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. 2003 Mar 4;129(3):165–174. doi: 10.1007/s00432-003-0420-3

Prostate cancer cell proliferation is strongly reduced by the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in vitro on human cell lines and primary cultures

Carlo Vicentini 1, Claudio Festuccia 2, Giovanni Luca Gravina 1, Adriano Angelucci 2, Angelo Marronaro 1, Mauro Bologna 2,3,✉
PMCID: PMC12161944  PMID: 12712332

Abstract

Purpose.

To investigate the effects of the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) ZD1839 ('Iressa') on the cellular proliferation of androgen-sensitive and androgen-independent human prostatic cancer cell lines and primary cultures in vitro.

Experimental design.

In this study, we investigated the effects of the quinazoline ZD1839, a potent, selective EGFR-TKI, on the EGFR autophosphorylation and cellular proliferation of androgen-sensitive (ND1, LNCaP, and ALVA-31) and androgen-independent (PC3, DU145, and TSU-Pr1) human prostatic cancer cell lines and 20 primary cultures derived from human prostatic cancer tissue.

Results.

EGFR was present and phosphorylated in all cell lines tested. ZD1839 reduced EGFR autophosphorylation in intact cell lines with IC50s of 0.46–0.97 μM, and inhibited cellular proliferation with IC50s of 0.37–1.03 μM. Constitutive EGFR autophosphorylation was low in primary cell cultures, but addition of EGF (50 ng/ml) caused marked EGFR autophosphorylation; cellular proliferation in the presence of EGF was inhibited by ZD1839 with a mean IC50 of 0.45 μM. At doses >1 μM, ZD1839 induced apoptosis in both androgen-dependent and androgen-independent PCa cell lines.

Conclusion.

Our experiments suggest that EGFR-TKIs such as ZD1839 may have potential in blocking the growth and progression of human prostatic cancers even in early phases of the disease.

Keywords: Prostate cancer therapy, EGFR, ZD1839, 'Iressa', Androgen-dependence

Introduction

The treatment of cancer mainly uses cytotoxic drugs that kill cells by acting on molecular targets common to both normal and malignant cells. Therapeutic regimens typically consist of a combination of relatively non-cross-resistant drugs. However, several cancer types are not sensitive, or become resistant, to treatment with cytotoxic drugs, resulting in disease relapse or progression on therapy. The rational search for novel drugs that could selectively interfere with the intracellular targets involved in these processes is in progress. Such drugs could be used in combination with conventional therapies.

Prostate cancer (PCa) is predominantly a tumor of older men. It may be treated surgically and cured when localized, but locally advanced cancer is not usually curable, and, although median survival may be as long as 5 years, a substantial fraction of patients will eventually die of their tumor. If the cancer has spread to distant organs, currently available therapies are not able to cure the disease although it usually shows some response to treatment with antiandrogens (Rambeaud 1999; Boyle and Severi 1999). The prostate gland requires androgens for cellular proliferation and maintenance of its function and a battery of other regulators is involved in fine-tuning prostatic growth and differentiation (Cunha 1994). Androgen ablation, the standard treatment for non-organ-confined PCa, aims to block the hormonal signaling cascade that leads to the activation of the androgen receptor. In the hormone-responsive stage of PCa, this treatment causes downregulation of androgen-responsive genes and leads to apoptosis. Although androgen ablation is initially successful, many tumors overcome androgen blockade and develop a hormone-unresponsive phenotype that is resistant to therapy. Progression to hormone-refractory PCa can, in part, be explained by the concept of hypersensitive tumor cells (Harris and Reese 2001), which are highly responsive to residual circulating androgens, growth factors, and other cellular regulators; the mitogenic effects of growth factors are believed to be of primary importance (Koutsilieris and Mitsiades 2001). Stromal prostate tissue has been demonstrated to support the growth of cancer cells predominantly by secreting growth factors (Festuccia et al. 1999; Rajan et al. 1996; Scott et al. 2001; Song et al. 2000). Epidermal growth factor (EGF) and transforming growth factor α (TGFα)—related polypeptide growth factors that consist of 53 and 50 amino acids, respectively, and have about 35% sequence homology—are potent mitogens that play a regulatory role in the proliferation of PCa cells and in tumor angiogenesis (Scher et al. 1995; Sherwood et al. 1998; Kim et al. 2001; Shaheen et al. 2001). The epidermal growth factor receptor (EGFR) family members, in particular those encoded by the proto-oncogenes c-erbB1/HER-1/EGFR and c-erbB2/HER-2/neu, represent important regulatory elements in the growth and progression of many epithelial-derived tumors including PCa (Maihle et al. 2002; Damstrup et al. 2002; Skacel et al. 2001; Suo et al. 2002; Esteva et al. 2001; Vizoso et al. 2000).

The EGFR comprises an extracellular binding domain and a cytoplasmic domain with tyrosine kinase activity, and can bind either EGF or TGFα. Upon binding of a ligand to EGFR, receptor dimerization occurs and the intracellular tyrosine kinase region is activated, causing receptor tyrosine autophosphorylation and transphosphorylation of another receptor monomer (Fantl et al. 1993; Ullrich and Schlessinger 1990). The phosphorylated tyrosine residues on EGFR become the 'docking' sites for various molecules containing the Src homology 2 domain. Subsequent tyrosine phosphorylation of various protein substrates, including phospholipase Cg, phosphatidylinositol 3-kinase (Esteva et al. 2001), GTPase-activating protein, Grb2 (Lotti et al. 1996), Shc (Esteva et al. 2001), p21ras (Masuda et al. 2001), mitogen-activated protein kinases (Kawanaka et al. 2001; Pichard et al. 2001), and c-Src (Li et al. 2001), transmits proliferative and survival signals downstream.

Various autocrine loops have been postulated in prostate cancer cells: EGF activates the androgen receptor in prostate cancer cells in the absence of androgens (Nazareth and Weigel 1996; Yeh et al. 1999) and TGFα may promote prostate cell metastasis (Cohen et al. 1994). Coexpression of EGFR and EGF or TGFα results in an autocrine stimulation loop. Bombesin can also play a role as a growth-promoting factor (Bologna et al. 1989).

Chemoattractant growth factors expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of PCa cells to these organs (Festuccia et al. 1999; Zolfaghari and Djakiew 1996). EGF immunoreactivity is localized in the stroma of the human prostate (De Miguel et al. 1999), in the osteogenic stroma of pelvic medullary bone, and in the stroma within the capsule and trabeculae of pelvic lymph nodes (Song et al. 2000). In vitro studies showing that EGFR inhibitors reduce the chemomigration of human PCa cells across Boyden chambers (Zolfaghari and Djakiew 1996) suggest that EGFR inhibition may be a useful strategy in the treatment of metastatic disease. The large body of experimental and clinical evidence for EGF and TGFα/EGFR autocrine and paracrine growth pathways in common human cancers makes blockade of this pathway a promising anticancer therapeutic strategy (Arteaga and Johnson 2001). Indeed, a number of protein tyrosine kinase inhibitors have been evaluated in experimental systems (Kobayashi et al. 2002; Schiffer 2001; Burke et al. 1991; Fry et al. 1994; Traxler et al. 1995), including flavonoids (quercetin), tyrphostins, lavendustin, erbstatin, and genistein.

ZD1839 ('Iressa') is an orally active, selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that blocks signal transduction pathways implicated in the proliferation and survival of cancer cells, and other host-dependent processes promoting tumor growth. It has demonstrated significant antitumor activity in vivo against a range of human tumor xenografts, including PCa tumors (Baselga and Averbuch 2000; Moulder et al. 2001; Ciardiello et al. 2001; Ciardiello et al. 2000), and was well tolerated and showed encouraging activity against a range of human cancers, including PCa, in phase I trials (Baselga and Averbuch 2000). In the present study, we investigate the effects of the novel EGFR-TKI ZD1839 on EGFR autophosphorylation and cellular proliferation of all the major human PCa cell lines, to confirm previous indications, and on PCa primary cell cultures, to gain further information relevant to the use of such compounds in different categories of patients with the disease.

Materials and methods

Reagents

All the materials for tissue culture were purchased from Hyclone (Cramlington, Northumberland, UK). Plasticware was obtained from Nunc (Roskilde, Denmark). EGF was purchased from Sigma Chemical (St. Louis, Mo., USA). Antibodies against EGFR (Ab type 1005) and p-EGFR (Ab type Tyr1173) and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-goat antibodies were purchased from Santa Cruz (Santa Cruz, Calif., USA). ZD1839 was kindly provided by AstraZeneca Italy, Basiglio, Milan; serial dilutions of a stock solution of ZD1839 were made in dimethylsulfoxide.

Cell lines

We used six human PCa cell lines: two androgen-receptor-positive and androgen-sensitive cell lines (ND1 and LNCaP); one androgen-receptor-positive and androgen-insensitive cell line (ALVA-31); and three androgen-receptor-negative cell lines (PC3, DU145, and TSU-Pr1). LNCaP, PC3, and DU145 were obtained from the American Tissue Culture Collection (ATCC, Rockville, Md., USA). ND1 was kindly provided by Dr R. Dahiya (Narayan and Dahiya 1992), TSU-Pr1 by Dr K. Takeda (Iizumi et al. 1987), and ALVA-31 by Dr B.A. Roos (Loop et al. 1993). Recently, a discussion has been opened about TSU-Pr1 cell line origin (van Bokhoven et al. 2001). Cells were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS).

Primary cultures from prostatic specimens

Tissue samples were collected by needle biopsy or radical prostatectomy from patients attending the urology clinic of the University of L'Aquila Medical School. The samples were minced and cultured in DMEM as previously described (Bologna et al. 1988). Cultured cells were analyzed for the presence of androgen receptors, EGFR, prostate-specific antigen, and telomerase activity (Vicentini et al., manuscript submitted 2002).

Autophosphorylation assay

For assessment of the effects of ZD1839 on EGF-induced EGFR autophosphorylation, cells were cultured to confluency in 100-mm dishes with DMEM plus 5% FCS, then ZD1839 was added at concentrations from 0 μM to 10 μM for 2 h; finally, EGF was added at 50 ng/ml for 10 min.

After EGF treatment, the cells were washed with cold phosphate-buffered saline (PBS) and immediately lysed with 1 ml of lysis buffer (50 mM HEPES at pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM sodium orthovanadate, 30 mM p-nitrophenyl phosphate, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Lysates were centrifuged at 10,000 ×g for 10 min. Aliquots of cell lysate supernatants containing 40 μg of protein/lane were separated by 7% SDS-PAGE. After electrophoresis, separated proteins were transferred to nitrocellulose and immunoblotted with a 1:1,000 dilution of goat-anti-human p-EGFR antibody. The same blot was then stripped and incubated with a 1:100 dilution of rabbit-anti-human EGFR antibody to determine the total EGFR level. The antibody-labeled protein bands were visualized using HRP-labeled anti-goat and anti-rabbit antibodies, and quantified by densitometry. The IC50s for ZD1839 inhibition of EGF-induced EGFR autophosphorylation relative to controls were calculated.

Growth curve analyses

A classical growth curve analysis was used to determine the effects of ZD1839 on cellular proliferation in the presence of EGF. Cells were cultured in DMEM plus 5% FCS, then trypsinized, counted using a hemocytometer, and adjusted to 2×104 cells per 50-mm diameter Petri dish in DMEM plus 5% FCS. After 24 h, three dishes were sacrificed for cell counting (time 0) to measure the baseline cell number. The remaining dishes received fresh medium containing 50 ng/ml EGF and ZD1839 at concentrations from 0.01 μM to 20 μM. The dishes were then sacrificed in triplicate every 24 h for 5 days for cell counting. Morphological checks were performed on the remaining dishes every day with an inverted phase-contrast photomicroscope (Nikon Diaphot, Tokyo, Japan).

For cell counting, cells were trypsinized, resuspended in 20 ml saline, and counted in a Coulter Counter (LabRecyclers, Gaithersburg, Md., USA). Five independent counts were made from each dish. Mean counts were plotted against time, and linear regression analysis was performed using the logarithmic tract of the growth curve. The parameters calculated were: dn/dt (the coefficient of variation of the number of cells in the unit of time), growth rate, and the mean doubling time (generation time). Statistical comparisons between the slopes of the logarithmic tracts of the growth curves were performed using the Student's t-test, with both intra- and inter-experimental evaluations (SPSS statistical package, SPSS, Ill., USA).

Inhibition of proliferation with ZD1839

For assessment of inhibition of cellular proliferation with ZD1839, aliquots of 2.5×103 cells were seeded into 96-well plates in their routine culture medium. After 16 h, the culture medium was replaced with DMEM plus 5% FCS, 50 ng/ml EGF, and ZD1839 at concentrations from 0 μM to 20 μM. At time points of 48 h, 72 h, and 96 h, 10 µl of Owen's reagent (MTS tetrazolium) was added to each well and plates were incubated for 4 h (CellTiter cell proliferation assay, Promega Madison, Wis., USA). The 96-well plates were then placed on a microplate shaker for 5 min, and the absorbance of the converted dye was measured at 490 nm using a BioRad multiscan plate reader (BioRad, Richmond, Calif., USA). Five replicate wells were used for each experiment. Optical density percentages for ZD1839 inhibition relative to untreated controls were determined, and the concentration that causes 50% inhibition (IC50) was calculated by GraFit (Erithacus Software Limited, Staines, UK). Statistical comparisons were based on Student's t-test, and the one-way analysis of variance (ANOVA) followed by a post hoc test for multiple comparison (Tukey's test).

Apoptosis and cell cycle analysis

For assessment of cellular apoptosis with ZD1839, aliquots of 1×106 cells were seeded into Petri dishes in their routine culture medium. After 16 h, the culture medium was replaced with DMEM plus 5% FCS, 50 ng/ml EGF, and ZD1839 at concentrations from 0 µM to 20 µM. After 24 h of treatment, cells were harvested, washed, and then fixed in ice-cold 70% ethanol while vortexing. After twice washing in PBS, fixed cells were treated with 50 µg/ml ribonuclease for 30 min at 37 °C and then incubated with 40 µl propidium iodide (50 µg/ml) for 1 h at room temperature. Flow cytometry was performed using a FACScan (Becton Dickinson, Franklin Lakes, N.J., USA), gating out doublets and clumps using pulse processing, and collecting fluorescence above 620 nm.

Results

Effect of ZD1839 on EGFR autophosphorylation in intact PCa cell lines

All six human PCa cell lines expressed functional EGFR, as determined by Western blot analysis using anti-EGFR and anti-p-EGFR antibodies (Fig. 1). The cell lines also expressed EGFR ligands and showed EGF-dependent growth. Levels of EGFR and its ligands were higher in the androgen-receptor-negative cell lines (PC3, DU145, TSU-Pr1) than in the androgen-receptor-positive cell lines (LNCaP, ND1, ALVA-31). EGFR was autophosphorylated in tyrosine residues after addition of EGF for 10 min (Fig. 2A). However, constitutive autophosphorylation of EGFR also occurred during routine cell culture, due to the secretion of EGFR ligands (our unpublished results; Torring et al. 2000; Rajan et al. 1996). Treatment with ZD1839 produced dose-dependent inhibition of EGF-induced autophosphorylation of EGFR, with different inhibition kinetics across the six human PCa cell lines: the IC50s for ZD1839 inhibition ranged from 0.46 μM (PC3) to 0.97 μM (ALVA-31) (Table 1). Figure 2B illustrates the inhibition of EGF-induced EGFR autophosphorylation by ZD1839 in the DU145 cell line.

Fig. 1.

Fig. 1.

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Basal expression of functional EGFR by human PCa cell lines. Western blot analysis of cell extracts (40 μg/lane) using anti-EGFR and anti-phosphorylated EGFR (p-EGFR) antibodies

Fig. 2. A.

Fig. 2. A

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Exogenous EGF (50 ng/ml) induces autophosphorylation of EGFR in DU145 cell line, as determined by Western blot analysis of extracts of untreated and treated cells (40 μg/lane) using anti-EGFR and anti-phosphorylated EGFR (p-EGFR) antibodies. B ZD1839 dose-dependently inhibits EGF-induced EGFR autophosphorylation, as determined by Western blot analysis of extracts of DU145 cells (40 μg/lane) treated with ZD1839 (0–10 μM) and EGF (50 ng/ml)

Table 1.

IC50 values calculated for ZD1839 inhibition of EGFR autophosphorylation and cellular proliferation in human PCa cell lines treated with EGF (50 ng/ml). Data are mean (SD)

IC50, µM
EGFR autophosphorylation Cellular proliferation
ND1 0.70 (0.02) 0.90 (0.06)
LNCaP 0.85 (0.07) 0.37 (0.06)
ALVA-31 0.97 (0.04) 0.89 (0.09)
PC3 0.46 (0.02) 0.55 (0.09)
DU145 0.47 (0.33) 0.60 (0.09)
TSU-Pr1 0.79 (0.27) 1.03 (0.24)
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Effect of ZD1839 on the proliferation of PCa cell lines

ZD1839 also dose-dependently inhibited the proliferation of the human PCa cell lines in the presence of EGF. Analysis of growth curves showed that ZD1839 caused a significant reduction in cellular proliferation rates at concentrations greater than 0.2–1 μM (Fig. 3; Tables 2 and 3).

Fig. 3.

Fig. 3.

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Growth curves of human PCa cell lines treated with EGF (50 ng/ml) and ZD1839 (0–20 μM). Each panel is representative of three different experiments; data are mean±SD

Table 2.

Growth parameters in androgen receptor-positive human PCa cell lines. (dn/dt the coefficient of variation of the number of cells in the unit of time, GR growth rate, GT generation time, NS not significant)

Cell line Parameter Control ZD1839 (µM)
0.01 0.1 0.2 0.5 1.0 5.0 10.0 20.0
ND1 dn/dt 2.04 1.96 1.77 1.72 1.69 1.67 1.55 1.39 1.26
GR 0.71 0.67 0.57 0.55 0.52 0.50 0.45 0.33 0.23
GT (h) 24.8 24.7 29.0 30.3 31.8 33.5 37.0 50.3 71.3
P a NS NS <0.05 <0.05 <0.05 <0.01 <0.001 <0.001
LNCaP dn/dt 1.77 1.67 1.58 1.53 1.35 1.30 1.28 1.26 1.20
GR 0.57 0.50 0.46 0.44 0.30 0.26 0.25 0.24 0.18
GT (h) 29.0 33.6 36.4 39.7 55.7 64.0 64.0 68.5 91.7
P a NS NS <0.05 <0.01 <0.001 <0.001 <0.001 <0.001
ALVA-31 dn/dt 2.03 1.93 1.86 1.82 1.77 1.56 1.41 1.37 1.32
GR 0.71 0.65 0.62 0.60 0.58 0.45 0.34 0.31 0.28
GT (h) 23.4 25.5 26.8 27.7 28.5 37.0 48.5 54.0 63.5
P a NS NS NS NS <0.05 <0.001 <0.001 <0.001
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aAll P values are statistically significant compared with control values (Student's t-test)

Table 3.

Growth parameters in androgen-receptor-negative human PCa cell lines. (dn/dt the coefficient of variation of the number of cells in the unit of time, GR growth rate, GT generation time, NS not significant)

Cell line Parameter Control ZD1839 (µM)
0.01 0.1 0.2 0.5 1.0 5.0 10.0 20.0
PC3 dn/dt 2.04 2.03 1.96 1.72 1.66 1.44 1.40 1.26 1.20
GR 0.71 0.71 0.70 0.54 0.50 0.36 0.32 0.24 0.18
GT (h 23.3 23.6 24.0 30.7 35.1 45.6 49.7 68.5 91.7
P a NS NS <0.05 <0.05 <0.01 <0.001 <0.001 <0.001
DU145 dn/dt 1.86 1.77 1.65 1.58 1.55 1.39 1.30 1.28 1.22
GR 0.62 0.59 0.55 0.47 0.44 0.34 0.25 0.24 0.21
GT (h) 28.3 30.5 35.4 35.4 38.0 44.8 64.8 66.5 87.5
P a NS NS NS <0.05 <0.001 <0.001 <0.001 <0.001
TSU-Pr1 dn/dt 2.06 2.00 1.75 1.77 1.69 1.68 1.60 1.44 1.26
GR 0.72 0.70 0.56 0.57 0.52 0.52 0.47 0.36 0.24
GT (h) 23.1 23.9 30.0 29.0 31.8 32.1 35.5 45.6 69.0
P a NS <0.05 <0.05 <0.05 <0.05 <0.01 <0.001 <0.001
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aAll P values are statistically significant compared with control values (Student's t-test)

IC50 values for ZD1839 inhibition of cellular proliferation in the presence of EGF were measured using the MTS technique (Fig. 4; Table 1). ZD1839 was effective in significantly reducing the proliferation rates at doses greater than 0.2 μM (P <0.001 for all cell lines by one-way ANOVA), and the IC50s ranged from 0.37 (LNCaP) to 1.03 (TSU-Pr1).

Fig. 4.

Fig. 4.

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ZD1839 inhibition of cellular proliferation of human prostatic carcinoma lines after 72 h of treatment with ZD1839 (0.01–20 μM). Measurement is by MTS, and inhibition is expressed as optical density percentages for ZD1839 inhibition relative to untreated control cells. Each value is representative of three different experiments; data are mean±SD

Effect of ZD1839 in primary cell cultures

Twenty primary cultures of telomerase-positive epithelial cells were derived from tissue samples of human PCas. In all cultures, EGF was a potent stimulator of cellular proliferation. The primary epithelial cells also responded to androgen stimulation in vitro (testosterone 0.01–1 nM, data not shown). ZD1839 produced a marked inhibition of proliferation in the presence of EGF, but not under basal conditions in the absence of EGF (Fig. 5, bottom panels). The mean IC50 value of ZD1839 inhibition of cellular proliferation in the presence of EGF across the primary cultures was 0.45 μM (SE 0.06). Figure 5 shows the growth curves of two of the primary cultures: PU 486 (panel A, Gleason score 2+2) and PU 418 (panel B, Gleason score 4+3). The tumors were characterized according to the tissue disorganization (assessed by the Gleason score) and invasion criteria, and broadly grouped into three categories for statistical analysis. Group I (Gleason scores of 2–4; n = 7) consisted of well-differentiated tumors showing little tumor disorganization and limited stromal invasion; group II (Gleason scores of 5–7; n = 6) tumors were less well-differentiated with low to moderate disorganization and a modest degree of stromal invasion; and group III (Gleason scores of 8–10; n = 7) consisted of poorly differentiated tumors showing high levels of disorganization and stromal invasion. The mean IC50 value for ZD1839 was highest in cultures derived from group I tumors and lowest in those from group III tumors (Table 4). ANOVA analysis showed significant differences between the groups with an F value of 15.75 (P <0.001).

Fig. 5.

Fig. 5.

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Growth curves of two representative human PCa primary cell cultures treated with EGF (50 ng/ml) and ZD1839 (0–20 μM): panel A shows the growth curve (top) and the percentage of inhibition versus control (bottom) of PU 486 (Gleason score 2+2) and panel B shows corresponding values for PU 418 (Gleason score 4+3)

Table 4.

IC50 values calculated for ZD1839 inhibition of cellular proliferation in human PCa primary cultures treated with EGF (50 ng/ml), according to Gleason score

Gleason score n IC50, µM mean (SE)
2–4 7 0.69 (0.08)
5–7 6 0.47 (0.09)
8–10 7 0.22 (0.03)
All 20 0.45 (0.06)
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Apoptosis and cell cycle analysis

Analysis of the effects of ZD1839 on the cell cycle stage of the human PCa cell lines cultured in the presence of EGF, by FACS analysis of cells stained with propidium iodide, revealed primarily a shift of stained cells from the G2 phase to the G1 phase visible within 24 h of treatment. ZD1839 also caused a 10-fold to 20-fold increase in the apoptotic cell death rate across the cell lines at doses greater than 0.1 μM after 24 h, as represented by peaks positioned on the left of the G1 peak on the FACS analyses. Figure 6 shows FACS analysis results from PC3 cells treated with ZD1839 for 24 h.

Fig. 6.

Fig. 6.

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FACS analysis with propidium iodide staining of PC3 cells treated with EGF (50 ng/ml) and ZD1839 (0–5 μM) for 24 h. Each panel is expressed using semilogarithmic scale to show ipodiploid peaks, representative of apoptotic cells

Discussion

EGF and TGFα, among other growth factors, have been studied extensively in the etiology of benign prostatic hyperplasia and PCa (Harris and Reese 2001; Koutsilieris and Mitsiades 2001; Scher et al. 1995), and have been found to play significant roles not only during glandular development but also during neoplastic transformation and tumor progression. Consequently, interruption of EGFR tyrosine kinase signaling can be considered as a potential strategy for the effective inhibition of tumor growth and metastasis. Broadly acting nonselective inhibitors may have advantages in overcoming redundancies in growth signaling pathways to arrest aggressively proliferating cells. However, in theory, selective EGFR-TKIs should be less likely to affect healthy cells, minimizing unwanted side effects. Most suppression of intracellular tyrosine phosphorylation by existing tyrosine kinase inhibitors has been shown to be due to inhibition of EGFR tyrosine kinase activity (Schiffer 2001; Burke et al. 1991; Fry et al. 1994; Traxler et al. 1995; Baselga and Averbuch 2000). The orally active, selective EGFR-TKI ZD1839 has demonstrated marked effects against the growth of a variety of human tumor xenografts in mice, including PCa tumors (Baselga and Averbuch 2000; Moulder et al. 2001; Ciardiello et al. 2001; Ciardiello et al. 2000). Furthermore, phase I trials of ZD1839 monotherapy have demonstrated antitumor activity in a range of tumor types, including PCa (Baselga et al. 1999).

In the present study, we have shown that ZD1839 causes cell cycle arrest and initiates apoptosis in EGFR-expressing human PCa cell lines. To investigate the therapeutic potential of ZD1839 in PCa, we evaluated its antiproliferative effects in all six of the most studied PCa cell lines with differing EGFR levels and androgen sensitivity. The androgen-receptor-negative cell lines (PC3, DU145, and TSU-Pr1) were found to express higher levels of EGFR and EGFR ligands than the androgen-receptor-positive cell lines (ND1, LNCaP, and ALVA-31). This finding supports the hypothesis that increased expression of EGFR may reflect a more malignant phenotype of PCa. Androgen-independent PCa cell lines are more aggressive than androgen-responsive ones (Cinar et al. 2001), and it is possible that increased expression of EGFR and its ligands may result in the enhancement of an autocrine mechanism through which these cells may overcome androgen dependence. There are data to support a similar mechanism for overcoming estrogen dependence in breast cancer cell lines (Yarden et al. 2001; Lundholt et al. 2001). However, there is evidence that the levels of EGFR and its ligands do not always correlate with androgen sensitivity. For example, we have found that the androgen-sensitive PCa cell line LNCaP is more responsive to exogenous EGF than the androgen-receptor-negative cell line DU145 (data not shown). In line with this observation, in the present study we found a lower IC50 value for ZD1839 inhibition of cellular proliferation in the presence of EGF in LNCaP cells than in DU145 cells.

The role of EGFR pathways in androgen-sensitive cell lines may be influenced by additional intracellular mechanisms that regulate the extent of EGF-mediated signal transduction (Koutsilieris and Mitsiades 2001; Nazareth and Weigel 1996; Yeh et al. 1999). EGFR signal transduction requires autophosphorylation of tyrosine residues by its tyrosine kinase domain. In the present study, we evaluated the effects of inhibition of EGFR tyrosine kinase activity by ZD1839 in the intact cell lines treated with EGF. Inhibition of autophosphorylation was rapid (within 10–30 min of treatment) across the cell lines, and the IC50s for inhibition of autophosphorylation were generally similar to those for inhibition of proliferation.

Our results obtained in vitro are comparable to those obtained with human PCa cell lines in vivo with other EGFR inhibitors including CP358,774 and PD153035 (Pollack et al. 1999; Moyer et al. 1997; Kunkel et al. 1996). In vivo, the results with EGFR inhibitors are limited by the effective duration of tyrosine kinase inhibition. In vitro, we have found that administration of ZD1839 can be maintained over time. Administration of the IC50 doses calculated for ZD1839 inhibition of autophosphorylation after 10 min of EGF stimulation resulted in >30% inhibition of autophosphorylation after 12–24 h in the PCa cell lines compared with untreated control cells, as demonstrated by Western blotting using the p-EGFR antibody (data not shown).

In the present study, we have also evaluated the effects of ZD1839 EGFR tyrosine kinase inhibition in 20 primary cultures derived from tissue samples of human PCa characterized as telomere positive, which means that they are of neoplastic origin (Vicentini et al. manuscript submitted 2002). In all 20 primary cultures, ZD1839 blocked cellular proliferation in the presence of EGF, but not under basal conditions in the absence of EGF. This finding suggests that autocrine secretion of EGFR ligands by these primary cultures is low.

In conclusion, our observations of ZD1839 inhibition of autophosphorylation and cellular proliferation of PCa cell lines in the presence of EGF suggest that EGFR tyrosine kinase inhibition may have potential in blocking tumor growth and progression in patients with PCa. EGF stimulates the cellular proliferation of PCa cells in primary culture, and may therefore have a role in cellular proliferation and migration at sites of tumor invasion.

We also have preliminary data to suggest possible cooperative effects with antiandrogens in androgen-responsive tumors (manuscript in preparation), through downmodulation of EGFR expression by androgen withdrawal and changes in the androgen susceptibility of androgen-responsive cells. Furthermore, in a study by Sirotnak and colleagues, ZD1839 markedly increased the antiproliferative action of the antiandrogen bicalutamide ('Casodex') in human PCa xenografts (Sirotnak et al. 2001). Further investigations will indicate the possible role of ZD1839 in the treatment of PCa either as a monotherapy or in combination with antiandrogens, due to the emerging interactions between EGFR and androgen stimulation pathways in prostatic epithelium with the possibility of enhanced EGFR activity in androgen-independent tumors.

Acknowledgments.

Dr. Adriano Angelucci was supported by a research fellowship by FIRC, Fondazione Italiana per la Ricerca sul Cancro, Milan, Italy. ALVA-31 cells were obtained via a collaborative arrangement with the Miami GRECC (Geriatric Research, Education and Clinical Center, Miami, Fla., USA). We thank Dr Demetrio Del Monaco, Senior Medical Adviser of AstraZeneca Italy (Basiglio, Milan, Italy) for his kind cooperation and scientific support. 'Iressa' and 'Casodex' are trademarks of the AstraZeneca group of companies.

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