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[Cancer Research 63, 7876-7882, November 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Free and N-(2-Hydroxypropyl)methacrylamide Copolymer-bound Geldanamycin Derivative Induce Different Stress Responses in A2780 Human Ovarian Carcinoma Cells

Nobuhiro Nishiyama, Aparna Nori, Alexander Malugin, Yuji Kasuya, Pavla Kopecková and Jindrich Kopecek

Department of Pharmaceutics and Pharmaceutical Chemistry/Center for Controlled Chemical Delivery (CCCD), University of Utah, Salt Lake City, Utah


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of geldanamycin (GA), 17-(3-aminopropylamino)-17-demethoxygeldanamycin (AP-GA), and N-(2-hydroxypropyl)methacrylamide copolymer-AP-GA conjugate [P(AP-GA)] on A2780 human ovarian carcinoma cells at an equitoxic dose (2x IC50) were compared by the gene expression array analysis. All treatments resulted in similar gene expression profiles up to 12 h (e.g., down-regulation of CDK4 and up-regulation of APAF-1), although P(AP-GA)-treated cells showed delayed gene expression because of time-dependent internalization of the conjugate and intracellular drug release from P(AP-GA). However, AP-GA-treated cells showed elevated expression of HSP70 and HSP27 after 6 h compared with that observed by GA and P(AP-GA) treatments. Depletion of C-Raf, an HSP90 client protein, was observed in all treatments up to 12 h. Confocal microscopy using mesochlorin e6 as a model drug revealed that drug release caused by the lysosomal cleavage of glycylphenylalanylleucylglycine oligopeptide spacer, used as GA derivative copolymer attachment/release point, was moderately fast. These results suggested that AP-GA treatment may activate stress-response pathways, whereas P(AP-GA) treatment may suppress them and trigger signaling pathways essential to cell growth arrest and death by inducing an HSP90-active factor. Although GA and P(AP-GA) treatments induced a time-dependent increase in HSP70 and HSP27 protein expression (detected by Western blotting analysis), AP-GA treatment resulted in more rapid and more intense expression of both proteins. Our results suggest that conjugation of AP-GA to N-(2-hydroxypropyl)methacrylamide copolymer may be able to modulate the cell stress responses induced by AP-GA because of differences in its internalization mechanism, subcellular localization, and intracellular concentration gradients.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Use of macromolecular carriers such as water-soluble polymer-drug conjugates has been recognized as a promising way to increase the therapeutic efficacy of low molecular weight drugs (1, 2, 3) . The main concept is based on improving pharmacokinetic properties of low molecular weight drugs and targeting the diseased site. It has been shown that macromolecular carriers can preferentially accumulate in solid tumors because of the so-called enhanced permeability and retention effect (4) . Hence, the use of macromolecular carriers has been attracting great interest in targeted cancer therapy. In addition, the advantages of macromolecular carriers may not be confined to improvements in biodistribution. Recently, our laboratory has demonstrated in vitro and in vivo that HPMA1 copolymer-DOX conjugate [P(DOX)] can trigger different signaling pathways (i.e., activation of the apoptotic pathways and impairment of the cell defensive mechanisms) compared with free DOX, yielding better pharmacological effects (5 , 6) . We hypothesize that such effects of HPMA copolymer-drug conjugate may be the result of difference in their pathways of internalization and intracellular trafficking. Indeed, a number of experiments have indicated that macromolecular drugs circumvent P-gp-associated drug efflux because of their endocytic internalization. This increases the antitumor activity of the loaded drug in multidrug-resistant cells (7 , 8) . Thus, there are other interests regarding modulation of the pharmacological effects by use of macromolecular carriers. However, little is known regarding general effects of macromolecular drugs at the pharmacological level. Recently, gene expression arrays were used to compare the gene expression profiles that resulted from treatment using free versus micellar cisplatin, and the feasibility of this technique to study the mechanism of action of macromolecular drugs has been demonstrated (9) .

GA is known to bind specifically to the ATP-binding site of HSP90 and related Grp94 and to inhibit their chaperone function (10 , 11) . As a result, client proteins of HSP90 such as Src, C-Raf, and p185erbB-2 are dominantly degraded via the ubiquitin-proteasome pathway, resulting in antiproliferative effects on the cell. The ability of GA to abrogate multiple cell survival and growth signaling pathways has attracted much attention to its use as an antitumor agent. It is anticipated that this quality will overcome the phenotypic heterogeneity of spontaneous tumors. In addition, the selective depletion of mutant p53 by GA may also sensitize tumor cells to other cytotoxic agents (12) . However, the development of GA as an antitumor agent has been hampered severely because of severe toxicity (13) . A large number of GA derivatives have been developed, and its structure-activity studies have revealed that modification of 17-position GA can improve the chemical and physicochemical properties (e.g., stability in serum and water solubility) while maintaining HSP90-inhibitory activity (14) . Recently, 17-allylamino-17-demethoxy-geldanamycin entered Phase I clinical study; however, significant hepatotoxicity was observed because of the lack of selectivity to tumor cells (11) . Hence, several attempts have been made recently to develop a tumor-selective delivery system for GA or its derivatives to increase the therapeutic window (15, 16, 17, 18) .

Recently, we modified GA to produce AP-GA, a derivative possessing a primary amine group applicable to polymer-drug conjugation (19) . AP-GA was then attached to HPMA copolymer via a lysosomally degradable oligopeptide (GFLG) spacer (15 , 16) . This spacer allows for the efficient release of drug in the lysosome after endocytosis but is stable in bloodstream. In this study, we evaluated the pharmacological effects of HPMA copolymer-AP-GA conjugate [P(AP-GA)] on A2780 human ovarian carcinoma cells by the gene expression array analysis. Using the array data and validation by RT-PCR and Western blotting analysis, we specifically evaluated the effect of HPMA copolymer-drug conjugate on cell stress-response signaling pathway.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Line.
The human ovarian carcinoma cell line A2780 was obtained from Dr. T. C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA). Cells were cultured in RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 10 µg/ml insulin (HyClone) under a humidified atmosphere containing 5% CO2 (v/v) at 37°C.

Drugs.
GA was kindly supplied by the National Cancer Institute. AP-GA and HPMA copolymer-AP-GA conjugate [P(AP-GA)] were synthesized according to the procedure reported previously (15 , 16 , 19) , and their chemical structures are shown in Fig. 1Citation . A lysosomally degradable GFLG spacer was used as a polymer-drug linker (20) . The average molecular weight, molecular weight distribution (Mw/Mn), and AP-GA content of the synthesized P(AP-GA) were determined to be Mw 17,600, 1.3 and 1.8 mol %, respectively. HPMA copolymer-Mce6 conjugate with a lysosomally degradable GFLG drug spacer (Mw ~22 000; Mw/Mn ~1.3; Mce6 content, 3.5 mol %) was prepared as reported previously (21) .



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  		Fig. 1. Chemical structures of GA (A), AP-GA (B), and P(AP-GA) (C).

 
Confocal Fluorescence Microscopy.
Fifty thousand A2780 cells were plated on a Lab-Tek II chamber slide (Nalge Nunc International, Rochester, NY). After 24 h, the cells were incubated with HPMA copolymer-Mce6 conjugate at an 800 µM Mce6-equivalent concentration for 1 h, washed twice with PBS, and then incubated further in fresh culture medium. The cells were fixed with 4% formaldehyde after a defined incubation period, and the slide samples for confocal microscopy were prepared by using a SlowFade Light antifade kit (Molecular Probes, Eugene, OR). Confocal microscopy was performed using a Zeiss LSM510 confocal imaging system (Carl Zeiss, Inc., Thornwood, NY) with a x63 plan-apo objective lens. Mce6 was excited at 400 nm by using an 800-nm two-photon infrared (IR) laser and detected using a 680-nm KP filter. The settings for the measurement of control cells were designed to minimize autofluorescence and were used for all of the measurements.

Cytotoxicity Assay.
The cytotoxicity of GA, AP-GA, and P(AP-GA) on A2780 cells was evaluated by the MTT (Sigma Chemical Co.) assay (22) . A2780 cells (10,000 cells) were exposed to each drug in the culture medium containing 0.5% DMSO for 72 h in 96-well multiplate. Cell viability was estimated based on the formed formazan absorbance at 570 nm.

RNA Isolation.
A2780 cells were exposed to each drug using 2-fold IC50, as estimated by MTT assay. After a defined period of drug exposure, total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA) based on phenol-chloroform extraction. Total RNA was purified further by RNeasy mini kit (Qiagen, Valencia, CA) before use. A260/A280 values of the purified total RNA was within a range of 1.79–1.85.

Gene Expression Array Analysis.
Gene expression array analysis was performed according to the manufacturer’s protocols (Atlas pure total RNA labeling system and Atlas human 1.2 cDNA expression array kit; BD Bioscience Clontech, Palo Alto, CA). The nylon array membranes consisted of 1176 genes including genes related to oncogenes, tumor supressors, cell cycle regulators, transporters, signal transductions, GDP/GTP exchangers and GTPase stimulators/inhibitors, apoptosis, transcription factors, cell signaling and extracellular communication, cell surface antigens, cell adhesion, receptors, stress response, and cell-cell communication. Fifty micrograms of total RNA were used for mRNA preparation, and the probe was synthesized using [{alpha}-32P]dATP (Amersham Biosciences, Piscataway, NJ).

The array image from the PhosphoImager screen (Amersham Biosciences) was exposed to the cDNA probe-hybridized membranes for 4 days and was scanned with a Storm PhosphoImager (Amersham Bioscicences) at a 100-µm resolution. The signal density of each spot was quantified using AtlasImage version 1.5 (BD Bioscience Clontech). The data were processed as: (a) the background adjustment was performed by using the minimum density value of all spots; (b) genes in which none of the membranes showed a visible spot were removed; (c) the global median adjustment was applied to the remaining genes; (d) the expression ratio of drug treated to untreated was calculated; and (e) genes in which none of the membranes showed >3.0 or <0.33 of expression ratio were removed, and the remaining genes were used for additional analysis. The hierarchical clustering analysis was performed by using Web-available software, "Cluster" and "Tree View," provided by Dr. M. Eisen.2

RT-PCR.
RT-PCR was performed by a one-step method using Ready-To-Go You-Prime first strand beads (Amersham Bioscicences). One microgram of total RNA and 50 ng of oligo(dT)12–18 primer were used for the synthesis of first-strand cDNA. After the addition of 0.5 µM of specific primers (Integrated DNA Technologies, Inc., Coralville, IA) and 0.025 units/µl TaqDNA polymerase (Invitrogen), the PCR reaction was performed using the PTC-100 or the PTC-200 block thermal cycler (M. J. Research, Inc., Watertown, MA). For the purpose of semiquantitative analysis, ß-actin (5'-GACAACGGCTCCGGCATGTGCA-3', 5'-TGAGGATGCCTCTCTTGCTCTG-3', PCR product 166 bp) was amplified as an internal standard. The PCR regimen was: CDK4 (5'-TGATGCGCCAGTTTCTAAGAGG-3', 5'-GGTCGGCTTCAGAGTTTCCACA-3', 308 bp): 96°C/3 min for 1 cycle; 94°C/30 s, 55°C/30 s, 72°C/1 min for 24 cycles; 72°C/5 min for 1 cycle; APAF-1 (5'-GAGGCCATCAGGAAACAGTG-3', 5'-CTGAAAGCGGAGCACACAAAT-3', 389 bp): 96°C/3 min for 1 cycle; 94°C/30 s, 53°C/30 s, 72°C/1 min for 32 cycles; 72°C/5 min for 1 cycle; HSP90 (5'-TGGAGGAGGAGGAGGTTGAGAC-3', 5'-TTCCACGACCCATAGGTTCACC-3', 518 bp): 96°C/3 min for 1 cycle; 94°C/30 s, 55°C/30 s, 72°C/1 min for 23 cycles; 72°C/5 min for 1 cycle; HSP70 (5'-CAGGTGATCAACGACGGAGACA-3', 5'-GTCGATCGTCAGGATGGACACG-3', 365 bp) and HSP27 (5'-AGGAGTGGTCGCAGTGGTTAGG-3', 5'-GGGACAGGGAGGAGGAAACTTG-3', 359 bp): 96°C/3 min for 1 cycle; 94°C/30 s, 55°C/30 s, 72°C/1 min for 25 cycles; 72°C/5 min for 1 cycle; HSC71 (5'-CTGTTCTTGGCGTGCTTCCAGT-3', 5'-GTGCTGGAAAACACCCACACAA-3', 450 bp): 96°C/3 min for 1 cycle; 94°C/30 s, 55°C/30 s, 72°C/1 min for 29 cycles; 72°C/5 min for 1 cycle. The PCR products were separated by agarose gel electrophoresis (2% w/v) in 1x Tris-Borate-EDTA (TBE) buffer, and the gels were stained with ethidium bromide (Invitrogen).

Western Blotting.
After a defined period of drug exposure, 1 x 106 cells were lyzed in 300 µl of Laemmli sample buffer containing a protease inhibitor mixture (Sigma Chemical Co.). Whole cell lysates were heated at 90°C for 2 min and then incubated on ice for 3 h. To remove cell debris, the lysates were centrifuged at 16,000 x g. The protein concentration of the cell lysates was determined using the microBCA protein assay kit (Pierce Chemical Co., Rockford, IL). After the adjustment of protein concentration, 1–2 mg of total proteins were separated on 12% (for C-Raf and HSP70) and 15% (for HSP27) SDS-polyacrylamide gels. Proteins were then transferred onto a polyvinylidene difluoride membrane (Sigma Chemical Co.) by electroblotting and kept overnight in a solution containing 5% nonfat dry milk, PBS (Sigma Chemical Co.), and 0.1% Tween 20 (Bio-Rad Laboratories, Hercules, CA) at 4°C. The membranes were reacted with the primary antibodies in PBS containing 0.1% Tween 20 and 0.1% BSA (Sigma) at room temperature for 1 h, washed in PBS containing 0.1% Tween 20, and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The membranes were washed in PBS containing 0.1% Tween 20 buffer, and antibody-labeled proteins were detected using FAST 3,3'-diaminobenzidine (Sigma Chemical Co.). The monoclonal antibodies used in this study were: anti-actin IgM antibodies kit (dilution, 1:1000 for both primary and secondary antibodies; Oncogene Research Products, Inc., Boston, MA), anti-C-Raf antibody (mouse; final concentration, 1.85 µg/ml; BD Transduction Laboratories, Inc., Palo Alto, CA), antihuman HSP70 and HSP27 antibodies (mouse; dilution, 1:1000; Calibiochem, San Diego, CA), and horseradish peroxidase-labeled antimouse IgG antibody (goat; Southern Biotechnology Associates, Inc., Birmingham, AL).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Preliminary Evaluation of Intracellular Oligopeptide Drug Spacer Cleavage.
P(AP-GA)-treated cells may show a delayed response because of two time-dependent processes: cellular uptake by endocytosis and intracellular drug release via oligopeptide spacer cleavage in the lysosome. The former is well studied and known to be a relatively fast process (23) . However, the latter is less well known and may be an important issue to be addressed. For this reason, intracellular drug release from HPMA copolymer was studied. It should be noted that the GFLG spacer, which is most susceptible to lysosomal cysteine proteinase cathepsin B, is expected to readily liberate the active drug after endocytosis (24) . Also, it has been demonstrated that the cleavage site of the GFLG drug spacer by cathepsin B is the bond between the last amino acid and the drug (24) . Because AP-GA is not fluorescent, fluorescent Mce6 was used as a model drug in this study. The time-dependent fluorescent images of A2780 cells are shown in Fig. 2Citation . After a 1-h drug exposure, the cells showed only faint and diffuse fluorescence. After a 5-h incubation following a 1-h drug exposure, however, the cells showed diffuse and punctuate fluorescence along with a significantly increased intensity. The diffuse and punctuate fluorescence correspond to the localization of Mce6 in the cytoplasm and endosome/lysosome, respectively. We reported previously that the cells treated with free Mce6 showed diffuse fluorescence in the cytoplasm and faint fluorescence in the nucleus (25 , 26) . The faint fluorescence after a 1-h drug exposure may be the results of fluorescent quenching of Mce6 in the endosomal/lysosomal compartments because of high concentrations. We have observed that free or polymer-bound Mce6 quenches above a 5–10 µM Mce6-equivalent concentration in PBS (pH 7.4) or acetic-buffered solution (pH 4.0; data not shown). With an increase in incubation time, punctuate fluorescence might be detectable and fluorescent intensity might increase because of the release of free Mce6 from HPMA copolymer. Therefore, our observations that the diffuse fluorescence was detected even after a 1-h drug exposure and punctuate fluorescence was clearly observable with considerably increased intensity after 5 h of additional incubation (Fig. 2)Citation suggest that the lysosomal cleavage of oligopeptide (GFLG) spacer may be moderately fast and free Mce6 may be readily released from HPMA copolymer after endocytosis. ubr et al. (27) reported that the GFLG spacer showed ~30% and 90% of drug (DOX and daunomycin) release from HPMA copolymer in isolated rat liver lysosomal enzymes (tritosomes) after 6 and 48 h, respectively. This report seems to be in agreement with our observation. In Fig. 2Citation , the image after an 11-h incubation following a 1-h drug exposure was not significantly different from that after the 5-h incubation, but the image after a 23-h incubation seemed to show more diffuse fluorescence compared with those after a 5- and 11-h incubation. What percentage of Mce6 can be released at each incubation period remains to be studied, but it may depend on the polymer-drug conjugate concentration at the incubation.



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  		Fig. 2. Fluorescence images of Mce6 in A2780 ovarian carcinoma cells after a 1-h exposure to HPMA copolymer-Mce6 conjugate. A, untreated cells. B, an image after a 1-h exposure to HPMA copolymer-Mce6 conjugate. C–E, images after a 5-h, 11-h, and 23-h incubation after a 1-h exposure to HPMA copolymer-Mce6 conjugate, respectively.

 
In Vitro Pharmacological Activity.
The growth-inhibitory activities of GA, AP-GA, and P(AP-GA) against A2780 ovarian carcinoma cells after a 72-h drug exposure are shown in Fig. 3Citation . IC50 values of GA, AP-GA, and P(AP-GA) were 0.0367, 0.811, and 47.2 µM (GA equivalent), respectively. The results indicated a 22 times lower cytotoxicity of AP-GA compared with GA, which is consistent with results reported previously using other cell lines (17 , 18) . P(AP-GA) exhibited an additional 58-fold lower cytotoxicity, however, this can be explained by differences in the mode of cell entry of P(AP-GA) (i.e., the cellular uptake of the polymer-drug conjugate by endocytosis versus uptake of low molecular weight drug by diffusion). We previously demonstrated that HPMA copolymer-DOX conjugate showed similar reduction of the cytotoxicity (~200-fold higher IC50) compared with free DOX; however, on the basis of intracellular drug concentration, the polymer-DOX conjugates were at least as effective as free DOX (5) . It is known that the main mechanism of the cytotoxicity of GA could be ascribed to the depletion of HSP90 client oncoproteins (28) . Because optimal effect of GA on the depletion of HSP90 client oncoproteins is observed at concentrations several times higher than the cytotoxic IC50 (28) , the C-Raf depletion activities of GA, AP-GA, and P(AP-GA) were examined using 2x IC50 in this study (Fig. 4)Citation . It was shown that the activity of GA toward one oncoprotein was equivalent to its activity toward other oncoproteins (28) , suggesting that the depletion of C-Raf might signify inhibition of HSP90. Exposure of A2780 cells to GA and AP-GA resulted in C-Raf depletion after 6 h (Fig. 4)Citation . In contrast, P(AP-GA) treatment exhibited the C-Raf depletion after a 12-h exposure, although it showed slightly decreased C-Raf levels after 6 h (Fig. 4)Citation . The delayed response of C-Raf depletion with P(AP-GA) may be because of the time required for endocytosis of P(AP-GA) as well as the intracellular drug release via cleavage of oligopeptide (GFLG) spacer in the lysosomal compartment. Incubation of cells with P(AP-GA) apparently results in the increase of the intracellular active AP-GA concentration with time. This seems to be consistent with the results in Fig. 2Citation . In this study, the gene expression array analysis was performed in A2780 cells treated with each drug at 2x IC50 for 6 and 12 h. To properly prove the mechanism of cytotoxicity, concentrations in which nearly all cells were killed was deemed preferable to the IC50 values.



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  		Fig. 3. Growth inhibition curves for A2780 ovarian carcinoma cells exposed to GA ({bullet}), AP-GA ({blacktriangleup}), and P(AP-GA) ({blacksquare}) for 72 h. Cell viability was assessed by MTT assay.

 


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  		Fig. 4. Depletion of C-Raf in A2780 ovarian carcinoma cells treated with GA, AP-GA, and P(AP-GA) for 6 or 12 h. Cells were exposed to each drug at 2x IC50 concentration. The expression of actin was measured as an internal standard.

 
Gene Expression Study.
The gene expression profiles of A2780 cells treated using GA or AP-GA for 6 h were compared. The genes in which the expression ratio was >3.0 or <0.33 are listed in Table 1Citation . Although both GA and AP-GA were assumed to possess the same molecular target, several genes were differentially expressed. In particular, differences in the expression of stress response-related genes were observed on the membrane array (see Fig. 6BCitation ).


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  		Table 1 List of genes differentially expressed between GA and AP-GA treatment for 6 h in A2780 cells

 


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  		Fig. 6. Gene expression of CDK4 and APAF-1 (A) and cell stress response-related proteins (B) in A2780 ovarian carcinoma cells treated with GA, AP-GA, and P(AP-GA) at 2x IC50 concentration for 6 or 12 h on the Atlas human 1.2 array.

 
The gene expression profiles of A2780 cells treated with AP-GA and P(AP-GA) were compared. Because P(AP-GA)-treated cells may exhibit delayed responses, longer exposure times (i.e., 6 and 12 h) were examined. The hierarchical clustering of the expression ratios of the selected genes (68 genes; the gene selection process is described in "Materials and Methods") is shown in Fig. 5Citation . Considerable similarities in the gene expression profiles were found after AP-GA and P(AP-GA) treatments. The vertical length of the dendritic diagram (Fig. 5)Citation corresponds to the similarity in the expression profiles. For example, the expression of CDK4, an enzyme with an important role in G1 growth arrest, was down-regulated by AP-GA and P(AP-GA) treatments for up to 6 h (Fig. 6A)Citation . The expression of APAF-1, a protein related to the mitochondrion-induced apoptosis, was up-regulated after 6 h of treatment with AP-GA and after 12 h of treatment with P(AP-GA; Fig. 6ACitation ). A similar down-regulation of CDK4 and up-regulation of APAF-1 expression, as observed after AP-GA exposure, was also observed after 6 h of exposure to GA. These results suggest that the signaling pathways induced by a HSP90-inhibitory agent may be seen for all agents used here, although the cells treated with P(AP-GA) may exhibit a delayed cellular response. This seems to be consistent with the delayed depletion of C-Raf by P(AP-GA) treatment (Fig. 4)Citation and moderately fast intracellular release of drug from HPMA copolymer (Fig. 2)Citation . In contrast, unlike the AP-GA treatment, P(AP-GA) treatment induced little expression in stress response-related genes even after 12 h (Fig. 6B)Citation , although P(AP-GA) seems to liberate AP-GA in the cell fast enough to deplete C-Raf (Fig. 4)Citation and trigger signaling pathways that may be essential to HSP90 inhibition-induced cell growth arrest and death (Figs. 5Citation and 6ACitation ). Because GA-treated cells exhibited little expression in stress response-related genes (Table 1Citation and Fig. 6BCitation ), the elevated expression of stress response-related genes after exposure of cells to AP-GA may not be directly related to cell death mechanism induced by HSP90 inhibition. Therefore, it is possible that P(AP-GA) may suppress the expression of stress response-related genes activated by AP-GA because of differences in its internalization mechanism, subcellular localization, and intracellular concentration gradients.



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  		Fig. 5. Hierarchical cluster analysis of the expression of the selected genes (68 genes) in A2780 ovarian carcinoma cells treated with AP-GA and P(AP-GA) at 2x IC50 concentration for 6 and 12 h. The Atlas human 1.2 cDNA expression array was used to analyze the gene expression profiles.

 
Because the gene expression array data may include possible variables, the expression of stress response-related genes and CDK4 and APAF-1 was further examined by semiquantitative RT-PCR (Fig. 7)Citation . Here, the down-regulation of CDK4 by all treatments, the up-regulation of APAF-1 by GA after 6 h, AP-GA after 6 h and P(AP-GA) after 12 h treatments, and the up-regulation of HSP70 and HSP27 by AP-GA after 6 h and 12 h treatments were confirmed.



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  		Fig. 7. Detection of the expression of the selected genes on the Atlas human 1.2 array (Fig. 5)Citation by RT-PCR. The expression of ß-actin was measured as an internal standard.

 
HSP70 and HSP27 Protein Expression.
The expression of HSP70 and HSP27 in A2780 cells treated with GA, AP-GA, and P(AP-GA) for 6 and 12 h was evaluated by Western blotting (Fig. 8A)Citation . In agreement with the gene expression, AP-GA-treated cells showed significant expression of HSP70 and HSP27 after 6 h. However, GA- and P(AP-GA)-treated cells also showed a time-dependent expression of these proteins, although the expression level of HSP70 seems to be lower than that resulting from the AP-GA treatment. The up-regulation of HSP70 and HSP27 associated with 12 h of treatment with P(AP-GA) may be under the detection limit of the gene expression array and RT-PCR analyses. It has been reported that GA-induced proteotoxic stress may induce the expression of HSP70 and HSP27 (29) . This is consistent with our observations. However, the expression levels and time profiles reported here seemed to be significantly different for GA versus AP-GA treatments, suggesting that the overexpression of HSP70 and HSP27 by AP-GA treatment may be attributed to mechanisms other than HSP90 inhibition. Because P(AP-GA)-treated cells may show a delayed cellular response because of an increase in the intracellular AP-GA concentration, a time-dependent expression of HSP70 by AP-GA and P(AP-GA) treatments up to 24 h was evaluated (Fig. 8B)Citation . Consequently, the expression of HSP70 after 24 h of exposure to P(AP-GA) reached a level comparable with that observed after a 6-h exposure to AP-GA.



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  		Fig. 8. Western blotting analysis of HSP70 and HSP27 expression in A2780 ovarian carcinoma cells treated with GA, AP-GA, and P(AP-GA) at 2x IC50 concentration for 6 and 12 h (A) and HSP70 expression in A2780 cells treated with AP-GA and P(AP-GA) at 2x IC50 concentration for 6, 12, and 24 h (B). The expression of actin was detected as an internal standard.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been demonstrated that the cytotoxicity induced by GA is attributed to degradation of HSP90 client proteins. An et al. (28) suggested the relationship between the cytotoxicity of 28 GA derivatives and their ability to down-regulate HSP90-client oncoproteins. In this study, AP-GA showed ~22 times lower cytotoxicity compared with GA (Fig. 3)Citation . This decrease in cytotoxicity is in agreement with a previous study using other cell lines (17 , 18) . However, Schnur et al. (14) reported that AP-GA has a HSP90-inhibitory activity comparable with GA; IC50 values of GA and AP-GA on the level of p185erbB2 in SKBr cells were 70 and 80 nM, respectively. Taken together, a different mechanism for AP-GA other than inhibition of HSP90 chaperoning may account for in its reduced cytotoxicity. GA derivatives may interact with other protein in addition to HSP90 (30) . GA and its derivatives contain quinone moieties that produce cytotoxic free radicals in reducing environments (i.e., the cytoplasm), possibly causing phospholipid peroxidation, cytoskeletal injury, DNA fragmentation, and so forth (30) . Kim et al. (29) previously reported that GA could induce the expression of HSP70 and HSP27 probably because of GA-induced proteotoxic stresses via inhibition of HSP90. Similarly, in this study, GA treatment for 12 h up-regulated HSP70 and HSP27 (Fig. 8A)Citation . However, AP-GA-treated cells showed more rapid and higher levels of HSP70 and HSP27 expression compared with GA at an equitoxic dose (i.e., 2x IC50; Table 1Citation and Figs. 6Citation , 7Citation , and 8ACitation ), although decreased expression of C-Raf (Fig. 4)Citation and other gene expression profiles (Figs. 6Citation and 7Citation ) were observed for both GA- and AP-GA-treated cells after 6 h. These results suggest that induction of HSP70 and HSP27 might not be directly correlated with the mechanism of antiproliferative action of HSP90-active agents and cell stress responses might be different between GA and AP-GA. AP-GA treatment may cause stronger cell stress responses than GA because of either alternative molecular interactions or higher drug concentrations.

In general, HSPs function as molecular chaperones to minimize protein misfolding and aggregation induced by various stressors (heat, environmental stress such as antibiotics, and others) to protect the cell (31 , 32) . Expression of HSPs is regulated by HSFs, which bind to heat shock elements in the promoter region of the HSP gene (31) . Under unstressed conditions, HSFs reside in the cytosol in an inactive form bound to HSPs such as HSP70 (33) and HSP90 (34) . Under stress conditions, however, HSFs become detached from HSPs and function as initiators of transcription of the HSP gene through a multistep process involving homotrimer formation, hyperphosphorylation, translocation into the nucleus, and binding to heat shock elements (31) . Because newly synthesized HSPs also bind to HSFs, the expression level of HSPs is self-regulating (31) . HSPs typically have a relatively long half-life [48 h in human epidermoid cells (31) and 20–24 h in human monoblastoid U937 cells (35) ]. It is known that HSP70 and HSP27 are the most stress-inducible members of the HSP families, whereas HSP90 is constitutively expressed and less stress inducible (32) . HSP70 and HSP27 have been suggested to have an important role in resistance to necrosis (36 , 37) and apoptosis (35 , 38) . Samali and Cotter (35) demonstrated that transfection with HSP70 or HSP27 increased resistance to cytotoxic drug-induced apoptosis in murine fibrosarcoma Wehi-s cells. Hout et al. (39) have observed a positive linear correlation between cell survival after exposure to DOX and other cytotoxic drugs (daunorubicin, colchicine, vincristine, hydrogen peroxide, sodium arsenite) and the expression level of HSP27 in HSP27-transfected Chinese hamster O23 cells. They demonstrated that HSP27-mediated cellular protection was not associated with either decreased drug accumulation or overexpression of P-gp. Although the mechanisms by which either of these genes participates in cell resistance to anticancer agents is unclear, HSP70 and HSP27 are associated with some type of acquired multiple drug resistance to structurally unrelated drugs, which is unrelated to drug efflux pumps (30) . Possibly, the overexpression of HSP70 and HSP27 may, in part, contribute to the aforementioned large difference between the HSP90-inhibitory activity and cytotoxic activity of AP-GA.

P(AP-GA)-treated cells showed lower expression of HSP70 and HSP27 compared with AP-GA up to 12 h (Figs. 6Citation 7Citation 8)Citation . P(AP-GA)-treated cells also showed similar gene expression profiles to AP-GA (Figs. 5Citation 6Citation 7)Citation , and exhibited depletion of C-Raf (Fig. 4)Citation and moderately fast intracellular drug release (Fig. 2)Citation . P(AP-GA) may suppress the activation of cell stress responses while triggering the cell growth arrest and death-signaling pathways. It is possible that internalization of HPMA copolymer-drug conjugate via endocytosis may circumvent interactions with external components of the cell, such as plasma membrane, which may be sensitive to stressors and environmental changes. Similarly, we observed previously that A2780 cells treated with HPMA copolymer-DOX conjugate showed a down-regulation of the HSP70 gene more pronounced than that observed in the cells treated with free DOX (5) . Therefore, HPMA copolymer-drug conjugate may bypass not only P-gp-associated but also other forms of multidrug resistance. In this study, however, the expression of HSP70 in the cells treated with P(AP-GA) at 24 h reached a level comparable with 12 h AP-GA treatment. Consequently, two factors may contribute to changes in HSP expression after cell exposure to P(AP-GA) when compared with AP-GA: expression modulation by the macromolecular form of the drug and different intracellular pharmacokinetics. To evaluate the participation of each factor in the final expression level, additional investigation is needed. The intracellular concentration of AP-GA increases with time, and AP-GA released from HPMA copolymer may ultimately distribute anywhere in the cell. It is possible that the time to release AP-GA from the copolymer may delay the cellular response. In contrast, because GA treatment also showed time-dependent increases in the expression of HSP70 and HSP27 (Fig. 8A)Citation , the proteotoxic stress via inhibition of HSP90 chaperoning may have resulted in the expression of HSP70 after 24 h of exposure to P(AP-GA) even if P(AP-GA) could suppress cell stress responses after shorter exposure intervals. Because gene expression array analysis is able to identify genome-wide changes in expression, we suggest that HPMA copolymer-drug conjugate may modulate cell stress responses.

In addition, this study describes a new way to evaluate the mechanism of action of macromolecular drugs. Compiling the data of HPMA copolymer conjugates with other antitumor drugs should lead to a better understanding of the universal effects of macromolecular therapeutics. This information will assist in the design of more effective polymer-drug conjugates.


   ACKNOWLEDGMENTS
 
We thank Jon Callahan, University of Utah, for carefully revising this manuscript.


   FOOTNOTES
 
Grant support: NIH Grant CA51578 from the National Cancer Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Jindich Kopecek, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 30 South 2000 East Room 301, Salt Lake City, Utah 84112. Phone: (801) 581-4532; Fax: (801) 581-3674; E-mail: Jindrich.Kopecek{at}m.cc.utah.edu

1 The abbreviations used are: HPMA copolymer, N-(2-hydroxypropyl)methacrylamide copolymer; GFLG spacer, glycylphenylalanylleucylglycine spacer; GA, geldanamycin; AP-GA, 17-(3-aminopropylamino)-17-demethoxygeldanamycin; P(AP-GA), AP-GA bound to HPMA copolymer with GFLG spacers; Mce6, mesochlorin e6 monoethylenediamine; CDK4, cyclin-dependent kinase 4; APAF-1, apoptotic protease activating factor-1; HSP90, heat shock protein 90 kDa; HSP70, heat shock protein 70 kDa; HSP27, heat shock protein 27 kDa; HSC71, heat shock cognate 71 kDa; DOX, doxorubicin; P-gp, P-glycoprotein; HSF, heat shock transcription factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. Back

2 Internet address: http://rana.lbl.gov/. Back

Received 5/22/03. Revised 8/ 1/03. Accepted 8/27/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Maeda H. Kabanov A. Kataoka K. Okano T. eds. . Polymer Drugs in the Clinical Stage: Advantages and Prospects, Kluwer Publishers New York, NY 2003.
  2. Kopecek J., Kopecková P., Minko T., Lu Z-R. HPMA copolymer-anticancer drug conjugate: design, activity, and mechanism of action. Eur. J. Pharm. Biopharm., 50: 61-81, 2000.[Medline]
  3. Duncan R. The dawning era of polymer therapeutics. Nat. Rev. Drug Disc., 2: 347-360, 2003.[Medline]
  4. Matsumura Y., Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent Smancs. Cancer Res., 46: 6387-6392, 1986.[Abstract/Free Full Text]
  5. Minko T., Kopecková P., Kopecek J. Comparison of the anticancer effect of free and HPMA copolymer-bound adriamycin in human ovarian carcinoma cells. Pharmaceutical Res., 16: 986-996, 1999.[Medline]
  6. Minko T., Kopecková P., Kopecek J. Efficacy of the chemotherapeutic action of HPMA copolymer-bound doxorubicin in a solid tumor model of ovarian carcinoma. Int. J. Cancer, 86: 108-117, 2000.[Medline]
  7. Omelyanenko V., Kopecková P., Gentry C., Kopeccek J. Targetable HPMA copolymer-adriamycin conjugates. recognition, internalization, and subcellular fate. J. Controlled Release, 53: 25-37, 1998.[Medline]
  8. Miyamoto Y., Maeda H. Comparison of the cytotoxic effects of the high-and low-molecular weight anticancer agents on multidrug-resistant Chinese hamster ovary cells in vitro. Cancer Res., 50: 1571-1575, 1990.[Abstract/Free Full Text]
  9. Nishiyama N., Koizumi F., Okazaki S., Matsumura Y., Nishio K., Kataoka K. Differential gene expression profile between PC-14 cells treated with free cisplatin- and cisplatin-incorporated polymeric micelles. Bioconjugate Chem., 14: 449-457, 2003.[Medline]
  10. Richter K., Buchner J. Hsp90: chaperoning signal transduction. J. Cell. Physiol., 188: 281-290, 2001.[Medline]
  11. Blagosklonny M. V. Hsp-90-associated oncoproteins: multiple targets of geldanamycin and its analogs. Leukemia (Baltimore), 16: 455-462, 2002.
  12. Blagosklonny M. V., Toretsky J., Necker L. Geldanamycin selectively destabilizes and conformationally alters mutated p53. Oncogene, 11: 933-939, 1995.[Medline]
  13. Supko J. G., Hickman L. R., Grever M. R., Malspeis L. Preclinical pharmacologic evaluation of geldanamycin as an antitumor agent. Cancer Chemother. Pharmacol., 36: 305-315, 1995.[Medline]
  14. Schnur R. C., Corman M. L., Gallaschun R. J., Cooper B. A., Dee M. F., Doty J. L., Muzzi M. L., Moyer J. D., DiOrio C. I., Barbacci E. G., Miller P. E., O’Brien A. T., Morin M. J., Foster B. A., Pollack V. A., Savage D. M., Sloan D. E., Pustilnik L. R., Moyer M. P. Inhibition of the oncogene product p185erbB-2 in vitro and in vivo by geldanamycin and dihydrogeldanamycin derivatives. J. Med. Chem., 38: 3806-3812, 1995.[Medline]
  15. Kasuya Y., Lu Z-R., Kopecková P., Minko T., Tabibi S. E., Kopecek J. Synthesis and characterization of HPMA copolymer-aminopropylgeldanamycin conjugates. J. Controlled Release, 74: 203-211, 2001.[Medline]
  16. Kasuya Y., Lu Z-R., Kopecková P., Tabibi S. E., Kopecek J. Influence of the structure of drug moieties on the in vitro efficacy of HPMA copolymer-geldanamycin derivative conjugates. Pharmaceutical Res., 19: 115-123, 2002.[Medline]
  17. Mandler R., Wu C., Sausville E. A., Roettinger A. J., Newman D. J., Ho D. K., King C. R., Yang D., Lippman M. E., Landolfi N. F., Dadachova E., Brechbiel M. W., Waldmann T. A. Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies: antiproliferative activity on human breast carcinoma cell lines. J. Natl. Cancer Inst., 92: 1573-1581, 2000.[Abstract/Free Full Text]
  18. Mandler R., Kobayashi H., Davis M. Y., Waldmann T. A., Brechbiel M. W. Modification in synthesis strategy improve the yield and efficacy of geldanamycin-herceptin immunoconjugates. Bioconjugate Chem., 13: 786-791, 2002.[Medline]
  19. Kasuya Y., Lu Z-R., Kopecková P., Kopecek J. Improved synthesis and evaluation of 17-substituted aminoalkylgeldanamycin derivatives applicable to drug delivery system. Bioorg. Med. Chem. Lett., 11: 2089-2091, 2001.[Medline]
  20. Rejmanová P., Pohl J., Baudys M., Kostka V., Kopecek J. Polymers containing enzymatically degradable bonds. 8. Degradation of oligopeptide sequences in N-(2-hydroxypropyl)methacrylamide copolymers by bovine spleen cathepsin B. Makromol. Chem., 184: 2009-2020, 1983.
  21. Tijerina M., Kopecková P., Kopecek J. The effects of subcellular localization of N-(2-hydroxypropyl)methacrylamide copolymer-Mce6 conjugates in a human ovarian carcinoma. J. Controlled Release, 74: 269-273, 2001.[Medline]
  22. Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65: 55-63, 1983.[Medline]
  23. Marecos E., Weissleder R., Bogdanov A., Jr. Antibody-mediated versus nontargeted delivery in a human small cell lung carcinoma model. Bioconjugate Chem., 9: 184-191, 1998.[Medline]
  24. Soyez H., Schacht E., Vanderkerken S. The crucial role of spacer groups in macromolecular prodrug design. Adv. Drug Deliv. Rev., 21: 81-106, 1996.
  25. Lu Z-R., Kopecková P., Kopecek J. Polymerizable Fab' antibody fragment for targeting of anticancer drugs. Nat. Biotechnol., 17: 1101-1104, 1999.[Medline]
  26. Tijerina M., Kopecková P., Kopecek J. Mechanisms of cytotoxicity in human ovarian carcinoma cells exposed to free Mce6 or HPMA copolymer-Mce6 conjugates. Photochem. Photobiol., 77: 645-652, 2003.[Medline]
  27. ubr V., Strohalm J., Ulbrich K., Duncan R., Hume I. C. Polymers containing enzymatically degradable bonds XII: effect of spacer structure on the rate of release of daunomycin and adriamycin from N-(2-hydroxypropyl) methacrylamide copolymer drug carriers in vitro and antitumour activity measured in vivo. J. Control. Rel., 18: 123-132, 1992.
  28. An W. G., Schnur R. C., Neckers L., Blagosklonny M. V. Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity. Cancer Chemother. Pharmacol., 40: 60-64, 1997.[Medline]
  29. Kim H. R., Kang H. S., Kim H. D. Geldamamycin induces heat shock protein expression through activation of HSF1 in K562 erythroleukemic cells. IUBMB Life, 48: 429-433, 1999.[Medline]
  30. Benchekroun M. N., Schneider E., Safa A. R., Townsend A. J., Sinha B. K. Mechanism of resistance to ansamycin antibiotics in human breast cancer cell lines. Mol. Pharmacol., 46: 677-684, 1994.[Abstract]
  31. Kiang J. G., Tsokos G. C. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol. Ther., 80: 183-201, 1998.[Medline]
  32. Creagh E. M., Sheehan D., Cotter T. G. Heat shock proteins-modulators of apoptosis in tumor cells. Leukemia (Baltimore), 14: 1161-1173, 2000.
  33. Morimoto R. I. Cells in stress: transcriptional activation of heat shock genes. Science (Wash. DC), 259: 1409-1410, 1993.[Free Full Text]
  34. Zou J., Guo Y., Guettouche T., Smith D. F., Voellmy R. Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell, 94: 471-480, 1998.[Medline]
  35. Samali A., Cotter T. G. Heat shock proteins increase resistance to apoptosis. Exp. Cell Res., 223: 163-170, 1996.[Medline]
  36. Gabai V. L., Kabanov A. E. Rise in heat-shock protein level confers tolerance to energy deprivation. FEBS Lett., 327: 247-250, 1993.[Medline]
  37. Kabanov A. E., Gabai V. L. Heat shock-induced accumulation of 70-kDa stress protein (HSP70) can protect ATP-depleted tumor cells from necrosis. Exp. Cell Res., 217: 15-21, 1995.[Medline]
  38. Gabai V. L., Zamulaeva I. V., Mosin A. F., Makarova Y. M., Mosina V. A., Budagova K. R., Malutina Y. V., Kabanov A. E. Resistance of Ehrich tumor cells to apoptosis can be due to accumulation of heat shock proteins. FEBS Lett., 375: 21-26, 1995.[Medline]
  39. Hout J., Roy G., Lambert H., Chretien P., Landry J. Increased survival after treatments with anticancer agents of Chinese hamster cells expressing the human Mr 27,000 heat shock protein. Cancer Res., 51: 5245-5252, 1991.[Abstract/Free Full Text]



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