Supplementary Information For s3

Supplementary Information for

Acta Pharmacologica Sinica advance online publication, 25 Apr 2011; doi: 10.1038/aps.2011.17 Epigallocatechin-3-gallate regulates cell growth, cell cycle and phosphorylated nuclear factor-κB in human dermal fibroblasts

Dong-Wook HAN1, #, Mi Hee LEE2, 3, #, Hak Hee KIM2, 4, Suong-Hyu HYON4, Jong-Chul

PARK2, 3, *

1Department of Nanomedical Engineering, College of Nanoscience & Nanotechnology,

Pusan National University, Busan 609-735, Korea; 2Cellbiocontrol Laboratory, Department of Medical Engineering, 3Brain Korea 21 Project for Medical Science, Yonsei University

College of Medicine, Seoul 120-752, Korea; 4Department of Medical Simulation

Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical

Sciences, Kyoto University, Kyoto 606-8507, Japan

# These authors contributed equally to this paper.

* To whom correspondence should be addressed: Prof Jong-Chul Park, Department of

Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong,

Seodaemun-gu, Seoul 120-752, Korea. Tel: +82 2 2228 1917; fax: +82 2 363 9923. E-mail address: [email protected]. Materials and methods

Preparation of fluorescent DNA probe and hybridization

Total RNA was extracted from the EGCG-treated and non-treated nHDFs using the TRIzol reagent

(MRC, Cincinnati, OH, USA). Fluorescence labeled cDNA probes were prepared form 30 g of total

RNA by oligo (dT)18-primed polymerization using SuperScript II reverse transcriptase (Invitrogen,

Grand Island, NY, USA) in a total reaction volume of 30 L. The reverse transcription mixture included 400 U Superscript RNase H-reverse transcriptase (Invitrogen), 15 mmol/L dATP, dTTP and dGTP, 0.6 mmol/L dCTP, and 3 mmol/L cyanine (Cy)3- or Cy5-labeled dCTP (NEN Life Science

Product Inc, Boston, MA, USA). After reverse transcription, the sample RNA was degraded by adding 5 L of stop solution (0.5 mol/L NaOH/50 mmol/L EDTA) and incubating at 65 °C for 10 min. The labeled cDNA mixture was then concentrated using ethanol precipitation method. The concentrated Cy3- and Cy5-labeled cDNAs were resuspended in 10 L of hybridization solution

(GenoCheck, Ansan, Gyunggi-do, Korea). After two labeled cDNAs were mixed, the mixture was denaturized at 95 °C for 2 min. The slides were hybridized for 12 h at 62 °C hybridization chamber.

The hybridized slides were washed in 2 SSC, 0.1 % SDS for 2 min, 1 SSC for 3 min, and then

0.2 SSC for 2 min at room temperature. The slides were centrifuged at 3000 revolutions per minute for 10 s to dry.

Scanning and image analysis

Hybridized slides were scanned with the Axon Instruments GenePix 4000B scanner and the scanned images were analyzed with the software program GenePix Pro 5.1 (Axon Instruments Inc, Foster

City, CA, USA) and GeneSpring 7.0 – R Integration Package (Sillicon Genetics, Redwood City, CA,

USA). In order to allow algorithm to eliminate all bad spots, no data points were eliminated by visual inspection from the initial GenePix image. For signal normalization, positive control genes (A. thaliana genes and Amp genes) were spotted onto each slide. The signals of these spots were used for

2 normalization. To determine the background signal intensity, the spotting solution was spotted on each slide. To filter out the unreliable data, spots with signal-to-noise (signal – background – background SD) below 100 were not included in the data. Data were normalized by Global, lowess, print-tip, and scale normalization for data reliability. Gene spots showing more than a 2-fold difference between the Cy3 and Cy5 signals were considered to be differentially expressed genes as a result of EGCG treatment. A comparison of the data analysis obtained from the two experiments indicated that both experiments were highly reproducible. Hybridization was performed three times for each sample. Average values were obtained.

Cell culture and conditions

Primary-cultured cells, including human articular chondrocytes (HACs), human dermal fibroblasts

(HDFs), human umbilical vein endothelial cells (HUVECs), and rat aortic smooth muscle cells

(RASMCs), were isolated from their corresponding tissues according to established procedures. As cell lines, murine fibroblastic (L-929) cells and human fibrosarcoma (HT-1080) cells were obtained from American Type Culture Collection (Rockville, MD, USA). HACs were cultured in Dulbecco's modified Eagle's medium/Ham’s F-12 (DMEM/F-12, Sigma-Aldrich Co, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich Co) and 1% antibiotic-antimycotic solution (including 10 000 units penicillin, 10 mg streptomycin and 25 g amphotericin B per mL,

Sigma-Aldrich Co) at 37 °C and 5% CO2 in a humid environment. HUVECs were cultured in endothelial cell basal medium-2 (Lonza, Walkersville, MD, USA) supplemented with 2% FBS

(Lonza), EC growth supplements (Lonza) and 1% antibiotic-antimycotic solution at the same conditions as mentioned above. The other cells including HDFs, RASMCs, L-929 and HT-1080 cells were routinely maintained in DMEM (Sigma-Aldrich Co) with 10% FBS (Sigma-Aldrich Co) and

1% antibiotic-antimycotic solution at the same conditions as mentioned above.

3 Cytotoxicity assay

The number of viable cells was quantified indirectly using a highly water soluble tetrazolium salt

[WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (Dojindo Lab., Kumamoto, Japan), reduced to formazan dye by mitochondrial dehydrogenases. Cell viability was found to be directly proportional to the metabolic reaction products obtained in WST-8. Briefly, WST-8 assays were conducted as follows. All cells were incubated with WST-8 for the last 4 h of the culture period (24 h) at 37 °C in the dark. In order to avoid a direct reaction between EGCG and WST-8, the cells were thoroughly washed with phosphate-buffered saline and refreshed with a fresh medium containing WST-8. Parallel sets of wells containing freshly cultured non-treated cells were regarded as the controls. Absorbance was determined at 450 nm using an ELISA reader (Spectra Max 340, Molecular Device Co., Sunnyvale,

CA, USA). The relative cell viability was determined as percentage of the optical densities in the medium containing serially diluted concentrations of EGCG to the optical densities in the fresh

control medium. The LC50 value, the concentration (mol/L or g/mL) inhibiting growth of cells by

50%, was estimated from the relative cell viability.

Confocal laser scanning microscopy

To examine cellular uptake of EGCG in L-929 cells, the cells were treated for 24 h with 100 or 400

mol/L FITC-conjugated EGCG (FITC-EGCG). After FITC-EGCG treatment, the cells were washed thoroughly with PBS, fixed with 3.5% paraformaldehyde (Sigma-Aldrich Co) in 0.1 mol/L phosphate buffer (pH 7) for 5 min at room temperature and immediately observed under a confocal laser scanning microscope (LSM 510, Carl Zeiss Advanced Imaging Microscopy, Jena, Germany).

Cell nuclei were counterstained with 5 mol/L PI directly before 3 ~ 5 min of observation.

Fluorescence microscopy

4 In order to examine the time course of the incorporation of FITC-EGCG into nHDFs, the cells were treated with 50 mol/L FITC-EGCG for 2 d. After FITC-EGCG treatment, the cells were thoroughly washed with PBS and further cultured with refreshment with fresh complete media. After 1, 3, 6 and

10 d of incubation, the cells were observed under a fluorescence microscope (Biozero – 8000,

Keyence, Osaka, Japan).

Results

Effect of EGCG on gene expression of nHDFs

We employed a cDNA microarray analysis to evaluate the effect of EGCG on the gene expression levels in nHDFs. The genechip containing 3096 human genes was used and included 50 cell cycle- related genes. We report here only the difference in gene expression levels that are statically significant. Out of 3096 probe sets, we focused on 1520 (49.1%) genes that received a present call which is statistically reliable with respect to the control. Among them, the expression levels of 84 genes (5.5%) were changed over 2-fold by EGCG treatment relative to control signals. We categorized changed genes according to specific functions, including cellular (927 genes, 61.0%), signal transduction (346 genes, 22.8%) processes and the others (247 genes, 16.2%). Cellular processes represent cell cycle (50 genes, 3.3%) and apoptosis (877 genes, 57.7%). Among 50 cell cycle-related genes, the expression levels of 10 genes (20.0%) were changed over 2-fold due to

EGCG treatment. Within these 10 genes, the expression of 9 genes was statistically down-regulated with EGCG treatment (Supplementary Table S1) and the rest, ie, only 1 genes statistically up- regulated. Among the 877 genes related to apoptotic process, the expression of 40 genes (4.6%) was statistically changed with EGCG treatment (Supplementary Table S2). In 346 genes correlated with signal transduction, the expression of 18 genes (5.2%) was changed over 2-fold with EGCG treatment (Supplementary Table S3).

5 Cytotoxicity profiles

Cytotoxicity profiles of EGCG were determined in various phenotypes of primary cells or cell lines by WST-8 assay. All cells exposed to increasing concentrations of EGCG for 24 h showed a dose-

dependent decrease in their relative cell viability. The LC50 valves of EGCG (mol/L and g/mL) according to the primary cells or cell lines were listed as shown in Supplementary Table 4. Among

these cells, HT-1080 cells showed relatively higher sensitivity to EGCG with 200 mol/L of LC50 valve, which was approximately 3.5-fold lower than that of L-929 cells. Importantly, this value was

3-fold lower than that of their normal counterpart, HDFs. In the case of the primary cells, RASMCs were least sensitive to EGCG than other primary cells and the value of LC50 was found to be about

1100 mol/L that was about 7.3-fold higher than that of HUVECs. This result implies that cellular sensitivities to EGCG are quite different between cells and the cytotoxicity profile of EGCG depends upon the cell phenotypes, cell-originated tissues and species.

Cellular uptake of FITC-EGCG in L-929 cells

The cellular uptake of FITC-EGCG in L-929 fibroblastic cells was clearly seen into both the cytoplasm and nucleus. These cells were highly sensitive to FITC-EGCG with the evidence that some cells showed typical apoptotic appearances, including blebbing, nucleus swelling, and changes to the cell membrane such as loss of membrane asymmetry and attachment, and that others were not subjected to the nuclear translocation of FITC-EGCG (Supplementary Figure S1A). Confocal micrographs of L-929 cells detached due to apoptosis mediated by higher dose (400 mol/L) of

FITC-EGCG showed that FITC-EGCG was still incorporated into the cytoplasm of the detached cells (Supplementary Figure S1B). Furthermore, both the membrane and nucleus of the cells were too damaged to delineate due to apoptosis.

Time course of cellular uptake of FITC-EGCG in nHDFs

6 Fluorescence microscopic observation demonstrated the time course of cellular uptake of FITC-

EGCG in cultured nHDFs. It was revealed that FITC-EGCG was clearly observed in both the membrane and cytoplasm of the cells until 10 d despite the removal of FITC-EGCG from culture media (Supplementary Figure S2). Although the exact half-life of FITC-EGCG was not determined, it seemed to be at least 10 d or longer even at 50 mol/L.

Discussion

Taking these results into consideration, a feasible action mechanism of EGCG for cell cycle- regulation and subsequent cytoprotection in nHDFs would be suggested as shown in a schematic illustration in Supplementary Figure S2. First, EGCG freely binds to membrane receptors, eg laminin receptors, on nHDFs, forming EGCG-receptor complexes. Next, it is internalized into the cytosol through unknown mechanism, followed by affecting various signal cascades responsible for the regulation of nuclear transcription factors, ie pNF-B. Afterwards, it is translocated into the nucleus via unknown pathways, which might be partly accounted by the fact that a phytoestrogen molecule, structurally related with catechin, binds to an estrogen receptor and then moves to the nucleus thru nuclear pores[1]. Finally, it regulates cell cycle-related genes, including CCNs and CDKs, reversibly.

Therefore, cell cycle in normal cells is delayed by the cytopretective activity of EGCG, whereas cell cycle in cancer cells is arrested by the cytotoxic effect of EGCG and the cells would be apoptosed.

Further study about what signal transduction pathways at upstream are involved in cell cycle delay is required. Moreover, these results do not rule out the possibility of transcription factors responsible for the EGCG-mediated reversible regulation of cell cycle, and the exact mechanism needs to be elucidated.

7 Reference

1 Gruber CJ, Tschugguel W, Schneeberger C, Huber JC. Production and actions of estrogens. N

Engl J Med 2002; 346: 340–52.

8 Supplementary Table S1. List of significantly down-regulated cell cycle-related genes (except for genes listed in Table

1) in nHDFs after EGCG treatment

Gene (symbol) Role Fold

Ubiquitin-like, containing PHD and RING finger Required during the cell cycle for DNA replication 0.19 domains, 1 (UHRF1) Ribonucleotide reductase M2 polypeptide (RRM2) Synthesis of M2 is regulated in a cell-cycle dependent fashion 0.28 Serine/threonine kinase 6 (STK6) Cell cycle-regulated kinase involved in microtubule formation 0.33 V-myb myeloblastosis viral oncogene homolog Nuclear protein involved in cell cycle progression 0.42 (avian)-like 2 (MYBL2)

9 Supplementary Table S2. List of significantly down-regulated/up-regulated apoptosis-related genes in nHDFs after

EGCG treatment

2', 5'-oligoadenylate synthetase 2 (OAS2) Inhibit cellular protein synthesis and viral infection resistance 0.08 2', 5'-oligoadenylate synthetase 1 (OAS1) 0.12 Inhibitor of DNA binding 1 (ID1) Play a role in cell growth, senescence and differentiation 0.14 Kinetochore associated 2 (KNTC2) Involved in spindle checkpoint signaling 0.17 Inhibitor of DNA binding 3 (ID3) Inhibit transcription through formation of nonfunctional dimmers 0.19 Centromere protein F (CENPF) Play a role in chromosome segregation during mitotis 0.24 MAD2 mitotic arrest deficient-like 1 (MAD2L1) Component of the mitotic spindle assembly checkpoint 0.27 Topoisomerase (DNA) II  170kDa (TOP2A) Control and alter the topologic states of DNA during transcription 0.30 Interferon induced with helicase C domain 1 (IFIH1) Involved in alteration of RNA secondary structure, embryogenesis, 0.31 spermatogenesis, and cellular growth and division Baculoviral IAP repeat-containing 5 (BIRC5) Encode negative regulatory proteins that prevent apoptotic cell death 0.32 Transgelin (TAGLN) Early and sensitive marker for the onset of transformation 0.36 Minichromosome maintenance deficient 5 (MCM5) Involved in the initiation of DNA replication 0.36 Matrix metalloproteinase 3 (MMP3) Involved in the breakdown of ECM in normal physiological processes 0.42 Fibronectin 1 (FN1) Involved in cell adhesion and migration processes 0.43 CSE1 chromosome segregation 1-like (CSE1L) Play a role both in apoptosis and in cell proliferation 0.43 Tripartite motif-containing 21 (TRIM21) Mediate intracellular immunity 0.45 Ectonucleotide pyrophosphatase/phosphodiesterase 1 Function to hydrolyze nucleoside 5' triphosphates to their corresponding 0.48 (ENPP1) monophosphates and may also hydrolyze diadenosine polyphosphates Thymidylate synthetase (TYMS) Considered as a target for cancer chemotherapeutic agents 0.49

10 Continued form Supplementary Table S2.

24-dehydrocholesterol reductase (DHCR24) Catalyze the reduction of double bond during cholesterol biosynthesis 3.02 Keratin 17 (KRT17) Encode the type I intermediate filament chain keratin 17 2.87 Insulin induced gene 1 (INSIG1) Play a critical role in regulating cholesterol concentrations in cells 2.86 Desmoglein 2 (DSG2) Ca-binding transmembrane glycoprotein component of desmosomes 2.47 Damage-specific DNA binding protein 2 (DDB1) Implicated in the etiology of Xeroderma Pigmentosum group E 2.27 Fatty acid synthase (FASN) Catalyze the synthesis of palmitate from acetyl-CoA and malonyl-CoA 2.18 Emopamil binding protein (EBP) Involved in the transport of cationic amphiphilics 2.12 Protein tyrosine phosphatase, receptor type, U Regulate a variety of cellular processes including cell growth, 2.02 (PTRU) differentiation, mitotic cycle, and oncogenic transformation Programmed cell death 4 (PDCD4) Play a role in apoptosis, but the specific role has not yet been determined 1.96 Tetraspanin 4 (TM4SF7) Play a role in the regulation of cell development, activation, growth and 1.96 motility

11 Supplementary Table S3. List of significantly down-regulated/up-regulated signal transduction-related genes in nHDFs after EGCG treatment

Myxovirus (influenza virus) resistance 1 (MX1) Responsible for a specific antiviral state against influenza virus infection 0.07 Guanylate binding protein 1 (GBP1) Bind guanine nucleotides (GMP, GDP and GTP) 0.26 RAN binding protein 1 (RANBP1) Act as a negative regulator of RCC1 by inhibiting RCC1-stimulated 0.40 guanine nucleotide release from RAN Stathmin 1/oncoprotein 18 (STMN1) Function as an intracellular relay integrating regulatory signals of the 0.40 cellular environment Fibroblast growth factor 18 (FGF18) Involved in embryonic development, cell growth, morphogenesis, 0.42 tissue repair, tumor growth, and invasion Dishevelled, dsh homolog 1 (DVL) Regulate cell proliferation, acting as a transducer molecule for 0.43 developmental processes, including segmentation and neuroblast specification

Growth differentiation factor 15 (GDF15) Regulate tissue differentiation and maintenance 2.78 Chloride channel 6 (CLCN6) Defined as voltage-dependent chloride channel genes 2.14 Bone morphogenetic protein 2 (BMP2) Induce bone formation 2.13 Tumor necrosis factor receptor superfamily, Function as an antagonistic receptor that protects cells from 2.00 member 10c (TNFRSF10C) TRAIL-induced apoptosis Lymphotoxin receptor (LTBR) Play a role in the development and organization of lymphoid tissue 1.92 and transformed cells

12 Supplementary Table S4. LC50 values of EGCG according to various phenotypes of primary cells or cell lines.

Cells tested LC50 value, mol/L (g/mL)

HACs 320 (147)

HDFs 600 (275)

HUVECs 150 (69)

RASMCs 1,100 (504)

L-929 cells 690 (316)

HT-1080 cells 200 (92)

13 Supplementary Figure S1. Cellular uptake of FITC-EGCG in L-929 cells. Cells were incubated with 100 mol/L (A) and 400 mol/L (B) FITC-EGCG for 24 h and then observed under a confocal laser scanning microscope. The micrographs shown in this figure are representative of 6 independent experiments, showing similar results [Original magnification: × 500 and × 1500

(A, upper and lower, respectively), and × 1800 (B)].

14 Supplementary Figure S2. Fluorescence micrographs for the time course of cellular uptake of FITC-EGCG in cultured nHDFs. After treated with 50 mol/L FITC-EGCG for 2 d, the cells were thoroughly washed with PBS, further incubated and then observed under a fluorescence microscope at 1, 3, 6 and 10 d. All the photographs shown in this figure are representative of 4 independent experiments, showing similar results (Original magnification, × 200).

15 Supplementary Figure S3. Schematic illustration of mechanism of cell cycle-regulation and subsequent cytoprotection mediated by EGCG in nHDFs.