Journal of Biomedical Science (2007) 14:87–105 DOI 10.1007/s11373-006-9130-6 Toxicogenomics of A375 human malignant melanoma cells treated with arbutin Sun-Long Cheng1, Rosa Huang Liu2, Jin-Nan Sheu1, Shui-Tein Chen3,4, Supachok Sinchaikul3 & Gregory Jiazer Tsay5,* 1Institute of Medicine, Chung Shan Medical University, Taichung, 40242, Taiwan; 2School of Nutrition, Chung Shan Medical University, Taichung, 40242, Taiwan; 3Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei, 11529, Taiwan; 4Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, 10617, Taiwan; 5Institute of Immunology, Chung Shan Medical University, 110 Sec. 1, Chien-Kuo N. Road, Taichung, 40242, Taiwan Received 17 August 2006; accepted 10 October 2006 Ó 2006 National Science Council, Taipei Key words: arbutin, A375 cells, toxicogenomics, DNA microarray, gene expression Summary Although arbutin is a natural product and widely used as an ingredient in skin care products, its effect on the gene expression level of human skin with malignant melanoma cells is rarely reported. We aim to investigate the genotoxic effect of arbutin on the differential gene expression profiling in A375 human malignant melanoma cells through its effect on tumorigenesis and related side-effect. The DNA microarray analysis provided the differential gene expression pattern of arbutin-treated A375 cells with the significant changes of 324 differentially expressed genes, containing 88 up-regulated genes and 236 down-regulated genes. The gene ontology of differentially expressed genes was classified as belonging to cellular component, molecular function and biological process. In addition, four down-regulated genes of AKT1, CLECSF7, FGFR3, and LRP6 served as candidate genes and correlated to suppress the biological processes in the cell cycle of cancer progression and in the downstream signaling pathways of malignancy of melanocytic tumorigenesis. Introduction study of its biological effect on human genomics level in the regulation of malignant melanogenesis Arbutin, a natural compound of beta-D-glucopyr- through the functional effect on carcinogenesis is anoside of hydroquinone, is widely used as an not clear and rarely reported. ingredient in skin care products [1]. It is effective in Recently, the high throughput technology of the treatment of various cutaneous hyperpigmen- DNA microarray has become important in geneo- tations and has the inhibitory effects on the mic research and used to analyze gene expression melanogenesis in melanoma cells [2–4]. However, profiling of human melanomas [5–7]. Since the recent findings have raised serious questions of study of gene expression profiling in human concern regarding both the safety and side-effect melanoma cells treated with arbutin has never of arbutin. Although some inhibitory effects of been reported, it would be very challenging to use arbutin on melanogenesis in melanoma cells and the DNA microarray in studying the biological its mechanism have been elucidated, the intensive effect of arbutin on malignant melanoma cells through its action on anti-cancer property. In this *To whom correspondence should be addressed. Tel: +886-4- study, we aim to investigate the genotoxic effect of 24738170; Fax: +886-4-23248172; E-mail: [email protected] arbutin on the differential gene expression profiling 88 in A375 human malignant melanoma cells through bicarbonate, 1 mM sodium pyruvate and 1% its effect on melanocytic tumorigenesis for cancer antibiotic-antimycotic in a humidified incubator therapy and other related side-effects. We used the with 5% CO2 and 95% air at 37 °C. Upon DNA microarray technology and bioinformatic confluence, the cells were detached by treatment tools to explore the differential gene expression with 0.05% trypsin and 0.53 mM EDTA. During profiling in arbutin-treated A375 cells and to subculture, the medium was replaced every classify the differentially expressed genes by Gene 2–3 days. Oncology, which provided more understanding of the genetic basis of metabolic and cellular Treatment with arbutin and cell viability test responses in tumergenesis of human skin cancer. In addition, we compared the biological effects To perform cell attachment, the cells were seeded of arbutin and kojic acid on differentially at 5.0 Â 103 cells/well in 96-well tissue culture- expressed genes in A375 cells and also proposed treated plates (NUNCTM, Roskilde, Denmark) in the hypothetical pathway of arbutin-responsive 100 ll of culture media. They were then washed genes correlated in the suppression of tumorigen- with PBS and were cultured either alone or in the esis. These candidate genes may lead to conse- presence of various concentrations of arbutin quently aid for early diagnostic and therapeutic (0.32, 1.6, 8, 40, 200 and 1000 lg/ml) for 72 h. applications. Arbutin was suspended in 0.01% DMSO, and the control was added with the same concentration of DMSO in order to reduce the variation of cell Materials and methods growth inhibition. Triplicate experiments were done for each pair group of arbutin concentration Materials and control. The cell viability was determined by MTT assay [8]. The percentage of cell growth Human malignant melanoma cell line, A375 inhibition was calculated as follows: Inhibition (CRL-1619), was obtained from the ATCC (Rock- (%) = [1-A550 nm (arbutin)/A550 nm (con- ville, MD, USA). Dulbecco’s Modified Eagle’s trol)] Â 100%. Medium (DMEM), D-PBS and Trypsin-EDTA were purchased from Atlanta Biologicals (Nor- RNA preparation and quantitative measurement cross, GA, USA). Fetal bovine serum, sodium pyruvate, antibiotic-antimycotic and TRIzol re- After the cells were treated with 8 lg/ml arbutin agent were purchased from Invitrogen (Carlsbad, for 24 h, RNA was extracted by a modified CA, USA). Arbutin, MTT (methylthiazoletetrazo- method using TRIzol combined with the RNeasy lium) and sodium bicarbonate were purchased Mini Kit. The total RNA was quantified by a UV from Sigma (St. Louis, MO, USA). RNeasy Mini spectrophotometer and RNA quality was evalu- Kit was purchased from Qiagen (Valencia, CA, ated by capillary electrophoresis on an Agilent USA). Cyanine 3- and 5-labeled CTP were pur- 2100 Bioanalyzer using RNA 6000 Nano LabChip chased from Perkin-Elemer/NEN Life Science Kit. For each paired sample, the number of (Boston, MS, USA). RNA 6000 Nano LabChip independent paired samples of cultured cells that Kit, Low RNA Input Fluorescent Linear Ampli- were treated with arbutin either present or absent fication Kit, Human 1A Oligo Microarray Kit was done in triplicate. (V2), in situ Hybridization Kit Plus, and Stabil- ization and Drying Solution were purchased from RNA amplification and labeling Agilent Technologies (Palo Alto, CA, USA). All other chemicals were purchased from Sigma Targets of cRNA were amplified and fluorescently (St. Loius, MO, USA). labeled from 0.5 lg total RNA in each reaction using the Agilent Low RNA Input Fluorescent Cell culture Linear Amplification kit. For each sample pair, the control sample was labeled with Cy3 and the A375 cells were cultured in DMEM supplemented treated sample was labeled with Cy5. After puri- with 10% fetal bovine serum, 1.5 g/l sodium fication using Qiagen’s RNeasy mini-spin 89 columns, the quantification, quality and size databases: NCBI (http://www.ncbi.nlm.nih.gov), distribution of the labeled cRNA targets were Ensembl (http://www.ensembl.org), TIGR (http:// then determined by ultraviolet (UV) spectropho- www.tigr.org) and GeneCards (http://www.gene tometry and RNA 6000 Nano LabChip Assay. cards.org). In addition, the category classification of gene expression was done by in-house Bulk Microarray hybridization Gene Search System for Java (BGSSJ) program that is a searching system accomplished by open Hybridization was performed following the Agi- database connectivity, UniGene database and lent oligonucleotide microarray hybridization Gene Ontology knowledgebase, and is available user’s manual and Agilent in situ Hybridization at http://servx8.sinica.edu.tw/bgss-cgi-bin/gene.pl. Kit Plus. Briefly, 2 lg of the labeled cRNA per On the other hand, the protein search program channel was mixed with 50 ll10Â control targets used the Swiss-Prot/TrEMBL (http://www.exp- and nuclease-free water to a final volume of 240 ll. asy.ch/sprot), Proteome (http://www.prote- Each sample tube was added with 10 llof ome.com/databases/HumanPD/reports) and 25Â fragmentation buffer and was incubated at PubMed (http://www.ncbi.nl.nih.gov/PubMed). 60 °C in a water bath in the dark for 30 min. The Moreover, the combining pathway databases of reaction was then terminated by adding 250 llof Biocarta (http://www.biocarta.com), KEGG 2Â hybridization buffer. A volume of 500 llof (http://www.genome.ad.jp/kegg/pathway.html) hybridization mix was applied to Agilent’s Human and the PubMed literature were used to search the 1A Oligonucleotide Microarray, containing 20,173 correlated signaling pathways and mechanisms in (60 mer) oligonucleotide probes, and hybridized in the malignant melanogenesis of A375 cells. a hybridization rotation oven at 60 °C for 17 h. The slides which disassembled in 6Â SSPE and Real-time quantitative PCR (RT-qPCR) 0.005% N-Lauroylsarcosine were washed with 6Â SSPE, 0.005% N-Lauroylsarcosine for 1 min at Specific oligonucleotide primer pairs were room temperature, then with 0.06Â SSPE, 0.005% designed using the analysis Beacon designer 4.00 N-Lauroylsarcosine for 1 min and with Stabiliza- (Premier Biosoft International) and were then used tion and Drying Solution for 30 s. for real-time quantitative PCR. The sequences of the primers used are: (1) AKT1 (NM_005163, Data analysis and bioinformatics 150 bp): forward 5¢-ACCATCACACCACCTG- ACCAAG-3¢ and reverse 5¢-CGCCTCTCCATC- The microarray chip was scanned using an Agilent CCTCCAAG-3¢; (2) CLECSF7 (NM_130441, G2565BA Microarray Scanner System, and the 184 bp): forward 5¢-ATCAACACCAGGGAAG- Agilent Feature Extraction software 7.5 used AACAGG-3¢ and reverse 5¢-TCGCACAACGCT- defaults for all parameters including a parameter- CAT CAAGG-3¢; (3) FGFR3 (NM_000142, ized error model to compute the significance 102 bp): forward 5¢-CTGCCAGCCGAGGAG- (p-values) of log ratios.