Inhibition of Lncap Prostate Cancer Cells by Means of Androgen Receptor Antisense Oligonucleotides

Inhibition of LNCaP prostate cancer cells by means of androgen receptor antisense oligonucleotides

Iris E. Eder,1 Zoran Culig,1 Reinhold Ramoner,1 Martin Thurnher,1 Thomas Putz,1 Claudia Nessler-Menardi,1 Martin Tiefenthaler,2 Georg Bartsch,1 and Helmut Klocker1 Departments of 1Urology and 2Internal Medicine, University of Innsbruck, Innsbruck, Austria.

Currently available methods for treatment of human prostatic carcinoma aim to inactivate the androgen receptor (AR) by androgen deprivation or blockade with anti-androgens. Failure of endocrine therapy and tumor progression is characterized by androgen- independent growth despite high levels of AR expression in metastatic disease. We inhibited AR expression in LNCaP prostate tumor cells by using antisense AR oligodeoxynucleotides (ODNs) and explored whether antisense AR treatment would be conceivable as a therapy for advanced prostate cancer. Among the various AR antisense ODNs tested, a 15-base ODN targeting the CAG repeats encoding the poly-glutamine region of the AR (as750/15) was found to be most effective. Treatment of LNCaP cells with as750/15 reduced AR expression to ϳ2% within 24 hours compared with mock-treated controls. AR down-regulation resulted in significant cell growth inhibition, strongly reduced secretion of the androgen-regulated prostate-specific antigen, reduction of epidermal growth factor receptor expression, and an increase in apoptotic cells. Mis-sense and mismatched control ODNs had no or only slight effects. Antisense inhibition was also very efficient in LNCaP-abl cells, a subline established after long-term androgen ablation of LNCaP cells, resulting in inhibition of AR expression and cell proliferation that was similar to that seen for parental LNCaP cells. This study shows that inhibition of AR expression by antisense AR ODNs may be a promising new approach for treatment of advanced human prostate cancer. Cancer Gene Therapy (2000) 7, 997–1007

Key words: Androgen receptor; antisense oligodeoxynucleotides; prostate cancer; LNCaP.

on-organ confined prostatic carcinoma is treated by gen-independent prostate tumor growth is not associ- Nsurgical or pharmacological castration. The aim of ated with loss of the AR. This knowledge is in contrast to therapy is to prevent androgenic stimulation of the findings that the two human metastatic prostate cancer tumor. Androgen ablation is often combined with block- cell lines PC-3 and DU-145 and advanced rat Dunning ade of the androgen receptor (AR) by various anti- tumors do not express the AR.8–10 Immunohistochemi- androgens.1 As androgens regulate growth and develop- cal results were further supported by the detection of 2 ment of the prostate gland, androgen deprivation AR gene amplification that occurs in about one-third of 3 results in induction of programmed cell death. How- tumors in conjunction with the development of therapy ever, most tumors, initially sensitive to such kind of resistance.11 In addition, these studies showed increased hormonal therapy, progress and become androgen-inde- AR mRNA levels in tumors with AR gene amplification pendent. Therefore, beneficial effects are only palliative and even in some tumors with a single AR gene.12 When and non-organ confined prostatic carcinoma is not cur- long-term androgen ablation therapy is simulated in the able. LNCaP tumor model, these cells also up-regulate the Recent findings led us to the assumption that the AR AR at the mRNA and protein level and become hyper- plays a critical role in hormonal therapy-resistant pros- sensitive to small concentrations of androgens.13,14 An- tate cancer and led us to inhibit AR expression by other important fact is the occurrence of AR gene antisense oligodeoxynucleotides (ODNs). The most im- mutations that can result in a promiscuous receptor with portant finding was the proof that the AR is expressed 4,5 a broad hormone binding and transactivation spec- not only in primary prostate tumors but also in their trum.15,16 Activation of mutated AR has been shown for corresponding metastatic lesions, as shown in vivo in 6,7 steroid hormones such as estrogens, progestins, adrenal immunohistochemical studies. It has become clear androgens, and dihydrotestosterone metabolites, and that progression from androgen-dependent to andro- even for the nonsteroidal anti-androgens flutamide and nilutamide.17–21 Received October 22, 1999; accepted February 11, 2000. Prostate tumor progression can also be accompanied Address correspondence and reprint requests to Dr. Klocker Helmut, by AR activation through growth factors and protein Department of Urology, University of Innsbruck, A-6020 Innsbruck, kinase A activators. Ligand-independent activation of Anichstr. 35, Austria. E-mail address: [email protected] the AR by various growth factors, such as insulin-like

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Table 1. Sequences of ODNs (nucleotide numbers according to Ref. 30)

Name of the ODN Sequence Target site

Antisense as750/15 5Ј-CTGCTGCTGCTGCTG-3Ј “CAG” repeat region (bases 703-750)

Mis-sense ms750/15 5Ј-ATCGTGGTGTTGATC-3Ј Control

Mismatched mm750/15 5Ј-TTGCAGCTGATGCTA-3Ј Control

Antisense as1944/15 5Ј-GCCGCCGCCGCCGCC-3Ј “GGC”repeat region (bases 1885-1947)

Mis-sense ms1944/15 5Ј-AGCTCTCAGATGACA-3Ј Control growth factor-I (IGF-I), keratinocyte growth factor, supplemented with penicillin/streptomycin and 10% charcoal- epidermal growth factor (EGF), cyclic adenosine mono- stripped FCS and routinely passaged with trypsin/EDTA. phosphate analogs, and forskolin, and by luteinizing hormone-releasing hormone has been described previ- Electroporation of oligonucleotides ously.22–24 Antisense phosphorothioate-ODNs were purchased from Eu- We reasoned that if AR expression could be pre- rogentec (Brussels, Belgium). Sequences of ODNs are summa- vented or at least reduced, this should be the most rized in Table 1. Mis-sense control ODNs had a random efficient blockade of the androgen-signaling cascade and sequence, whereas mismatched control ODNs had the se- therefore should be a promising new approach for quence of the antisense ODN with four bases replaced to therapy of hormone-resistant prostatic carcinoma. eliminate antisense effects. All ODNs consisted of 15 bases with thioate modifications at each nucleotide. LNCaP cells in Herein, we describe for the first time the use of AR log phase were trypsinized, and 9 ϫ 105 cells were suspended antisense ODNs to inhibit AR expression in LNCaP and in 400 ␮L Hank’s balanced salt solution (Boehringer In- LNCaP-abl prostate tumor cells. LNCaP cells, derived gelheim, Heidelberg) supplemented with 1 mM spermidine from a lymph node from a patient with carcinoma of the (Sigma, St. Louis, Mo). ODNs were added to a final concen- prostate, express AR and secrete prostate-specific anti- tration of 7.5 ␮M. Cell suspensions were placed into a 0.4-cm gen (PSA).25,26 The subline LNCaP-abl was established electroporation chamber and electroporated using an Easyject by long-time growth of LNCaP cells in steroid-deprived Cell Porator Electroporation System (Eurogentec). Optimal medium.14 These cells display increased AR protein electroporation parameters were worked out to yield a high cell viability and transfection efficiency in LNCaP cells: a levels associated with androgen hypersensitivity and ␮ ⍀ growth in castrated mice. low-voltage pulse (220 V, 1050 F, 201 ) immediately followed a high-voltage pulse (650 V, 25 ␮F, 99 ⍀). After We tested ODNs targeting either the translation start electroporation, cells were quickly transferred into RPMI 1640 site or the two triplet repeats within the mRNA region 27,28 containing 10% FCS, centrifuged (1000 rpm, 3 minutes) to encoding the AR N-terminus. The latter antisense eliminate remaining ODNs, and seeded into 6-well plates in oligonucleotides proved to be highly effective in suppres- culture medium. In control experiments, efficient uptake of sion of AR expression and inhibition of the growth of ODNs was demonstrated by means of fluorescence-activated LNCaP and LNCaP-abl cells. cell sorter analysis and a fluorescence-labeled control ODN (data not shown).

MATERIALS AND METHODS Reverse transcriptase polymerase chain reaction (RT-PCR) Cell culture Total RNA was isolated from cells using a Total RNA LNCaP cells were obtained from the American Type Culture Isolation System (Promega, Madison, Wis). After denaturation Collection (Manassas, Va). Cells were maintained at 37°C in a (94°C, 2 minutes), 500 ng of RNA was transcribed into cDNA humidiﬁed atmosphere of 5% CO2 in RPMI 1640 (Hyclone, using a Ready-To-Go kit (You-Prime-First-Strand-Beads; Logan, Utah) supplemented with penicillin/streptomycin (100 Pharmacia, Uppsala, Sweden). cDNA fragments of AR and ␮ ␤ ␤ international units/mL and 100 g/mL, respectively) and 10% 2-microglobulin ( 2m) were ampliﬁed using the following fetal calf serum (FCS) (PAA Laboratories, Linz, Austria). primers purchased from Pharmacia: sense AR 2047/20 (5Ј- Passaging was routinely performed with trypsin/ethylenediami- ATGGTGAGCAGAGTGCCCTA-3Ј), antisense AR 2474/20 Ј Ј ␤ Ј netetraacetic acid (EDTA) (0.05% and 0.02%, respectively; (5 -GTGGTGCTGGAAGCCTCTCC-3 ), 2m sense (5 -AT- Ј ␤ Ј PAA Laboratories). GCCTGCCGTGTGAACCATGT-3 ), and 2m antisense (5 - LNCaP-abl cells were recently established in our laboratory AGAGCTACCTGTGGAGCAACCT-3Ј). A total of 2 ␮Lof by long-term culture of LNCaP cells in steroid-deprived me- cDNA solution was added to 50 ␮L of PCR mix containing dium.14 The AR protein level is increased ϳ3-fold in these 10ϫ Finnzyme buffer F-511 (Finnzyme; Biotrade, Vienna, cells. They respond in a hypersensitive manner to androgen Austria), 200 ␮M deoxynucleoside triphosphates, 2% dimeth- ␤ stimulation and, in contrast to parental LNCaP cells, grow in ylsulfoxide, 250 pmol of each primer (AR 2047/2474 and 2m castrated mice. LNCaP-abl cells were grown in RPMI 1640 sense/antisense), and1UofF-501 L DynaZyme DNA poly-

Cancer Gene Therapy, Vol 7, No 7, 2000 EDER, CULIG, RAMONER, ET AL: AR ANTISENSE AND PROSTATE CANCER 999 merase (Finnzyme). Duplex amplification was performed on a additional 24 hours using an annexin V detection kit (Gen- thermocycler 60 (Biometra, Go¨ttingen, Germany) for 26 cycles zyme, Cambridge, Mass). Briefly, cells were trypsinized, according to the following program: 25 seconds at 94°C, 1 washed twice with PBS, and stained with annexin V/propidium minute at 57°C, and 30 seconds at 73°C. PCR fragments were iodide for 15 minutes at room temperature. Annexin V binding separated electrophoretically in a 2% agarose gel in TAE was assessed by flow cytometry (FACScalibur; Becton Dickin- buffer (40 mM tris(hydroxymethyl)aminomethane (Tris)/HCl, son) within 1 hour. 20 mM sodium acetate, 1 mM EDTA (pH 7.8)). Bands were ␤ visualized by ethidium bromide staining. 2m served as an EGF receptor (EGFR) enzyme-linked immunosorbent internal standard. assay (ELISA) Immunoblotting Cells were scraped off, washed once with PBS, and resus- pended in PBS. The total cell number was determined with a Cell pellets were suspended in lysis buffer (20 mM NaH2PO4 Coulter counter (Coulter, Hialeah, Fla). After centrifugation (pH 7.5), 1 mM EDTA, 10% glycerol, and 0.1% monothio- (1800 rpm, 5 minutes), pellets were suspended in 100 ␮LofTE glycerol) and lysed by ultrasound for 5 minutes and by three buffer (50 mM Tris, 5 mM EDTA (pH 7.4)) and lysed with 20 subsequent cycles of freezing in liquid nitrogen and thawing on ␮L of antigen extraction agent for 30 minutes on ice. Another ice water. Total amounts of protein were determined by the centrifugation step was performed to remove cell debris 29 method of Bradford. A total of 20 ␮g of total protein were (10,000 rpm, 5 minutes). Supernatants were frozen at Ϫ80°C then mixed with 5ϫ sample buffer (2.5 mL of 1.25 M Tris (pH until measurement. For quantitation of EGFR, samples were 6.8), 5 mL of glycerin, 2.5 mL of 2-mercaptoethanol, 4 mg of diluted 1/6 in sample diluent. We used a commercial ELISA kit bromophenol blue, and1gofsodium dodecyl sulfate), dena- (Oncogene Research Products, Cambridge, Mass) according tured at 90°C for 2 minutes, and separated on an 8% poly- to the manufacturer’s instructions. Amounts of EGFR (fmol/ acrylamide gel. Proteins were transferred onto an Immo- mL) were corrected for cell number and indicated as femto- bilon-P membrane (Millipore, Bedford, Mass). Blots were moles of EGFR per 105 cells. incubated sequentially with 1% nonfat dry milk at room temperature for at least 1 hour, diluted 1/500 with mouse Statistical analysis monoclonal anti-AR antibody (Genentec, San Francisco, Calif) at 4°C overnight, diluted 1/2200 with secondary horse- Results were expressed as means Ϯ SD. P values of Ͻ0.05 were radish peroxidase-conjugated anti mouse immunoglobulin G considered statistically significant. Statistical significance be- (Santa Cruz Biotechnology, Santa Cruz, Calif) for 1 hour at tween groups was assessed by the Mann-Whitney U test room temperature, and evaluated using the enhanced chemi- (unpaired t test, two-sided). luminescence system from Amersham (Little Chalfont, UK). AR protein band intensities were determined densitometri- cally using a video imaging system and IPLab Gel 2.0 program. RESULTS Cell proliferation assay Antisense-mediated inhibition of AR expression in LNCaP cells Cells were electroporated with or without ODNs and seeded into 24-well plates (Sarstedt, Newton, NC). Cell mass was We tested the efficacy of several phosphorothioate anti- determined by a colorimetric EZ4U 3-(4,5-dimethylthiazol-2- sense ODNs targeting various regions of the human AR yl)-2,5-dimethyltetrazolium bromide (MTT) assay (Biomedica, in diminishing AR expression. ODNs directed against Vienna, Austria) according to the manufacturer’s instructions. the translation start region of the AR mRNA comprising Briefly, cells were washed once with phosphate-buffered saline nucleotides 529–548 and 549–568, (nucleotide numbers (PBS) (PAA Laboratories) and subsequently incubated for 2 according to Ref. 30) revealed only a slight down- hours at 37°C in a substrate solution containing 60 ␮Lof substrate in 600 ␮L of PBS per well. Units of absorption (UA) regulation of the AR protein level and nonspecific side were measured on a photometer at 550 nm. effects of random control ODNs (data not shown). Therefore, we tested ODNs that are directed to the Quantitation of PSA in cell culture supernatants CAG and the GGC repeats encoding the N-terminal poly-glutamine (as750/15) and the poly-glycine (as1944/ Amounts of PSA were determined in cell culture supernatants 15) regions of the human AR, respectively. ODNs were using a microparticle enzyme immunoassay (MEIA) kit (Ab- delivered by electroporation, as simple incubation of bott Laboratories, Abbott Park, Ill). PSA levels were related to cell mass as determined by MTT assay and were expressed as cells with ODNs alone did not change AR expression nanograms of PSA per UA. levels (data not shown). As demonstrated in Figure 1, electroporation of LNCaP cells with 7.5 ␮M of either Apoptosis as750/15 (Fig 1A) or as1944/15 (Fig 1B) resulted in a marked reduction of AR mRNA levels within 6–24 Nonelectroporated control cells were seeded on 6-well-plates hours after electroporation. (Falcon Primaria; Becton Dickinson, Franklin Lakes, NJ) at a ϫ 5 Similarly, AR protein levels were reduced by as750/15 density of 1.5 10 cells/well in normal growth medium (10% as well as by as1944/15 ODNs, as determined by immu- FCS, RPMI 1640). Electroporation with or without ODNs was noblot analysis (Fig 2). AR protein levels were down- performed as described above and cells were seeded onto Ͻ 6-well plates immediately afterward. After 24 hours, medium regulated to 2% within 24 hours after treatment with was replaced by serum-reduced RPMI 1640 (1% FCS). At this as750/15 (Fig 2A) or as1944/15 (Fig 2B) compared with time, mibolerone was added to a final concentration of 5 nM. mock-treated controls. Down-regulation of AR protein The percentage of apoptotic cells was determined after an persisted for at least 72 hours, although maximal effects

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Figure 1. Reduced expression of AR mRNA in LNCaP cells treated with AR antisense ODNs. Cells were electroporated in the absence (control) ␮ ␤ or presence of ODNs at a concentration of 7.5 M. RNA was prepared and analyzed 6 and 24 hours later. AR and 2m cDNA fragments were ␤ ␮ coamplified in a duplex PCR. 2m served as an internal standard. A: Aliquots (10 L) of PCR samples were separated in a 2% agarose gel and stained with ethidium bromide. RT-PCR results are shown for cells electroporated with the antisense ODN as750/15 and the corresponding controls (control, ms750/15, mm750/15), respectively. B: RT-PCR results of cells electroporated with and without the antisense ODN as1944/15 and corresponding controls (control, ms1944/15). were observed 24 hours after electroporation (Fig 2, A whereas the as1944/15 ODN elicited its effect 48 hours and B). after electroporation. After these timepoints, there was To ensure specificity of antisense AR ODNs, we have a complete arrest of growth in the antisense ODN- chosen random control ODNs (ms1944/15, ms750/15) as treated cells. Mock-treated cells that were incubated well as a mismatched control analog (mm750/15). No with 1 ␮M of the nonsteroidal anti-androgen bicaluta- effects on AR expression were observed on mRNA (Fig mide (Casodex) for 72 hours showed only ϳ24% growth 1) or on protein levels (Fig 2) when cells were electro- retardation. porated in the presence of 7.5 ␮M control ODNs. This To further investigate AR specificity, we tested the result further shows that the exchange of four nucleo- ODNs in DU-145 cells. These prostate cancer cells, tides eliminates the antisense effect of the as750/15 which are derived from a brain metastasis, do not ODN. express the AR and grow independent of androgen. As shown in Figure 4, DU-145 cells were slightly inhibited Growth inhibition of LNCaP cells treated with AR (19%) by electroporation with as750/15 compared with antisense ODNs mock-treated controls; however, growth inhibition was not statistically significant (P Ͼ .05). Moreover, the To determine whether AR down-regulation resulted in anti-androgen bicalutamide (1 ␮M) also resulted in a an inhibition of prostate tumor cell growth, cell mass was slight but insignificant inhibition of DU-145 cells (14% evaluated at 8, 24, 48, and 72 hours after electroporation compared with mock-treated controls). Corresponding in the absence or presence of ODNs using a MTT cell control ODNs (mm750/15 and ms750/15) did not inhibit viability assay (Fig 3). Treatment with either as750/15 or DU-145 cell proliferation. as1944/15 yielded a significant inhibition of LNCaP cell growth by 73% and 61%, respectively, within 72 hours Reduced androgen-mediated growth stimulation and after electroporation (P Ͻ .01) compared with mock- PSA secretion treated controls. The control ODNs ms750/15 and mm750/15, respectively, did not affect cell growth. The LNCaP cell growth and PSA production are stimulated control ODN ms1944/15 had a small but insignificant by androgens.25 To investigate the effects of AR down- effect. The antiproliferative effect of the antisense ODN regulation on androgen-mediated cell proliferation and as750/15 was evident already 24 hours after treatment, PSA secretion, cells were electroporated with or without

Figure 2. Inhibition of AR protein expression in antisense-treated LNCaP cells. Cells were electroporated in the absence (electroporation control) or presence of ODNs at a concentration of 7.5 ␮M. Protein extracts were prepared 24 and 72 hours later and analyzed by immunoblotting using the enhanced chemiluminescence system. For detection of AR protein, we used a monoclonal mouse anti-human AR antibody. AR bands in the ﬂuorogram were quantiﬁed by densitometric evaluation. A: Immunoblot analysis of the AR in untreated cells, mock-electroporated controls, and in cells electroporated with ODNs (ms750/15, mm750/15, and as750/15). B: AR protein levels in LNCaP cells after electroporation with the antisense ODN as1944/15 or corresponding controls (electroporation control, ms1944/15).

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Figure 3. Antiproliferative effects of as750/15 and as1944/15 in Figure 4. Effects of antisense AR ODN treatment in DU-145 prostate LNCaP cells in comparison with bicalutamide- or mock-treated carcinoma cells. DU-145 cells lack AR expression and therefore controls. Control cells were set at 100% cell mass 8 hours after should not be inhibited by target-specific antisense AR ODNs. Cells electroporation in the absence of any ODNs. LNCaP cells were were electroporated with or without ODNs (ms750/15, mm750/15, electroporated as indicated, seeded into 24-well tissue culture as750/15, and mock controls) at a concentration of 7.5 ␮M and plates and grown in RPMI 1640 supplemented with 10% FCS. Cell incubated for 72 hours. Another control sample was treated with the mass was determined by MTT assay at 8, 24, 48, and 72 hours after anti-androgen bicalutamide. Bicalutamide (1 ␮M) was added imme- Ⅺ F electroporation without any ODN ( ) or with as750/15 ( ), diately after electroporation. Cell mass was evaluated by MTT Œ ‚ छ as1944/15 ( ), missense controls ( , ms1944/15; , ms750/15), or assay. The percentage of inhibition was calculated by comparison E ␮ a mismatched control ( , mm750/15) at a concentration of 7.5 M. with mock-treated controls, set as 100%. Each bar represents the ␮ Bicalutamide-treated cells were mock-electroporated, and 1 M mean of at least four measurements, with SD indicated as error µ bicalutamide was added immediately thereafter ( ). Each point bars. represents the mean percent cell mass of at least eight measurements, with SD indicated as error bars. 29.14 Ϯ 4.71 ng/UA with a 72-hour incubation with 2 nM mibolerone (Fig 5B). As750/15-treated cells produced ODNs (ms750/15, mm750/15, as750/15). After 24 hours, significantly less PSA (13.17 Ϯ 3.21 ng/UA; P ϭ .006). medium was changed into RPMI 1640 with 10% char- PSA secretion in cells treated with mismatched or coal-stripped FCS, supplemented with increasing con- mis-sense control ODNs was not different from control centrations of the synthetic androgen mibolerone (1 pM LNCaP cells. to 2 nM). Cells were incubated for another 72 hours. Similar results were obtained when PSA secretion was Thereafter, cell mass was determined by MTT assay and stimulated by IGF-I, a ligand-independent activator of PSA levels were measured by MEIA in cell culture the AR.22,31 In control cells (mock electroporation, supernatants. After a 72-hour incubation without mibo- mm750/15) 50 ng/mL IGF-I treatment for 72 hours lerone, there was already a significant difference in cell significantly increased PSA levels by 3.8- and 3.0-fold, mass between controls and antisense-treated cells (P Ͻ respectively (P ϭ .02). In contrast, PSA secretion was .001) (Fig 5A). With increasing concentrations of mibo- not increased in as750/15-treated cells (1.1-fold induc- lerone, both controls and as750/15-treated cells showed tion). the biphasic growth curve that is typical for LNCaP cells, with maximal stimulation at androgen concentrations of Treatment with antisense AR ODNs induces apoptosis 0.1–2 nM. However, at all androgen concentrations, cell in LNCaP cells masses of antisense-treated samples were significantly lower compared with mock-treated controls (P Ͻ .05). Does antisense down-regulation of AR expression in- Maximal androgen stimulation of antisense-treated cells hibit LNCaP cells solely by inhibition of proliferation, or for 72 hours with 1 nM mibolerone increased cell mass does it also have an effect on the rate of apoptosis? To by 63% (P ϭ .02), whereas the increase was 80% in answer this question, LNCaP cells were treated with stimulated control oligonucleotide-treated cells. Fur- control and antisense ODNs or cultured in the presence thermore, maximal stimulation at 1 nM mibolerone was or absence of FCS. Apoptosis was determined after 48 significantly lower in antisense-treated cells (63% in- hours by measurement of annexin V binding (Fig 6). creased cell mass; P ϭ .02) compared with mock-treated Under standard culture conditions (RPMI 1640 plus controls (80% increased cell mass). 10% FCS), a spontaneous apoptosis rate of ϳ4% was LNCaP cells secrete PSA after activation of the PSA observed. Electroporation (mock treatment) increased gene by androgens. PSA levels in supernatants of mock- the percentage of apoptotic cells under normal culture electroporated control cells reached a maximum of conditions to ϳ6%. Treatment with the antisense AR

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Figure 5. Proliferation and PSA secretion after stimulation with androgen for 72 hours. LNCaP cells were electroporated in the absence (Ⅺ, electroporation control) or presence of the antisense ODN as750/15 (Œ) or control ODNs (छ, ms750/15; E, mm750/15). ODNs were administered at a concentration of 7.5 ␮M. Culture media were supplemented with increasing concentrations of mibolerone (0–2 nM) 24 hours after electroporation. A: After another 72 hours with or without androgen, viable cell mass was determined by MTT assay and calculated in a percentage of the mock-electroporated, unstimulated cells. B: PSA levels in the supernatants were measured by MEIA as described in Materials and Methods. PSA levels were corrected for cell mass and indicated as nanograms of PSA per UA. Each point represents the mean of at least four measurements, with SDs indicated as error bars.

ODN as750/15 (7.5 ␮M) was associated with induction Serum deprivation (RPMI 1640, 1% FCS) increased of apoptosis to 26% of the cells 48 hours after electro- apoptosis in mock-electroporated LNCaP cells to poration. The mismatched control ODN (mm750/15) ϳ12%, whereas the addition of mibolerone (5 nM) also increased the rate of apoptosis (21%), but this signiﬁcantly reduced the number of apoptotic cells to increase was less than that seen for the antisense ODN. ϳ3%. Mibolerone (5 nM) failed to overcome the effect

Figure 6. Induction of apoptosis in antisense-treated LNCaP cells. LNCaP cells were electroporated in the absence (mock treatment) or presence of mismatched control ODNs (mm750/15) and antisense ODNs (as750/15), respectively. Cells were cultured in normal growth medium (RPMI 1640, 10% FCS) or in serum-reduced medium (RPMI 1640, 1% FCS) either alone or supplemented with 5 nM mibolerone. After 2 days, the percentage of apoptotic cells was assessed by ﬂuorescence-activated cell sorter analysis after staining with ﬂuorescent-labeled annexin V and propidium iodide.

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Figure 7. Effects of AR antisense treatment on EGFR expression. LNCaP cells were electroporated with or without ODNs and seeded onto 6-well plates (Falcon Primaria). After 24 hours, EGF (20 ng/mL) was added to serum-reduced medium (RPMI 1640, 1% FCS). After an additional 48 hours, cells were scraped off, counted, and lysed. EGFR levels were measured in cell extracts by ELISA and calculated as femtomoles of EGFR per 105 cells. of as750/15 under serum-reduced conditions (RPMI LNCaP-abl cells were electroporated with AR anti- 1640, 1% FCS). sense (as750/15), with AR missense (ms750/15), or with mismatched control ODNs (mm750/15); results were Reduced EGFR expression after treatment with compared with mock-treated controls. Immunoblot antisense AR ODNs analysis revealed that AR protein levels could be effec- tively reduced by antisense treatment (Fig 8). This There is evidence that androgens up-regulate EGFR down-regulation of AR protein was associated with expression in prostate cancer cells.26,32 Therefore, we significant growth inhibition (P ϭ .003) (Fig 9). Mis- were interested in determining whether AR antisense matched and mis-sense control ODNs (mm750/15, treatment of LNCaP cells has any effects on the expres- ms750/15), respectively, again had no effect on AR sion of EGFR. For this purpose, LNCaP cells were protein expression or on cell growth of LNCaP-abl. electroporated and grown in serum-reduced medium (RPMI 1640, 1% FCS) in the absence (control medium) or presence of 20 ng/mL EGF. EGFR levels were DISCUSSION measured 48 hours afterward (Fig 7). Treatment of cells with as750/15 caused a significant decrease in EGFR In the present study, we have investigated the efficiency levels to about two-thirds of that seen for mock-electro- of AR antisense treatment for inhibition of prostate porated controls (P ϭ .021). Stimulation with EGF (20 cancer cells. We demonstrate that the specific down- ng/mL) also lowered EGFR levels significantly in all regulation of AR with antisense ODNs results in pros- cultures (P ϭ .020). Again, the lowest levels were tate tumor cell growth inhibition, reduced androgen- measured in the AR antisense-treated cells. In these mediated growth stimulation and PSA secretion, cultures, EGFR levels reached only half of the levels of mock-electroporated and control ODN-electroporated cells.

AR antisense-mediated growth inhibition of long-term androgen-ablated LNCaP-abl cells Recently, we reported on the establishment of a new LNCaP subline, LNCaP-abl, that was cultured over 10 months in androgen-depleted medium. LNCaP-abl cells display ϳ4-fold higher AR protein levels than normal controls, a hypersensitive response to androgenic stimulation, and a 30-fold higher basal AR transcriptional Figure 8. Reduced AR protein expression in LNCaP-abl cells after activity than LNCaP cells. In contrast to parental AR antisense treatment. Cells were electroporated with or without LNCaP cells, LNCaP-abl cells also grow in castrated ODNs (7.5 ␮M) and analyzed 24 hours afterward. AR levels from 14 nude mice. We tested the efﬁcacy of AR antisense antisense AR-treated cultures (as750/15) were compared with those treatment in LNCaP-abl cells, as these cells are a model from mock-treated (mock control) and control ODN-treated for prostate cancer after long-term androgen ablation. (mm750/15) cells, respectively.

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Because AR protein was maximally reduced already after 24 hours and growth arrest occurred between 24 and 48 hours after antisense treatment, effects of AR down-regulation seem to be delayed in comparison with gene expression. As expected, androgen-induced PSA secretion as well as growth stimulation were distinctly reduced compared with mock-electroporated controls. Although antisense-treated cells also showed the typical biphasic growth pattern, similar to untreated LNCaP cells, in response to stimulation with androgen, the number of these cells was significantly lower at all androgen concentrations tested compared with the mock control. Similar results were obtained after incubation of cells with IGF-I. As shown in recent studies, IGF-I is a ligand-independent activator of the AR, resulting in stimulation of PSA secretion even in the Figure 9. Growth inhibition of LNCaP-abl cells by antisense AR absence of androgens.22,31 As expected, down-regulation treatment. Cells were electroporated in the absence (Ⅺ, mock of the AR in LNCaP cells also inhibited IGF-I-mediated treatment) or presence of control ODNs (‚, ms750/15; छ, mm750/ PSA secretion. Œ 15) or AR antisense ODNs ( , as750/15) and seeded into 24-well Additional control experiments were performed with plates. Cell proliferation was assessed by MTT assay 8, 24, 48, and 72 hours afterward. DU-145 cells that lack AR expression and grow inde- pendently of androgen in vitro.40,41 Mis-sense and mismatched control ODNs (ms750/15, mm750/15) did not increased apoptosis, and decreased EGFR expression. inhibit DU-145 cell proliferation. In contrast, the We tested several ODNs directed against the N-terminal as750/15 antisense ODN led to a slight inhibition of CAG and the GGC repeat regions and the translation DU-145 cells; however, this effect was not statistically start region of the human AR mRNA and found as750/ significant. 15, a 15-base phosphorothioate ODN targeting the CAG Blockade of androgen stimulation by hormonal cas- repeat region, to be the most potent and AR-specific tration is thought to lead to apoptosis of prostate antisense ODN. epithelial cells that mostly results in a reduction of To our knowledge, this is the first report on inhibition tumor volume. Serum starvation was associated with a of human AR expression by antisense AR ODNs in cells moderate induction of apoptosis in LNCaP cells, expressing a functional human AR in vitro. The ODNs whereas the addition of androgen prevented cells from used in our study were only active when delivered by programmed cell death. This is consistent with observa- electroporation. Although several studies have reported tions made by Raffo et al.42 Antisense AR treatment also satisfying antisense effects by simple incubation of cells induced apoptosis. This effect was more pronounced with ODN-supplemented medium, other investigators than that observed with serum deprivation and could not postulated that permeabilization techniques such as the be abrogated by mibolerone. Our observation that AR use of cationic liposomes33–35 or electroporation36,37 are down-regulation is associated with induction of apopto- absolutely necessary for ODN uptake. This process is sis supports data from Gao et al,43 who reported that obviously dependent upon the structure, type, length, retinoic acid-induced apoptosis leads to a reduction of and sequence of the ODNs, as well as on cell type and AR protein levels. culture conditions,38 and might explain the variability of Another promising result was the observation that the experimental designs. long-term androgen-ablated LNCaP subline, LNCaP- ODNs directed against the start of the translation site abl, which was recently established in our lab,14 was of the AR were ineffective, whereas ODNs targeted to significantly growth inhibited by antisense AR treat- the N-terminal CAG and GGC repeat regions of the ment. Although these cells express increased levels of AR, respectively, exerted a strong antisense effect. We AR, are hypersensitive to androgen, and have a higher assume that ODNs that are directed against a triplet AR transactivation activity, they respond to antisense repeat sequence have a greater chance of binding to AR treatment with significant growth retardation in their respective target RNA, and are therefore more comparison with mock-treated controls. effective. Down-regulation of AR protein with antisense Although the effects of our AR antisense ODNs are AR ODNs persisted for at least 72 hours in our exper- promising, one could argue that growth inhibition might iments, although AR levels increased slowly after a result from unspecific side effects rather than from AR minimum at 24 hours. The gradual abatement of the down-regulation. It has been shown recently that degra- antisense effect may be explained by inactivation of dation products of ODNs, nucleosides and nucleotides, ODNs by enzymatic degradation or by the release of can affect cell proliferation and differentiation (reviewed ODNs by exocytosis.39 Proliferation of antisense-treated in Ref. 35). To demonstrate that the ODNs do in fact LNCaP cells was completely inhibited compared with exert specific AR antisense effects, we used different mock-treated controls after a 24- to 48-hour lag-phase. control ODNs, including mis-sense ODNs with random

Cancer Gene Therapy, Vol 7, No 7, 2000 EDER, CULIG, RAMONER, ET AL: AR ANTISENSE AND PROSTATE CANCER 1005 sequences as well as mismatched ODNs in which 4 of 15 levels. This is in contrast to the current situation in nucleotides from the original antisense sequence were breast cancer, where EGFR expression is inversely cor- exchanged. These control ODNs did not have a signifi- related with expression of the estrogen receptor.49 Es- cant effect on AR expression or on cell proliferation. trogen receptor-negative breast tumors that express high However, one cannot rule out that in addition to the levels of EGFR also tend to have a higher risk for specific antisense effect, other mechanisms, such as progression; therefore, determination of EGFR expres- direct toxicity or cleavage of nontargeted mRNA, may sion is even used as a prognostic marker.50,51 Our contribute to the inhibitory effects seen with the anti- findings suggest that, with respect to EGFR regulation sense oligonucleotide. Such side effects would explain by steroid receptors, mammary and prostate tumors are the increase in apoptotic cells seen after treatment with different. the mismatched control ODN mm750/15. Electropora- The present study points out the possibility that tion is a harsh method of DNA delivery into cells and prostate tumor cell growth can be inhibited by the also increases apoptosis when applied alone. Therefore, application of antisense AR ODNs. Nevertheless, one the increased levels of apoptotic cells in the mismatched has to keep in mind that the use of oligonucleotides control group may result from a toxic effect of the ODN might also have effects that cannot be evaluated by in in combination with electroporation. This might lead to vitro experiments, even by the use of different control a state of early apoptosis that is detected by annexin V ODNs. Therefore, in vivo studies are planned to explore binding. the utility of this antisense technology for treatment of Comparative studies with the nonsteroidal anti-andro- non-organ confined metastatic prostate cancer. It is also gen bicalutamide, a “pure” anti-androgen that inhibits conceivable that antisense AR ODNs could be com- androgen-induced gene expression and cell growth,44 bined with any of the currently available forms of revealed that the AR antisense ODN as750/15 is more androgen ablation therapy to affect all of the heteroge- effective in tumor cell growth inhibition than this com- nous tumor cell populations present within an individual monly used nonsteroidal anti-androgen. These findings patient with prostate cancer. As AR down-regulation further demonstrate that down-regulation of AR expres- also induces a pronounced reduction of EGFR levels, sion by AR antisense ODNs might be more effective in one can suppose that AR down-regulation also influ- tumor growth inhibition than AR inactivation by anti- ences the expression of many other genes either directly androgen treatment. Anti-androgens such as bicaluta- or indirectly. In this respect, the application of antisense mide and flutamide have been developed to overcome ODNs to down-regulate AR expression raises the excit- the problems of androgen withdrawal therapies that ing possibility of gaining a better understanding about result only in a partial androgen deprivation. Nonsteroi- the role of the AR in gene regulation and critical dal anti-androgens were intended to block androgen molecular events responsible for prostatic disease. action by inhibiting AR function as a transcription factor, thus preventing androgens from the adrenal glands from stimulating prostate tumor growth. How- ACKNOWLEDGMENTS ever, studies in the last few years have shown that in some patients, nonsteroidal anti-androgens act as ago- We thank Drs. K. Parczyk, D. Zopf, M. Schneider, and J. nists, especially in the context of mutated ARs.15 Flut- Hoffmann for helpful discussions and T. Sierek, G. Ho¨lzl, and amide and nilutamide, for example, activate the AR of P. Dertschnig for excellent technical assistance. Financial LNCaP cells,17,18 which contains a point mutation in the support from Schering AG (Berlin, Germany) and the Aus- ligand-binding domain.17 In contrast, bicalutamide was trian Research Foundation (FWF SFB 002F203) is gratefully acknowledged. shown to retain its antagonistic effects in LNCaP cells.45 In recent years, prostate cancer cell growth has been associated with various growth factors playing a major REFERENCES role in the shift from androgen dependence to androgen independence. In addition, androgen-mediated cell 1. Crawford ED, Eisenberger MA, McLeod DG, et al. A growth seems to be influenced, at least in part, by growth 46 controlled trial of leuprolide with and without flutamide in factors and their respective receptors. The EGF and its prostatic carcinoma. N Engl J Med. 1989;321:419–424. corresponding receptor (EGFR) in particular have been 2. Wilding G. Endocrine control of prostate cancer. 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