The genome-wide expression profile of Paeonia suffruticosa-treated cisplatin-stimulated HEK 293 cells

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Environmental Toxicology and Pharmacology 28 (2009) 453–458

Contents lists available at ScienceDirect

Environmental Toxicology and Pharmacology

journa l homepage: www.e lsev ier .com/ locate /e tap

he genome-wide expression profile of Paeonia suffruticosa-treatedisplatin-stimulated HEK 293 cells

ung-Hwa Sohna, Eunjung Koa, Yunju Joa, Sung-Hoon Kimb, Yangseok Kima, Minkyu Shina,oochang Honga, Hyunsu Baea,∗

Department of Physiology, College of Oriental Medicine, Kyung Hee University, 1, Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of KoreaDepartment of Oriental Pathology, College of Oriental Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea

r t i c l e i n f o

rticle history:eceived 17 April 2009eceived in revised form 24 July 2009ccepted 4 August 2009vailable online 11 August 2009

a b s t r a c t

The root cortex of Paeonia suffruticosa Andrews (PS) is a crude Chinese drug used in many traditionalprescriptions. This study was conducted to evaluate the recovery mechanisms induced by PS in HEK 293cells treated with cisplatin. Previous studies revealed that 1 �g/ml PS treatment showed greater than20% recovery of 14 �M cisplatin-induced 50% loss of cell proliferation (IC50) determined by mitochon-

eywords:isplatinaeonia suffruticosaene expression profileEK 293 cells

drial activity MTS assay in HEK 293 cells (Sohn et al., 2009). In the present study, the effects of PS on geneexpression profiles in stimulated HEK 293 cells were evaluated by oligonucleotide microarray and real-time RT-PCR. The results of the microarray analysis and real-time RT-PCR revealed that DNA repair- andcell proliferation-related genes were up-regulated in PS-treated HEK 293 cells. The mechanism respon-sible for the effects of PS on HEK 293 cells is closely associated with the regulation of DNA repair andcell proliferation. Thus, PS possesses novel therapeutic potential that could be used for the prevention or

uced

treatment of cisplatin-ind

. Introduction

Cisplatin (cis-diamine II dichloroplatinum) is a DNA-damagingolecule that is widely used as an antineoplastic drug (Brabec

nd Kasparkova, 2002). Indeed, cisplatin is frequently used toreat malignancies such as cancer of the testis, metastatic ovar-an tumors, lung cancer, advanced bladder cancer and many otherypes of solid tumors. Although high doses of cisplatin effectivelyuppress malignancies (Siddik, 2003; Iraz et al., 2006; Pratibha etl., 2006; Hsu et al., 2007; Martins et al., 2008), dose-dependentumulative nephrotoxicity is the major limitation of this com-ound, sometimes requiring a reduction in dose or discontinuationf treatment (Schrier, 2002).

Nephrotoxicity is of critical concern during the early stages ofrug development when selecting new drug candidates (Uehara etl., 2007). Due to its unique metabolism, the kidney is an impor-ant target of the toxic effects of drugs, xenobiotics, and oxidativetress. Therefore, during the screening of products produced by

edicinal plants for use as new therapeutic agents for the treat-ent of renal diseases, the recovery activities of 239 spray-dried

xtracts of herbal medicines on cisplatin-induced cytotoxicity inEK 293 cells were evaluated. Among these extracts, Paeonia suf-

∗ Corresponding author. Tel.: +82 2 961 0323; fax: +82 2 962 9316.E-mail address: [email protected] (H. Bae).

382-6689/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.etap.2009.08.001

nephrotoxicity.© 2009 Elsevier B.V. All rights reserved.

fruticosa (PS) showed the highest recovery and was thus selected forthis study (Sohn et al., 2009). The root cortex of PS is a well-knowntraditional Chinese medicine that has been widely used as a seda-tive, analgesic, anti-diabetic agent, anti-inflammatory agent andas a remedy for several conditions, such as cardiovascular diseaseand female genital diseases (Lin et al., 1998; Stickel and Schuppan,2007; Wang et al., 2008). Microarray analysis is a molecular tech-nique that enables the parallel analysis of gene expression by avery large number of genes encompassing a significant fraction ofthe human genome. This method is both qualitative and quantita-tive because it is able to detect changes in the levels of expressionin treated cells based on comparison with control samples (Kim etal., 2006; Wang et al., 2006). Therefore, the use of microarray anal-ysis can enable the development of more advanced therapies forthe treatment of renal disease using naturally derived products.

The goal of this study was to elucidate the possible mechanismsby which PS extract improved the recovery of HEK 293 cells underpressure from cisplatin-induced cytotoxicity.

2. Materials and methods

2.1. Preparation of PS and cisplatin

PS was purchased from Sun Ten Pharmaceutical (Taipei, Taiwan), powdered to0.1 g and then extracted by stirring in 10 ml of DW (distilled water) overnight at roomtemperature using a stirrer. The sample was then centrifuged for 10 min at 191 × g,after which the supernatant was removed and sterilized by passing it through a0.22 �m syringe filter (Sartorius Stedium Biotech GmbH 37070, Goettingen, Ger-

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any). The filtered extract was then used for the experiments. A voucher specimenas deposited in the Herbarium of the College of Oriental Medicine, Kyung Hee Uni-

ersity, Korea. Dr. Minkyu Shin, the director of the herbarium, confirmed the identityf the plants and assigned a herbarium sheet number (No. PMP0081). Cisplatin wasissolved in 0.1% DMSO and used as a positive control.

.2. Cell culture

HEK 293 cells, which represent a primary embryonal kidney cell line that haseen transfected with adenovirus 5 DNA, were obtained from the Korean Cell Lineank (KCLBTM). The HEK 293 cells were grown and maintained at 37 ◦C and 100%umidity under 5% CO2 in Dulbecco’s modified Eagles’s medium (DMEM) supple-ented with 10% fetal bovine serum (FBS), streptomycin, and penicillin (Invitrogen

ife Technologies, Rockville, MD, USA). The HEK 293 cells were then seeded intoissue culture flasks (T-75 cm2) that contained hormonally defined DMEM media atdensity of 1 × 107/ml as described previously. The medium was changed every 3ays until the cells became 80–90% confluent, at which point they were used for thexperiments.

.3. RNA preparation

HEK 293 cells were initially cultured in a 100 mm dish (2 × 106/ml) for 24 h at

7 ◦C, after which they were treated with 14 �M cisplatin for 1 h. Next, 0 or 1 �g/mlS was added and the cells were then incubated at 37 ◦C for 23 h. The total RNAas then isolated from the HEK 293 cells using an Rneasy® mini kit (Qiagen GmbH,ilden, Germany) according to the manufacturer’s instructions and subsequentlyuantified using a 1000 NanoDrop spectrophotometer (NanoDrop Technologies Inc.,ilmington, DE, USA).

ig. 1. Clustergram of up- and down-regulated genes in HEK 293 cell line. Microarray datlus PS-treated HEK 293 cells) groups were combined and clustered. There were 3 indelustered boxes, and each experimental sample is represented by a single column. Each camples. The expression level of each gene was visualized in color. A color bar indicates hompared with those in the common reference RNA. Experimental conditions (NC; nonamples) are shown at the top of the data matrix. (For interpretation of the references to

nd Pharmacology 28 (2009) 453–458

2.4. Oligonucleotide chip microarray

Microarray analysis was conducted using single round RNA amplification pro-tocols, following the Affymetrix specifications (Affymetrix GeneChip ExpressionAnalysis Technical Manual). Briefly, 3 �g of total RNA were used to synthesize first-strand cDNA using oligonucleotide probes with 24oligo-dT plus T7 promoter as aprimer (Proligo LLC, Boulder, CO) and the Superscript Choice System (InvitrogenLife Technologies, Rockville, USA). Double-stranded cDNA was then synthesized andpurified by phenol–chloroform extraction, and biotinylated antisense complimen-tary RNA (cRNA) was generated through in vitro transcription using the BioArrayRNA High-Yield Transcript Labeling kit (ENZO Life Sciences Inc., Farmingdale, NY).The biotinylated cRNA was then fragmented, after which 10 �g of total fragmentedcRNA was hybridized to the Affymetrix Human Genome U133 Plus 2.0 GeneChiparray (P/N900470, Affymetrix Inc., Santa Clara, CA, USA) and washed, stained andscanned according to the manufacturer’s instructions. Next, the fluorescence wasdetected using the Genechip System Confocal Scanner (Hewlett-Packard, SantaClara, CA, USA), after which analysis of each GeneChip was conducted with theGeneChip 3.1 software produced by Affymetrix, using the standard default settings.To compare different chips, global scaling was used, with all probe sets being scaledto a user-defined target intensity of 150.

2.5. Data analysis

The MAS5 algorithm was used to evaluate the expression signals generated bythe GeneChip. Global scaling normalization was then performed, after which thenormalized data were log-transformed using base2. Next, fold change was appliedto select the differentially expressed genes (DEGs) using a fold change thresholdof 2.0 and a p < 0.05 to indicate significance. Each probe set used in the GeneChip

a from control (non-treated HEK 293 cells) and experimental (cisplatin or cisplatinpendent samples for each treatment. Each gene is represented by a single row ofolumn represents the average expression levels of all genes in three hybridizationigher levels of gene expression in the sample in red and lower levels in green when-treated control, PC; cisplatin-treated control, and PS; Paeonia suffruticosa-treatedcolor in this figure legend, the reader is referred to the web version of the article.)

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Table 1Down-regulation of genes based on comparison of gene expression between experimental (Paeonia suffruticosa (PS)-, cisplatin (PC), PC plus PS-treated) and control (non-treated (NC)) HEK 293 cells.

Genes Probe ID Symbol Regulation profile and fold change

NC PC PC + PS PS

Immune and inflammatory response-related genesComplement component 8, gamma

polypeptide210324 at C8G 1 30 8.5 1.3

Chemokine (C-C motif) receptor 2 206978 at CCR2 1 6 1.3 3.7Dual oxidase 1 1565795 at DUOX1 1 1.8 0.5 0.8Immunoglobulin heavy constant mu 212827 at IGHM 1 1.1 0.3 0.72′-5′-Oligoadenylate synthetase-like 205660 at OASL 1 2.8 1.0 3.2Prostaglandin-endoperoxide synthase 2

(prostaglandin G/H synthase andcyclooxygenase)

1554997 a at PTGS2 1 8.4 3.8 6.3

Apoptosis-related genesFission 1 (mitochondrial outer membrane)

homolog244082 at FIS1 1 1.6 0.6 1.1

Table 2Up-regulation of genes based on comparison of gene expression between experimental (Paeonia suffruticosa (PS)-, cisplatin (PC), PC plus PS-treated) and control (non-treated(NC)) HEK 293 cells.

Genes Probe ID Symbol Regulation profile and fold change

NC PC PC + PS PS

DNA repair-related genesAtaxia telangiectasia mutated 208442 s at ATM 1 0.9 1.7 1.4Excision repair cross-complementing rodent

repair deficiency, complementation group 4210158 at ERCC4 1 2.3 6.5 4.7

PI-3-kinase-related kinase SMG-1 214984 at DKFZp547E087 1 2.0 5.9 1.6

Cell proliferation-related genesBUB1 budding uninhibited by

benzimidazoles 1 homolog215508 at BUB1 1 0.3 2.1 2.1

v-erb-a erythroblastic leukemia viraloncogene homolog 4

206794 at ERBB4 1 0.8 3.7 3.5

PDS5, regulator of cohesion maintenance, 1570119 at PDS5B 1 0.3 1.2 1.4

PL

pcrftGlwh 52).

2

t(tAqa9etgt(5((cttie

homolog B

Negative chemotaxis-related genesPlexin A3 1567519 at

roduces a detection call, with P (present call) indicating good quality, M (marginalall) indicating intermediate quality and A (absent call) indicating relatively loweliability. Therefore, probe sets that resulted in A calls were removed to filteralse positives. Gene ontology significance analysis was conducted to investigatehe functional relationships among the 2.0-fold DEGs using high-throughputoMiner (Zeeberg et al., 2003). The gene list was selected based on fold changes

arger than 2.0-fold (log2). Among these genes, a candidate gene list was selectedith the perl 5.10.0 program using CTD (the Comparative Toxicogenomics Database,ttp://ctd.mdibl.org/downloads/;jsessionid=1D66F5393023B6D4369AE7656531CB

.6. Real-time RT-PCR (RTRP) analysis

Microarray verification was performed by RTRP analysis of selected genes usinghe SYBR Green I Master Mix (Applied Biosystems, Foster City, CA, USA) and primersGenotech Inc., Korea). cDNA was synthesized using 2 �g of RNA in a reverseranscription reaction. RTRP quantitative mRNA analyses were performed with anpplied Biosystems 7300 Real-time PCR System using the SYBR Green fluorescenceuantification system (Applied Biosystems, Foster City, CA, USA) to quantify themplicons. PCR was conducted by subjecting the reaction mixture to 40 cycles of5 ◦C for 15 s and 60 ◦C for 1 min, after which a standard denaturation curve was gen-rated. The sequences of the human primers were as follows: BUB1 (FW 5′-gat gcatt gaa gcc cag tt-3′; RW 5′-atg cat tcc ctg cct tta tg-3′), PDS5B (FW 5′-ggt gga atg attcc tca gt-3′; RW 5′-aag aag gag gat gct aaa aat gg-3′), ATM (forward primer (FW) 5′-ga aca ggg caa aat cct tc-3′; reverse primer (RW) 5′-tcc atg gtt ttc tca cag ca-3′), CCR2FW 5′-tcc att ctc tca ggc ttg ct-3′; RW 5′-aag gac atc tgc gaa gca ct-3′), ERBB4 (FW′-aac ctt gca acg gta tcc ag-3′; RW 5′-cat gga att caa gcc aat cc-3′), DKFZp547E087FW 5′-tgc ggt ctc ttt ctt ctt cc-3′; RW 5′-tcc agt ccc aag tat ttc aga t-3′), and GAPDH

FW 5′-ttc acc acc atg gag aag gc-3′; RW 5′-ggc atg gac tgt ggt cat ga-3′). The PCRonditions for each target were optimized according to the primer concentration,he absence of primer dimer formation, and the efficiency of amplification of bothhe target genes and the housekeeping gene control. PCR reactions were conductedn a total volume of 20 �l in PCR master mix containing 10 �l 2× SYBR Green, 5 �Mach of sense and antisense primer, and 2 �l of 1:2 diluted cDNA diluted to 20 �l

XNA3 1 0.4 1.9 1.3

with DEPC-treated H2O. To normalize the cDNA content of the samples, we usedthe comparative threshold (CT) cycle method, which consists of the normalizationof the number of target gene copies vs. the endogenous reference gene, GAPDH. TheCT is defined as the fractional cycle number at which the fluorescence generated bycleavage of the probe passes a fixed threshold baseline when amplification of thePCR product is first detected.

2.7. Statistical analysis

Statistical analysis of the data was conducted using Prism 4.02 software (Graph-icPad Software Inc., CA, USA). Data were analyzed by one-way analysis of variance(ANOVA) for Tukey’s multiple comparisons. Results with a p-value < 0.05 were con-sidered statistically significant.

3. Results

3.1. Gene expression profiles in HEK 293 cells

Gene expression profiles were significantly up- or down-regulated in the experimental groups (PS, cisplatin-treated (PC) orcisplatin plus PS-treated (PC + PS) HEK 293 cells) when comparedwith the controls (non-treated (NC)). A total of 180 differentiallyexpressed genes (128 up- and 52 down-regulated) were detectedin the experimental group using approximately 54,600 oligonu-cleotide probes. A hierarchical clustering algorithm was used to

group the genes on the basis of similar expression patterns (Fig. 1).To assess the reliability of our microarray technique, we calculatedthe microarray reproducibility between triplicate RNA samplesfrom three independent cell cultures. A correlation matrix plot wasgenerated based on triplicate samples of the control (NC or PC)

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nd the cisplatin plus PS-treated HEK 293 cells. Reproducibilityhowed an R2 value. The R2 value was determined based on thentensity of signals from all probe sets on the Affymetrix Humanenome U133 Plus 2.0 GeneChip array. Based on the R2 values, theicroarray hybridization patterns were highly consistent among

amples. The average R2 values were 0.97 (NC), 0.99 (PC), 0.99PC + PS), and 0.91 (PS). A perfect relationship among the samplesould produce a slope of 1. For the experimental group, genes

howing highly altered expression levels were aligned accordingo the magnitude of the altered expression. DNA repair, cell pro-iferation, negative chemotaxis, and immune and inflammatoryesponse-related genes were then selected (based on the degreef differential expression) (Tables 1 and 2).

.2. Validation of the selected genes by real-time RT-PCR

We normalized the relative expression levels of each gene byividing the expression of the target genes by the expressionf GAPDH following real-time PCR analysis. GAPDH was cho-

ig. 2. Validation of gene expression by microarray. The results are normalized as a ratio ond the values expressed. This real-time RT-PCR data indicated agreement with the microC; non-treated control, PC; cisplatin-treated control, PS; Paeonia suffruticosa. Data are p

o control.

nd Pharmacology 28 (2009) 453–458

sen to normalize the samples because it was the most stablyexpressed housekeeping gene when the microarray analysis wasconducted. The mean intensities of GAPDH in NC, PS, PC, and PC + PSwere 20,035 ± 1208, 22,873 ± 269, 21,602 ± 629, and 23,680 ± 721,respectively. A SYBR Green assay was used to confirm changes inthe expression of four selected genes identified in the microarrayanalysis of the PS-treated HEK 293 cells, BUB1, PDS5B, ATM, andCCR2. The results of the real-time RT-PCR assay were in agreementwith the microarray results (Fig. 2).

4. Discussion

In a previous study, cisplatin was found to inhibit HEK 293cell proliferation at low micromolar concentrations (Sohn et al.,

2009). Therefore, the recovery activities of herbal medicines againstcisplatin-induced cytotoxicity in HEK 293 cells were evaluatedin an effort to find new therapeutic agents for the treatment ofrenal diseases. Indeed, PS showed greater than 20% recovery ofcisplatin-induced loss of cell proliferation. To evaluate the mecha-

f each specific mRNA signal to the GAPDH gene signal within the same RNA samplearray data. (a) CCR2, (b) ATM, (c) DKFZp547E087, (d) BUB1, (e) ERBB4, and (f) PDS5B.

resented as the mean ± S.E.M. (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 compared

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ism responsible for the action of PS, the gene expression profilesf HEK 293 cells treated with PS were evaluated. We found spe-ific and significant differences in the expression profiles producedy PS-treated HEK 293 cells when compared to untreated HEK 293ells (Tables 1 and 2).

Recently, several studies have shown that cisplatin causesephrotoxicity through multiple mechanisms, including hypoxia,he generation of free radicals, inflammation, and apoptosis (Kangt al., 2004). Specifically, cisplatin induces the expression ofnflammatory cytokines and chemokines and intracellular adhe-ion molecule 1 following cisplatin-induced renal injury (Rameshnd Reeves, 2002, 2003; Faubel et al., 2007; Suzuki et al., 2008).hese cytokines and chemokines are known to contribute to theevelopment and progression of renal injury with renal leukocyte

nfiltration. For example, chemokine (C-C motif) receptor 2 (CCR2)cts as a potent chemoattractant for monocytes and macrophageso promote migration from the peripheral circulation to the sitesf inflammation (Matsushima et al., 1989; Yoshimura et al., 1989;haro et al., 1994). In the present study, our results showed thatCR2 expression was decreased under PC plus PS vs. PC condi-ion by real-time RT-PCR and microarray analysis (Table 1, Fig. 2a).n addition, dual oxidase 1 (Duox1) mRNA expression is knowno be induced by the Th2-specific cytokines interleukin-4 andnterleukin-13 (Harper et al., 2005). This gene is known to mediate

ucus hypersecretion (Shao and Nadel, 2005) and smooth mus-le cell hyperplasia (Suh et al., 1999; Arnold et al., 2001; Arbisert al., 2002). In the present experiment, our results indicated thatuox1 expression was decreased under PC plus PS vs. PC conditiony oligonucleotide microarray (Table 1).

Cisplatin acts as a DNA alkylator and which forms cross-linksetween guanine bases. It is believed that these cisplatin-DNAdducts initiate apoptosis in treated cells (Weiss and Christian,993). A comet assay was used to detect genotoxic effects of cis-latin in the cell cultures of HEK 293 cells (Follmann and Birkner,008). The cisplatin exhibited an increase in the median tail length.revious studies revealed that 1 �g/ml PS exerted a significantecrease in the median tail length by comet assay (Sohn et al.,009). In the present study, our results showed that ATM, ERCC4,nd DKFZp547E087 expression were increased in PC plus PS con-ition compared with that in PC condition by real-time RT-PCRnd microarray analysis (Table 2, Fig. 2b and c). The study showedhat ATM, ERCC4, and DKFZp547E087 genes are DNA repair-relatedenes. Apoptosis is an important mode of cell death in cisplatinephrotoxicity, and many studies have demonstrated that renalubular cell apoptosis occurs after cisplatin treatment (Tsuruya etl., 2003). In the present study, accordant with real-time RT-PCRnd microarray analysis showed that an apoptosis-related geneuch as FIS1 expression was decreased in PC in the presence of PSompared with PC alone, whereas cell proliferation-related genesuch as BUB1, ERBB4, and PDS5B expression were both increasedn PC plus PS compared with that of PC alone (Tables 1 and 2,ig. 2d, e, and f). Besides, PS extract itself affected many pathways.S-treated samples showed down-regulation of genes involved indipocytokine, calcium and MAPK signaling pathways, as well asytokine-cytokine receptor interaction and Toll-like receptor sig-aling pathways. Up-regulation of genes in the presence of PS also

nvolved the citrate cycle, DNA replication and repair, as well as theell cycle and cellular proliferation (Supplement tables).

Taken together, the identification of DNA repair- and cellroliferation-related genes will help us to understand the processesy which cisplatin exerts its toxicity to open a new route for the

herapeutic amelioration of cisplatin-induced nephrotoxicity.

onflict of interest

This manuscript is inapplicable to conflict of interest statement.

nd Pharmacology 28 (2009) 453–458 457

Acknowledgements

This work was supported by the Korea Science and Engineer-ing Foundation (KOSEF) grant funded by the Korean government(MEST) (No. 2009-0063466).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.etap.2009.08.001.

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