Differential Gene Expression Profile between PC-14 Cells Treated

National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. Received June 2, 2002; Revised Manuscript Received ...
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Bioconjugate Chem. 2003, 14, 449−457

449

Differential Gene Expression Profile between PC-14 Cells Treated with Free Cisplatin and Cisplatin-Incorporated Polymeric Micelles Nobuhiro Nishiyama,† Fumiaki Koizumi,‡ Soichiro Okazaki,† Yasuhiro Matsumura,§ Kazuto Nishio,‡ and Kazunori Kataoka*,† Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, Pharmacology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan, and Investigative Treatment Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. Received June 2, 2002; Revised Manuscript Received November 17, 2002

Cisplatin (CDDP)-incorporated polymeric micelles (CDDP/m) are a macromolecular carrier system possessing a time-modulated decaying property accompanied by sustained release of free drug. The gene expression profiles in nonsmall cell lung cancer PC-14 cells treated with free CDDP and CDDP/m were evaluated by a cDNA expression array for 807 genes. Although the total gene expression profile of the cells treated with CDDP/m approximated that of free CDDP in the hierarchical clustering analysis, a number of genes showed differential expression according to whether the cells had been treated with CDDP or CDDP/m. Ultimately, 50 genes with significant differential expression between cells treated with CDDP and CDDP/m were selected by principal component (PC) analysis and the unpaired t-test. The genes selected, including genes related to cell cycle regulation, apoptosis-related proteins, detoxification, and DNA repair enzymes, were considered to be related to CDDP-induced cytotoxicity. Interestingly, CDDP/m down-regulated the genes encoding integrins and matrix metalloproteinases (MMPs), which play an integral role in tumor invasion, metastasis, and angiogenesis, whereas free CDDP up-regulated them. The results suggest that use of the macromolecular carriers may yield additional therapeutic effects over free drug.

INTRODUCTION

A variety of macromolecular carrier systems have been developed for the use in the diverse field of chemotherapy, particularly for cancer treatment, and this technology will continue to receive growing attention as a key to the development of novel therapeutic agents (14). Macromolecular carriers that provide increased accumulation at target sites and reduced accumulation at side effect sites should yield maximal therapeutic efficacy. Polymer-drug conjugates and colloidal drug carrier systems, such as liposomes and micelles, in particular, have been attracting great interest in tumor targeting therapy because of their preferential accumulation in solid tumors (5, 6). The preferential tumor accumulation of macromolecular drug carrier systems is explained by the so-called “enhanced permeability and retention (EPR)” effect, which accounts for the unique characteristics of solid tumors with enhanced permeability in tumor vasculature (7). Hence, some formulations have yielded remarkable antitumor activity and are being assessed in clinical studies (8, 9). In addition to controlled biodistribution, macromolecular carrier systems allow controlled drug release and * To whom correspondence should be addressed. Kazunori Kataoka, Ph.D., Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656. Japan. Phone: +81-3-5841-7138. Fax: +81-3-5841-7139. E-mail: kataoka@ bmw.t.u-tokyo.ac.jp. † The University of Tokyo. ‡ National Cancer Center Research Institute. § National Cancer Center Research Institute East.

alteration of the internalization routes and subcellular localization of drugs. The control of drug release and internalization routes by macromolecular carriers may lead additional or better therapeutic effects. Indeed, there is benefit of macromolecular drugs that will circumvent drug efflux, e.g., resistance against P-glycoprotein dependent efflux in multidrug resistant cells (10-12). In addition, we hypothesized that alteration of the modality of cell entry, leading to different subcellular drug localization, drug-concentration gradients, and controlled drug release, will regulate gene and protein expression in a manner different from exposure to the free drug, resulting in additional or better therapeutic effects. Indeed, Kopecek et al. have suggested that N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates (PK-1), which are internalized via endocytosis, and whose entry into cells is followed by lysosomal enzyme-specific drug release, regulates different genes from free Dox and thereby overcomes the Dox-resistance of tumor cells both in vitro and in vivo (13, 14). In this study, we used cDNA expression array techniques (15, 16) to evaluate differences in gene expression between human nonsmall cell lung cancer (NSCLC) cells treated with cisplatin (CDDP) and CDDP-incorporated polymeric micelles (CDDP/m). CDDP-incorporated micelles formed via a ligand exchange reaction of CDDP from the chloride to the carboxylate side chain of poly(ethylene glycol)-poly(amino acid) block copolymer was found to be very stable when diluted with distilled water, whereas they dissociated into unimers with sustained release of the regenerated CDDP via an inverse ligand exchange from carboxylate to

10.1021/bc025555t CCC: $25.00 © 2003 American Chemical Society Published on Web 02/22/2003

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chloride in physiological saline at 37 °C (17). CDDPincorporated micelles also yielded prolonged blood levels in murine Lewis lung carcinoma (LLC)-bearing mice, resulting in preferential and enhanced accumulation in tumors (18). These CDDP-incorporated micelles are also expected to allow different internalization into cells and subcellular drug distribution, and they can be expected to exert the novel biological actions. MATERIALS AND METHODS

Materials. CDDP [cis-diamminedichloroplatinum(II): CDDP] was purchased from Aldrich Chemical Co., Inc., Milwaukee, WI. R-Methoxy-ω-aminopoly(ethylene glycol) (CH3O-PEG-NH2; Mw ) 12 000) was donated by Nippon Oil Fats Co., Ltd., Tokyo, Japan. 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Isogen were obtained from Wako Pure Chemical Industries, Co., Ltd., Osaka, Japan, and Nippon Gene Co., Ltd, Tokyo, Japan, respectively. An Atlas Pure Total RNA Labeling System and Atlas cDNA Expression Array were purchased from BD Bioscience Clontech Co., Inc., (Palo Alto, CA,). [R-32P]dATP (110 TBq/mmol) was obtained from Amersham Bioscicences Co, Tokyo, Japan. Preparation of CDDP-Incorporated Micelles. A micelle-forming poly(ethylene glycol)-poly(glutamic acid) block copolymer [PEG-P(Glu)] was prepared according to a previously reported procedure (19). Briefly, poly(ethylene glycol)-poly(β-benzyl L-glutamate) block copolymer (PEG-PBLG) was synthesized by ring-opening polymerization of the N-carboxy anhydride of β-benzyl L-glutamate (BLG-NCA) initiated by CH3O-PEG-NH2 in dimethylformamide (DMF), followed by alkali hydrolysis of β-benzyl group to prepare PEG-P(Glu). The degree of polymerization of P(Glu) segment was determined to be 35 by 1H NMR measurement, and a narrow distribution of the obtained block copolymer was confirmed by gel-permeation chromatography (GPC) measurements (Mw/Mn ∼ 1.06). CDDP-incorporated micelles were prepared by simple mixing of CDDP and PEG-P (Glu) in distilled water at a mixing molar ratio of CDDP to Glu residue of 1. Purification of CDDP-incorporated micelles was carried out by ultrafiltration (MWCO: 100 000, membrane: Millipore PT series). Characterization of CDDP-Incorporated Micelles. The size of the CDDP-incorporated micelles and their CDDP content were determined by dynamic light scattering (DLS) and atomic absorption spectroscopy (AAS) measurements, respectively. The release of CDDP from CDDP-incorporated micelles in physiological saline at 37 °C (pH 5.2 and 7.4, 150 mM NaCl) was evaluated by the dialysis method (Spectra/Por-6 (MWCO: 1000), Spectrum Laboratories, Inc. Rnacho Dominguez, CA), and concomitant decay of the micellar structure was estimated by static light scattering (SLS) based on a change in the Rayleigh ratio at a scattering angle of 90° (R(90°)) (17). The R(90°) value should decrease with micelle dissociation in tandem with a decrease in apparent molecular weight of the micelles (Mw,app) and in micelle concentration. The R(90°) value of the micelles in distilled water at 37 °C was used as the control in this measurement. MTT Assay. The growth-inhibitory activity of free CDDP and CDDP-incorporated micelles on human nonsmall cell lung cancer cell line PC-14 was evaluated by MTT assay (20). PC-14 cells (5000 cells) were cultured in RPMI 1640 medium (Sigma Biosciences Co.) containing 10% fetal bovine serum in 96-well multiplate. The cells were then exposed to each preparation (free CDDP

Figure 1. Release of Pt from CDDP-incorporated micelles (CDDP/m) at pH 5.2 (closed triangle) and pH 7.4 (open triangle) and change in relative scattering light intensity of CDDP/m (closed square) in physiological saline at 37 °C.

or CDDP-incorporated micelles) for 72 h, and MTT solution was added. Cell viability was estimated by formazan absorbance at 570 nm. RNA Isolation. PC-14 cells were exposed to each preparation at the 90% cell growth-inhibitory concentration (IC90), which was estimated based on MTT assay after 72 h incubation. Following 6- or 12-h drug exposure, total RNA was isolated with Isogen based on phenolchloroform extraction. mRNA was prepared with an Atlas Pure Total RNA Labeling System according to the manufacturer’s protocol. cDNA Expression Array. cDNA expression array analysis was carried out by using an Atlas cDNA expression array customized in our laboratory. The customized array consists of 807 genes including genes related to cell cycle regulation, signal/oncogenes, angiogenesis/adhesion/ cell-cell interaction, growth factors, cytokines, apoptosisrelated, DNA transcription factors/damage response/ repair, and recombination, metabolism, translation/ protein turnover/detoxification enzymes, transporters/ nucleocytoplasmic transporters/symporter, and antiporters/ cytoskeletal proteins (see Figure 2a). The experiment was performed according to the manufacturer’s protocol Data Analysis. The image on the imaging plate (BAS-III2040; Fuji Film Co., Tokyo, Japan) that was exposed to the cDNA-hybridized membranes for 3-4 days was scanned with a BAS-2000II image scanner (Fuji Film Co.). The signal density of each spot was quantified by Array Gauge ver. 1.2 software (Fuji Film Co.), and the data were subjected to background adjustment by using the minimum density value of all spots and the global median normalization. The expression ratio (log2 (drugtreated)/(drug untreated)) was used for further analysis. The hierarchical clustering analysis was carried out by using Web-available software, “Cluster” and “Tree View,” provided by M. Eisen (http://rana.lbl.gov/). Principal component (PC) analysis with Simca-P ver. 8.0 (Umetrics Co. Ume, Sweden) followed by the unpaired t-test was performed to select genes differentially expressed by cells treated with CDDP and CDDP-incorporated micelles. RESULTS

Formation of CDDP-incorporated micelles (CDDP/m) having a diameter of 27.9 nm and a narrow size distribution was confirmed by DLS measurement. AAS measurement yielded a CDDP content of CDDP/m of 31% (w/w), which corresponds to a [CDDP]/[Glu] ratio of 0.61. Since CDDP/m showed a decrease in relative scattering light intensity and sustained release of CDDP in physiological saline (150 mM NaCl), as previously reported (Figure 1)

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Figure 2. (a) Coordinates of the customized Atlas gene expression array composed of 807 genes including (A) genes encoding factors related to cell cycle regulation, (B) signal/oncogenes, (C) angiogenesis/adhesion/cell-cell interaction, (D) growth factors, (E) cytokines, (F) apoptosis-related genes, (G) DNA transcription factors/damage response/repair and recombination, (H) metabolism, (I) translation/ protein turnover/detoxification enzymes, (J) transporters/nucleocytoplasmic transporters/symporter & antiporters/cytoskeletal proteins, (K) other genes. (b) gene expression images of untreated, CDDP 6 h-, CDDP 12 h-, CDDP/m 6 h-, CDDP 12 h-treated PC-14 cells. Table 1. Cytotoxic Activity of CDDP and CDDP/m against PC-14 Cells IC50a CDDP CDDP/m

IC90b

48 h

72 h

48 h

72 h

1.88 5.49

1.45 4.82

25.3 48.7

10.8 28.3

a,b 50% and 90% cell growth-inhibitory concentration determined by MTT assay (unit: µg/mL).

(17), CDDP/m is a novel type macromolecular carrier system exhibiting time-modulated decay of carrier as well as sustained drug release (18). Interestingly, the release of CDDP was accelerated by decreasing the pH of the media from 7.4 to 5.2, suggesting accelerated drug release from CDDP/m in the endosomal/lysosomal environment when whole CDDP/m is internalized into a cell. The growth-inhibitory effect of CDDP and CDDP/m on PC-14 cells was evaluated by MTT assay. The 50% and 90% cell growth-inhibitory concentrations (IC50 and IC90) after 48 and 72 h drug exposure are shown in Table 1. The increased IC50 value of CDDP/m may be consistent with the hypothesis that the CDDP released from the micelles is mainly responsible for the cytotoxic activity of CDDP/m. Indeed, we previously reported that preincubation of CDDP/m in physiological saline disrupting the micellar structure displayed comparable cytotoxicity to free CDDP (18). In this study, PC-14 cells were exposed to each drug at the IC90, which was estimated based on 72 h drug exposure (Table 1) to isolate total RNA. The gene expression profiles of PC-14 cells were examined by using the customized Atlas cDNA expression array. The customized array consists of 807 genes,

and their coordinates are shown in Figure 2a. To investigate time-dependent gene expression, total RNA was isolated after 6 and 12 h of drug-exposure and followed by mRNA isolation and synthesis of 32P-labeled cDNA. The array images after hybridization of cDNA from untreated, CDDP 6 h-, CDDP 12 h-, CDDP/m 6h-, and CDDP/m 12 h-treated PC-14 cells are shown in Figure 2b. The signal intensity of each spot was quantified by Array Gauge ver. 1.1, followed by background adjustment and global median normalization. The expression ratio [log2 (drug-treated)/(drug untreated)] was used for further analysis. Hierarchical clustering of all 807 genes was performed and is shown in Figure 3. Although CDDP 6 and 12 h showed clustering into the closest branch, as expected, CDDP/m 12 h was clustered into the branch closer to CDDP 6 and 12 h rather than to CDDP/m 6 h. The clustering results suggest that regulation of total gene expression by CDDP/m may approximate that of free CDDP. This seems to be consistent with the aforementioned hypothesis that the CDDP released from micelles plays a major role of the cytotoxic activity of CDDP/m. Nevertheless, it is of great interest that a number of genes were differentially expressed by cells treated with CDDP/m in comparison to CDDP (Figure 3). Principal component (PC) analysis by Simca-P 8.0 was performed to investigate the relation between important sets of variables (samples). PC modeling in Simca-P is programmed to calculate two or three score vectors for genes that summarize all the variables entered into the analysis and plot them against each other (score plot: Figure 4(B)). PC analysis by Simca-P also provides values

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The unpaired t-test between CDDP- and CDDP/mtreatment was further carried out for the aforementioned 105 candidate genes, and 50 significant genes were selected as a result (P < 0.05) (Table 2). The 50 genes include the most important genes for the action of CDDP, such as the genes encoding check point kinase 1 (Chk1), phospholipase C delta (PLC-delta), p53-binding mouse double minute 2 homologue (MDM2), caspase-9 precursor, glutathione S-transferase (GST), glutathione reductase (GSR), and damage response and repair-related proteins, as well as interesting genes involved in angiogenesis and cell adhesion factors, such as integrins and matrix metalloproteinases (MMPs). The results of reclustering the 50 genes selected are shown in Figure 5 and provide easy-to-follow graphical profiles of differential gene expression by genes treated with CDDP and CDDP/m. DISCUSSION

Figure 3. Hierarchical cluster analysis of the expression of all genes (807 genes) on the Atlas gene expression array.

for so-called loading vectors, showing how the variables are combined to form the scores. The plot of loading vectors (loading plot: Figure 4A) indicates which of the variables are important and corresponds to the directions in the score plot. Importantly, the opposite direction of the loading vector p[1], the first principal component, was observed between CDDP and CDDP/m treatment. It is worth noting that the first principal component possessed a clearly larger fraction of sum of squares (SS) (0.526), which is also computed by Simca-P, than the next component (0.274). Since the position of an observation in a score plot is influenced by genes lying in the same direction in the loading plot, genes with an absolute score vector value (|t[1]|) above 0.04 were extracted to select genes differentially regulated in cells treated with CDDP and CDDP/m. As a result, we selected 105 candidate genes.

CDDP is one of the most important antitumor drugs in clinical use and is widely used for the treatment of many malignancies, including testicular, ovarian, bladder, head and neck, stomach, esophagus, and small-cell and non-small-cell lung cancers (21). However, it is wellknown that CDDP has significant toxic side effects, such as nephrotoxicity and neurotoxicity, limiting its therapeutic window (22). Since intravenously administered CDDP is rapidly eliminated from the blood circulation by glomerular excretion (23), a large number of macromolecular carriers for CDDP have been developed with the aim of increasing its bioavailability and achieving selective targeting of solid tumors (24-27). We also prepared CDDP-incorporated polymeric micelles (CDDP/ m) via polymer-metal complex formation between CDDP and PEG-poly (amino acid) block copolymers containing a carboxyl group and found that CDDP/m is a promising formulation for macromolecular carrier systems with both prolonged blood circulation and preferential accumulation in solid tumors (17, 18). In addition, CDDP/m exhibited the time-modulated decaying of a carrier and the sustained drug release at an increased rate in an acidic environment (Figure 1), which may lead to accelerated drug release from CDDP/m in the endosomal/ lysosomal environment after internalization via an endocytic pathway. We hypothesized that the aforementioned characteristics of CDDP/m alter the pharmacological effect of free CDDP in addition to improving its biodistribution. In this study, we used cDNA expression array techniques to evaluate the biological effect of CDDP/m on PC-14 cells from the standpoint of the gene expression profiles.

Figure 4. Loading plot (A) and score plot (B) in the principal component (PC) analysis computed by Simca-P 8.0. The 105 genes most likely to influence the loading vector p[1] were selected in the Score plot.

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Table 2. List of Genes Differentially Expressed between CDDP- and CDDP/m-Treatment in PC-14 Cells (50 genes) coordinate

GenBank assoc no.

A04j A04k

AF016582 L25676

A05m

Z12020; M92424 L36870 mitogen-activated protein kinase kinase 4 (MAP kinase kinase 4; MKK4; PRKMK4); c-jun N-terminal kinase kinase 1 (JNKK1); JNK-activating kinase 1; SAPK/ERK kinase 1 (SERK1; SEK1) M65066 cAMP-dependent protein kinase type I beta regulatory subunit (PRKAR1B) D10495 protein kinase C delta (PKC-delta) M22430; membrane-associated phospholipase A2 group 2A precursor J04704 (PLA2G2A); PLA2B; PLA2L; synovial group II phospholipase A2; phosphatidylcholine 2-acylhydrolase; RASFA U09117 phospholipase C delta-1 (PLC-delta 1; PLCD1); PLC-III; 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase delta 1 X60957; tyrosine kinase receptor tie-1 precursor S89716 M15042 carcinoembryonic antigen precursor (CEA); meconium antigen 100; CD66E antigen M35296 Abelson murine leukemia viral oncogene homologue 2 (ABL2); Abelson-related gene (ARG) X95456 Rho7 protein X61587 rho-related GTP-binding protein (RHOG); ARHG L20688 rho GDP dissociation inihibitor 2 (RHO GDI2; RHO-GDI beta); LY-GDI; ARHGDIB; GDID4 L36531 integrin alpha 8 (ITGA8) D25303; integrin alpha 9 (ITGA9); integrin alpha-RLC L24158 M34189 integrin beta 1 (ITGB1) M62880; integrin beta 7 precursor (ITGB7) S80335 M73780 integrin beta 8 precursor (ITGB8) X89576 matrix metalloproteinase 17 (MMP17); membrane-type matrix metalloproteinase 4 (MT-MMP4) J03210; matrix metalloproteinase 2 (MMP2); 72-kDa gelatinase A; J05471 72-kDa type IV collagenase precursor (CLG4A); H-ras-transformed bronchial epithelial cells protein (TBE1) U14394 tissue inhibitor of metalloproteinase 3 precursor (TIMP3) M62403 insulin-like growth factor binding protein 4 precursor (IGF-binding protein 4; IGFBP4; IBP4) X63454 fibroblast growth factor 6 precursor (FGF6); HBGF6; HST2 U31628 interleukin 15 receptor alpha subunit precursor X01394 tumor necrosis factor alpha precursor (TNF-alpha; TNFA); cachectin X01992; interferon gamma precursor (IFN-gamma; IFNG); immune interferon M29383 X86779 fas-activated serine/threonine kinase (FAST) U56390; caspase 9 precursor (CASP9); MCH6; ICE-like apoptotic protease 6 U60521 (ICE-LAP6); apoptotic protease activating factor 3 (APAF3) U18321 + ionizing radiation resistance-conferring protein + death-associated ×83544 protein 3 (DAP3) X65372 polypyrimidine tract-binding protein (PTB); heterogeneous nuclear ribonucleoprotein I (HNRNP I); 57-kDa RNA-binding protein PPTB-1 L16785; nucleoside diphosphate kinase B (NDP kinase B; NDKB); M36981 expressed in nonmetastatic cells 2 protein (NME2); myc purine-binding transcription factor (PUF); NM23B M58603 nuclear factor kappa-B DNA binding subunit (NF-kappaB; NFKB) M11722; DNA nucleotidylexotransferase; terminal addition enzyme; K01919 terminal deoxynucleotidyltransferase (DNTT; TDT) M29971 6-O-methylguanine-DNA methyltransferase (MGMT); methylated-DNA-protein-cysteine methyltransferase X02308 thymidylate synthase (TYMS; TS) AF005482 histone deaceytlase 3C (HD3C) X98743 RNA helicase X95694 AP-2 BETA TRANSCRIPTION FACTOR X16135 heterogeneous nuclear ribonucleoprotein L (HNRNPL) U39487 xanthine dehydrogenase/oxidase U89507 UDP-glucuronosyltransferase 1A7 D29013 DNA polymerase beta (POLB) M81882 glutamate decarboxylase 65-kDa isoform; 65-kDa glutamic acid decarboxylase (GAD-65); GAD2 X68676; glutathione S-transferase mu1 (GSTM1; GST1); HB subunit 4; S01719 GTH4 X15722 glutathione reductase (GRase; GSR; GR) AF098951 ATP-binding cassette subfamily G (white) member 2 (ABCG2); breast cancer resistance protein (BCRP); mitoxantrone resistance protein 1 (MXR1) L06237 microtubule-associated protein 1B X03212 cytoskeletal keratin 7 (KRT7); cytokeratin 7 (CK7) AF007567 insulin receptor substrate 4

A13j B03k B04f B04m B05h B09g C02h C02i C03g C03h C03k C08l C08m C09d C09g C09h C10i C10k C12h D07e D09b D10j D11k D13j E02i E03e E04f E05g E06g E06h E07g E07h E09e E09k E10b E10c E10i F01d F02f F02j F02l F05g F05l F06i F07h F08d F10i a

GENES checkpoint kinase 1 (CHK1) cell division protein kinase 9 (CDK9); serine/threonine protein kinase PITALRE p53-binding mouse double minute 2 homologue (MDM2)

log-transformed gene expression ratio (log2 (drug-treated)/(drug untreated)).

CDDP/m CDDP/m 6 ha 12 ha

P value

0.3589 0.9808

-0.5818 0.0236

-0.5492 -0.0425

0.0254 0.0056

-0.4768 -0.3122

0.7938

0.7725

0.0413

-0.4867 -0.2272

-1.3349

-1.0977

0.0400

CDDP 6 ha 0.2526 0.8879

CDDP 12 ha

4.1849

4.1084

-0.5778

-0.1806

0.0229

0.8123 1.7693

1.1251 1.4086

-1.1035 -0.0236

-0.9485 0.3154

0.0209 0.0284

1.3815

1.0711

-1.2880

-1.5894

0.0065

1.2182

1.0839

0.5231

0.6724

0.0322

2.3447

2.0317

-0.5250

0.0159

0.0290

4.0238

3.9180

0.2762

0.0546

0.0053

1.1885 1.1718 2.1443

0.9857 1.6974 2.0033

-0.2615 -1.5768 0.1056

-0.5891 -1.0904 0.3166

0.0260 0.0166 0.0078

1.4232 1.0698

1.8722 1.2810

-0.0612 -0.3565

-0.3279 -0.2194

0.0328 0.0124

2.1738 3.8544

2.1555 4.4260

0.1417 0.6540

0.0746 -0.0444

0.0061 0.0152

3.8738 0.9206

3.5687 0.7022

0.1551 -1.3248

-0.2853 -0.8462

0.0078 0.0449

1.4994

1.9333

-0.9108

-0.7919

0.0401

2.2666 1.6132 -1.0697 -0.9829

-0.0050 0.6220

-0.5752 0.3936

0.0372 0.0267

1.1856 2.2810 2.1371 4.7192

0.3042 -0.2602 0.6883 2.2839

0.2158 0.4647 1.0533 2.0270

0.0054 0.0495 0.0355 0.0056

-2.2257 -1.7354 1.7003 2.3393

0.2259 -0.6017

-0.3990 -0.1793

0.0459 0.0341

-3.4638 -3.2228

-1.6241

-1.6746

0.0374

-3.1025 -3.2089

-1.5117

-1.2152

0.0322

-3.8504 -4.2303

-1.6370

-2.2779

0.0474

-1.9854 -2.1616 -2.1594 -2.3056

-0.9785 -1.3909

-0.8970 -1.1817

0.0225 0.0239

-2.3814 -2.5830

-1.2392

-1.2036

0.0447

-2.1649 4.0119 -2.3798 -1.4885 -1.2177 -0.3445 -0.2770 -0.4463 4.3263

-2.0495 3.3157 -1.7416 -1.2814 -1.3347 -0.4636 -0.4710 -0.3087 3.8605

-0.3644 0.0742 0.1599 0.1142 -0.1598 0.5293 0.4563 0.5213 1.6765

-0.2277 0.3781 -0.2631 -0.1966 -0.3418 0.5462 0.3379 0.4079 1.2696

0.0028 0.0347 0.0460 0.0269 0.0178 0.0367 0.0336 0.0124 0.0144

-0.8445 -1.0977

0.2464

0.0912

0.0274

-0.6048 -0.3911 0.0774 0.1956

0.4085 0.7054

0.5739 0.5973

0.0214 0.0238

0.2454 0.6525 0.4567

0.0838 0.8723 0.3861

0.0236 0.0450 0.0274

1.1213 2.6822 2.5606 5.0160

1.0748 1.6226 1.1887

1.3015 1.7168 1.3401

454 Bioconjugate Chem., Vol. 14, No. 2, 2003 Nishiyama et al.

Figure 5. Hierarchical cluster analysis of the expression of 50 genes selected by PC analysis followed by the unpaired t-test (P < 0.05).

Differential Gene Expression Profile

It is well-known that CDDP is a DNA-damaging agent that reacts with genome DNA to form interstrand and intrastrand DNA cross-links and DNA-protein crosslinks, and that the formation of CDDP-DNA adducts is essential to its cytotoxic activity (28, 29). Although the mechanism whereby the CDDP-induced DNA damage leads to cell death is not fully understood, there is evidence that an intrastrand cross-link between adjacent guanines (1,2-d(GpG)-CDDP adduct) forms more frequently and is more refractory to excision repair (29). The use of the macromolecular carriers might not change this essential subcellular target of CDDP (i.e., 1,2-d(GpG)), but may change the drug’s modality of cellular uptake, its subcellular localization and concentration gradient, and interaction with subcellular molecules, such as lipids and proteins. It has been suggested that approximately one-half of free CDDP enters cells by passive diffusion and the other half by an unidentified pump (29). The internalized CDDP may interact with many cellular components with nucleophilic sites, especially being inactivated by binding to glutathione (γ-glutamylcysteinylglycine, GSH), and only approximately 1% of the internalized CDDP may interact with genomic DNA (28, 29). CDDP/m, on the other hand, is internalized via endocytosis, allowing intracellular drug release. Actually, the results of the hierarchical clustering analysis suggested that the total gene expression profile of CDDP/m may approximate that of free CDDP with free drug release from the micelles (Figure 3). Nevertheless, a large number of genes were found to be differentially expressed by cells treated with CDDP and CDDP/m (Figure 3). Indeed, the first principal component in the PC analysis was most likely to discriminate between CDDP and CDDP/m (Figure 4). As a result, 50 genes that significantly differed between cells treated with CDDP and CDDP/m were selected (P < 0.05) (Table 2). It is generally accepted that CDDP-induced DNA damage is detected by several families of DNA excision and mismatch repair proteins, leading to cell cycle arrest or induction of apoptosis depending on the level of CDDP-DNA adducts (28, 29). If the level of CDDP-DNA adducts is high enough to be irreparable during the G2 phase, apoptosis is triggered. Chk1 has been proposed to constitute signaling pathways that respond to structures characteristic of DNA damage and/or incomplete DNA replication and regulate the G2 checkpoint (30, 31). The p53 tumor suppressor protein is also thought to influence CDDP-induced apoptosis (32). In response to DNA damage, p53 is activated and functions as a transcription factor to up-regulate gene products necessary for G1 phase cell cycle arrest or apoptosis (33). One of the genes up-regulated by p53, is mdm2, and MDM2 protein shuttles p53 through MDM2-p53 complex formation, promoting the degradation of p53 via polyubiquitination (34, 35). Thus, MDM2 constitutes a negative autoregulatory feedback of p53, preventing p53-dependent apoptosis. The caspase family was activated by CDDP treatment (28). Procaspase-9 is transformed into caspase-9 through the association initiated by binding of the apoptotic protease-activating factor-1 (Apaf-1) in the presence of ATP and cytochrome c, and activated caspase-9 sequentially activates caspase-3 leading to apoptosis (36, 37). Indeed, it has been suggested that the mitochondrial release of cytochrome c may be involved in CDDP-induced apoptosis (38). On the other hand, CDDP treatment may activate JNK kinase activity and induce c-jun expression (39, 40). Depending on the cell type, c-jun has been shown to act either as an effector of apoptosis or as an antiapoptotic factor after stress induced injury (41). c-Abl is a

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nonreceptor tyrosine kinase that may be translated in response to CDDP-induced DNA damage (29) and may promote CDDP-induced apoptosis by unknown mechanism involving the DNA-dependent protein kinase and ataxia telangiectasia mutated (ATM) gene product (42, 43). Abl2 is a null mutation of c-Abl with an almost identical phenotype (44). Although genomic DNA is generally accepted as the critical target for CDDPinduced cytotoxicity, there is evidence that other cellular targets may be involved in the cytotoxicity (28). The phosphatidylcholine-specific phospholipase C (PC-PLC) may be activated prior to DNA-CDDP adduct formation during CDDP treatment, and this early event may play an important role in CDDP-induced cytotoxicity (45). The data in Table 2 suggest that CDDP/m activates the aforementioned genes toward anti-apoptosis, whereas CDDP seems to activate them toward apoptosis. Glutathione S-transferase (GST) and glutathione reductase (GSR) are responsible for detoxification of intracellular CDDP in a cell defense mechanism against CDDP toxicity, while several families of nucleotide excision repair enzymes function in removal of the platinum adducts from DNA (28, 29). We observed several differences in the expression of such genes between cells treated with CDDP and CDDP/m (Table 2). Interestingly, CDDP/m down-regulated the gene expression of integrin and matrix metalloproteases (MMPs) families, whereas free CDDP up-regulated them (Table 2), and it has been suggested that the cell adhesion status and composition of cytoskeletal components may be associated with the antitumor effect of CDDP/m. The family of integrins, heterodimeric transmembrane glycoproteins, links the intracellular skeketon to the extracellular matrix (ECM) proteins, affecting cell adhesion and migratory activity. Some members of the integrin family have been reported to be overexpressed or differently expressed in chemotherapeutic agent-resistant cells compared with the sensitive parental cells (46-48). MMPs, a family of enzymes contributing to the degradation of ECM, play an integral role in tumor invasion, metastasis, and angiogenesis. MMPs are possible targets related to angiogenesis and metastasis for cancer treatment (49, 50). The down-regulation of integrin and MMPs genes by CDDP/m may lead to additional therapeutic effects. In this study, cDNA expression array techniques revealed differential gene expression between PC-14 cells treated with CDDP and CDDP/m. These results may involve the effect of the delayed cellular response to the sustained release of free drug from the micelles leading to a lag in gene expression. In addition, it will be necessary to investigate the transcriptional and translational levels of the selected genes by conventional techniques, such as Northern blotting and Western blotting, respectively. Furthermore, the hypothesized mechanism of CDDP/m needs to be confirmed by in vivo animal model experiment in the future work. Nevertheless, it is of a great interest that the expression of important sets of genes significantly differed between cells treated with CDDP and CDDP/m, and the difference in expression may affect the pharmacological effects of the drug. For example, we have often observed that macromolecular carriers containing antitumor drugs did not parallel with an equimolar dose of free drug in vitro (18, 51, 52) and in vivo (18, 53) that cannot be completely explained based on its pharmacodynamic and pharmacokinetic properties, and gene expression altered by macromolecular carrier systems may be involved in this unexplained cytotoxic

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activity. On the other hand, since integrin and MMP family genes were down-regulated by CDDP/m, gene expression altered by macromolecular carrier systems may confer additional therapeutic effect. Thus, macromolecular carrier systems may provide technology that modulates not only pharmacokinetics in the body but therapeutic effects at diseased sites, and it may be necessary to take the latter effect into consideration in addition to optimization of pharmacokinetics when designing novel macromolecular carriers. In the future, more information regarding the relationships between structural, physicochemical, and functional properties of macromolecular carriers affecting drug release, cell entry, and subcellular localization and their pharmacological effects, including transcriptional and translational regulation, as well as interaction with intracellular molecules will provide better understanding of the signaling pathways specific to macromolecular carrier systems, leading to the design of clinically useful macromolecular carrier systems. Advanced bioinformatics technologies, such as gene expression and proteome analysis, will shed new light on such studies. ACKNOWLEDGMENT

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