,

Chem. Res. Toxicol., Vol. 22, No. 12, 2009 2019 the organic solvent was removed by rotary evaporation for 60 min at 50 °C to gain a homogeneous thin film. The thin film was kept dried under vacuum for an additional 20 min to remove the residual trace amount of the organic solvent and then was hydrated in pure water for 20 min at 60 °C. After hydration, the vesicle diameter was reduced through a high-intensity ultrasonic instrument (Sonicator3000, combined with a cup horn, Misonix Inc., United States) without directly contacting the titanium probe for 40 min at 60 °C. The total final concentration of the liposomal composition without AE was fixed at 5 mM. Particle Size and ζ-Potential Distributions of Liposome-AE. The particle size distributions of liposome-AE (average hydrodynamic diameter, Dz, and polydispersity, P.I.) were determined by a 90 Plus Nanoparticle Size Analyzer (Brookhaven Instrument Corp., Holtsville, NY) equipped with a laser (λ ) 570 nm, 35 mW) and a scatter angle of 90° detection optic. Furthermore, the ζ-potential of the liposome dispersion was also measured at 25 °C by means of the same instrument upgraded with a ζ-potential analyzer. All samples were further diluted in an appropriate multiple (×10) for each particle size and ζ-potential measurement. In Vitro Skin Permeation and Skin Deposition Studies of Liposome-AE, Free AE Dispersion, and Empty Liposomes with Free AE. The 6 week old ICR mice (25-30 g) were sacrificed by means of ether, and the full thickness skin was excised from the dorsal region. Skin permeations were performed by using locally made Franz diffusion cells with an effective permeation area and receptor cell volume of 0.785 cm2 and 4 mL, respectively. The temperature was maintained at 37 °C. The receptor was filled with a 0.9% NaCl solution and was constantly stirred by a magnetic stirrer at 600 rpm. A skin was mounted on a receptor compartment with the stratum corneum side facing upward into the donor compartment. The 1 mL of sample was applied on the skin in the donor chamber, which was then covered with a parafilm to avoid any evaporation occurrence. At predesigned time intervals, the receptor medium of 300 µL was withdrawn and immediately replenished with an equal volume of fresh medium. The amount of AE was determined by the HPLC method (6). Triplicate experiments were performed for each study. The amount of AE retained in the skin was determined at the end of each in vitro permeation experiment (7 h). The skin was washed 10 times using a cotton cloth immersed in methanol. A sample of skin was weighed, cut with scissors, placed into a glass homogenizer containing 1 mL of methanol, and homogenized for 5 min with an electric stirrer. The resulting solution was centrifuged at 8000 rpm for 10 min. The supernatant was analyzed for AE content by HPLC. HPLC Assay. The HPLC analysis for AE was carried out by using a mobile phase of methanol-water-acetic acid (65:35:0.2) at a flow rate of 1.2 mL/min. The HPLC equipment, comprised of a model L2200 autosampler, a model L2100 pump, and a fluorescence detector model L2485, was obtained from Hitachi (Hitachi High-Technologies Corp., Japan). Twenty microliters of injection volume was eluted in Nucleosil 100-5 C18 (4.6 mm × 250 mm, 5 µm particle sizes) column (MACHEREY-NAGEL GmbH & Co., KG, Germany) at room temperature. The peak was detected with a fluorescence detection at λex ) 410 nm and λem ) 510 nm. Encapsulation Efficiency of Liposome-AE. Prepared liposomeAE was isolated from the free AE by a gel filtration with Sephadex G 50 (Sigma-Aldrich Inc.). An aliquot of liposomes (100 µL) was loaded, a fraction was collected every 1 mL, and then, the drug signal in each tube was determined by a fluorescence detector as described above. The concentration of free drug fraction dissolved in methanol was measured by HPLC. The AE concentration in liposome was also analyzed by HPLC after liposomal fractions dissolved in pure methanol. It was found that AE of 5 mol % (AE/ total lipids) can be completely encapsulated into the liposomal formulation (HSPC/Chol/DHDAB ) 7/3/1) used in this study. Combination Data Analysis by the Isobologram Method. The data obtained from the MTS assays in response to the combinations of 5-Fu with AE were subjected to isobologram analysis (16). The

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Figure 1. Dose and time dependency of the inhibition of cell viability by treating nonmelanoma skin cancer cells with AE. (A) Cell viability of AE in skin cancer (A431, SCC25, and A375 cells) and noncancerous (HaCaT and Hs68) cells for 72 h. (B) The IC20, IC50, and IC80 (µM) values obtained from MTS assays of various skin cell lines. (C) Time course of cell death after treatment with constant amounts of AE (IC20, IC50, and IC80) in A431 and SCC25 cells. The percentage of cell viability under AE-mediated cytotoxicity was determined by MTS assay. Each value is presented as the mean ( SD of the percentage cell proliferation determined in triplicate experiments. (D) Morphological changes in A431 and SCC25 cells after AE (IC20, IC50, and IC80) treatment. The change was inspected by light microscopy (200×).

isobologram method relies on the calculation of the combined concentrations of 5-Fu and AE that cause a given effect like 20, 50, and 80% of growth inhibition. For each combination of drug concentrations (D5-FuX, DAEX), producing in combination the effect X, the combination index (CI) was calculated as follows: CI ) D5-FuX/ICX,5-Fu + DAEX/ICX,AE, where ICX,5-Fu and ICX,AE are the concentrations of each individual drug that would produce the effect X if given alone. In this study, the CI values obtained from all experiments with a given cell line were pooled, and the mean and variance were calculated at the 20, 50, and 80% cell survival levels. A CI value 1 indicates antagonism. Statistical Analysis. The experimental results were presented as means ( SDs. Statistically significant differences were determined using independent and paired Student’s t test on unpaired and paired samples, respectively. When a control group was compared with more than one experimental group, one-way analysis of variance (ANOVA) or two-way repeated measures of ANOVA were used. Whenever ANOVA data revealed statistical differences, Dunnett’s test or of the Student-Newman-Keuls test was performed. P < 0.05 was regarded as significant in all experiments. Data were analyzed, and relevant figures were plotted using SigmaPlot software, Version 8.0 (San Rafael, CA).

Results Cytotoxic Activity of AE in Skin Cancer Cells. The cytotoxic potential of AE at increasing concentrations of 0-200 µM was evaluated on cells growing exponentially over a period of 72 h by the MTS assay. The AE had a specific dose-dependent cytotoxic effect on human epidermoid carcinoma A431 cells and human head and neck SCC SCC25 cells (Figure 1A). The

proliferation of skin cancer cell lines was specifically inhibited; the AE concentrations that induced cell death by 20, 50, and 80% (IC20, IC50, and IC80) were approximately 14.4 ( 2.1, 25.9 ( 1.7, and 87.9 ( 3.4 µM for A431 and 6.8 ( 1.5, 19.3 ( 1.7, and 45.3 ( 2.3 µM for SCC25 cells, respectively (Figure 1B). Conversely, human skin melanoma A375 cells, human immortal keratinocytes HaCaT, and nontransformed human skin fibroblast Hs68 cells were largely refractory to AE treatment, and IC50 values ranged between 84.8 and >200 µM (Figure 1B), indicating that AE inhibition of cell growth is more significant for human skin epidermoid and SCC cells than melanoma and noncancerous cells. Treatment of A431 and SCC25 cells with serial AE concentrations (IC20, IC50, and IC80) for 24, 48, and 72 h demonstrated that inhibition of cell proliferation was timedependent (Figure 1C). Additionally, after treatment with IC20, IC50, and IC80 AE concentrations for 72 h, cells had morphological features typical of apoptotic cell death, such as cell shrinkage, rounded cell bodies, membrane blebbing, and nuclear condensation (Figure 1D), indicating cell death by apoptosis. Effect of AE on Cell Cycle and Apoptosis. To determine the stage at which AE-induced growth inhibition occurs in cell cycle progression, apoptosis was further confirmed by the detection of a sub-G1 peak by flow cytometry after AE treatment for 24, 48, and 72 h (IC50 and IC80 concentrations). Treating cells with IC50 and IC80 AE concentrations suggests that the main characteristic of apoptosis is cleavage of nuclear DNA into small fragments, causing a dose-dependent increase in the sub-G1 phase (Figure 2A). Additionally, Figure 2B shows the relative proportion of cell cycle distribution. The increases in

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Figure 2. Cell cycle distribution and apoptotic nonmelanoma skin cancer cells after AE treatments. (A) Flow cytometric analysis of cell cycle progression of A431 and SCC25 cells after treatment with IC50 and IC80 concentrations of AE for 24, 48, and 72 h. The M1 region represents signals from apoptotic cells with reduced DNA content sub-G1 peak; the M2, M3, and M4 regions are signals from cells in the G0/G1, S, and G2/M phases, respectively. (B) The percentage cell distribution in each phase (sub-G1, G0/G1, S, and G2/M) of the cell cycle was determined using WinMDI software. Data are given as means ( SDs from three independent experiments.

Figure 3. AE up-regulated the expressions of receptor-mediated death pathways in nonmelanoma skin cancer cells. (A) The A431 and SCC25 cells were treated with a constant amount of AE (IC50) for 72 h. The expressions of TNF-R, TNF-R1, TNF-R2, FasL, Fas, TRADD, and FADD in cells were determined by immunofluorescent analysis and observed using a fluorescent microscope (200×). Nuclei were visualized by staining DNA with Hoechst 33342 (blue). Protein expressions were stained with FITC (green). For quantitative analysis of receptor-operated death proteins, the IC50 and IC80 concentrations of AE were added to A431 and SCC25 cells for 72 h, and the levels of fluorescent-stained receptor-mediated protein expression were determined by a fluorescent microplate reader (B) and flow cytometry (C). The light gray line implied the negative control, the deep gray line is the control, and the black line is AE treatment. *P < 0.05 as compared to control values.

AE-induced (IC50 concentration) sub-G1 of A431 and SCC25 cells were 19.2-36.8 and 20.3-49.5%, and those for AEinduced (IC80 concentration) sub-G1 peaks were 19.9-85.8 and 30.7-75.0% at 24, 48, and 72 h. Moreover, changes to other cell cycle phases were observed. The relative proportion of both cells in the S-G2/M phase increased with a marked apoptotic

sub-G1 peak after treatment with an IC50 concentration of AE for 24 and 48 h (Figure 2B), indicating that a low concentration of AE and short exposure time (24 and 48 h) may arrest the cell cycle during the S-G2/M phase and induce apoptosis of G0/ G1 cells. However, the cell population in the G0/G1 and S-G2/M phases was reduced concurrently as the sub-G1 population in

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Figure 4. AE induces nonmelanoma skin cancer cell apoptosis through the mitochondria-dependent oxidative stress signaling pathway. (A) A constant amount of AE (IC50) was added to A431 and SCC25 cells for 72 h. The expressions of p53, cytochrome c, Bax, and Bcl-2 in cells were determined by immunofluorescent analysis and using a fluorescent microscope (200×). Nuclei were visualized by staining DNA with Hoechst 33342 (blue). Protein expressions were stained with FITC (green). For quantitative analysis of mitochondria-mediated protein expressions, the IC50 and IC80 concentrations of AE were added to A431 and SCC25 cells for 72 h, and the percentage of fluorescent-stained mitochondria-mediated protein expression levels was analyzed by a fluorescent plate reader (B) and flow cytometry (C). The light gray line implies the negative control, the deep gray line is the control, and the black line is AE treatment. To determine the effects of AE on intracellular ROS activities and GSH production in A431 and SCC25 cells, the IC50 and IC80 concentrations of AE were treated with cells for 24, 48, and 72 h, and the fluorescence percentage was defined by fluorescent DCF/Hoechst 33342 (nucleus) intensity (D) and GSH/Hoechst 33342 (nucleus) intensity (E). Treatment with IC80 concentrations of AE for 72 h elevated ROS and GSH expressions in A431 and SCC25 cells and were observed by fluorescence microscopy (200×). *P < 0.05 as compared to control values.

both cells increased after incubation with an IC80 concentration of AE for 72 h. This experimental finding implies that a high AE concentration and long incubation time (72 h) may cause cell cycle sensitivity during the S-G2/M phase, generally inducing cell apoptosis. AE Induces Apoptosis of Skin Cancer Cells Via the Receptor- and Mitochondria-Dependent Pathways. Notably, AE is capable of inducing apoptosis in human skin cancer cells (4). Here, the cell death signaling pathway is further investigated. After A431 and SCC25 cells were treated with IC50 and IC80 concentrations of AE for 72 h, AE dose dependently upregulated receptor-mediated pathway protein expressions by death ligands TNF-R and FasL to their receptors, TNFRs and Fas, and triggered apoptosis by recruiting adaptor proteins TRADD and FADD, as determined by fluorescent immunocytochemistry (Figure 3A), quantitative analysis of fluorescent staining (Figure 3B), and flow cytometry (Figure 3C). The tumor suppressor p53 protein is a critical checkpoint for activation of the intrinsic pathway: p53 responds to various cellular stresses by arresting cell cycle progression (17). In the context of extensive damage that cells cannot repair, p53 promotes apoptosis via expression of apoptotic proteins (e.g., Bax) or antiapoptotic proteins (e.g., Bcl-2) (18). Furthermore, the ROS generated may contribute to mitochondrial damage, reduction of mitochondrial transmembrane potential, release of cytochrome c, and subsequent caspase activation and apoptosis (9). To determine the degree of mitochondrial involvement in the pathway leading to AE-induced apoptosis, A431 and SCC25

cells were exposed to IC50 and IC80 AE concentrations for 72 h, resulting in up-regulation of p53, cytochrome c, and apoptotic Bax and down-regulation of antiapoptotic Bcl-2 expression, as determined by fluorescent immunocytochemistry (Figure 4A), quantitative analysis of fluorescent staining (Figure 4B), and flow cytometry (Figure 4C). These analytical findings suggest that p53 up-regulation is involved in AE-induced G2/M cellcycle arrest in A431 and SCC25 cells (Figure 2B). To demonstrate the role ROS play in AE-induced apoptosis of A431 and SCC25 cells, production of ROS was examined using an oxidant-sensitive fluorescent probe, CM-H2DCFDA. Experimental data demonstrate that treatment with IC50 and IC80 concentrations of AE significantly increased the intensity of the DCF signal, as compared with those in the control after treating A431 cells with AE for 24, 48, and 72 h (Figure 4D). Therefore, treatment with IC80 concentrations of AE for 72 h elevated the expression of ROS in A431 and SCC25 cells, as observed by fluorescence microscopy. However, the initial phase of the AE response had a markedly greater increase in ROS activity as compared with those of control cells at 24 h as AE treatment of SCC25 cells for 48 and 72 h (Figure 4D). Moreover, treating A431 and SCC25 cells with IC50 and IC80 concentrations of AE for 24 h did not influence the intracellular GSH concentration; however, GSH activity decreased as treatment time increased to 48 and 72 h (Figure 4E). This GSH depletion is clearly in agreement with high ROS levels detected after treating A431 and SCC25 cells with AE. Experimental results indicate

AE/Liposome-AE Interacted with Nonmelanoma Skin Cancer Cells

Chem. Res. Toxicol., Vol. 22, No. 12, 2009 2023

Figure 5. AE-induced apoptosis involved the caspases pathway in nonmelanoma skin cancer cells. (A) The A431 and SCC25 cells were treated with IC50 concentrations of AE for 72 h, and the expressions of caspase-8, -9, and -3 were determined by immunofluorescent analysis and using a fluorescent microscope (200×). Nuclei were visualized by staining DNA with Hoechst 33342 (blue). Caspase expressions were stained with FITC (green). The IC50 and IC80 concentrations of AE were added to both cells for 72 h. The percentage of fluorescent-stained caspase expression levels was analyzed by quantitative analysis of fluorescent staining (B) and flow cytometry (C). The light gray line implies the negative control, the deep gray line is the control, and the black line is AE treatment. *P < 0.05 as compared to control values.

that AE induced apoptosis by generating oxidative stress signaling pathways in skin cancer cells. Caspase, a family of cysteine proteases, profoundly impacts apoptosis. Caspase-8 and -9 have been proposed as initiator caspases, which directly or indirectly activate caspase-3 (19). Exposure of A431 and SCC25 cells to IC50 and IC80 concentrations of AE for 72 h significantly increased caspase-8, -9, and -3 activities, as determined by fluorescent immunocytochemistry (Figure 5A), quantitative analysis of fluorescent staining (Figure 5B), and flow cytometry (Figure 5C). These analytical results suggest that AE may create cross-links between apoptotic pathways via both the signaling pathways of death receptors and the mitochondria-operated caspases. Effect of 5-Fu Combined with AE on A431 and SCC25 Cells. The effect of 5-Fu (0-200 µM) on the growth inhibition of A431 and SCC25 cells for 72 h was elucidated by the MTS assay (Figure 6A). The 5-Fu concentrations that induced cell death by 20, 50, and 80% (IC20, IC50, and IC80) were approximately 0.7 ( 5.3, 9.8 ( 4.2, and 128.5 ( 9.8 µM for A431 cells and 4.1 ( 3.2, 57.7 ( 2.6, and 259.0 ( 3.7 µM for SCC25 cells, respectively (Figure 6B). Moreover, increasing doses of 5-Fu plus AE at IC20, IC50, and IC80 concentrations were added to cells for 24, 48, and 72 h to investigate the effect of AE on 5-Fu-induced death of A431 and SCC25 cells. The combination of 5-Fu and AE markedly enhanced the cytotoxicity to both A431 and SCC25 cells (Figure 6C). Drug interactions were assessed using the CI value, which was calculated using the concentrations of drug pairs that inhibited cell viability by 20, 50, and 80%. After treatment for 72 h, the combination of 5-Fu plus AE at IC20, IC50, and IC80 concentrations applied to A431 and SCC25 cells on growth inhibition and the CI20, CI50, and CI80 values of 5-Fu plus AE are approximately 0.76, 0.60, and

0.68 for A431 cells and 0.95, 0.86, and 1.11 for SCC25 cells, respectively. The CI20 and CI50 values of this drug combination for both skin cancer cells were