April 2006 Biol. Pharm. Bull. 29(4) 655—669 (2006) 655

Toxicogenomics of Kojic Acid on Gene Expression Profiling of A375 Human Malignant Melanoma Cells

a b a c,d c Sun-Long CHENG, Rosa HUANG LIU, Jin-Nan SHEU, Shui-Tein CHEN, Supachok SINCHAIKUL, and ,e Gregory Jiazer TSAY* a Institute of Medicine, Chung Shan Medical University; Taichung, 402, Taiwan: b School of Nutrition, Chung Shan Medical University; Taichung, 402, Taiwan: c Institute of Biological Chemistry and Genomics Research Center, Academia Sinica; Taipei, 115, Taiwan: d Institute of Biochemical Sciences, College of Life Science, National Taiwan University; Taipei, 106, Taiwan: and e Institute of Immunology, Chung Shan Medical University; Taichung, 402, Taiwan. Received November 5, 2005; accepted December 27, 2005

Kojic acid is a natural product and normally used as a food additive and preservative, a skin-whitening agent in cosmetics, a plant growth regulator and a chemical intermediate. Using DNA microarray technology, the overall biological effects of kojic acid on the gene expression profiling of a human skin A375 malignant melanoma cells were examined. After treatment with kojic acid, a total of 361 differentially expressed genes were distinctively changed with 136 up-regulated genes and 225 down-regulated genes. We used the bioinformatics tool to search the gene ontology and category classification of differentially expressed genes that provided the useful information of expressed genes belonging to cellular component, molecular function and biological process in regulation of melanogenesis. Seven down-regulated genes of APOBEC1, ARHGEF16, CD22, FGFR3, GALNT1, UNC5C and ZNF146 that were typically validated by the real-time quantitative PCR (RT-qPCR) analysis technology showed to be the tumor suppressor genes in melanoma cancer cells. Thus, microarray tech- nology coupled with RT-qPCR offered a high throughput method to explore the number of differentially ex- pressed genes responding to kojic acid and their biological functions, and led to more understanding of kojic acid effects on skin cancer therapy and related side effects. Moreover, the differentially expressed genes may be- come useful markers of skin malignant melanoma for further diagnostic and therapeutic applications. Key words kojic acid; A375 cell; melanoma; DNA microarray; gene expression

Kojic acid (5-hydroxy-2-hydroxymethyl-1,4-pyrone) is a been studied. secondary metabolic product and widely used as a food addi- Recently a high throughput DNA microarray technique tive for preventing enzymatic browning of raw crabs and has an important role for genomics research by allowing the shrimps,1—3) and as a skin lightening or bleaching agent for a study of the function of thousands of genes simultaneously, long time in cosmetic preparations.4—7) Used as an important showing differential gene expression profiling, and opening material in antibiotic and pesticide productions,3,8,9) kojic the door to the discovery the biomarkers or special gene acid has been shown to act as a competitive and reversible in- markers that intend to use for pharmaceutical applications hibitor of animal and plant polyphenol oxidases, xanthine ox- and disease therapy.26—30) In this study, we used the DNA mi- 10—16) idase, and D- and some L-amino acid oxidases. About croarray technology to investigate the biological effects of the toxicological aspect, the acute or subchronic toxicity re- kojic acid on the differential gene expression profiling of sulting from an oral dose of kojic acid has never been re- A375-malignant melanoma cells as a model of human skin ported, but convulsions may occur if kojic acid is injected. cells in order to understand the genetic basis of metabolic Continuous administration of high doses of kojic acid in and cellular responses to human skin and to discover the tar- mice resulted in induction of thyroid adenomas in both get-based drug of new genes leading to a systematic drug de- sexes.8,16,17) Moreover, some reports showed the evaluation of velopment approach. In addition, we used the bioinformatics the tumorigenic potential of kojic acid and study the geno- tool to search the gene ontology that provided the useful in- toxic risk for humans using kojic acid as a skin-lightening formation of category classification, such as cellular compo- agent.11,18—20) On the other hand, kojic acid at high dose has nent, molecular function and biological process. Moreover, some side effects; for example, it affected thyroidal function we found that seven significantly expressed genes play an im- when given at a massive dose or in a long administration pe- portant role in the cancer suppression in human skin riod by inhibiting iodine organification in the thyroid, leading melanoma cells that may become the useful markers for di- to decreases in triiodothyronine (T3) and thyroxine (T4) and agnostic and therapeutic applications. increase in thyroid-stimulating hormone (TSH).21—24) Re- cently, kojic acid is found to be a tumor promoter and can MATERIALS AND METHODS also enhance the hepatocarcinogenesis in rat as well as in mice.25) Moreover, the topical use of kojic acid as a skin Materials Human malignant melanoma cell line A375 lightening agent results in minimal exposure that poses no or (CRL-1619) was obtained from ATCC (Rockville, MD, negligible risk of genotoxicity or toxicity to the consumer.18) U.S.A.). Dulbecco’s Modified Eagle’s Medium (DMEM), D- Although kojic acid has been widely studied in the tumori- PBS and Trypsin-EDTA were purchased from Atlanta Bio- genic potential, genotoxic risk for humans and other applica- logicals (Norcross, GA, U.S.A.). Fetal bovine serum, sodium tions, the mechanism of kojic acid on the gene expression pyruvate, antibiotic-antimycotic and TRIzol reagent were and regulation in normal and/or diseased cells has rarely purchased from Invitrogen (Carlsbad, CA, U.S.A.). Kojic

∗ To whom correspondence should be addressed. e-mail: [email protected] © 2006 Pharmaceutical Society of Japan 656 Vol. 29, No. 4 acid, MTT (methylthiazoletetrazolium) and sodium bicarbon- RNA Amplification and Labeling Each total RNA ate were purchased from Sigma (MO, U.S.A.). RNeasy Mini from each paired sample treatment was pooled and amplified Kit was purchased from Qiagen. Cyanine 3- and 5-labeled to obtain the cRNA in order to avoid variations of each ex- CTP were purchased from Perkin-Elemer/NEN Life Science. periment in sample preparations or hybridization steps. RNA 6000 Nano LabChip Kit, Low RNA Input Fluorescent Briefly, targets of cRNA were amplified and fluorescently la- Linear Amplification Kit, Human 1A Oligo Microarray Kit beled from 0.5 mg total RNA in each reaction using the Agi- (V2), in situ Hybridization Kit Plus, and Stabilization and lent Low RNA Input Fluorescent Linear Amplification kit by Drying Solution were purchased from Agilent Technologies following the protocol described in the user’s manual. For (Palo Alto, CA, U.S.A.). All other chemicals were purchased each sample pair, control sample was labeled with Cy3 and from Sigma (MO, U.S.A.). treated with Cy5. Briefly, 1.2 ml of T7 promoter primer was Cell Culture A375, human malignant melanoma cells, added to a 1.5 ml microcentrifuge tube containing 0.5 mg were cultured in Dulbecco’s Modified Eagle’s Medium total RNA and final volume was adjusted to 11.5 ml with nu- (DMEM) supplemented with 10% fetal bovine serum, 1.5 g/l clease-free water. The primer and the RNA template were de- sodium bicarbonate, 1 mM sodium pyruvate and 1% antibi- natured at 65 °C in a heating block for 10 min and cooled on otic-antimycotic in a humidified incubator with 5% CO2 and ice for 5 min. A volume of 8.5 ml of cDNA master mix was 95% air at 37 °C. After reaching confluence, the cells were added to each reaction tube. The cDNA synthesis reaction detached by treatment with 0.05% trypsin–0.53 mM EDTA. mix contains 4 ml of 5 first strand buffer, 2 ml of 0.1 M DTT, During subculture, the medium was replaced every 2 to 3 d. 1 ml of 10 mM dNTP mix, 1 ml of MMLV-RT, and 0.5 ml of Pretreatment with Kojic Acid and Cell Viability Test RNaseOUT. Samples were incubated at 40 °C in a circulating A375 cells were re-suspended at 5.0104 cells/ml in culture water bath for 2 h, heated at 65 °C for 15 min (to inactivate medium, and seeded at 5.0103 cells/well in 96-well tissue MMLV-RT) and cooled on ice for 5 min. A volume of 2.4 ml TM culture-treated plates (NUNC , Roskilde, Denmark) in of 10 mM cyanine 3-CTP or cyanine 5-CTP was added to 100 ml of culture media for an overnight in order to perform each sample tube, followed by the addition of 57.6 ml of tran- cell attachment. The cells were then washed with PBS and scription master mix. The transcription master mix contained cultured either alone or in the presence of various concentra- 15.3 ml of nuclease-free water, 20 ml of 4 transcription tions of kojic acid (0.32, 1.6, 8, 40, 200, 1000 mg/ml) for buffer, 6 ml of 0.1 M DTT, 8 ml of NTP mix, 6.4 ml of 50% 72 h. The number of independent paired samples of cultured PEG, 0.5 ml of RNaseOUT, 0.6 ml of inorganic pyrophos- cells that were treated with kojic acid either present or absent phatase and 0.8 ml of T7 RNA polymerase. The reaction mix- was done in triplicate for each paired sample. MTT was used ture was then incubated at 40 °C for 2 h. Following the Qia- to assess the viability of cells following treatment.31) gen protocol; labeled cRNA was purified using Qiagen’s Aliquots of 10 ml of stock MTT solution (5 mg/ml) were RNeasy mini-spin columns. The cRNA product was eluted added to each well containing 100 ml of culture medium and with nuclease-free water from the column in 230 ml of final incubated with cells for 4 h. Following incubation, the volume. The quality and size distribution of cRNA targets medium was removed and 100 ml DMSO were added to dis- were determined by RNA 6000 Nano LabChip Assay, and solve the precipitates of reduced MTT. The absorbances at quantitation was determined by ultraviolet (UV) spectropho- 550 and 690 nm as reference were read on the VERSAmaxTM tometry. tunable microplate reader (Molecular Devices, Sunnyvale, Microarray and Hybridization Hybridization was per- CA, U.S.A.). The percentage of cell growth inhibition was formed following the Agilent oligonucleotide microarray hy- calculated as follows: inhibition (%) [1 A550 nm (kojic bridization user’s manual and Agilent in situ Hybridization acid)/A550 nm (control)] 100%. Kit Plus. Briefly, 2 mg of labeled cRNA per channel was RNA Preparation and Quantitative Measurement mixed with 50 ml 10 control targets and nuclease-free A375 cells were seeded at 1.0106 cells/dish in 10 cm tissue water to a final volume of 240 ml. Then 10 ml of 25 frag- culture dish (NUNCTM, Roskilde, Denmark) in 10 ml of cul- mentation buffer was added to each sample tube and reac- ture media for an overnight in order to perform cell attach- tions were incubated at 60 °C in a water bath, in the dark for ment. The cells were then washed with PBS and treated with 30 min. The addition of 250 ml of 2 hybridization buffer vehicle (DMSO, 0.01%) and kojic acid (8 mg/ml) for 24 h. terminated the reaction. A volume of 500 ml of hybridization RNA was extracted by a modified method using TRIzol (In- mix was applied to each of Agilent’s human 1A oligonu- vitrogen, Carlsbad, CA, U.S.A.) combined with RNeasy Mini cleotide microarray, containing approximately 20173 (60- Kit (Qiagen, Valencia, CA, U.S.A.). Briefly, total RNA was mer) oligonucleotide probes, which span conserved exons extracted with 1 ml of TRIzol reagent per 1106 cells or across the transcripts of 18716 targeted full-length genes 10 cm cell culture dish following the manufacturer’s instruc- (Agilent Tech., Mississauga, ON, Canada) and hybridized in tions. For each ml of TRIzol, the samples were added with a hybridization rotation oven at 60 °C for 17 h. The slides, 0.2 ml of chloroform, vigorously shaken for 15 s and then in- disassembled in 6 SSPE and 0.005% N-lauroylsarcosine, cubated at 15 to 30 °C for 2 to 3 min. The aqueous phase was were washed first with 6 SSPE, 0.005% N-lauroylsarcosine separated by centrifugation at 12000 g for 15 min at 4 °C. for 1 min at room temperature, then with 0.06 SSPE and The supernatant was used as the input material for the 0.005% N-lauroylsarcosine for 1 min and with Stabilization RNeasy Mini Kit and the total RNA was isolated according and Drying Solution for 30 s. to the manufacturer’s protocol. The total RNA was quantified Scanning and Gene Expression Analysis The microar- by a UV spectrophotometer and RNA quality was evaluated ray chip was scanned using Agilent G2565BA Microarray by capillary electrophoresis on an Agilent 2100 Bioanalyzer Scanner System. The arrays were scanned using the Agilent using RNA 6000 Nano LabChip Kit. dual laser DNA microarray scanner with SureScan technol- April 2006 657

Table1. Primers Used in RT-qPCR Analysis

Gene product Accession No. Log ratio Product size (bp) Primer sequence

1. APOBEC1 NM_001644 0.655 141 Forward 5-TGGATGATGTTGTACGCACTGG-3 Reverse 5-TGGCGGAATCGTTTGGTAATGG-3 2. ARHGEF16 NM_014448 0.302 182 Forward 5-CGGGACGGAGAGACGGGATG-3 Reverse 5-GAGCCAGGCACGACCACTTG-3 3. CD22 NM_001771 0.267 199 Forward 5-ACCCTCCCGTCTCCCACTAC-3 Reverse 5-CGCCTGCCGATGGTCTCC-3 4. FGFR3 NM_000142 0.385 102 Forward 5-CTGCCAGCCGAGGAGGAG-3 Reverse 5-CACCACCAGGATGAACAGGAAG-3 5. GALNT1 NM_020474 0.212 155 Forward 5-GCCACAGAAGAGGATAGCCAGG-3 Reverse 5-GCTGAGGTAGCCCAGTCAATCC-3 6. UNC5C NM_003728 0.337 125 Forward 5-CCTGCGAACACCATCACCAC-3 Reverse 5-GCCAGCATCCTCCAGTCATG-3 7. ZNF146 NM_007145 0.212 100 Forward 5-ACGGATTGGAAGGATACAGACC-3 Reverse 5-TAACCAATACCCATCCCACCTC-3 ogy. The information was extracted from images by Agilent’s the LightCycler instrument 1.5 (Roche) using LightCycler® Feature Extraction software 7.5 using defaults for all parame- FastStart DNA MasterPLUS SYBR Green I kit (Roche Applied ters. Following to the instruction protocol of Agilent Science). Briefly, 10 ml reactions contained 2 ml Master Mix, G2567AA Feature Extraction software (v.7.1), the signifi- 2 ml of 0.25 m M gene specific primer, and 6 ml of cDNA, gen- cance (P-values) of log ratios was computed by using a para- erated as described above. Each sample, which consisted of meterized error model. The image quantities of interest pro- cDNA generated from all the samples as detailed above, was duced by the image analysis methods were the (R, G) fluo- run in duplicate. The PCR parameters were 95 °C for 10 s, 45 rescence intensity pairs for each gene on each array probe, cycles of 95 °C for 10 s, 60 °C for 3 s and 72 °C for 10 s. At where Rred for Cy5 and Ggreen for Cy3. An ‘MA-plot’ the end of the program a melt curve analysis was made. At was used to represent the normalized (R, G) data, where the end of each PCR run, the data were automatically ana- Mlog R/G and Alog √RG.32,33) lyzed by the system and an amplification plot was generated For the bioinformatics tool used for searching gene ontol- for each cDNA sample. From each of these plots, the Light- ogy, we used the combination of databases to access the in- Cycler software calculated the threshold cycle (Cp), defined formation such as gene name, gene symbol, subcellular loca- as the fractional cycle number at which the fluorescence tion, family and superfamily classification, chromosome map reached the baseline. The fold expression or repression of the location, similar gene, molecular function, biochemical target gene relative to b-actin in each sample was then calcu- DDCp function related protein and references. The gene search lated by the formula: 2 where DDCp DCpkojic acid sample programs were used the following sequential order of DCpcontrol sample, DCpkojic acid sample Cpkojic acid-stimulated target gene databases: NCBI (http://www.ncbi.nlm.nih.gov), Ensembl Cpkojic acid-stimulated b-actin and DCpcontrol sample Cpcontrol target gene (http://www.ensembl.org), TIGR (http://www. tigr.org), and Cpcontrol b-actin. GeneCards (http://bioinfo.weizmann.ac.it/cards/index.shtml). In addition, the category classification of gene expression RESULTS was done by in-house BGSSJ (Bulk Gene Search System for Java; http://servx8.sinica.edu.tw/bgss-cgi-bin/gene.pl) pro- Inhibition Effect of Kojic Acid on A375 Melanoma gram which is a searching system accomplished by open Cells The examination of kojic acid effect on cell growth database connectivity that integrates UniGene, Swiss-Prot, inhibition of A375 melanoma cells showed that the cell NCBI and PubMed databases to correlate and simplify data growth of A375 melanoma cells was directly inhibited by the analysis of gene ontology. On the other hand, the protein increase of kojic acid concentrations. After treatment with search programs were used the following databases: Swiss- kojic acid for 72 h, the highest concentration of 1000 mg/ml Prot/TrEMBL (http://www.expasy.ch/sprot), Proteome (http: kojic acid allowed the inhibition of A375 cells less than 40% //www.proteome.com/databases/HumanPD/ reports) and while the lower concentrations of 0.32, 1.6, 8 and 40 mg/ml PubMed (http://www.ncbi.nlm. nih.gov/PubMed). kojic acid only allowed the inhibition of A375 cells less than Real-Time Quantitative PCR (RT-qPCR) Analysis 20% (Fig. 1). It indicated that the cell growth inhibition of Specific oligonucleotide primer pairs were designed with the A375 cells was not strongly effected by all concentrations of analysis Beacon designer 4.00 (Premier Biosoft Interna- kojic acid. In addition, there was no morphological change of tional) and used for real-time PCR. The sequences of the A375 cells along 24 h in the presence of 8 mg/ml kojic acid, primers used are shown in Table 1. The specificity of each which was referred as the mild concentration or recom- primer pair was tested by using common reference RNA mended dosage of kojic acid for human skin safety.3) In order (Stratagene) to perform RT-PCR reaction and followed by to study the early-stage of stimulation effect of kojic acid on DNA 500 chip run on Bioanalyzer 2100 (Agilent Technolo- gene expression profiling of A375 cells, compatible with the gies) to check the size of the PCR product. Primer pairs of human skin therapy, we decided to choose the time point at production predicted product size and no other side-product 24 h-treatment for the following microarray analysis. were chosen to conduct the following SYBR reaction. Microarray Analysis of Differential Gene Expression in Real-time quantitative PCR (RT-qPCR) was performed on A375 Cells after Kojic Acid Treatment Messenger RNA 658 Vol. 29, No. 4

Fig. 1. Effect of Kojic Acid Concentrations on the Growth Inhibition of A375 Cells at 72 h The concentrations of kojic acid used were 0.32, 1.6, 8, 40, 200 and 1000 mg/ml.

Fig. 2. Microarray Data Represents M-A Plot of Differentially Expressed Probes of Kojic Acid Responded Genes in A375 Cells cRNA targets were amplified and labeled using total RNA from A375 cells treated with 8 mg/ml kojic acid and the Agilent Low RNA Input Fluorescent Linear Amplification Kit. Cyanine 3- and cyanine 5-labeled targets were then combined and hybridized to an Agilent Human 1A oligonucleotide (V2) microarray using the Agilent in situ Hybridization Kit. M-A plot for a representative probe of comparative experiments (normal mRNA labeled with Cy3 and kojic acid-treated mRNA labeled with Cy5) where Rlog(Cy5/Cy3) repre- sents the common log ratio of the two dyes and A[log(Cy5)log(Cy3)]/2 is the average logarithmic fluorescence intensities of both channels. The gene expression pattern shows over 433 significantly differentially expressed probes (p0.01). Blue represents any data point whose log ratio is not significantly different from 0; redand greenrepresent data points whose log ratios are greater or less than 0, respectively (p0.01). from control and kojic acid-treated A375 cells were labeled bioinformatics tools combining NCBI, Ensembl, TIGR and with Cy3- or Cy5-dCTP, respectively, using a single round of GeneCards database to access the gene information that pro- reverse transcription and hybridization with the oligonu- vided the description of the expressed genes from the mi- cleotide microarray chip. The microarray results showed dif- croarray data. Moreover, we also used the in-house BGSSJ ferent fluorescence intensities of Cy3 and Cy5, depending on program to search the gene ontology that classified the gene the differential gene expression level. The comparative fluo- category into three main groups according to cellular compo- rescence intensities of Cy3 and Cy5 are represented in the nent, molecular function and biological process. The sum- MA-plot, in which those spots show markedly significant mary of category classification of differentially expressed genes between the Cy3 and Cy5 signals in P-value less than genes in kojic acid-stimulated A375 cells is shown in Fig. 3. 0.01 (p0.01) (Fig. 2). Analysis of gene expression changes Only 75 up-regulated genes and 123 down-regulated genes in kojic acid-stimulated A375 cells at the RNA level using were classified by BGSSJ program and showed the different human 1A oligonucleotide microarray complementary to ratios of subcategories in each group of expressed genes. 20173 (60-mer) oligonucleotide probes showed the total Some of genes could be classified into more than two cate- number of 433 differentially expressed probes that were gories, depending on their cellular component, molecular matched with the gene list approximately 361 genes, contain- function and biological process. The expressed genes were ing 136 up-regulated genes (Table 2) and 225 down-regulated found to locate in the subcellular region rather than or- genes (Table 3). The 24-h kojic acid treatment was the early- ganelle, extracellular region, protein complex and extracellu- phase stimulated time scale for gene to turn on and the mi- lar matrix, respectively. It may indicate the signal transduc- croarray data represented the differential expression genes in tion pathway occurred from extracellular region to intracellu- kojic acid-stimulated A375 cells. In addition, we used the lar region because some of genes were also found in both re- April 2006 659

Table2. Up-Regulated Genes (136 Genes) in Kojic Acid-Stimulated A375 Cells

Gene name Accession No. Ratio Description

A_23_P130496 A_23_P130496 3.39 Unknown A_23_P134704 A_23_P134704 4.30 Unknown A_23_P13535 A_23_P13535 6.18 Unknown A_23_P17152 A_23_P17152 1.51 Unknown A_23_P251953 A_23_P251953 2.42 Unknown A_23_P48430 A_23_P48430 6.49 Unknown A_23_P72800 A_23_P72800 3.69 Unknown AK094187 AK094187 1.55 cDNA FLJ36868 fis, clone ASTRO2016681 AK124775 AK124775 1.52 cDNA FLJ42785 fis, clone BRAWH3006548 Amyloid beta (A4) precursor protein (protease nexin-II, Alzheimer disease) (APP), APP NM_000484 1.76 transcript variant 1, mRNA AQP1 NM_000385 4.50 Aquaporin 1 (channel-forming integral protein, 28 kDa) (AQP1), transcript variant 2, mRNA ARMC4 AK001238 2.57 cDNA FLJ10376 fis, clone NT2RM2001982 BC031966 BC031966 8.53 cDNA clone MGC:43036 IMAGE:4839025, complete cds. BCL2A1 NM_004049 1.37 BCL2-related protein A1 (BCL2A1), mRNA C11orf11 NM_006133 1.69 Chromosome 11 open reading frame 11 (C11orf11), mRNA C14orf130 NM_175748 1.59 Chromosome 14 open reading frame 130 (C14orf130), mRNA C3orf6 NM_178335 1.38 Chromosome 3 open reading frame 6 (C3orf6), transcript variant 2, mRNA C6orf128 NM_145316 2.97 Chromosome 6 open reading frame 128 (C6orf128), mRNA C9orf42 NM_138333 1.37 Chromosome 9 open reading frame 42 (C9orf42), mRNA CA14 NM_012113 1.67 Carbonic anhydrase XIV (CA14), mRNA CAB39L NM_030925 2.54 Calcium binding protein 39-like (CAB39L), mRNA CABC1 NM_020247 1.49 Chaperone, ABC1 activity of bc1 complex like (S. pombe) (CABC1), mRNA CCAR1 NM_018237 2.91 Cell division cycle and apoptosis regulator 1 (CCAR1), mRNA CEBPD NM_005195 1.39 CCAAT/enhancer binding protein (C/EBP), delta (CEBPD), mRNA CENTG3 NM_031946 1.42 Centaurin, gamma 3 (CENTG3), mRNA Cep192 AK074074 1.79 mRNA for FLJ00145 protein Cep72 NM_018140 1.39 Hypothetical protein FLJ10565 (FLJ10565), mRNA CGI-94 NM_016037 1.37 Comparative gene identification transcript 94 (CGI-94), mRNA CNIH NM_005776 1.62 Cornichon homolog (Drosophila) (CNIH), mRNA Collagen-like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase COLQ NM_080542 2.49 (COLQ), transcript variant VI, mRNA CPE NM_001873 2.98 Carboxypeptidase E (CPE), mRNA CPNE5 NM_020939 2.11 Copine V (CPNE5), mRNA CRSP2 NM_004229 6.23 Cofactor required for Sp1 transcriptional activation, subunit 2, 150 kDa (CRSP2), mRNA CTSZ NM_001336 1.54 Cathepsin Z (CTSZ), mRNA CYP26B1 NM_019885 1.68 Cytochrome P450, family 26, subfamily B, polypeptide 1 (CYP26B1), mRNA DAZAP2 NM_014764 1.53 DAZ associated protein 2 (DAZAP2), mRNA DLGAP4 NM_014902 1.78 Discs, large (Drosophila) homolog-associated protein 4 (DLGAP4), transcript variant 1, mRNA DOCK4 AB018259 2.14 mRNA for KIAA0716 protein, partial cds. DSTN NM_006870 1.38 Destrin (actin depolymerizing factor) (DSTN), mRNA E2F3 NM_001949 1.42 E2F transcription factor 3 (E2F3), mRNA EIF5A2 NM_020390 1.37 Eukaryotic translation initiation factor 5A2 (EIF5A2), mRNA ENST00000307033 ENST00000307033 2.42 Olfactory receptor 4D5. [Source:SWISSPROT;Acc:Q8NGN0] ENST00000316004 ENST00000316004 5.75 Olfactory receptor 4H12 (Fragment). [Source:SWISSPROT;Acc:Q8NG74] ENST00000316643 ENST00000316643 1.71 Hypothetical protein MGC11082, mRNA (cDNA clone IMAGE:3689882), partial cds. ENST00000319799 ENST00000319799 1.75 Olfactory receptor 4A4. [Source:SWISSPROT;Acc:Q8NGN8] ENST00000335297 ENST00000335297 1.60 Clone IMAGE:4754514, mRNA ENST00000335397 ENST00000335397 1.96 Olfactory receptor 9Q1. [Source:SWISSPROT;Acc:Q8NGQ5] EP300 NM_001429 1.39 E1A binding protein p300 (EP300), mRNA EPHA4 NM_004438 2.26 EphA4 (EPHA4), mRNA ESAM NM_138961 1.71 Endothelial cell adhesion molecule (ESAM), mRNA F10 NM_000504 2.80 Coagulation factor X (F10), mRNA FBN2 NM_001999 2.86 Fibrillin 2 (congenital contractural arachnodactyly) (FBN2), mRNA FBXO16 NM_172366 4.21 F-box only protein 16 (FBXO16), mRNA FGF12 NM_004113 5.95 Fibroblast growth factor 12 (FGF12), transcript variant 2, mRNA FKBP10 NM_021939 1.61 FK506 binding protein 10, 65 kDa (FKBP10), mRNA FLJ13089 NM_024953 2.54 Hypothetical protein FLJ13089 (FLJ13089), mRNA FLJ20171 NM_017697 1.72 Hypothetical protein FLJ20171 (FLJ20171), mRNA FLJ23018 NM_024810 3.32 Hypothetical protein FLJ23018 (FLJ23018), mRNA FLJ23221 NM_024579 1.75 Hypothetical protein FLJ23221 (FLJ23221), mRNA FLJ23311 NM_024680 2.41 FLJ23311 protein (FLJ23311), mRNA FLJ32452 NM_144576 1.38 Hypothetical protein FLJ32452 (FLJ32452), mRNA FLJ39370 NM_152400 1.52 Hypothetical protein FLJ39370 (FLJ39370), mRNA FLJ46156 NM_198499 5.78 FLJ46156 protein (FLJ46156), mRNA FZD8 NM_031866 1.44 Frizzled homolog 8 (Drosophila) (FZD8), mRNA GAJ NM_032117 1.51 GAJ protein (GAJ), mRNA GNB1 NM_002074 1.45 Guanine nucleotide binding protein (G protein), beta polypeptide 1 (GNB1), mRNA GPR32 NM_001506 2.79 G protein-coupled receptor 32 (GPR32), mRNA HAND1 NM_004821 1.86 Heart and neural crest derivatives expressed 1 (HAND1), mRNA HH114 NM_032499 2.23 Hypothetical protein HH114 (HH114), mRNA HINT3 NM_138571 1.45 Histidine triad nucleotide binding protein 3 (HINT3), mRNA HIP1 NM_005338 2.64 Huntingtin interacting protein 1 (HIP1), mRNA 660 Vol. 29, No. 4

Table2. (Continued)

Gene name Accession No. Ratio Description

HIST1H1A NM_005325 1.46 Histone 1, H1a (HIST1H1A), mRNA HIST1H2AC NM_003512 3.34 Histone 1, H2ac (HIST1H2AC), mRNA HIST1H2AJ NM_021066 1.44 Histone 1, H2aj (HIST1H2AJ), mRNA HNRPH2 NM_019597 1.36 Heterogeneous nuclear ribonucleoprotein H2 (H’) (HNRPH2), mRNA IL22RA1 NM_021258 2.85 Interleukin 22 receptor, alpha 1 (IL22RA1), mRNA INCENP NM_020238 1.48 Inner centromere protein antigens 135/155 kDa (INCENP), mRNA IQCB1 NM_014642 1.44 IQ calmodulin-binding motif containing 1 (IQCB1), mRNA JMJD2D NM_018039 2.61 Jumonji domain containing 2D (JMJD2D), mRNA JWA NM_006407 1.67 Cytoskeleton related vitamin A responsive protein (JWA), mRNA KDELC1 NM_024089 1.44 KDEL (Lys-Asp-Glu-Leu) containing 1 (KDELC1), mRNA KLF5 NM_001730 1.50 Kruppel-like factor 5 (intestinal) (KLF5), mRNA KLHL13 NM_033495 10.13 Kelch-like 13 (Drosophila) (KLHL13), mRNA KRT7 NM_005556 10.71 Keratin 7 (KRT7), mRNA LOC137392 NM_145269 1.41 Similar to CG6405 gene product (LOC137392), mRNA LOC220929 NM_182755 2.34 Hypothetical protein LOC220929 (LOC220929), mRNA MAEA NM_005882 1.37 Macrophage erythroblast attacher (MAEA), mRNA MAGEC2 NM_016249 1.44 Melanoma antigen, family E, 1, cancer/testis specific (MAGEE1), mRNA MAGEF1 NM_022149 1.39 Melanoma antigen, family F, 1 (MAGEF1), mRNA MGC35118 NM_152453 5.07 Hypothetical protein MGC35118 (MGC35118), mRNA MGC4606 NM_024516 1.47 Hypothetical protein MGC4606 (MGC4606), mRNA NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5 kDa (NDUFA1), nuclear gene NDUFA1 NM_004541 1.41 encoding mitochondrial protein, mRNA NFIL3 NM_005384 1.41 Nuclear factor, interleukin 3 regulated (NFIL3), mRNA Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon (NFKBIE), NFKBIE NM_004556 1.63 mRNA OTX1 NM_014562 2.51 Orthodenticle homolog 1 (Drosophila) (OTX1), mRNA PARG NM_003631 4.05 Poly (ADP-ribose) glycohydrolase (PARG), mRNA PDCD1LG1 NM_014143 2.65 Programmed cell death 1 ligand 1 (PDCD1LG1), mRNA PDCL NM_005388 2.44 Phosducin-like (PDCL), mRNA PDHA1 NM_000284 1.37 Pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), mRNA PDLIM2 NM_021630 1.36 PDZ and LIM domain 2 (mystique) (PDLIM2), transcript variant 2, mRNA PGM2 NM_018290 1.41 Phosphoglucomutase 2 (PGM2), mRNA PHCA NM_018367 4.47 Phytoceramidase, alkaline (PHCA), mRNA PITPNB NM_012399 1.48 Phosphotidylinositol transfer protein, beta (PITPNB), mRNA PLAGL1 NM_002656 5.14 Pleiomorphic adenoma gene-like 1 (PLAGL1), transcript variant 1, mRNA PPP4R1 NM_005134 3.01 Protein phosphatase 4, regulatory subunit 1 (PPP4R1), mRNA PRPS2 NM_002765 1.71 Phosphoribosyl pyrophosphate synthetase 2 (PRPS2), mRNA PSCD3 NM_004227 3.49 Pleckstrin homology, Sec7 and coiled-coil domains 3 (PSCD3), mRNA PSCDBP NM_004288 15.34 Pleckstrin homology, Sec7 and coiled-coil domains, binding protein (PSCDBP), mRNA PTMAP7 AF170294 1.43 Prothymosin a14 mRNA, complete cds. PVRL4 NM_030916 1.79 Poliovirus receptor-related 4 (PVRL4), mRNA RAB3GAP D31886 1.79 KIAA0066 mRNA, partial cds. RAD1 NM_133377 1.38 RAD1 homolog (S. pombe) (RAD1), transcript variant 3, mRNA RASD1 NM_016084 1.35 RAS, dexamethasone-induced 1 (RASD1), mRNA RASSF3 AK129920 2.39 cDNA FLJ26410 fis, clone HRT09622 RKHD3 AB095929 1.94 mRNA for KIAA2009 protein SEC24B NM_006323 1.56 SEC24 related gene family, member B (S. cerevisiae) (SEC24B), mRNA SEC5L1 NM_018303 1.77 SEC5-like 1 (S. cerevisiae) (SEC5L1), mRNA SFRS2 NM_003016 1.38 Splicing factor, arginine/serine-rich 2 (SFRS2), mRNA SHCBP1 NM_024745 1.69 Likely ortholog of mouse Shc SH2-domain binding protein 1 (SHCBP1), mRNA SOAT2 NM_003578 4.02 Sterol O-acyltransferase 2 (SOAT2), mRNA Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) SPP1 NM_000582 5.38 (SPP1), mRNA SPTLC1 NM_006415 1.41 Serine palmitoyltransferase, long chain base subunit 1 (SPTLC1), transcript variant 1, mRNA SSX1 NM_005635 1.37 Synovial sarcoma, X breakpoint 1 (SSX1), mRNA STX6 NM_005819 1.67 Syntaxin 6 (STX6), mRNA SWAP70 NM_015055 1.90 SWAP-70 protein (SWAP70), mRNA SYNCRIP NM_006372 1.39 Synaptotagmin binding, cytoplasmic RNA interacting protein (SYNCRIP), mRNA PWP1_HUMAN Periodic tryptophan protein 1 homolog (Keratinocyte protein IEF SSP 9502). THC1805956 THC1805956 1.62 [Human] {Homo sapiens}, complete AF244540 immunodominant membrane protein precursor {Aster yellows phytoplasma}, partial THC1991976 THC1991976 3.67 (6%) TM4SF3 NM_004616 10.99 Transmembrane 4 superfamily member 3 (TM4SF3), mRNA TP53BP2 NM_005426 1.59 Tumor protein p53 binding protein, 2 (TP53BP2), mRNA TPCN1 AB032995 1.69 mRNA for KIAA1169 protein, partial cds. TTC9 D86980 4.83 mRNA for KIAA0227 gene, partial cds. UFD1L NM_005659 1.42 Ubiquitin fusion degradation 1-like (UFD1L), mRNA WBSCR19 NM_175064 9.58 Williams Beuren syndrome chromosome region 19 (WBSCR19), mRNA XTP2 NM_015172 1.47 HBxAg transactivated protein 2 (XTP2), mRNA YEATS2 NM_018023 1.36 Hypothetical protein FLJ10201 (FLJ10201), mRNA April 2006 661

Table3.Down-Regulated Genes (225 Genes) in Kojic Acid-Stimulated A375 Cells

Gene name Accession No. Ratio Description

A_23_P122323 A_23_P122323 0.32 Unknown A_23_P129645 A_23_P129645 0.49 Unknown A_23_P161049 A_23_P161049 0.44 Unknown A_23_P161835 A_23_P161835 0.27 Unknown A_23_P202572 A_23_P202572 0.21 Unknown A_23_P253353 A_23_P253353 0.29 Unknown A_23_P29907 A_23_P29907 0.28 Unknown A_23_P395326 A_23_P395326 0.32 Unknown A_23_P86779 A_23_P86779 0.44 Unknown A_23_P89080 A_23_P89080 0.72 Unknown A_23_P89369 A_23_P89369 0.49 Unknown A_23_P89841 A_23_P89841 0.29 Unknown A_23_P9270 A_23_P9270 0.60 Unknown AB063751 AB063751 0.47 IGH mRNA for immunoglobulin heavy chain VHDJ region, partial cds, clone: H101 ABCA13 NM_152701 0.29 ATP binding cassette gene, sub-family A (ABC1), member 13 (ABCA13), mRNA ATP-binding cassette, sub-family C (CFTR/MRP), member 13 (ABCC13), transcript variant 1, ABCC13 NM_138726 0.29 mRNA ACMSD NM_138326 0.22 Aminocarboxymuconate semialdehyde decarboxylase (ACMSD), mRNA ACVRL1 NM_000020 0.45 Activin A receptor type II-like 1 (ACVRL1), mRNA ADAM20 NM_003814 0.29 A disintegrin and metalloproteinase domain 20 (ADAM20), mRNA ADRB3 NM_000025 0.59 Adrenergic, beta-3-, receptor (ADRB3), mRNA AF090929 AF090929 0.29 Clone HQ0477 PRO0477p mRNA, complete cds. AK096677 AK096677 0.19 cDNA FLJ39358 fis, clone PEBLM2004015. AKT1 NM_005163 0.44 v-akt murine thymoma viral oncogene homolog 1 (AKT1), mRNA ALPI NM_001631 0.40 Alkaline phosphatase, intestinal (ALPI), mRNA AMAC NM_054028 0.46 Acyl-malonyl condensing enzyme (AMAC), mRNA ANXA1 NM_000700 0.71 Annexin A1 (ANXA1), mRNA AP1S1 NM_057089 0.72 Adaptor-related protein complex 1, sigma 1 subunit (AP1S1), transcript variant 2, mRNA Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (APOBEC1), transcript variant 1, APOBEC1 NM_001644 0.22 mRNA APTX NM_175073 0.64 Aprataxin (APTX), transcript variant 1, mRNA ARFIP2 NM_012402 0.72 ADP-ribosylation factor interacting protein 2 (arfaptin 2) (ARFIP2), mRNA ARHGEF16 NM_014448 0.50 Rho guanine exchange factor (GEF) 16 (ARHGEF16), mRNA ASAM NM_024769 0.48 Adipocyte-specific adhesion molecule (ASAM), mRNA AY032661 AY032661 0.51 Lung squamous cell carcinoma related protein-2 mRNA, complete cds. BAG5 NM_004873 0.46 BCL2-associated athanogene 5 (BAG5), mRNA BC030623 BC030623 0.64 Similar to L1 repeat, Tf subfamily, member 14, clone IMAGE:4820809, mRNA BC032472 BC032472 0.18 Similar to caspase 4, apoptosis-related cysteine protease, clone IMAGE:5216666, mRNA BC036198 BC036198 0.34 Clone IMAGE:5298326, mRNA BC039714 BC039714 0.36 cDNA clone MGC:47794 IMAGE:5585073, complete cds. Beta4GalNAc-T4 NM_178537 0.53 Hypothetical protein FLJ25045 (FLJ25045), mRNA BIRC2 NM_001166 0.67 Baculoviral IAP repeat-containing 2 (BIRC2), mRNA BMP5 NM_021073 0.46 Bone morphogenetic protein 5 (BMP5), mRNA BST2 NM_004335 0.47 Bone marrow stromal cell antigen 2 (BST2), mRNA C10orf117 NM_022451 0.73 AD24 protein (AD24), mRNA C12orf4 NM_020374 0.60 Chromosome 12 open reading frame 4 (C12orf4), mRNA C19orf4 NM_012109 0.47 Chromosome 19 open reading frame 4 (C19orf4), mRNA C1orf1 U88965 0.43 PO42 gene, complete cds. C20orf165 NM_080608 0.50 Chromosome 20 open reading frame 165 (C20orf165), mRNA C6orf139 NM_018132 0.73 Chromosome 6 open reading frame 139 (C6orf139), mRNA C6orf162 NM_020425 0.61 Chromosome 6 open reading frame 162 (C6orf162), mRNA CCPG1 NM_004748 0.61 Cell cycle progression 8 protein (CPR8), mRNA CCR4 NM_005508 0.32 Chemokine (C-C motif) receptor 4 (CCR4), mRNA CD22 NM_001771 0.54 CD22 antigen (CD22), mRNA CD3G NM_000073 0.38 CD3G antigen, gamma polypeptide (TiT3 complex) (CD3G), mRNA CDC10 NM_001788 0.66 CDC10 cell division cycle 10 homolog (S. cerevisiae) (CDC10), mRNA CDK10 NM_052987 0.53 Cyclin-dependent kinase (CDC2-like) 10 (CDK10), transcript variant 2, mRNA Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) (CEACAM1), CEACAM1 NM_001712 0.53 mRNA CLDN3 NM_001306 0.65 Claudin 3 (CLDN3), mRNA C-type (calcium dependent, carbohydrate-recognition domain) lectin, superfamily member 7 CLECSF7 NM_130441 0.46 (CLECSF7), transcript variant 1, mRNA CNKSR2 AF418270 0.40 Connector enhancer of KSR2B mRNA, complete cds. COL17A1 NM_000494 0.41 Collagen, type XVII, alpha 1 (COL17A1), transcript variant long, mRNA COL24A1 NM_152890 0.40 Collagen, type XXIV, alpha 1 (COL24A1), mRNA CPB1 NM_001871 0.25 Carboxypeptidase B1 (tissue) (CPB1), mRNA CREBL1 NM_004381 0.54 cAMP responsive element binding protein-like 1 (CREBL1), mRNA CSE1L NM_001316 0.66 CSE1 chromosome segregation 1-like (yeast) (CSE1L), transcript variant 1, mRNA CTNNAL1 NM_003798 0.68 Catenin (cadherin-associated protein), alpha-like 1 (CTNNAL1), mRNA 662 Vol. 29, No. 4

Table3. (Continued)

Gene name Accession No. Ratio Description

D82326 D82326 0.29 mRNA for Na+-independent neutral and basic amino acid transporter, complete cds. DEK NM_003472 0.71 DEK oncogene (DNA binding) (DEK), mRNA DKFZP434G1415 NM_031292 0.49 Hypothetical protein DKFZp434G1415 (DKFZP434G1415), mRNA DKFZp547B1713 NM_152379 0.59 Hypothetical protein DKFZp547B1713 (DKFZp547B1713), mRNA DKFZP564O0463 NM_015420 0.74 DKFZP564O0463 protein (DKFZP564O0463), mRNA DKFZP586L0724 NM_015462 0.72 DKFZP586L0724 protein (DKFZP586L0724), mRNA DLX1 NM_178120 0.42 Distal-less homeo box 1 (DLX1), mRNA DMXL1 NM_005509 0.16 Dmx-like 1 (DMXL1), mRNA DOK4 NM_018110 0.59 Docking protein 4 (DOK4), mRNA DSPP NM_014208 0.17 Dentin sialophosphoprotein (DSPP), mRNA EFNA3 NM_004952 0.44 Ephrin-A3 (EFNA3), mRNA EIF4E NM_001968 0.69 Eukaryotic translation initiation factor 4E (EIF4E), mRNA ELA2 NM_001972 0.34 Elastase 2, neutrophil (ELA2), mRNA ENST00000222567 ENST00000222567 0.63 Clone IMAGE:4340767, mRNA ENST00000329697 ENST00000329697 0.59 Xenobiotic/medium-chain fatty acid : CoA ligase [Source:RefSeq;Acc:NM_182617] ERBP NM_014597 0.59 Acidic 82 kDa protein mRNA (HSU15552), mRNA FGF5 NM_004464 0.22 Fibroblast growth factor 5 (FGF5), transcript variant 1, mRNA Fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) (FGFR3), transcript FGFR3 NM_000142 0.41 variant 1, mRNA FIBCD1 NM_032843 0.61 Fibrinogen C domain containing 1 (FIBCD1), mRNA FLJ11535 NM_024888 0.55 Hypothetical protein FLJ11535 (FLJ11535), mRNA FLJ12242 NM_024681 0.52 Hypothetical protein FLJ12242 (FLJ12242), mRNA FLJ14007 NM_024699 0.62 Hypothetical protein FLJ14007 (FLJ14007), mRNA FLJ14297 NM_024903 0.20 Hypothetical protein FLJ14297 (FLJ14297), mRNA FLJ14442 NM_032785 0.38 Hypothetical protein FLJ14442 (FLJ14442), mRNA FLJ14503 NM_152780 0.28 Hypothetical protein FLJ14503 (FLJ14503), mRNA FLJ20245 NM_017723 0.46 Hypothetical protein FLJ20245 (FLJ20245), mRNA FLJ25952 NM_153251 0.61 Hypothetical protein FLJ25952 (FLJ25952), mRNA FLJ32675 NM_173811 0.49 Hypothetical protein FLJ32675 (FLJ32675), mRNA FLJ32803 AK057365 0.35 cDNA FLJ32803 fis, clone TESTI2002632. FLJ33655 NM_173641 0.52 Hypothetical protein FLJ33655 (FLJ33655), mRNA FNBP3 AF151059 0.68 HSPC225 mRNA, complete cds. FPGT NM_003838 0.42 Fucose-1-phosphate guanylyltransferase (FPGT), mRNA UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl transferase 1 GALNT1 NM_020474 0.61 (GalNAc-T1) (GALNT1), mRNA GFOD1 NM_018988 0.59 Glucose-fructose oxidoreductase domain containing 1 (GFOD1), mRNA GLE1L NM_001499 0.61 GLE1 RNA export mediator-like (yeast) (GLE1L), mRNA GMPR NM_006877 0.52 Guanosine monophosphate reductase (GMPR), mRNA GPR146 NM_138445 0.34 G protein-coupled receptor 146 (GPR146), mRNA GPR26 NM_153442 0.59 G protein-coupled receptor 26 (GPR26), mRNA GPR61 NM_031936 0.33 G protein-coupled receptor 61 (GPR61), mRNA GSDM1 NM_178171 0.40 Gasdermin (GSDM), mRNA GUCY1A2 NM_000855 0.32 Guanylate cyclase 1, soluble, alpha 2 (GUCY1A2), mRNA HDCMA18P NM_016648 0.62 HDCMA18P protein (HDCMA18P), mRNA HIBCH NM_014362 0.74 3-Hydroxyisobutyryl-Coenzyme A hydrolase (HIBCH), transcript variant 1, mRNA HLF NM_002126 0.21 Hepatic leukemia factor (HLF), mRNA HMMR NM_012484 0.67 Hyaluronan-mediated motility receptor (RHAMM) (HMMR), transcript variant 1, mRNA HPS3 NM_032383 0.35 Hermansky-Pudlak syndrome 3 (HPS3), mRNA HRMT1L1 NM_001535 0.53 HMT1 hnRNP methyltransferase-like 1 (S. cerevisiae) (HRMT1L1), transcript variant 2, mRNA IL6ST NM_002184 0.18 Interleukin 6 signal transducer (gp130, oncostatin M receptor) (IL6ST), transcript variant 1, mRNA JJAZ1 NM_015355 0.71 Joined to JAZF1 (JJAZ1), mRNA KARS NM_005548 0.72 Lysyl-tRNA synthetase (KARS), mRNA KCNAB3 NM_004732 0.36 Potassium voltage-gated channel, shaker-related subfamily, beta member 3 (KCNAB3), mRNA KIAA0052 NM_015360 0.69 KIAA0052 protein (KIAA0052), mRNA KIAA0409 NM_015324 0.55 KIAA0409 protein (KIAA0409), mRNA KIAA0565 AB011137 0.24 mRNA for KIAA0565 protein, partial cds. KIAA1811 NM_032430 0.46 KIAA1811 protein (KIAA1811), mRNA KLKB1 NM_000892 0.51 Kallikrein B, plasma (Fletcher factor) 1 (KLKB1), mRNA KTN1 NM_182926 0.71 Kinectin 1 (kinesin receptor) (KTN1), mRNA LEREPO4 NM_018471 0.70 Likely ortholog of mouse immediate early response, erythropoietin 4 (LEREPO4), mRNA Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 5 LILRB5 NM_006840 0.29 (LILRB5), mRNA LMO6 NM_006150 0.56 LIM domain only 6 (LMO6), mRNA LOC124220 NM_145252 0.49 Similar to common salivary protein 1 (LOC124220), mRNA LOC221442 AK126092 0.30 cDNA FLJ44104 fis, clone TESTI4044123 LOC51619 NM_015983 0.43 Ubiquitin-conjugating enzyme HBUCE1 (LOC51619), mRNA LRP6 NM_002336 0.41 Low density lipoprotein receptor-related protein 6 (LRP6), mRNA LRRC4 NM_022143 0.37 Leucine rich repeat containing 4 (LRRC4), mRNA MAFK NM_002360 0.49 v-maf musculoaponeurotic fibrosarcoma oncogene homolog K (avian) (MAFK), mRNA April 2006 663

Table3. (Continued)

Gene name Accession No. Ratio Description

MAPK8 NM_002750 0.51 Mitogen-activated protein kinase 8 (MAPK8), transcript variant 2, mRNA MBNL1 NM_021038 0.70 Muscleblind-like (Drosophila) (MBNL1), transcript variant 1, mRNA MDFI NM_005586 0.51 MyoD family inhibitor (MDFI), mRNA Malic enzyme 3, NADP(+)-dependent, mitochondrial (ME3), nuclear gene encoding mitochondrial ME3 NM_006680 0.58 protein, mRNA MFAP4 NM_002404 0.29 Microfibrillar-associated protein 4 (MFAP4), mRNA MGC13275 AK123855 0.39 cDNA FLJ41861 fis, clone NTONG2008672 MGC15716 NM_032370 0.48 Hypothetical protein MGC15716 (MGC15716), mRNA MGC17791 NM_152362 0.54 Hypothetical protein MGC17791 (MGC17791), mRNA MGC33407 NM_178525 0.55 Hypothetical protein MGC33407 (MGC33407), mRNA MGC33637 NM_152596 0.41 Hypothetical protein MGC33637 (MGC33637), mRNA MGC33839 NM_152353 0.51 Hypothetical protein MGC33839 (MGC33839), mRNA MGC33867 BC004501 0.72 Hypothetical protein MGC33867, mRNA (cDNA clone IMAGE:3835289), partial cds. MGC34837 NM_152377 0.25 Hypothetical protein MGC34837 (MGC34837), mRNA MGC35308 NM_175922 0.52 Hypothetical protein MGC35308 (MGC35308), mRNA MGC5395 NM_024060 0.66 Hypothetical protein MGC5395 (MGC5395), mRNA MIDN AK075506 0.50 cDNA PSEC0204 fis, clone HEMBA1002706 Mitochondrial ribosomal protein L30 (MRPL30), nuclear gene encoding mitochondrial protein, MRPL30 NM_145212 0.47 transcript variant 1, mRNA NCALD NM_032041 0.58 Neurocalcin delta (NCALD), mRNA NKX6-1 NM_006168 0.62 NK6 transcription factor related, locus 1 (Drosophila) (NKX6-1), mRNA NLGN2 NM_020795 0.66 Neuroligin 2 (NLGN2), mRNA NM_017571 NM_017571 0.41 Hypothetical protein LOC55580 (LOC55580), mRNA NS3TP1 NM_019048 0.61 HCV NS3-transactivated protein 1 (NS3TP1), mRNA NUDT10 NM_153183 0.16 Nudix (nucleoside diphosphate linked moiety X)-type motif 10 (NUDT10), mRNA OLFML1 NM_198474 0.57 MVAL564 (UNQ564), mRNA OR51B4 NM_033179 0.39 Olfactory receptor, family 51, subfamily B, member 4 (OR51B4), mRNA OSBP2 AF288741 0.40 Oxysterol binding protein 2 (OSBP2) mRNA, complete cds. OSTM1 NM_014028 0.68 Osteopetrosis associated transmembrane protein 1 (OSTM1), mRNA P29 NM_015484 0.73 GCIP-interacting protein p29 (P29), transcript variant 1, mRNA PAH NM_000277 0.27 Phenylalanine hydroxylase (PAH), mRNA PAPD5 NM_022447 0.74 PAP associated domain containing 5 (PAPD5), mRNA PCNXL3 BX640978 0.55 mRNA; cDNA DKFZp686M14190 (from clone DKFZp686M14190) PCSK4 NM_017573 0.60 Proprotein convertase subtilisin/kexin type 4 (PCSK4), mRNA PDZK10 AB002314 0.29 mRNA for KIAA0316 protein, partial cds. PEX5L NM_016559 0.18 Pex5p-related protein (PEX5R), mRNA PIGQ NM_004204 0.61 Phosphatidylinositol glycan, class Q (PIGQ), transcript variant 2, mRNA POU2F3 NM_014352 0.43 POU domain, class 2, transcription factor 3 (POU2F3), mRNA PRIM1 NM_000946 0.72 Primase, polypeptide 1, 49kDa (PRIM1), mRNA PRRX1 NM_006902 0.59 Paired related homeobox 1 (PRRX1), transcript variant pmx-1a, mRNA PSMC6 NM_002806 0.72 Proteasome (prosome, macropain) 26S subunit, ATPase, 6 (PSMC6), mRNA PSMD3 NM_002809 0.73 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 3 (PSMD3), mRNA PTK9 NM_002822 0.67 PTK9 protein tyrosine kinase 9 (PTK9), transcript variant 1, mRNA PTX1 NM_016570 0.54 PTX1 protein (PTX1), mRNA RAB6B NM_016577 0.59 RAB6B, member RAS oncogene family (RAB6B), mRNA RASGEF1B NM_152545 0.38 RasGEF domain family, member 1B (RASGEF1B), mRNA RB1CC1 NM_014781 0.62 RB1-inducible coiled-coil 1 (RB1CC1), mRNA RECQL NM_032941 0.63 RecQ protein-like (DNA helicase Q1-like) (RECQL), transcript variant 2, mRNA RG9MTD1 NM_017819 0.73 RNA (guanine-9-) methyltransferase domain containing 1 (RG9MTD1), mRNA RIMS2 NM_014677 0.35 Regulating synaptic membrane exocytosis 2 (RIMS2), mRNA RNPC1 NM_017495 0.60 RNA-binding region (RNP1, RRM) containing 1 (RNPC1), transcript variant 1, mRNA RSBN1 NM_018364 0.33 Hypothetical protein FLJ11220 (FLJ11220), mRNA SAA4 NM_006512 0.17 Serum amyloid A4, constitutive (SAA4), mRNA SCN10A NM_006514 0.43 Sodium channel, voltage-gated, type X, alpha (SCN10A), mRNA SCP2 NM_002979 0.71 Sterol carrier protein 2 (SCP2), mRNA SDC4 NM_002999 0.72 Syndecan 4 (amphiglycan, ryudocan) (SDC4), mRNA SECTM1 NM_003004 0.47 Secreted and transmembrane 1 (SECTM1), mRNA Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5 SERPINA5 NM_000624 0.48 (SERPINA5), mRNA SIN3B AB014600 0.23 mRNA for KIAA0700 protein, partial cds. SLC17A2 NM_005835 0.25 Solute carrier family 17 (sodium phosphate), member 2 (SLC17A2), mRNA 0.44 Solute carrier family 25 (mitochondrial carrier; ornithine transporter) member 2 (SLC25A2), SLC25A2 NM_031947 mRNA SLC38A4 NM_018018 0.37 Solute carrier family 38, member 4 (SLC38A4), mRNA SMC6L1 NM_024624 0.44 SMC6 structural maintenance of chromosomes 6-like 1 (yeast) (SMC6L1), mRNA SNX10 NM_013322 0.72 Sorting nexin 10 (SNX10), mRNA ST18 NM_014682 0.29 Suppression of tumorigenicity 18 (breast carcinoma) (zinc finger protein) (ST18), mRNA STARD8 NM_014725 0.41 START domain containing 8 (STARD8), mRNA SYMPK NM_004819 0.59 Symplekin (SYMPK), mRNA 664 Vol. 29, No. 4

Table3. (Continued)

Gene name Accession No. Ratio Description

TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 68 kDa (TAF15), TAF15 NM_139215 0.74 transcript variant 1, mRNA TAL1 NM_003189 0.25 T-cell acute lymphocytic leukemia 1 (TAL1), mRNA TCF21 NM_003206 0.40 Transcription factor 21 (TCF21), transcript variant 2, mRNA TCN1 NM_001062 0.29 Transcobalamin I (vitamin B12 binding protein, R binder family) (TCN1), mRNA T-complex-associated-testis-expressed 1-like, mRNA (cDNA clone MGC:5007 IMAGE:3448623), TCTE1L BC000968 0.64 complete cds. Thyroid hormone receptor, beta (erythroblastic leukemia viral (v-erb-a) oncogene homolog 2, avian) THRB NM_000461 0.20 (THRB), mRNA Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (TIE), TIE NM_005424 0.54 mRNA Toll-interleukin 1 receptor (TIR) domain containing adaptor protein (TIRAP), transcript variant 2, TIRAP NM_148910 0.49 mRNA TOPK NM_018492 0.73 T-LAK cell-originated protein kinase (TOPK), mRNA TRAM1 NM_014294 0.71 Translocation associated membrane protein 1 (TRAM1), mRNA TRAM2 D31762 0.36 KIAA0057 mRNA, partial cds. TREM1 NM_018643 0.38 Triggering receptor expressed on myeloid cells 1 (TREM1), mRNA TRIP15 NM_004236 0.59 Thyroid receptor interacting protein 15 (TRIP15), mRNA TTK NM_003318 0.48 TTK protein kinase (TTK), mRNA TULP3 AF045583 0.49 Tubby like protein 3 (TULP3) mRNA, complete cds. TXNDC NM_030755 0.70 Thioredoxin domain containing (TXNDC), mRNA Ubiquitin-conjugating enzyme E2E 1 (UBC4/5 homolog, yeast) (UBE2E1), transcript variant 1, UBE2E1 NM_003341 0.61 mRNA UBE2M NM_003969 0.69 Ubiquitin-conjugating enzyme E2M (UBC12 homolog, yeast) (UBE2M), mRNA UNC5C NM_003728 0.46 Unc-5 homolog C (C. elegans) (UNC5C), mRNA UNQ689 AY358528 0.33 Clone DNA66660 RSTI689 (UNQ689) mRNA, complete cds. USP1 NM_003368 0.69 Uubiquitin specific protease 1 (USP1), mRNA USP44 NM_032147 0.26 Ubiquitin specific protease 44 (USP44), mRNA WNT10A NM_025216 0.41 Wingless-type MMTV integration site family, member 10A (WNT10A), mRNA ZMYND15 NM_032265 0.60 Zinc finger, MYND domain containing 15 (ZMYND15), mRNA ZNF146 NM_007145 0.61 Zinc finger protein 146 (ZNF146), mRNA ZNF192 NM_006298 0.26 Zinc finger protein 192 (ZNF192), mRNA ZNF483 AB075842 0.44 mRNA for KIAA1962 protein. ZNF497 NM_198458 0.39 Zinc finger protein 497 (ZNF497), mRNA ZP2 NM_003460 0.29 Zona pellucida glycoprotein 2 (sperm receptor) (ZP2), mRNA gions. Otherwise, they possessed the molecular functions in kojic acid-stimulated A375 cells in agreement with the DNA different ratios of binding, catalytic activity, signal transducer microarray expression data. Seven genes for RT-qPCR exam- activity, transcription regulator activity, enzyme regulator ac- ination were apolipoprotein B RNA editing deaminase gene tivity, transporter activity, structural molecule activity and (APOBEC1),34) Rho guanine exchange factor (GEF) 16 translation regulator activity. The different ratio of biological (ARHGEF16),35) CD22 antigen (CD22),36) fibroblast growth 37—40) functions of expressed genes may indicate the correlated factor receptor 3 (FGFR3), UDP-N-acetyl-alpha-D- function in regulation of melanogenesis. Moreover, the ex- galactosamine: polypeptide N-acetylgalactosaminyl trans- pressed genes involved some biological processes, such as ferase I (GALNT1),41,42) netrin receptor UNC5C precursor physiological process, cellular process, regulation of biologi- (UNC5C)43) and zinc finger protein 146 (ZNF146).44—46) The cal process and development, leading to understand the re- comparison of gene expression level of seven differentially lated malignancy processes in melanogenesis. Thus, this cat- expressed genes by microarray and RT-qPCR is shown in egory classification provided more useful information of Table 4. Those genes were down-regulated in kojic acid-stim- gene ontology in response to kojic acid stimulation and indi- ulated A375 cells and possessed the function of tumor sup- cated that all of differentially expressed genes are regulated pressor in the regulation of carcinogenesis mechanism. In ad- by various gene networks and play the related molecular dition, seven of differentially expressed genes were also clas- functions involving biological processes in the regulation of sified by BGSSJ program and showed the localization in sub- melanogenesis. cellular and/or extracellular region, where FGFR3, GALNT1 RT-qPCR Validation of Array Analysis The significant and UNC5C genes may occur the signal transduction from expressed genes chosen for RT-qPCR examination were de- extracellular region into cellular region (Fig. 4). They play termined a mean degree of regulation with p0.01 by array the molecular functions in binding, catalytic activity and sig- analysis. The differentially expressed genes were searched nal transducer activity and also involved in the biological the biological functions correlated with carcinogenesis and processes, such as physiological process, cellular process, selected the significant expressed genes following to the regulation of biological process and development, which may order of P values. The selected genes were validated by RT- regulate the malignancy of melanogenesis. qPCR analysis to confirm the result of gene expression with microarray data. The RT-qPCR results showed the perfectly matched gene expression levels of seven expressed genes in April 2006 665

Fig. 3. Category Classification of Up- and Down-Regulated Genes in Kojic Acid-Stimulated A375 Cells by Using In-House BGSSJ (Bulk Gene Search System for Java) Program BGSSJ program is available at http://servx8.sinica.edu.tw/bgss-cgi-bin/gene.pl. The up- and down-regulated genes are classified to cellular component (A, B), molecular func- tion (C, D), and biological process (E, F), respectively. The following numbers are the classified genes and percentage of genes in each category.

Table4. Comparison of Gene Expression Level of Seven Differentially Expressed Genes in Kojic Acid-Stimulated A375 Cells by Microarray and RT- qPCR Analysis

Gene name Accessions Description P value Microarray RT-qPCR

1. APOBEC1 NM_001644 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 0.0001 0.22 0.12 (APOBEC1) 2. ARHGEF16 NM_014448 Rho guanine exchange factor (GEF) 16 (ARHGEF16) 0.001 0.50 0.47 3. CD22 NM_001771 CD22 antigen (CD22) 0.001 0.54 0.80 4. FGFR3 NM_000142 Fibroblast growth factor receptor 3 (achondroplasia, thanatophoric 0.001 0.41 0.76 dwarfism) (FGFR3), transcript variant 1 5. GALNT1 NM_020474 UDP-N-acetyl-alpha-D-galactosamine:polypeptide 0.001 0.61 0.61 N-acetylgalactosaminyltransferase 1 (GalNAc-T1) (GALNT1) 6. UNC5C NM_003728 Unc-5 homolog C (C. elegans) (UNC5C) 0.0001 0.46 0.62 7. ZNF146 NM_007145 Zinc finger protein 146 (ZNF146) 0.01 0.61 0.51

DISCUSSION fect. To examine the genotoxicity effect of kojic acid on gene expression profiling of A375 cells, the microarray technol- Although some of the biological applications and potential ogy providing the high throughput method for easily screen- effects of kojic acid have been partially elucidated,19,25,47,48) ing the number of differentially expressed genes was used. It the intensive study of kojic acid effect on human genomics has been used for analysis of gene expression in dermatology level including gene regulatory mechanism is not clear and including human melanomas,49—54) however, the study of rarely reported. In this study, we used the human skin A375 gene expression profiling in kojic acid-stimulated A375 cells malignant melanoma cells to examine the genotoxicity effect by DNA microarray has never been reported. Therefore, it is of kojic acid on carcinoma therapy, except skin whitening ef- very challenging to use the high throughput technology of 666 Vol. 29, No. 4

Fig. 4. Category Classification of Seven Differentially Expressed Genes (APOBEC1, ARHGEF16, CD22, FGFR3, GALNT1, UNC5C and ZNF146) in Kojic Acid-Stimulated A375 Cells by Using In-House BGSSJ Program

DNA microarray for studying the effect of kojic acid on the According to the microarray data, we used the bioinfor- large numbers of differentially expressed genes of A375 matics tools to identify the differentially expressed genes and melanoma cells and functional regulations on anti-tumorige- search the gene ontology that provided the useful informa- nesis in malignancy of melanoma. In addition, the reasons tion of category classification consisting of cellular compo- that used the human 1A oligonucleotide microarray to ana- nent, molecular function and biological process. The ob- lyze gene expression of human skin A375 melanoma cells tained information can be used as indicators for studying the were: (i) Human 1A oligonucleotide microarray is comprised further regulatory mechanisms and applications of gene of 20173 oligonucleotide probes, which span conserved markers for early cancer diagnostics and therapy. For the exons across the transcripts of the 18716 human genes. This number of differentially expressed genes, we have selected human gene analysis system is compatible with the human the significant genes correlated to the carcinogenesis and skin A375 melanoma cells and could screen the large num- also used RT-qPCR for validating gene expression changes, bers of human differentially expressed genes in once time. comparing microarray analysis data. RT-qPCR offered the (ii) Human 1A oligonucleotide microarray used convenient confirmatory quantitative results under stringent condition two-color labeling procedure that reduces experimental vari- and showed the quantitative of kojic acid-responded genes in ability by allowing biological samples to be directly com- agreement with the DNA microarray expression data. It indi- pared with each other on the same microarray after undergo- cated that our validated genes in the robust biomarkers lists ing the same hybridization incubation. Thus, this microarray obtained precise data, in which the validated genes may be- platform allows to analysis of kojic acid-responded genes in come the useful biomarkers to understand the biochemistry A375 cells that may become the gene markers in the malig- of drug-molecular response in the future clinical researches. nancy of melanoma for diagnostic and therapeutic applica- Interestingly, we found seven significant kojic acid-re- tions on human skin. sponded genes that were down-regulated in kojic acid-stimu- For the cell treatment, we examined the various concentra- lated A375 cells. Most of genes are highly related to the reg- tions (0.32, 1.6, 8, 40, 200, 1000 mg/ml) of kojic acid on the ulation of carcinogenesis process. growth inhibition of A375 cells. All of kojic acid concentra- APOBEC1 (Apolipoprotein B mRNA editing enzyme, cat- tions did not strongly inhibit the cell growth of A375 cells alytic polypeptide 1) is the central component of an RNA ed- even though the incubation time was longer (72 h). By using itosome whose physiological role clearly lies in deamination microarray technology to study the overall effects of kojic of apoB mRNA C6666 to U in gastrointestinal tissues, acid on whole gene expression of A375 cells, we used the thereby creating a premature stop codon.55,56) Uncontrolled kojic acid at the concentration of 8 mg/ml (0.8%) because cytidine deamination could generate inappropriately folded this is the mild concentration that could inhibit A375 cells polypeptides, dominant-negative proteins, or mutations in with the inhibition less than 20% and avoid the differential tumor suppressor genes and thus contribute to tumor forma- gene expression data resulting from cell death response of tion process.57,58) Then, the overexpression of APOBEC1 has cytotoxicity of high concentration of kojic acid. Moreover, led to the development of a number of different cancers, such the safety of kojic acid for human skin care products is rec- as gastrointestinal cancer, colon cancer and heptocellular ommended for general use at a concentration up to 1% with- cancer, in a variety of tissues. It implies that any inappropri- out prescription.3) Under this consideration, the concentration ate expression of a member of the DNA deaminase family of 8 mg/ml kojic acid (0.8%) is safe for human skin and can can have potential oncogenic activity.59,60) Furthermore, most be used to study the gene expression profiling in A375 cells of human carcinomas were caused by APOBEC1 mediated and through the regulatory functions of differentially ex- mRNA editing.61—63) Thus, the down-regulated APOBEC1 pressed genes on carcinogenesis therapy. may potentially allow deactivating the tumor progression of April 2006 667

A375 melanoma cells. underlies the mechanism responsible for altered carbohydrate ARHGEF16 (Rho guanine exchange factor (GEF) 16) is antigen expression observed in gastric, colorectal and epithe- one of the multiple GEF members that have a crucial role in lial cancers.41,42,74) Thus, the down-regulated GALNT1 by activating small GTPase by exchanging GDP for GTP and kojic acid also potentially allows deactivating the tumor pro- regulate various cellular functions including actin cytoskele- gression of A375 melanoma cells. ton organization, activation of kinase cascades, mitogenesis, UNC5C as netrin-1 receptor was initially proposed as me- transcriptional activation and stimulation of DNA synthe- diator of the chemopulsive effect of netrin-1 on specific sis.35) Although the specific function of this gene is not yet axons. The expression of the human UNC5C is down-regu- known, it is regarded to be involved in protein–protein and lated in multiple cancers including colorectal, breast, ovary, protein–lipid interactions. In addition, some reports in cancer uterus, stomach, lung, or kidney cancers.75—77) Interestingly, research showed that Rho signaling and PI 3-kinase activa- our finding showed that this gene was strongly down-regu- tion play an important role in regulating breast tumor pro- lated in kojic acid-stimulated A375 cells, rather than the nor- gression.64,65) Moreover, a novel putative human Rho GEF mally untreated A375 cells. Although the signal downstream designated Src homology 3 (SH3) domain-containing gua- of the UNC5C receptor in either axon guidance or apoptosis nine nucleotide exchange factor (SGEF) provides a direct is not clear, most of published papers showed that the UNC5 link for cross-talk between the androgen receptor and Rho family acts as putative tumor suppressor because of their fre- GTPase signaling pathways in prostate cancer cells.66) In quent alteration in various human cancers.77,78) Thus, we ex- consequence, the down-regulated ARHGEF16 gene by kojic pected that the expression reduction of this gene in kojic acid may suppress or deactivate the tumor progression of acid-stimulated A375 cells might promote the tumor-sup- A375 melanoma cancer cells. pressor-gene in deactivation of carcinogenesis process. Like- CD22 encoding CD22 antigen, which is a type I integral wise, it has been reported that the removal of the netrin bind- membrane sialoglycoprotein with a molecular weight of 135 ing second Ig domains of UNC5C caused an increase of kDa, is found specifically on cancerous B-lymphocytes UNC5C in the basal tyrosine phosphorylation, occurring dur- where it is initially present in the cytoplasm of developing B- ing melanoma progression at the stage when cells first be- cells and later expressed on the cell surface of mature B- come competent for metastasis.79,80) cells.36,67—69) Preclinical CD22 has been shown to be an ef- ZNF146 or human OZF gene, located in chromosome fective target for immunotherapy of B-cell malignancies band 19q13.1, is overexpressed in the majority of pancreatic using either “naked” or toxin-labeled or radio-labeled mono- cancers with more than 80% of colorectal cancers.44—46) clonal antibodies. CD22 is strongly expressed in hairy cell ZNF146 encoding protein consisted of ten Kruppel zinc fin- leukemia and variants and anti-CD22 therapy has been ger motifs, called zinc finger protein OZF. It has been re- shown to have a high response rate in refractory hairy cell ported that the high-level expression of ZNF146 is frequent leukemia.70,71) The down-regulated CD22 by kojic acid may in pancreatic carcinomas and may contribute to the develop- suppress or deactivate the tumor progression of melanoma ment or progression of this tumor. Thus, the down-regulated cancer cells and be related to the immune response of human ZNF146 gene by kojic acid may allow deactivating the tumor skin cell. progression in A375 melanoma cells. FGFR3 (fibroblast growth factor receptor 3) is one of four In conclusion, we used the high throughput analysis of high affinity tyrosine kinase receptors for the FGF family of DNA microarray and RT-qPCR for global analysis of differ- ligands. On ligand stimulation, FGFR3 undergoes dimeriza- entially expressed genes responding to kojic acid in A375 tion and tyrosine autophosphorylation that result in cell pro- human melanoma cells. The differentially expressed genes liferation or differentiation, depending on the cell context. were classified the category, depending on cellular compo- The results are processed through the mitogen-activated pro- nent, molecular function and biological process, and led to tein kinase (MAPK) and phospholipase Cg signal transduc- explore the more valuable data in the regulation of melanoma tion pathways.40) The extracellular portion of the protein en- carcinogenesis against kojic acid. In addition, seven down- coded by FGFR3 gene interacts with fibroblast growth fac- regulated genes showed significantly differential expression tors, sets in motion an acidic and basic fibroblast growth hor- in A375 cells after kojic acid treatment and played the func- mone and plays a role in bone development and mainte- tion as tumor suppressors that may deactivate the regulation nance.72,73) The inhibition of FGFR3 also induced differentia- of melanoma tumorigenesis in human malignant melanoma tion and apoptosis in multiple myeloma activated FGFR3 is cells. These genes may become the useful markers for further also an oncogene that contributes to tumor progression in diagnostic and therapeutic applications. Moreover, our find- multiple myeloma including human breast cancer.37—39) Ac- ings also showed the probably side effects or disadvantages cording to our finding of down-regulated FGFR3 gene, it is of kojic acid on the dysfunction of immune system and bone supported that the down-regulation of FGFR3 may deacti- development and maintenance. However, we will further vate the tumor progression of malignant transformation in study the effects of kojic acid on biological and molecular melanoma cells. mechanisms of human melanoma skin cells, including other GALNT1 (UDP-N-acetyl-alpha-D-galactosamine: polypep- parts of human body, and also examine other biological char- tide N-acetylgalactosaminyl transferase 1, GalNAc-T1) trans- acterization of kojic acid in the cosmetic and/or therapeutic fers an N-acetyl galactosamine (GalNAc) to a serine or threo- applications. nine residue in the first step of O-linked oligosaccharide biosynthesis.41,42) GALNT1 has been reported to have a ubiq- REFERENCES uitous expression in all gastric carcinoma cell lines. It sug- gests that the differential expression of GalNAc transferases 1) Kwak M. Y., Rhee J. S., Biotechnol. Bioeng., 39, 903—906 (1992). 668 Vol. 29, No. 4

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