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Originally published In Press as doi:10.1074/jbc.M605569200 on September 6, 2006

J. Biol. Chem., Vol. 281, Issue 49, 37330-37344, December 8, 2006
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Molecular Mechanism of Adaphostin-mediated G1 Arrest in Prostate Cancer (PC-3) Cells

SIGNALING EVENTS MEDIATED BY HEPATOCYTE GROWTH FACTOR RECEPTOR, c-Met, AND p38 MAPK PATHWAYS*

Indranil Mukhopadhyay{ddagger}1, Edward A. Sausville§, James H. Doroshow{ddagger}, and Krishnendu K. Roy{ddagger}2

From the {ddagger}Laboratory of Clinical Trials Unit, Division of Cancer Treatment and Diagnosis, NCI, National Institutes of Health, Bethesda, Maryland 20892 and §Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received for publication, June 9, 2006 , and in revised form, July 31, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Adaphostin (NSC680410), a small molecule congener of tyrphostin AG957, has been demonstrated previously to have significant anti-proliferative effects in several leukemia models. However, this effect of adaphostin in adherent cells/solid tumor models has not been examined. In this study, we investigated the anti-proliferative effects of adaphostin in the human prostate cancer cell line PC-3. Specifically, we explored the potential molecular mechanism(s) by which adaphostin elicits its anti-proliferative effect(s). We demonstrate that adaphostin inhibits the proliferation of PC-3 cells by inducing a G1 phase cell cycle arrest. This adaphostin-induced G1 arrest was associated with an increase in the expression of p21 and p27 and a decrease in the expression of G1-specific cyclins (cyclin A, D1, and D3) and cyclin-dependent kinases 4 and 6. Consequently, a dramatic decrease in the phosphorylation of retinoblastoma protein was also observed. Additionally, we found that adaphostin treatment induced a decrease in the phosphorylation of nucleophosmin, a major nuclear phosphoprotein, and that this decreased phosphorylation was a result of the p21- and p27-mediated inactivation of cyclin E-cyclin-dependent kinase 2 complex kinase activity. Furthermore, we have determined that the adaphostin-mediated cell cycle arrest of PC-3 cells is dependent upon activation of the p38 MAPK. We also demonstrate that the hepatocyte growth factor receptor-c-Met is involved in the adaphostin-mediated signaling events that regulate p38 MAPK. Taken together, these results identify for the first time a signaling cascade of adaphostin-mediated G1 phase-specific cell cycle arrest in PC-3 cells. These findings suggest that the tyrphostin member has a broader spectrum of activity than originally predicted.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
One hallmark of cancer is the deregulation of cell cycle progression. A large number of proteins are involved in the processes by which a cell chooses between growth arrest and proliferation. The mechanisms through which the epigenetic alterations in human cancers affect these decisions are not well understood. This lack of understanding impedes the development of new, more efficient anti-cancer therapies that rely on the identification of new therapeutic targets and the exploration of the mechanisms of resistance to existing therapies (1).

Protein-tyrosine kinases mediate the transduction and processing of many extra- and intracellular signals. The critical role of protein-tyrosine kinases in regulating cell proliferation and their involvement in cancer development is widely accepted (2, 3). In fact, the design of specific protein-tyrosine kinase inhibitors is considered to be an emerging new paradigm in the development of cancer therapies. Consequently, over the last 2 decades, many receptor and nonreceptor tyrosine kinases have emerged as clinically useful drug targets for the treatment of certain types of cancer (4). Tyrosine kinases play a central role in signal transduction pathways by relaying the signals of a complicated network of interdependent signaling molecules. These pathways ultimately affect nuclear processes, such as gene replication and transcription. Often, key tyrosine kinases in tumor cells are no longer properly regulated, resulting in excessive phosphorylation that maintains signal transduction pathways in an activated state. A greater understanding of the molecular mechanisms leading to the establishment and progression of cancer will allow not only for the re-assessment of existing cancer therapies but also the development of new cancer treatments involving tyrosine kinase inhibitors (4).

The receptor tyrosine kinase c-Met has been shown to be overexpressed in prostate cancer (5). The signaling events mediated by c-Met and the subsequent biological effects of hepatocyte growth factor (HGF)3 have been shown to be important in many key cellular functions as follows: epithelial-mesenchymal interactions; the regulation of cell migration, invasion, proliferation, and survival; angiogenesis; and morphogenic differentiation (6). The c-Met receptor, a disulfide-linked heterodimeric glycoprotein, is composed of a transmembrane beta-chain that contains the kinase domain and a unique multifunctional docking site. Ligand HGF-mediated tyrosine phosphorylation of the receptor c-Met recruits various signal transducers and adaptor molecules such as GAB1, SHC, and GRB2 (7–9). Ultimately, this leads to the activation of several MAPK pathways that elicit various biological responses (7, 9, 10).

Adaphostin (NSC680410), an adamantyl ester of AG957, is a recently identified and novel member of the tyrphostin family of protein-tyrosine kinase signaling inhibitors. These molecules function by interfering with the peptide substrate-binding site of the tyrosine kinases rather than the ATP-binding site of the respective tyrosine kinases. They have been shown to possess significant anti-proliferative activity, both in vivo and in vitro (11–14). Because of its low potency in vivo, adaphostin, which is considered to be an improved version of the original compound AG957, is currently under preclinical evaluations, including preclinical toxicology studies for potential human clinical trials (12, 15). Originally, adaphostin was identified as an inhibitor of p210 BCR/ABL kinase and a potent inducer of myeloid cell death in p210 BCR/ABL-positive K562 cells. Subsequently, adaphostin was also shown to efficiently induce apoptosis in p210 BCR/ABL-negative cell lines (16, 17) possibly by triggering redox-related cell killing mechanisms in addition to inhibition of tyrosine kinase functions (18). However, very little is known about the various effects of adaphostin on signaling pathways in adherent as well as suspension cell lines.

Because adaphostin has recently shown promise as an anti-cancer compound in several leukemia model systems (14, 16), it would therefore be intriguing to explore the effects of this compound in adherent cell lines. The study presented here was designed to examine the effects of adaphostin on various tyrosine kinase-dependent signaling and cell cycle regulatory pathways in human prostate cancer-derived PC-3 cell lines. Our results demonstrate that adaphostin induces a G1 phase-specific cell cycle arrest by altering HGF/c-Met associated cell survival signaling events that require activation of p38 MAPK. Additionally, we show that the p38 MAPK signaling pathway is involved in adaphostin-mediated reduced phosphorylation of NPM leading to the inhibition of G1 phase-specific cell cycle arrest in PC-3 cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Drug—Adaphostin (NSC680410) was derived and obtained through the Developmental Therapeutics Program, NCI, National Institutes of Health.

Cell Culture—All cell lines used for IC50 determination were obtained from the American Type Culture Collection (Manassas, VA) and maintained in their respective growth media as suggested by ATCC. Further experiments were performed in the human prostate adenocarcinoma cell line (PC-3) and maintained in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen) in a humidified incubator in an atmosphere of 5% CO2 in air at 37 °C. Cells were then treated with different concentrations of adaphostin for various time points, depending upon experimental design. The treated cells were then trypsinized and washed with PBS, and the experiments were performed.

Establishment of Adaphostin-resistant Cell Line—Resistant cell lines were selected in vitro for resistance to adaphostin by continuous exposure of the parental cell line (PC-3) to medium supplemented with adaphostin. The drug-resistant lines were established by exposing the PC-3 cells to stepwise increments of adaphostin at concentrations ranging from 1 to 10 µM, yielding the following two variant cell lines displaying a significant degree of resistance to the cytotoxic action of adaphostin: PC-3-ADP-R5, resistant to 5 µM, and PC-3-ADP-R10, resistant to 10 µM adaphostin. The cell lines were maintained in RPMI medium supplemented with 10% fetal bovine serum and an antibiotic solution of penicillin and streptomycin (50 units per ml) and gentamicin (25 µg per ml) and were stored at 37 °C in a humidified incubator containing 5% CO2. Various experiments were performed to discern molecular aberrations between the parental and resistant cell line.

Growth Inhibition Assay—PC-3 cells and two adaphostin-resistant PC-3 cell lines (PC-3-ADP-R5 and PC-3-ADP-R10) were seeded in 60-mm plastic tissue culture plates (Falcon Labware) in a concentration such that 30–40% confluency would be achieved after 24 h. Different concentrations of adaphostin were then added to these exponentially growing cells, and IC50 value was determined after 48 h following methods described previously (19).

Cell Cycle Analysis—A single cell suspension of PC-3 cells was obtained with trypsin-EDTA after which the cells were washed once in ice-cold PBS and fixed in chilled 70% ethanol for 4 h. The cells were then washed once in ice-cold PBS and resuspended in propidium iodide staining solution (propidium iodide 10 µg/ml; RNase 50 µg/ml in PBS) for 45 min at room temperature in the dark (20). Fluorescence was measured using a FACScan flow cytometer (BD Biosciences) and analyzed for cell cycle distribution by Flo Jo software (TreeStar, San Carlos, CA).

Bromodeoxyuridine (BrdUrd) Assay—S phase determination of cells was obtained using the fluorescein isothiocyanate-BrdUrd kit (BD Biosciences) following the manufacturer's protocol. Briefly, plated cells were harvested in 25 µM BrdUrd for 3 h; subsequently, cells were washed, trypsinized, and resuspended in Cytofix/Cytoperm buffer followed by incubation with DNase, fluorescein isothiocyanate-BrdUrd antibody, and 7-amino-actinimycin D as recommended by the manufacturer. Fluorescence was acquired and analyzed in a FACScan flow cytometer (BD Biosciences).

Assessment of Cell Morphology—PC-3 cells were plated onto chambered slides for 24 h followed by treatment with adaphostin for the desired time. They were then washed twice in PBS, fixed in 4% paraformaldehyde for 15 min, and then washed 2–3 times in PBS and mounted on a clean slide over mounting media. The cells were examined in an inverted microscope (Olympus) under a x40 magnification objective. Phase contrast images of the cells were captured under the same magnification.

Protein Extraction and Immunoblot Analysis—Cell lysates for immunoblotting were prepared as described previously (19). Briefly, total cell extracts were obtained using ice-cold cell lysis buffer (50 mM HEPES, 20 mM EDTA, 0.5 mM sodium orthovanadate, 10 mM sodium glycerophosphate, 1 mM sodium fluoride, 10% glycerol, 0.5% Nonidet P-40, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride). After 20 min of incubation of the cell extracts on ice, they were centrifuged for 30 min at 12,000 x g at 4 °C, and the supernatant was collected. Proteins were quantified with the BCA protein assay kit (Pierce). Lysates containing equal amounts of total protein were resolved by 4–20% Tris-glycine SDS-polyacrylamide gel and transferred overnight onto a polyvinylidene fluoride membrane (Immobilon-P, Millipore, Billerica, MA). The membrane was incubated for 1 h in blocking solution containing 5% nonfat dry milk to inhibit nonspecific binding. The membrane was then incubated with primary antibodies at optimal concentrations for 3 h followed by incubation in horseradish peroxidase-conjugated specific secondary antibodies for 45 min. The membrane was then developed using the ECL plus Western blotting detection system (Amersham Biosciences) according to the manufacturer's instructions. The antibodies against p21 (CS 2946), cyclin B1 (CS 4135), CDK1 (CS 9116), CDK4 (CS 2906), p-Rb (CS 9308), Rb (CS 9309), p-NPM (CS 3541), p-JNK (CS 9251), c-Met (CS 3127), p-Met (Tyr-1234/1235) (CS 3126), p-GAB1 (CS 3231), GAB1 (CS 3232), p-GAB2 (CS 3881), p-SHC (CS 2431), SHC (CS 2432), p38 (CS 9212), p-p38 (CS 9211), and p-Erk1/2 (CS 9101) were purchased from Cell Signaling Technology. The cyclin A (sc-239), CDK6 (sc-7961), and GAB2 (sc-25498) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies against p27 (BD 610242), cyclin D3 (BD 554195), cyclin E (BD 554182), and CDK2 (BD 610145) were acquired from Pharmingen. p-c-Met (Tyr-1349) (residues 44–892) antibody was purchased from BIOSOURCE. Antibody against cyclin D1 (C-7464) was from Sigma, and antibody against NPM (catalog number 32-500) was purchased from Zymed Laboratories Inc. Secondary antibodies goat anti-rabbit (sc-2054) and goat anti-mouse (sc-2055) were from Santa Cruz Biotechnology.

Immunoprecipitation and Kinase Assay—For immunoprecipitation, cells were washed in PBS, lysed using cold lysis buffer as mentioned above, and centrifuged. Protein estimation was performed with the supernatant, and 1000 µg of protein was taken per 1.5-ml microcentrifuge tube and incubated overnight at 4 °C with the respective antibodies (see figure legends). The immunocomplexes were absorbed onto protein G-Sepharose beads, washed five times with lysis buffer, denatured, and subjected to electrophoresis on a 4–20% Tris-glycine SDS-polyacrylamide gel. For kinase assay, immunoprecipitates with CDK2 were resuspended in kinase buffer, and kinase assays were performed as previously described (21) using 2 µg of histone H1 (Upstate%20Biotechnology">Upstate Biotechnology) as substrate.

Transient siRNA-mediated Gene Silencing—The control siRNA and individual double-stranded RNA oligonucleotides to p21, p27, and c-Met were purchased from Santa Cruz Biotechnology (sc-29427, sc-29429 and sc-29397, respectively), whereas siRNA for p38 was purchased from Dharmacon RNA Technologies (SMART pool plus; M-003556-00-05) and resuspended in reagents provided by the manufacturer. PC-3 cells were transfected in 60-mm tissue culture plates using Lipofectamine reagent (Invitrogen) with 200 nM concentrations each of double-stranded oligonucleotide as per the manufacturer's instructions. After 48 h of siRNA treatment, media were replaced with fresh warm media followed by adaphostin treatment for the desired time. Cell lysates were then prepared as mentioned before, and Western blotting was performed with appropriate antibodies.

Transfection of Plasmid DNAs—DNA encoding wild type cyclin E and CDK2 was obtained from Dr. Ed Harlow. Wild type human c-Met DNA was a kind gift from Dr. Toshikazu Nakamura. PC-3 cells were transfected in 60-mm tissue culture plates using Lipofectamine reagent (Invitrogen) with 5 µg of plasmid as per the manufacturer's instructions. After 24 h of transfection, media were replaced with fresh warm media followed by adaphostin treatment for the desired concentration and time. Cell lysates were then prepared as mentioned before, and Western blotting was performed with appropriate antibodies.

Reverse Transcription-PCR—PC-3 cells were cultured overnight on 100-mm tissue culture plates and then treated with adaphostin. Following treatment for the desired period of time, total RNA was isolated by using the RNeasy mini kit (Qiagen), and first strand cDNA was synthesized from 5 µg of total RNA using Superscript First-strand synthesis system for RT-PCR (Invitrogen) according to the manufacturer's instructions. PCR was performed in a final volume of 25 µl containing dNTPs (2 mM each), 1x PCR buffer, 2 µl of the above cDNA product, 50 units/ml Platinum Taq DNA polymerase (Invitrogen), and 1 µl each of the forward and reverse primers (0.2 µM each). Primers to specifically amplify the genes interested are as follows: forward, 5'-TTA GCA GCG GAA CAA GGA GT 3', and reverse, 5'-ATT CAG CAT TGT GGG AGG AG 3' for p21Waf1/Cip1 gene; forward, 5'-TTC TTT TCA CTT CGG GCT GT 3', and reverse, 5'-CAC AAA ACA TGC CAC TTT GG 3' for p27 gene; forward 5'-CAT GAA GAA GCA GCC AGA CA 3', and reverse, 5'-GTT CTC AGT CCT GAC GCA CA 3' for cyclin A gene; forward, 5'-AGA TGT TTC ACA CCG GAA GG 3', and reverse, 5'-CAT GCC TGT CCA ATC AGA TG 3' for cyclin D1 gene; forward, 5'-AGG CTG ATG GGA CAG AAT TG 3', and reverse, 5'-AGC TGA GCA GAA AGC AAA GC 3' for cyclin D3 gene; forward, 5'-AGC GGT AAG AAG CAG AGC AG 3', and reverse, 5'-AGC ACC TTC CAT AGC AGC AT 3' for cyclin E gene. For internal control, glyceraldehyde-3-phosphate dehydrogenase gene was used. The PCR products were separated by electrophoresis on 1% agarose gel and visualized by ethidium bromide staining.

Analysis of p21 and p27 Promoter Activity—The p21 promoter-luciferase reporter constructs p21P, p21P {Delta}p53, p21P {Delta}400, p21P {Delta}800, p21P {Delta}2.1, p21P {Delta}2.3, p21P Sma, and p21P Sma{Delta}1 were a kind gift from Dr. Xiao-Fan Wang (22). The p27 promoter-luciferase reporter constructs p27PF, p27 KpnI, p27 ApaI, p27MB-435, and p27 SacII were a kind gift from Dr. Toshiyuki Sakai (23). PC-3 cells were transfected with 0.5 µgof p21 or p27 promoter-luciferase plasmid along with beta-galactosidase (for normalizing luciferase activity) using Lipofectamine reagent (Invitrogen) following the manufacturer's instructions. After 24 h of transfection, media were replaced with fresh warm media and PC-3 cells were further incubated for 24 h in the presence or absence of 2.5 µM adaphostin. The cells were then collected, extracted, and analyzed for luciferase activity using dual-luciferase reporter assay system (Promega).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Adaphostin Treatment Affects Tumor Cell Proliferation—The structure of adaphostin, an adamantyl congener of tyrphostin AG957, is depicted in Fig. 1A. To examine the ability of adaphostin to alter the growth of tumor cells, its IC50 concentration (the concentration that inhibits growth by 50%) in various tumor cell lines was determined using a cell-based trypan blue dye exclusion assay. The results (Table 1) demonstrate that adaphostin inhibits the growth of many of the adherent and suspension cell lines tested. The IC50 values ranged from 50 to 200 nM (for CCRF-CEM and Jurkat E6-1 cells), to 2–6 µM (for PC-3, DU-145, MCF-7, BxPC-3, SW620, KM-12, K-562, HT, and Carpus cells), and 7–10 µM (for HT-29 and CLL-B cells). The human prostate cancer cell line PC-3 was used to further examine the effects of adaphostin on tumor cell growth. As shown in Fig. 1B, adaphostin treatment results in a dose-dependent inhibition PC-3 cell growth. After 48 h of treatment, the IC50 concentration of adaphostin in these cells was determined to be 2.5 ± 0.04 µM. Continued treatment with this concentration of adaphostin causes a 50% growth inhibition in these cells relative to control untreated PC-3 cells (Fig. 1C).


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  		TABLE 1
IC50 of adaphostin in different cancer cell lines

 


Figure 1
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  		FIGURE 1.
Adaphostin inhibits the proliferation of PC-3 cells. A, structure of adaphostin. B, PC-3 cells were exposed to adaphostin (0–6 µM) for 48 h. C, in a second set of experiments, PC-3 cells were exposed to 2.5 µM adaphostin for 48 h. Cells were then trypsinized, and the number of live cells was counted using a trypan blue dye exclusion assay. The results shown are mean of three independent experiments. Error Bars represent standard errors.

 
Adaphostin Induces a Strong G1 Phase Cell Cycle Arrest in PC-3 Cells—To explore the potential mechanisms through which adaphostin mediates its growth inhibitory effect(s), we examined whether it altered the cell cycle progression of PC-3 cells before and after adaphostin treatment. For these experiments, PC-3 cells were exposed to increasing concentrations of adaphostin for 0, 24, and 48 h. Following this treatment, the cells were stained with propidium iodide (which stains total cellular DNA) and evaluated by flow cytometry. This analysis allowed for the identification of the percentage of cells in G0/G1, S, and G2/M phases. A dose-dependent increase in the percentage of cells in G1 phase was observed after 24 h of treatment (Fig. 2A). For example, in the absence of treatment, 39% of the cells were in G0/G1 phase, but following treatment with 0.5, 1.0, or 2.5 µM adaphostin, the percentage of cells in G0/G1 phase increased to 44, 63, and 79%, respectively. This increase in G1 phase cells corresponded to a significant decrease in S phase cells (Fig. 2B) and a minimal decrease in G2-M cells (data not shown). The percentage of cells in G1 increased even further after 48 h of treatment at lower concentrations. Interestingly, at the highest concentration of adaphostin (5.0 µM), the percentage of cells in G1 actually decreased after 24 and 48 h of exposure to adaphostin relative to the percentage of G1 phase cells treated with 2.5 µM adaphostin. This result may be due to increased cell death. A more pronounced G1 phase cell cycle arrest was observed in PC-3 cells treated with 2.5 µM adaphostin for 24 h if the cells were first synchronized in G1 using aphidicolin (data not shown). In other experiments, the percentage of PC-3 cells in S phase following treatment with adaphostin for 24 h was determined by BrdUrd incorporation. Consistently, a significant (p < 0.05) dose-dependent decrease in the S phase population of these cells was observed (Fig. 2B). Therefore, adaphostin induces a G1 phase cell cycle arrest in PC-3 cells.


Figure 2
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  		FIGURE 2.
Adaphostin arrests PC-3 cells at G1-S phase of cell cycle. A, exponentially growing PC-3 cells were treated with increasing concentrations of adaphostin (0–5 µM) for 0, 24, or 48 h. The cells were then harvested, fixed, RNase-treated, and stained with propidium iodide. Samples were analyzed for DNA content by flow cytometry. The relative number of G1 phase cells for each condition of adaphostin treatment was assayed by Modfit analysis. B, percent of cells in S phase was estimated by BrdUrd incorporation analysis according to the manufacturer's protocol (BD Biosciences). The relative results of three independent experiments are shown. *, p < 0.05 versus 0 µM adaphostin.

 
Effects of Adaphostin on Cell Cycle Regulatory Proteins—To investigate the mechanisms by which adaphostin induces a G1 phase cell cycle arrest, we examined the expression profile of a number of cell cycle regulatory proteins (Fig. 3, A–D). For this analysis, PC-3 cells were treated with different concentrations of adaphostin (0, 1.0, 2.5, and 5.0 µM) for 24 h (left panels) or with 2.5 µM adaphostin for different lengths of time (0, 24, and 48 h) (right panels). Total cellular lysates were prepared from the cells, and Western blot analysis was performed on these lysates. The results demonstrate that after 24 h of treatment with 1.0 µM adaphostin, there is a decrease in the levels of cyclins A, D1, and D3 relative to control cells (Fig. 3A, left panel). This decrease was even greater in cells treated with higher concentrations (2.5 and 5.0 µM) of adaphostin. Furthermore, a time-dependent decrease in the levels of these proteins was also observed (Fig. 3A, right panel). No significant changes in the levels of cyclin B1 or cyclin E were observed (Fig. 3A). Adaphostin treatment also caused a moderate dose- and time-dependent decrease in the expression levels of CDK4 and CDK6 proteins. No significant reduction in the levels of CDK1 and CDK2 was observed (Fig. 3B). Next, we analyzed the effect of adaphostin on protein expression levels of CDKI. Western blot analysis demonstrated a strong dose- and time-dependent increase in the expression levels of p21 and p27 (Fig. 3C). No change was observed in the protein levels of p15 and p18 (Fig. 3C). Additionally, although the expression level of Rb did not change in response to adaphostin treatment, a dramatic dose- and time-dependent decrease in the Rb phosphorylation level was observed (Fig. 3D). In all of the Western blots, the membranes were also independently probed for beta-actin as a loading control.

Mechanistic Basis of Adaphostin Action on Cell Cycle Regulatory Proteins—We next examined whether the above-described decrease in the expression of cyclins A, D1, and D3 and increase in the expression of p21 and p27 was because of alterations in gene transcription. Time course studies of RNA transcript levels demonstrated a marked increase in the levels of both p21 and p27 mRNA after 12 h of adaphostin treatment (Fig. 4A). This result suggests that the adaphostin-mediated increase in p21 and p27 protein levels occurs via a transcriptional or post-transcriptional mechanism. To explore the mechanism further, we examined the effects of adaphostin treatment on p21 and p27 mRNA levels following adaphostin treatment in the presence and absence of actinomycin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor). As shown in Fig. 4B (left panel), adaphostin-mediated increase in the p21 and p27 mRNA and protein levels was significantly reduced in the presence of actinomycin D. However, although cycloheximide was able to block the adaphostin-mediated increase in p21 and p27 protein levels, it did not affect the increase in p21 and p27 mRNA levels (Fig. 4B, right panel). These suggest that an increase in p21 and p27 may be a transcriptional event. To establish that up-regulation of p21 and p27 mRNA is purely a transcriptional event and not because of the stability of the respective mRNAs, we investigated transcriptional regulation of their respective gene promoters. PC-3 cells were transiently transfected with a series of processive 5' promoter deletion mutants of p21 (22) and p27 (23), and luciferase activity was measured in untreated control cells and in cells treated with 2.5 µM adaphostin for 24 h. As shown in Fig. 4C, all the deletion mutants exhibited similar increases in luciferase activity following adaphostin treatment except p21P Sma{Delta}1 construct. p21P Sma{Delta}1 was created by digesting p21P with SmaI and religation and contains only 61 bp proximal to the transcription start site (22). This result suggests that the adaphostin-responsive site is located within this 50-bp SmaI fragment of the p21 promoter sequence. The promoter activities of full size p27 promoter construct (p27PF) following adaphostin treatment was similar to the deleted constructs of p27 KpnI and p27 ApaI (Fig. 4D). However, the promoter activities of p27 MB-435 and p27 SacII was significantly decreased compared with that of p27 ApaI construct. This suggests that the essential site for adaphostin-induced p27 transcription up-regulation may exist in this region. Collectively, these results confirmed that the adaphostin-mediated increase in p21 and p27 protein levels is because of an induction in transcription. In contrast to the results found for the CDKI, no change in the levels of cyclin A, D1, D3, or cyclin E mRNA was observed (Fig. 4A). This result suggests that the adaphostin-mediated decrease in cyclin A, D1, and D3 protein levels occurs via a translational or post-translational mechanism. Therefore, we next examined whether the adaphostin-mediated decrease in cyclin protein levels involves the proteasome, a multiprotein complex that is responsible for the degradation of many proteins that play roles in signal transduction, cell cycle regulation, and other critical cellular functions (24). However, when PC-3 cells were pretreated with MG-132 and lactacystin, two proteasome inhibitors, treatment with adaphostin still was able to mediate a decrease in the protein levels of cyclins A, D1, and D3 (Fig. 4E). This finding suggests that the adaphostin-mediated decrease in the expression level of these cyclins is not dependent upon the proteasome pathway.


Figure 3
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  		FIGURE 3.
Adaphostin alters the expression of cell cycle regulatory proteins. A–D, adaphostin-mediated changes in the protein levels of cyclins (A), CDKs (B), CDKI (C), and Rb (D). PC-3 cells were exposed to 0–5 µM of adaphostin for 24 h (left panel) or to 2.5 µM of adaphostin for 0, 24, or 48 h (right panel). Protein lysates were then prepared from these cells and separated by SDS-PAGE. Immunoblot analysis using specific antibodies (as described under "Experimental Procedures") was then performed. Actin was used as a loading control. Results shown are representative of three independent experiments.

 
Adaphostin-mediated Increase in the Expression of p21 and p27 Leads to an Inhibition of CDK2-Cyclin E-associated Kinase Activity—As described above, adaphostin treatment induces an increase in the expression of p21 and p27; we next wanted to determine the significance of these alterations in the regulation of the cell cycle. It is known that active CDK2-cyclin E complexes are required during late G1 phase (25) and that their activity is regulated by the binding of CDKI. Because the levels of CDK1, CDK2, and cyclin E are unaltered by adaphostin treatment, we investigated whether the adaphostin-induced expression of the CDKI p21 and p27 altered the association between these CDKI and the CDK2-cyclin E complexes. To do this, we specifically immunoprecipitated p21, p27, and CDK2 from PC-3 cells treated with 2.5 µM adaphostin for 24 h. The proteins present in these immunoprecipitates were then examined by immunoblotting. As shown in Fig. 5 (A–C), treatment with adaphostin did not affect the immunoprecipitation of the CDK2 protein or its association with cyclin E. However, there was a marked increase in the association between CDK2 and either p21 or p27 (Fig. 5C). The results were similar whether CDK2, p21, or p27 was specifically immunoprecipitated. Consistent with the adaphostin-mediated increase in p21 and p27 expression described above, we detected an increase in the amount of these proteins immunoprecipitated from adaphostin-treated cells (Fig. 5, A and B). Interestingly, we detected an association of p21 and p27 with both cyclin E and CDK2, but not with CDK1 (Fig. 5, A and B), following adaphostin treatment. In vitro kinase assays demonstrated a dramatic decrease in CDK2-associated kinase activity following adaphostin treatment (Fig. 5D). Collectively, these data suggest that a decrease in CDK2-cyclin E kinase activity may be one of the contributing factors responsible for G1-S cell cycle arrest following adaphostin treatment.


Figure 4
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  		FIGURE 4.
Adaphostin up-regulates p21 and p27 at the transcriptional level. A, PC-3 cells were treated with 5 µM adaphostin for 0, 12, or 24 h. Total RNA was isolated, and mRNA levels were examined by RT-PCR. B, PC-3 cells were treated with 5µM adaphostin for 12 or 15 h. Actinomycin D (ActD) (1.0µg/ml) was added after 9 h of adaphostin treatment (left panel). Cycloheximide (Chx) (1.0µg/ml) was added for 1 or 2 h (i.e. after 11 or 10 h of adaphostin treatment, respectively) (right panel). Total RNA was then isolated, and the levels of p21 and p27 mRNA transcripts were examined by RT-PCR. Additionally, total protein was isolated from these cells, and immunoblots analysis for p21 and p27 protein was performed using appropriate antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as internal controls for RT-PCR and Western blotting, respectively. C and D, transcriptional activation of p21 (C) and p27 (D) promoter by adaphostin. The p21 and p27 promoter constructs fused to the luciferase (LUC) gene were transiently transfected into PC-3 cells. The cells were further incubated for 24 h in the presence or absence of 2.5 µM adaphostin. The luciferase activity was normalized by beta-galactosidase activity. The results are expressed as mean ± S.E. from three separate experiments. E, PC-3 cells were pretreated with the proteasomal inhibitors MG-132 (500 nM) or lactacystin (500 nM) for 2 h and then treated with adaphostin (2.5 µM) for 20 h. Western blotting was then performed for analysis of the indicated proteins using appropriate antibodies. Data shown are representative of three independent experiments.

 
Adaphostin-mediated Inhibition of CDK2-Cyclin E Kinase Activity Leads to a Decrease in the Phosphorylation of Nucleophosmin—NPM is a major nuclear phosphoprotein that is bound to centrosomes. It is phosphorylated by the CDK2-cyclin E complex, and this phosphorylation allows NPM to dissociate from the centrosomes, an essential step in the initiation of DNA replication (26). Given the above-described adaphostin-mediated inhibition of the CDK2-cyclin E complex, we examined the phosphorylation status of NPM following adaphostin treatment. Immunoblot analysis of whole cell lysates demonstrated that although there was no detectable change in the level of total NPM protein following adaphostin treatment, there was a dose- and time-dependent decrease in its phosphorylation status at residue Thr-199 (Fig. 6A). Next, we immunoprecipitated NPM and CDK2 from extracts of PC-3 cells treated with 2.5 µM adaphostin for 24 h or untreated controls to examine protein-binding partners (Fig. 6B). In the absence of adaphostin treatment, NPM strongly associated with cyclin E and CDK2; however, this interaction was dramatically diminished following adaphostin treatment (Fig. 6B, left panel). No association between NPM and either p21 or p27 was detected in the presence or absence of adaphostin (data not shown). Similarly, a high level of NPM co-precipitated with CDK2 in extracts from the control PC-3 cells (i.e. without adaphostin), but this interaction was dramatically inhibited by adaphostin treatment (Fig. 6B, right panel). These results suggest that the adaphostin-mediated up-regulation of p21 and p27, which leads to an inhibition of CDK2-cyclin E complex kinase activity, ultimately results in dissociation between the CDK2-cyclin E complex and NPM. This dissociation results in a decrease in NPM phosphorylation. To determine whether the adaphostin-mediated up-regulation of p21 and p27 was required for the adaphostin-mediated down-regulation of NPM phosphorylation, we transfected p21- and p27-specific siRNA along with control scrambled siRNA into PC-3 cells in the presence or absence of adaphostin. As shown in Fig. 6C, transfection with p21 and p27 siRNAs dramatically reduced the level of their respective proteins even in the presence of adaphostin. Additionally, adaphostin treatment did not induce the down-regulation of NPM (Thr-199) phosphorylation (Fig. 6C). NPM is known to function as a molecular chaperone by resolving protein aggregation, protecting enzymes during thermal denaturation, and facilitating the renaturation of chemically denatured proteins (27). Because we observed a dramatic decrease in the proteins levels of cyclins and CDKs following exposure to adaphostin, we hypothesized that a decrease in phosphorylation of NPM may affect its chaperoning activity on the cyclins and CDKs. To test this possibility, we examined whether the overexpression of both cyclin E and CDK2, components of the protein complex responsible for NPM phosphorylation, would affect the adaphostin-mediated decrease in NPM phosphorylation, thereby preventing the degradation of the cyclin proteins. As depicted in Fig. 6D, when PC-3 cells were transfected with vector expressing cyclin E and CDK2 to high levels, the cells were significantly protected from the adaphostin-mediated decrease in NPM phosphorylation. This overexpression also helped to prevent the adaphostin-mediated decrease in the protein levels of cyclin A, cyclin D1 (Fig. 6D), and cyclin D3 (data not shown).


Figure 5
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  		FIGURE 5.
Adaphostin treatment induces the inactivation of cyclin E-CDK2 complex. Lysates from untreated or adaphostin-treated PC-3 cells were immunoprecipitated (IP) with antibodies specific for p21 (A), p27 (B), or CDK2 (C), and kinase assay with immunoprecipitated CDK2 using histone H1 as substrate (D). The immunoprecipitates were then resolved on denaturing polyacrylamide gels and immunoblotted for analysis of the indicated proteins using appropriate antibodies. The data shown are representative of three independent experiments.

 


Figure 6
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  		FIGURE 6.
Adaphostin alters the phosphorylation of NPM in PC-3 cells. A, adaphostin induces a decrease in the phosphorylation status of NPM. Western blot analysis of total cell lysates from PC-3 cells examined the levels of total and phosphorylated NPM protein. B, adaphostin promotes the dissociation of the NPM and cyclin E-CDK2 complex. Cell lysates from untreated or adaphostin-treated PC-3 cells were immunoprecipitated (IP) with antibodies specific for NPM (left panel) or CDK2 (right panel). The immunoprecipitates were then resolved on denaturing polyacrylamide gels and immunoblotted (IB) for analysis of the indicated proteins using appropriate antibodies. C, adaphostin-mediated decrease in the phosphorylation of NPM is rescued by p21 and p27 knockdown. PC-3 cells were either transfected or mock-transfected with p21- or p27-specific siRNA and then treated with 2.5 µM adaphostin for 24 h. Immunoblots of cell lysates using antibodies specific for p21, p27, p-NPM, and actin were performed to examine respective protein levels. D, overexpression of cyclin E and CDK2 prevents adaphostin-mediated decrease in the phosphorylation of NPM and partially rescues adaphostin-mediated decrease in cyclin A and cyclin D1 protein levels. PC-3 cells were transfected with wild type cyclin E and CDK2 expression vectors (pcDNA 3.1/cyclin E and pcDNA 3.1/CDK2, respectively) or empty vector (pcDNA 3.1) for 24 h and then exposed to 2.5 µM adaphostin for an additional 24 h. Cells were then washed, and total cell lysates were collected. Western blot analysis was then performed to examine the expression levels of the indicated proteins using appropriate antibodies.

 


Figure 7
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  		FIGURE 7.
Adaphostin-induced cell cycle arrest is mediated by the p38 MAPK pathway. A, adaphostin induces an increase in the phosphorylation of p38 MAPK. Western blot analysis of total cell lysates from PC-3 cells examined the expression pattern of p38 MAPK, p-p38, p-ERK, and p-JNK. B, p38-specific siRNA rescues the adaphostin-induced cell cycle arrest. PC-3 cells were either mock-transfected with control siRNA or transfected with p38 MAPK-specific siRNA and then treated with 2.5 or 5.0 µM adaphostin for 24 h. Western blot analysis of total cell lysates was then performed to examine the expression levels of the indicated proteins using appropriate antibodies.

 
Adaphostin-induced Cell Cycle Arrest Involves p38 MAPK—Recent studies have demonstrated that the MAPK cascade plays an essential role in the regulation of cell cycle progression (28). To determine whether MAPK signaling plays a role in the above-described adaphostin-induced cell cycle arrest, we examined the MAPK signaling events induced by adaphostin treatment on PC-3 cells. Following adaphostin treatment (at various doses and for various times), cell lysates were prepared and the activation of various MAPKs was determined by immunoblot analysis using antibodies specific for ERK, JNK, or p38 MAPKs. As shown in Fig. 7A, a dose- and time-dependent increase in the activation of p38 MAPK was observed, as demonstrated by the increase in the level of phosphorylated p38 MAPK. In contrast, adaphostin treatment had no effect on the activities of ERK and JNK as none of their hyperphosphorylated forms were detected (Fig. 7A). The total protein expression levels of the p38 MAPK, ERK, and JNK were unaffected by adaphostin treatment (Fig. 7A; data for ERK and JNK not shown). Previous studies have correlated increased p38 MAPK activity with increased p21 and p27 expression (29, 30). To explore a possible link between the adaphostin-induced activation of p38 MAPK and the increased expression of p21 and p27, we used siRNA to knock down the expression of p38 MAPK and monitored the expression of p21 and p27 by Western blotting (Fig. 7B). The results demonstrated that the adaphostin-mediated increase in p21 and p27 expression was significantly inhibited by the expression of p38-specific siRNA. Moreover, the expression of this p38-specific siRNA was also able to rescue the adaphostin-mediated decrease in NPM phosphorylation (Fig. 7B). Together, these findings suggest that the adaphostin-induced cell cycle arrest of PC-3 cells is mediated by the p38 MAPK pathway.

Adaphostin Treatment Inhibits c-Met Phosphorylation and Blocks c-Met-dependent Signaling—Cellular signaling via the hepatocyte growth factor/scatter factor tyrosine kinase receptor, c-Met, is critical for normal physiological processes. This signaling cascade is initiated by HGF/scatter factor binding to and stimulating c-Met. This stimulation results in the activation of c-Met, which then goes on to induce several cellular responses, such as proliferation (6). The deregulation of c-Met signaling has been found to contribute to the development and growth of primary tumors and their metastases (31). To determine whether the c-Met signaling cascade plays a role in the adaphostin-mediated cell cycle arrest, PC-3 cells nonstarved and prestarved for 24 h were exposed to various concentrations of adaphostin for 4–5 h. Following adaphostin treatment, prestarved cells were stimulated with HGF (40 ng/ml) for 15 min. The ability of adaphostin to inhibit the activation of c-Met was examined by Western blotting. As shown in Fig. 8A (left panel), treatment with 5.0 µM adaphostin for 4 h resulted in a decrease in the phosphorylation (i.e. activation) of c-Met and its associated docking proteins (such as GAB1, GAB2, and SHC) that contribute to c-Met-dependent downstream signaling. This inhibition of phosphorylation was observed even after HGF stimulation that typically leads to the tyrosine phosphorylation of the c-Met kinase domain at residues Tyr-1234/Tyr-1235 and Tyr-1349 (Fig. 8A, right panel). To further examine the adaphostin-mediated inhibition of c-Met in PC-3 cells, we overexpressed c-Met in PC-3 cells by transient transfection and monitored the effects of adaphostin treatment on c-Met downstream signaling. As shown in Fig. 8B, the above-described adaphostin-mediated increase in the phosphorylation of p38 MAPK was dramatically inhibited by the overexpression of c-Met. Furthermore, c-Met overexpression also reduced the adaphostin-mediated increase in p21 and p27 expression (Fig. 8B). Next, we used siRNA to knock down the expression of c-Met to determine whether the inhibitory effect of adaphostin treatment coupled with the reduced expression of c-Met enhanced adaphostin-mediated cellular perturbations. c-Met protein expression was decreased by 70–80% following transient transfection with c-Met-specific siRNA (Fig. 8C). Phosphorylation activation of c-Met (Tyr(P)-1234/1235 and Tyr(P)-1349) was also substantially inhibited (data not shown). The siRNA-mediated knockdown of c-Met expression caused an increase in the phosphorylation of the p38 MAPK, and this effect was further increased by adaphostin treatment (Fig. 8C). Expression of the c-Met-specific siRNA also resulted in a partial increase in the expression level of p21 and p27 in PC-3 cells not treated with adaphostin (relative to negative control cells transfected with a mock siRNA), and adaphostin treatment further increased the levels of these proteins (Fig. 8C). Given the above-described ability of adaphostin to inhibit c-Met signal transduction, we next examined its ability to block the cellular functions mediated by c-Met signaling, specifically cellular proliferation. As shown in Fig. 8D, adaphostin treatment inhibited the HGF-induced BrdUrd incorporation in PC-3 cells in a dose-dependent manner. Together, these data strongly suggest that the c-Met receptor tyrosine kinase is involved in the adaphostin-induced signaling cascade.


Figure 8
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  		FIGURE 8.
Adaphostin-mediated signaling cascade involves the hepatocyte growth factor receptor Met. A, effect of adaphostin on c-Met phosphorylation in PC-3 cells. Cells were either nonstarved (left panel) or prestarved (right panel) for HGF and then exposed to various concentrations of adaphostin for 4–5 h. Prestarved cells were stimulated with HGF (40 ng/ml) for 15 min. B, effect of Met overexpression on adaphostin-treated PC-3 cells. PC-3 cells were transfected with an empty vector (pcDNA 3.1) or a Met expression vector (pcDNA 3.1/Met) for 24 h and then exposed to 5 µM adaphostin for an additional 24 h. C, effect of Met-specific siRNA expression on adaphostin-treated PC-3 cells. PC-3 cells were either mock-transfected with control siRNA or transfected with c-Met-specific siRNA and then treated with 5.0 µM adaphostin for 24 h. Cells from A–C were then washed, and total cell lysates were collected. Western blot analysis was then performed to examine the expression of the indicated proteins using appropriate antibodies. D, adaphostin inhibits HGF-mediated BrdUrd incorporation in PC-3 cells. PC-3 cells were serum-starved overnight, and then stimulated with HGF (40 ng/ml) in the presence of various concentrations of adaphostin for 24 h. The amount of BrdUrd incorporation was determined by flow cytometric analysis according to the manufacturer's protocol (BD Biosciences). The results shown are the relative values from three independent experiments. Error bars represent standard errors.

 
Establishment and Characterization of Adaphostin-resistant PC-3 Cells—Drug resistance is a major obstacle for successful chemotherapies and occurs with the most commonly used anti-tumor drugs as well as with newer target-oriented anti-cancer agents (32). To examine the mechanism of action of adaphostin in PC-3 cells, we developed derivatives of the PC-3 cell line (PC-3-ADP-R5 and PC-3-ADP-R10) that were resistant to 5 and 10 µM adaphostin, respectively, by continuously exposing the cells to increasing concentrations of the compound. These resistant cell lines were maintained under constant respective concentrations of adaphostin pressure except at the time of the experiment. Although the PC-3-ADP-R cell lines represent in vitro models that are far from the human physiological drug resistance occurrence conditions, they can be potentially useful for studying the mechanism(s) of action of adaphostin. The IC50 value of adaphostin after a 48-h exposure was estimated to be ~17 and 31 µM for PC-3-ADP-R5 and PC-3-ADP-R10, respectively (Fig. 9A). Next, we examined the morphology and cell cycle distribution of the PC-3-ADP-R10 cell line relative to those of the parental PC-3 cell line and with PC-3 cells treated with adaphostin (Fig. 9, B–D). The morphology of the PC-3-ADP-R and untreated parental PC-3 cell lines was comparable (Fig. 9B), and they grew with similar doubling times (about 16–20 h when growing exponentially). In contrast, the adaphostin-treated PC-3 cells exhibited a completely different cellular morphology. These cells appeared to grow much slower and were more rounded and had no sprouting (Fig. 9B). Flow cytometric analysis of the cells following propidium iodide staining of total cellular DNA (Fig. 9C) and BrdUrd incorporation (Fig. 9D) did not reveal any differences in the cell cycle profile and S phase distribution, respectively, between the PC-3-ADP-R cell lines and the untreated parental PC-3 cells. However, following adaphostin treatment of PC-3 cells, a prominent G1-specific cell cycle arrest coupled with a decline in the S phase population of cells was observed (Fig. 9, C–D). Finally, we examined the expression and/or phosphorylation status of the various marker proteins believed to be involved in the adaphostin-mediated signaling cascade. As shown in Fig. 9E, the PC-3-ADP-R cell lines did not exhibit any increase in the protein levels of p21 and p27, whereas there was a significant increase in each of these two proteins in the parental PC-3 cells treated with 5 µM adaphostin. The phosphorylation status of NPM in the resistant cell lines was similar to that of parental PC-3 control (Fig. 9E). Additionally, the phosphorylation status of p38 MAPK and c-Met in the adaphostin-resistant cell lines was comparable with that of the parental PC-3 cells (Fig. 9E). Collectively, these data demonstrate the involvement of all of these proteins in adaphostin-mediated cell cycle arrest.


Figure 9
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  		FIGURE 9.
Comparison of parental PC-3 and PC-3-ADP-R cells. A, PC-3 cells resistant to adaphostin have higher IC50 values. PC-3-ADP-R cells were exposed to adaphostin (0–40 µM) for 48 h. Cells were then trypsinized, and the number of live cells was counted by trypan blue dye exclusion assay. The results are the mean of three independent experiments. B–D, comparison of the PC-3 parental cell line (left), PC-3 cells treated with 2.5 or 5.0 µM adaphostin for 24 h (middle), and PC-3-ADP-10R cells (right) with respect to cell morphology (B), cell cycle distribution (determined by FACS analysis of propidium iodide-stained cells) (C), and S phase distribution (determined by FACS analysis of BrdUrd stained cells) (D). E, Western blot analysis of total cell lysates of parental PC-3 cells, PC-3 cells treated with 5 µM adaphostin for 24 h, and PC-3-ADP5R and PC-3ADP10R cell lines. The data shown are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Constitutively active receptor tyrosine kinases have been described in cancer patients (33, 34). In vitro tyrosine phosphorylation of such receptor tyrosine kinases supports the evidence of their transforming activity (35, 36). Recently, small molecule agents that reduce the signaling of these receptor tyrosine kinases have become an important tool in the treatment of cancer patients (37, 38). In this view, the tyrphostin family analogue adaphostin represents an attractive anti-cancer candidate for inhibiting receptor tyrosine kinase-mediated signaling events because it acts by preventing the ability of the tyrosine kinases to bind their peptide substrates rather than by preventing the binding of ATP of the tyrosine kinases (15). The present study was aimed at investigating the antiproliferative potential of adaphostin, an adamantyl ester of AG957, on human prostate cancer (PC-3) cells.

Recent studies have examined the cellular effects of adaphostin. Hose et al. (39) demonstrated that adaphostin induces the rapid release of free iron, an effect related to reactive oxygen species production. Yu et al. (12) demonstrated that adaphostin treatment induces oxidative injury in a leukemia model system via the Raf-1/MEK/ERK1/2 and Akt pathways. A few other studies have successfully employed adaphostin in combination with either anti-angiogenesis drugs, DNA-damaging cytotoxic drugs (such as fludarabine) (40), or proteasome inhibitor drugs in BCR/ABL-positive and/or negative myelogenous leukemia cells lines (14, 41). However, despite our growing knowledge of the cellular activities of adaphostin, its primary cellular target and specific mechanism of action have not yet been identified in either suspension or solid cell culture model systems.

This study describes the in vitro anti-proliferative effects of adaphostin in PC-3 cells. The results of this study clearly demonstrate that adaphostin induces a G1 phase-specific cell cycle arrest via the induction of p21 and p27 and the degradation of G1 phase-specific cell cycle regulatory proteins. Importantly, we show for the first time that adaphostin induces a decrease in NPM phosphorylation by inducing the expression of p21 and p27, which ultimately leads to the inhibition of cyclin E-CDK2 complex activity. Normally, the phosphorylation of NPM by the cyclin E-CDK2 complex is an essential step required for the centrosome to initiate DNA replication. Additionally, we showed that p38 MAPK is a key regulator in the adaphostin-mediated cell cycle arrest and that it is involved in the adaphostin-mediated reduction in NPM phosphorylation. Finally, we provide evidence suggesting the involvement of c-Met, the hepatocyte growth factor receptor, in the adaphostin-mediated cellular perturbations, with p38 MAPK acting as an intermediate signaling sensor. Although the details of the signaling pathway involved in the transfer of signal from c-Met to p38 MAPK following adaphostin treatment remain to be elucidated, preliminary evidence suggests that GAB1 and GAB2 may play a role in the signaling event(s). A schematic representation of the hypothesized effects of adaphostin on the proliferation of PC-3 cells is shown in Fig. 10.


Figure 10
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  		FIGURE 10.
Schematic diagram of the suggested effects of adaphostin on the proliferation of PC-3 cells.

 
Normal eukaryotic cell cycle progression is regulated by the expression and sequential activation of various cell cycle-dependent cyclins, CDKs, and CDKI (42–44). Isoforms of the cyclin D proteins associate with CDK4 and CDK6 kinases, and this interaction leads to the activation of their kinase activity, which in turn mediates transition from early G1 to mid G1 phase of cell cycle. Cyclin E on the other hand associates with, and thereby activates, CDK2, and this active kinase complex plays a role in the transition from mid-G1 to S phase of the cell cycle (45). In this study, we demonstrate a dose- and time-dependent decrease in the levels of cyclin A, D1, and D3 proteins following adaphostin treatment. A moderate decrease in the levels of CDK4 and CDK6 proteins was also observed. Interestingly, no change in the levels of either cyclin E or CDK2 was observed following adaphostin treatment. However, adaphostin was shown to strongly inhibit CDK2-associated kinase activity. Also, adaphostin was shown to induce a decrease in the phosphorylation status of the Rb protein in a dose- and time-dependent manner. Collectively, these data demonstrate that adaphostin induces a G1 phase cell cycle arrest by decreasing the expression of G1 phase-specific cyclins (cyclin A, D1, and D3) and CDKs (CDK4 and CDK6), inhibiting CDK2 kinase activity, and thus inhibiting the hyperphosphorylation of Rb.

CDKI are tumor suppressor proteins that arrest cell cycle progression by binding to active CDK-cyclin complexes thereby inhibiting their activities (42, 45). The important members of the CDKI family include p21 and p27, and their expression regulates G1 phase CDKs (46, 47). In this study, we show that adaphostin treatment induces the expression of p21 and p27 in PC-3 cells. This increased expression occurs at the transcriptional level and activates p21 and p27 gene promoters. Because PC-3 cells contain a mutation in the p53 gene, this adaphostin-mediated induction of p21 and p27 is independent of p53. Similar p53-independent induction of p21 and p27 expression has been described previously (48–50). We also observed a significant increase in the association of p21 and p27 with the cyclin E-CDK2 complex. Therefore, it is likely that the adaphostin-mediated induction of p21 and p27 expression is responsible for the decreased CDK2 kinase activity observed following adaphostin treatment.

Growing evidence suggests that NPM, a nucleolar phosphoprotein, is a multifunctional protein involved in ribosome biogenesis, nucleocytoplasmic trafficking, and DNA and centrosome duplication (27, 51). Additionally, NPM has been shown to function as a chaperone for cell cycle regulatory proteins (52). The stress response-mediated degradation of key cell cycle regulatory proteins is known to be dependent on the phosphorylation status of NPM (51). For example, NPM is typically bound to centrosomes. During the initiation of centrosome duplication, the activated CDK2-cyclin E kinase complex phosphorylates NPM. The phosphorylated NPM then dissociates from the centrosome and triggers the initiation of centrosome duplication (26, 53). By this mechanism, NPM provides one of the licensing systems controlling centrosome duplication and ensures the coordination of centrosome and DNA duplication, events important for limiting duplication to once per cell cycle (26). Previous reports have documented altered NPM expression in prostate cancer (54). Our results demonstrate that although adaphostin does not alter NPM expression, it does induce a dose- and time-dependent decrease in the phosphorylation of NPM. This result prompted us to hypothesize that the p21- and p27-mediated inactivation of the CDK2-cyclin E complex induced by adaphostin treatment is responsible for this observed decrease in the phosphorylation of NPM. This hypothesis is supported by the finding that the siRNA-mediated knockdown of p21 and p27 expression significantly rescues the adaphostin-mediated decrease in NPM phosphorylation. Furthermore, the overexpression of cyclin E and CDK2 rescues not only the adaphostin-mediated decrease in NPM phosphorylation, but also the adaphostin-mediated decrease in the expression of cyclins A, D1, and D3, a result that may be attributed to the chaperoning activity of NPM. NPM is known to continually shuttle between the nucleolus and the cytoplasm (55). Specifically, NPM has been shown to bind to proteins containing nuclear localization signals and stabilize them for their transport (52, 56). Our results support this role for NPM and demonstrate that, at least in adaphostin-treated PC-3 cells, the CDK2-cyclin E complex regulates NPM function by altering its post-translational phosphorylation status and in turn NPM may stabilize the degradation of cyclins.

The c-met oncogene encodes for the receptor tyrosine kinase for HGF. It regulates several signaling pathways that lead to cell growth, invasion, and protection from apoptosis (3). Upon HGF stimulation, the c-Met receptor is phosphorylated, and this phosphorylation leads to the recruitment of a group of signaling molecules and/or adaptor proteins to the cytoplasmic domain and docking sites of c-Met. These events result in the activation of several downstream signaling cascades, including p38 MAPK (9). It has been reported previously that c-Met is crucial for PC-3 tumor cell survival and that the expression of c-Met is frequently elevated during prostate tumor progression (57, 58). In this study, we demonstrate that adaphostin treatment decreases the tyrosine phosphorylation status of the c-Met activation loop (Tyr(P)-1234/1235) and multifunctional docking site (Tyr(P)-1349), even under growth factor (HGF)-stimulated conditions. This decrease in c-Met phosphorylation leads to a reduction in the total tyrosine phosphorylation of GAB1, an adaptor protein that binds to the c-Met multifunctional docking site. It is interesting that, even under similar HGF-stimulated conditions, we observed a decrease in the phosphorylation status of additional adaptor molecules, such as SHC and GAB2 (GRB-associated binder 2), following the adaphostin treatment of PC-3 cells. This modulation of GAB1 tyrosine phosphorylation by adaphostin is consistent with the previously reported requirement for GAB1 in the mediation of critical c-Met-dependent cellular signaling (59). Previous studies have suggested the involvement of GRB2 and SHC in several survival pathways, and a decrease in the tyrosine phosphorylation status of these proteins inactivates these functions (60, 61). Therefore, it is likely that the inactivation of GRB2 and SHC also contributes to the deregulation of the critical survival pathways involved in prostate tumor growth and progression.

Consistently, the ectopic overexpression of c-Met is able to rescue PC-3 cells from the adaphostin-mediated perturbations in cellular signaling. Specifically, overexpression of wild type c-Met by transient transfection protected these cells from the adaphostin-mediated decrease in c-Met phosphorylation. Additionally, we found that c-Met overexpression significantly repressed the adaphostin-mediated induction of p21 and p27 expression. Furthermore, the adaphostin-mediated increase in the phosphorylation of p38 MAPK was dramatically inhibited by c-Met overexpression. We initially found that adaphostin treatment increases the phosphorylation of p38 MAPK in a dose- and time-dependent manner, whereas other MAPK kinases (such as ERK and JNK) were not affected (Fig. 7, A and B). This finding prompted our biochemical analysis of the effects of adaphostin treatment on cell cycle regulating proteins, particularly p21 and p27. We demonstrated that the siRNA-mediated knockdown of p38 MAPK expression reversed not only the adaphostin-mediated changes in the expression levels of p21 and p27 but also the decreased phosphorylation of NPM. Additionally, we showed that the siRNA-mediated knockdown of c-Met expression enhanced the adaphostin-mediated increase in the phosphorylation of p38 MAPK and p21 and p27 expression. Collectively, these data provide a link between c-Met receptor activation and increased phosphorylation of p38 MAPK and therefore the subsequent induction of p21 and p27 expression. These findings are consistent with those of Kim et al. (57) who demonstrated the ability of p38 MAPK to regulate p21 expression in prostate cancer cells and Wang et al. (62) who demonstrated that c-Met induced cell death primarily via the p38 MAPK pathway.

The MAPK superfamily is known to play a vital role in eukaryotic cell proliferation, differentiation, and apoptotic responses to a wide range of extracellular stimuli (63). Several studies have documented the involvement of the p38 MAPK pathway in the regulation of cell cycle progression, specifically the G1 phase (28, 64). Our results demonstrate that the adaphostin-mediated activation of the p38 MAPK pathway leads to the transcriptional up-regulation of p21 and p27. These data indicate that the adaphostin-mediated activation of p38 MAPK acts as a sensor for the transcriptional up-regulation of p21 and p27 and suggest a possible molecular link between c-Met/p38 MAPK signal transduction and the p21 and p27 transcriptional induction in response to adaphostin treatment. However, the transcriptional factors involved in this transcriptional induction and their cognate cis-sequence(s) remain to be elucidated. On a similar note, quite interestingly the literature documented that adaphostin regulates the Akt cellular survival pathway in leukemia cells (12, 39). Contrary to these reports, we did not observe any change in the phosphorylation and/or total protein of Akt following adaphostin treatment in PC-3 cells (data not shown).

The development of cell lines with resistance to specific drugs and the examination of the molecular, biological, and biochemical properties of those cell lines provide a useful approach for elucidating the mechanism of action of that specific drug (32, 65). In this study, we generated two adaphostin-resistant PC-3 cell lines (called PC-3-ADP-R) and characterized the cellular and biochemical properties of these cells. These two PC-3-ADP-R cell lines were not found to be resistant to other commonly tested anti-cancer drugs, such as perifosine, geldanamycin (17dMAG), UCN-01, rapamycin, and miltefosine (data not shown). Additionally, these cell lines were not shown to have multidrug resistance phenotypes and do not exhibited any change(s) in the proteins level of any multidrug resistance genes tested (data not shown). This finding demonstrates that the mechanism of adaphostin resistance in these cell lines differs from the mechanism of multidrug resistance in PC-3 cells. Contrary to the results from wild type PC-3 cells, the adaphostin-resistant PC-3 cell lines exhibit very high levels of c-Met phosphorylation and very low levels of p38 MAPK phosphorylation. Moreover, the resistant cell lines failed to induce a cell cycle arrest. Therefore, the PC-3-ADP-R cells could represent a potentially useful tool for examining the mode of action of adaphostin in PC-3 cells. They could also be used for the selection of noncross-resistant structural analogues of adaphostin and for the investigation and development of methods for preventing resistance to adaphostin.

Several studies have suggested that the hormone refractory, advanced prostate cancer phenotype is associated with many cellular changes, such as the deregulation of cell cycle progression and cell survival signaling, and the inactivation of p53 (66), changes like those present in PC-3 cells. Therefore, the findings presented here could be clinically significant considering that adaphostin treatment induces the p53-independent up-regulation of p21 and p27. Moreover, conventional treatments, such as taxane-based chemotherapies, are currently found to be an ineffective treatment for patients with advanced disease. Therefore, the identification of novel targets and new treatment regimens are critical for the future control of advanced prostate cancer.

In conclusion, the findings presented here demonstrate the in vitro anti-cancer potential of adaphostin. The ability of adaphostin to induce a cell cycle arrest in the human prostate cancer cell line, PC-3, strongly suggests the need for further in vivo efficacy studies in preclinical human prostate cancer models. We have identified a new cellular target for an interesting compound that possesses a wide range of interesting biological activities, including significant antiproliferative activity. The results of the study presented here provide a strong basis for the further development of adaphostin as a novel agent that, in combination with other compounds, may be useful for human prostate cancer therapy and/or prevention.


   FOOTNOTES
 
* 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. Back

1 Present address: Molecular Signal Transduction Section, Laboratory of Allergic Diseases, NIAID, National Institutes of Health, Bethesda, MD 20892. Back

2 To whom correspondence should be addressed: NCI, National Institutes of Health, 37 Convent Dr., Bldg. 37, Rm. 1052, Bethesda, MD 20892. Tel.: 301-496-4119; Fax: 301-480-7456; E-mail: kr91w{at}nih.gov.

3 The abbreviations used are: HGF, hepatocyte growth factor; PBS, phosphate-buffered saline; CDKI, cyclin-dependent kinase inhibitor; CDK, cyclin-dependent kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; NPM, nucleoplasmin; BrdUrd, 5-bromo-2-deoxyuridine; RT, reverse transcription; FACS, fluorescence-activated cell sorter; siRNA, short interfering RNA; Rb, retinoblastoma. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Toshikazu Nakamura (Osaka University Graduate School of Medicine, Osaka, Japan) for providing wild type human c-Met construct and Dr. Ed Harlow (Laboratory of Molecular Oncology, Massachusetts General Hospital Cancer Center) for human cyclin E and CDK2 constructs. We thank Dr. Xiao-Fan Wang (Duke University Medical Center, North Carolina) for p21 promoter-luciferase reporter constructs and Dr. Toshiyuki Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan) for p27 promoter-luciferase reporter constructs. We thank Dr. Vikas Rishi, Dr. Sabarni Chatterjee, and Robert Robey for critically reading the manuscript. We also thank Rituparna Mukherjee, Amanda Wood, and Jessie Eagleton for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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