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. 2007 Dec;9(12):1030–1037. doi: 10.1593/neo.07778

In Vivo Evaluation of AT-101 (R-(-)-Gossypol Acetic Acid) in Androgen-Independent Growth of VCaP Prostate Cancer Cells in Combination with Surgical Castration

Robert D Loberg 1,†, Natalie McGregor †, Chi Ying 2†, Erin Sargent †, Kenneth J Pienta *,†
PMCID: PMC2134900  PMID: 18084610

Abstract

PURPOSE

Upregulation of Bcl-2 family members is a well-established mechanism in the development of androgen-independent prostate cancer. Inhibition of the antiapoptotic proteins Bcl-2 and Mcl-1 may delay the transition to androgen-independent growth.

EXPERIMENTAL DESIGN

We have established a prostate cancer model with VCaP prostate cancer cells in vivo to study the transition to androgen independence. Here, we investigated the efficacy of AT-101 (R-(-)-gossypol acetic acid; a pan small molecule inhibitor of Bcl-2, Bcl-xL, and Mcl-1) in combination with surgical castration to delay the onset of androgen-independent growth in vivo.

RESULTS

AT-101 (15 mg/kg, per os (p.o.) 5 days/week) in combination with surgical castration delayed the onset of androgen-independent prostate cancer growth in vivo. In addition, we demonstrate the induction of caspase-9-and caspase-3-dependent induction of apoptosis following AT-101 treatment in vitro which was accompanied by an AT-101-induced downregulation of Bcl-2 and Mcl-1 mRNA and protein expression.

CONCLUSIONS

We conclude that AT-101 in combination with surgical castration delays the onset of androgen-independent prostate cancer in vivo by disrupting the antiapoptotic activity of Bcl-2 upregulation during the transition to androgen independence. Further studies are needed to define the mechanism of action by which AT-101 attenuates the expression of Bcl-2 and Mcl-1 and to characterize the potential for AT-101 in combination with hormone therapy.

Keywords: Apoptosis, Bcl-2, BH3 domain, hormone refractory, orchiectomy

Introduction

The primary treatment for patients with metastatic prostate cancer is androgen deprivation therapy (ADT). The majority of advanced prostate cancer tumors that recur are initially responsive to androgen blockade; however, nearly 100% of patients undergoing ADT will fail hormone therapy and progress to an androgen-independent phenotype. The current paradigm is that androgen-independent growth is due to a clonal expansion of prostate cancer cells that are able to survive and proliferate in an androgen-depleted milieu. The role of Bcl-2 in androgen-independent prostate cancer is well documented and has been shown to be a prominent mechanism by which cells adapt to an androgen-independent environment following ADT [1]. Bcl-2 is an antiapoptotic protein that prevents the release from mitochondria by sequestering Bax, a proapoptotic Bcl-2 family member protein. Bcl-2 was originally identified in Burkitt lymphoma as a result of a chromosome translocation t(14;18) prevalent in B cells [2]. The Bcl-2 family of proteins now includes both proapoptotic (i.e., Bax, Bak, Bok [Mtd], Bad, Bid, Bim, Bik, Hrk, Bcl-Xs, APR [Noxa], p193, Bcl-G, Nip3, and Nix [BNIP]) and antiapoptotic (i.e., Bcl-2, Bcl-xL, Bcl-W, MCL-1, Bfl-1 [A1], BOO [Diva], and NR-13) proteins [3]. The ratios and interactions of anti- and proapoptotic Bcl-2 family proteins are known to be intimately involved in the sensitivity or resistance of cells to apoptotic stimuli. Alterations in the amounts of these proteins have been associated with a variety of pathologic conditions, including cancer [4]. Specifically, proapoptotic members (e.g., Bax) homodimerize when released from the mitochondrial membrane and induce a loss in membrane potential of the mitochondria leading to cytochrome C release. Antiapoptotic members (e.g., Bcl-2) heterodimerize with proapoptotic members and prevent the loss in mitochondrial membrane potential and release of cytochrome C.

AT-101 (R-(-)-gossypol acetic acid) is a polyphenolic compound isolated from cottonseeds; it inhibits Bcl-2 by acting as a BH3 mimetic and disrupts the heterodimerization of Bcl-2 with proapoptotic family members. AT-101 was found to mimic the proapoptosis proteins and is able to bind to the BH3 domains of Bcl-2, Mcl-1, and Bcl-xL [5]. AT-101 has been shown to be therapeutically active as a single agent in several mouse models including breast, prostate, colon, and non-small cell lung cancer [6–9]. Here, we demonstrate that AT-101 administration delays the onset of androgen-independent prostate cancer in vivo. Recently, VCaP prostate cancer cells have been shown to express the wild-type androgen receptor (AR) and, as xenografts, develop an androgen-independent phenotype following castration in mice [10]. Previously, we demonstrated that Bcl-2 expression in VCaP androgen-independent cells was significantly upregulated in comparison to VCaP androgen-dependent cells [10]. The use of AT-101 along with ADT may provide a means to increase the time between remission and reoccurrence of androgen-dependant prostate cancer.

Materials and Methods

Materials

AT-101 was kindly provided by Ascenta Therapeutics (San Diego, CA). Dihydrotestosterone (DHT) was purchased from Sigma-Aldrich (St. Louis, MO) and WST-1 and the Cell Death Detection ELISA were purchased from Roche Inc. (Basel, Switzerland). Anti-Bcl-2, anti-Mcl-1, anti-Bcl-xL, anti-Bak, anti-Bad, anti-Bax, anti-caspase-9, anti-caspase-3, anti-caspase-7, anti-caspase-6, anti-poly(ADP-ribose) polymerase (PARP), anti-AR, and anti-β-actin were purchased from Cell Signaling Technology (Danvers, MA).

Cell Culture

VCaP cells were obtained from a lumbar vertebral metastatic lesion through the Rapid Autopsy program at the University of Michigan [6]. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimicotic and incubated at 37°C. PC-3 prostate cancer cells were obtained from the American Type Culture Corporation (Manassas, VA) and maintained in RPMI supplemented with 10% fetal bovine serum and 1% antibiotic/antimicotic.

Proliferation Assay

VCaP cells were seeded at a density of 8 x 104 cells/well in a 96-well plate with complete media, whereas PC-3 cells were seeded at a density of 1 x 104 cells/well in a 96-well plate also with complete media. The plates were then incubated for 24 hours and then treated for 72 hours. WST-1 was added to each well at a 1:10 concentration, incubated for 2 hours then read in a microplate reader (VersaMax Tunable; Molecular Devices, Sunnyvale, CA) at 440 nm.

Western Blot

Treated cells were lysed in RIPA buffer (120 mM NaCl, 50 mM Tris, pH 7.6, 0.05% NP-40, 1 mM EGTA, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM PMSF, 1 µM NaVO3, 1 µg/ml pepstatin, and 1 µg/ml okadaic acid). Lysates were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA). Membranes were blocked using 5% milk in TBS + 0.5% Tween for 1 hour at room temperature. The membranes were then incubated in primary antibody either overnight at 4°C or for 90 minutes at room temperature. They were then washed three times in TBS + 0.5% Tween then incubated for 60 minutes in HRPconjugated secondary antibody (Cell Signaling). Presence of the proteins was determined using Immobilon Western Chemiluminescent HRP Substrate (Millipore).

Cell Death Detection ELISA

VCaP cells were seeded at a density of 8 x 104 cells/well in a 96-well plate with complete Dulbecco's modified Eagle's medium, whereas PC-3 cells were seeded at a density of 1 x 104 cells/well in a 96-well plate with complete RPMI. The plates were incubated for 24 hours and then treated for 72 hours. After incubation, the cells were lysed and the ELISA was conducted according the protocol supplied by the manufacturer (Roche).

Subcutaneous Injection of VCaP Cells

Xenograft tumors were established as previously described [10]. Briefly, male severe combined immunodeficiency disease (SCID) mice (5–6 weeks) were injected subcutaneously with 1 x 106 VCaP prostate cancer cells in 200 µl of Matrigel (BD Biosciences, Inc., San Jose, CA). Tumor volumes were calculated by caliper measurement performed weekly to monitor and track tumor growth (tumor volume = L x W x W x 0.56). Once tumor volumes reached 250 mm3, mice were allotted into groups as follows: 1) intact, 2) AT-101-only (15 mg/kg, per os (p.o.), 5 days/week), 3) castration, and 4) castration + AT-101 (15 mg/kg, p.o., 5 days/week). AT-101 treatment was initiated 24 hours postsurgery.

Preparation of AT-101

A 0.5% carboxymethylcellulose stock was prepared to be used in the oral administration of the drug. A polytron homogenizer (Biospec Products, Bartlesville, OK) was used for 10 seconds to completely dissolve 1.0 g of carboxymethylcellulose in 200 ml of purified water. This solution was stored at 4°C for up to 1 month. The drug was prepared weekly and kept refrigerated for no longer than 7 days. Mice were fed 100 µl daily, of 0.375 mg/ml AT-101 homogenized in carboxymethylcellulose.

Quantitative Polymerase Chain Reaction (QPCR) Assay

VCaP cells were cultured to 80% confluence. Total RNA was isolated from cell lines using a reagent (Trizol; Invitrogen Corp, Carlsbad, CA) following the manufacturer's specifications. Purified RNA (5 µg) was converted to cDNA using Super Script II reverse transcriptase (Invitrogen) following the manufacturer's instructions and used for gene expression analysis by real-time polymerase chain reaction (PCR) using a thermocycler (ABI Prism7900HT). Primers and probes were purchased from Applied Biosystems, Inc. (Foster City, CA) and used with TaqMan Universal PCR Master Mix, No AmpErase UNG. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize and compare each sample. Cycle conditions for real-time PCR were 95°C (15 seconds), 60°C (1 minute), and 72°C (1 minute) for 40 cycles. Threshold cycle number for each sample was normalized to GAPDH for that sample and expressed on a log scale relative to GAPDH expression.

Statistical Analysis

Data were analyzed with GraphPad Prism software (San Diego, CA). A one-way analysis of variance was used with Bonferroni's post hoc analysis for comparison between multiple groups. Student's t test was used for comparison between two groups. Significance was defined as a P value of < .05.

Results

Expression of the Bcl-2 Family Members in Androgen Sensitive versus Androgen Independent VCaP Prostate Cancer Cells

To determine the mechanism mitigating the transition to androgen-independent prostate cancer, we assessed the expression profile of the Bcl-2 family by QPCR and compared the parental VCaP androgen-sensitive prostate cancer line to a daughter VCaP androgen-independent prostate cancer line. VCaP cells were implanted subcutaneously in male SCID mice and tumor growth was monitored by caliper measurement. To induce the androgen-independent phenotype, mice were surgically castrated or left intact when the tumors reached 250 mm3 as previously described [10]. Tumors were collected once they reached the 1000-mm3 critical mass and Bcl-2 family member expression was analyzed. Analysis revealed a significant upregulation of Bcl-2, Bcl-xL, Bcl-W, and Mcl-1 (antiapoptotic) in the VCaP tumors from castrated mice compared to VCaP tumors from intact mice (Figure 1). In addition, there was a significant downregulation of Bax, Bak, and Bad (proapoptotic) in the VCaP tumors from castrated mice compared to VCaP tumors from intact mice (Figure 1). No significant change in expression of Bid, Bim, Puma, Noxa, CIAP-2, CIAP-1, ML-IAP, and survivin was observed.

Figure 1.

Figure 1

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Upregulation of proapoptotic Bcl-2 family members in androgen-independent VCaP prostate cancer cells. Differential Bcl-2 family mRNA expression was determined by quantitative real-time PCR in androgen-sensitive VCaP cells (VCaP AS) and androgen-independent VCaP cells (VCaP AI). mRNA expression was determined and normalized to GAPDH levels using the Δ-C T method. Samples were run in triplicate in each of three independent experiments (mean ± SD, *P < .01).

AT-101 in Combination with Surgical Castration Attenuates Androgen-Independent Growth of Prostate Cancer

We next evaluated the efficacy of inhibiting the related antiapoptotic Bcl-2 family members that were upregulated following surgical castration. AT-101 inhibits Bcl-2 family proteins by binding to their BH3 domains and disrupts the heterodimerization of Bcl-2, Bcl-xL, Bcl-W, and Mcl-1 with proapoptotic members of the Bcl-2 family at submicromolar affinity [11,12]. In vivo administration of AT-101 (15 mg/kg, p.o., 5 days/week) as a single agent in intact mice significantly reduced the development of VCaP tumor growth compared to untreated tumors at weeks 2 to 6 (P < .001 and each week by unpaired t test) (Figure 2). No difference was seen between the AT-101-only and castration-only groups; however, AT-101 in combination with surgical castration delayed the onset of androgen-independent VCaP tumor growth compared to castration-only or AT-101-only groups in vivo (castration vs castration + AT-101: week 4, P = .0012; week 5, P = .172; week 6, P = .0245) (Figure 2). Tumor xenografts were isolated for histology when the tumors reached ∼ 1000 mm3. Expression of Bcl-2 and the AR were visualized by immunohistochemistry analysis (Figure 2, B–I). By qualitative analysis, AR expression was upregulated in the castration group compared to the intact group but the AR expression was localized to the cytoplasm (Figure 2, B and C). AT-101-only had no effect on AR expression compared to the intact group (Figure 2, B and D). Combination of AT-101 with surgical castration maintained the upregulation of AR; however, AR became localized primarily to the nucleus compared to the castration-only group (Figure 2, C and E). Similarly, Bcl-2 expression was upregulated on surgical castration (Figure 2, F and G) and this upregulation was attenuated by AT-101 treatment in combination with castration (Figure 2, G and I).

Figure 2.

Figure 2

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In vivo administration of AT-101 in combination with surgical castration delays the onset of androgen-independent prostate cancer. (A) VCaP cells were implanted subcutaneously in male SCID mice and tumor growth monitored by weekly caliper measurements. Mice were surgically castrated or left intact when tumors reached 200 mm3 and AT-101 (15 mg/kg, p.o. 5 days/week) was administered by gavage 24 hours postcastration [intact (gray line), castrate (solid line, ▴), AT-101 (dashed line, □), and AT-101 + castration (dashed line, ▵)]. (B–I) Tumor sections were analyzed for AR and Bcl-2 expression by immunohistochemistry.

AT-101 Induces Apoptosis Through Activation of Caspase-9, -3, and -7 in VCaP Cells

To determine the efficacy of AT-101 antitumor activity in VCaP cells directly, we assessed the effects of AT-101 on VCaP cell viability and apoptosis in vitro. VCaP parental cell viability was assessed using the WST-1 cell viability assay as previously described [10]. VCaP cells were treated with increasing concentrations of AT-101 (1–10 µM) and cell viability was measured at 72 hours posttreatment (Figure 3A). Treatment with AT-101 (1–10 µM) resulted in a dose-dependent decrease in VCaP cell viability after 72 hours compared to the vehicle control (VCaP cells: vehicle, 1.034 ± 0.45; AT-101 1 µM, 0.707 ± 0.019; AT-101 5 µM, 0.491 ± 0.015; AT-101 10 µM, 0.410 ± 0.01; mean ± SD, *P < .05 and **P < .001). Furthermore, we compared the antitumor activity of AT-101 on VCaP cell viability to PC-3 cell viability and found that VCaP cells are more sensitive to AT-101 in vitro (PC-3 cells: vehicle, 1.250 ± 0.099; AT-101 1 µM, 1.238 ± 0.037; AT-101 5 µM, 0.92 ± 0.04; AT-101 10 µM, 0.368 ± 0.005; mean ± SD, *P < .05 and **P < .01). To determine if the mechanisms of AT-101 on VCaP viability/proliferation were due to induction of apoptosis, VCaP cells were treated with increasing concentrations of AT-101 (1–10 µM) and apoptosis was quantified using the Cell Death Detection ELISA (Roche) which measures histone-complexed DNA fragments released from cells undergoing apoptosis (Figure 3B). Treatment with AT-101 (1–10 µM) resulted in a dose-dependent increase in VCaP cell apoptosis after 72 hours compared to the vehicle control (VCaP cells: vehicle, 0.66 ± 0.41; AT-101 1 µM, 1.064 ± 0.9; AT-101 5 µM, 1.615 ± 0.18; AT-101 10 µM, 1.985 ± 0.11; mean ± SD, *P < .05 and **P < .01). Activation of apoptosis in VCaP cells by AT-101 was assessed by immunoblot analysis for caspase activation. Treatment of VCaP cells with AT-101 (5 µM) induced activation of caspase-9, -3, and -7 and cleavage of PARP (Figure 3C).

Figure 3.

Figure 3

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AT-101 induces apoptosis in vitro. (A) Effects of AT-101 [0-10 µM] for 72 hours was determined on VCaP (open bars) and PC-3 cells (closed bars). Data are presented as mean ± SD from four independent experiments (*P < .05, **P < .001). (B) Treatment of VCaP cells with AT-101 [0–10 µM] for 72 hours resulted in a dose-dependent increase in apoptosis as detected by ELISA. Data are presented as mean ± SD from three independent experiments (*P < .05, **P < .01). (C) AT-101 [5 µM] induced caspase-9, -3, and -7 activation and PARP cleavage as detected by immunoblot analysis. Cells were treated with AT-101 [5 µM] for 24, 48, or 72 hours and duplicate samples for each treatment are representatives of four independent experiments. β-Actin was used as a loading control for each sample.

AT-101 Decreases Bcl-2 and Mcl-1 Expression in VCaP Cells In Vitro

To further determine the mechanism of AT-101-induced apoptosis in vitro, VCaP cells were treated with increasing concentrations of AT-101 (1–10 µM) for 24, 48, and 72 hours. Bcl-2 and Mcl-1 expression was determined by immunoblot analysis and revealed that AT-101 treatment of VCaP cells resulted in a dose-dependent downregulation of Bcl-2 expression at 24, 48, and 72 hours (Figure 4A). In addition, Mcl-1 expression was similarly downregulated at 24, 48, and 72 hours with AT-101 at 5 and 10 µM. The dose-dependent downregulation of Bcl-2 and Mcl-1 in response to AT-101 treatment was confirmed by QPCR (Figure 4B).

Figure 4.

Figure 4

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AT-101 downregulates Bcl-2 and Mcl-1 in VCaP cells. (A) The effects of AT-101 on Bcl-2 and Mcl-1 protein expression were determined by immunoblot analysis. VCaP cells were treated with AT-101 [1–10 µM] for 24, 48, or 72 hours and compared to DMSO vehicle-treated cells (control). Data are representative of three independent experiments and β-actin was used as a loading control. (B and C) AT-101-induced decrease in Bcl-2 and Mcl-1 expression was confirmed by QPCR. VCaP cells were treated with AT-101 [1–10 µM] for 24 (-□-), 48 (-○-), or 72 (-▴-) hours before quantitative analysis of Bcl-2 and Mcl-1 mRNA expression. Data were normalized to GAPDH levels and graphed as a percent of DMSO vehicle-treated levels.

Inhibition of AT-101-Induced Apoptosis By DHT

Next, the ability of AR activation with DHT to attenuate AT-101-mediated apoptosis in VCaP cell in vitro was determined. Stimulation of VCaP cells placed in androgen-depleted media with AT-101 (5 µM) for 72 hours resulted in an induction of apoptosis compared to vehicle-treated control cells (Figure 5A). The presence of DHT (100 nM) significantly attenuated the AT-101-mediated induction of apoptosis (vehicle, 0.261 ± 0.14; AT-101 5 µM, 1.529 ± 0.45; DHT 100 nM, 0.58 ± 0.18; AT-101 5 µM + DHT 100 nM, 0.631 ± 0.12; mean ± SD, *P < .001 and **P < .01). In contrast, the presence of DHT did not have an effect on AT-101-mediated apoptosis in PC-3 cells placed in androgen-depleted media (vehicle, 0.222 ± 0.02; AT-101 5 µM, 0.93 ± 0.05; DHT 100 nM, 0.277 ± 0.013; AT-101 5 µM + DHT 100 nM, 0.924 ± 0.01; mean ± SD, *P < .05) (Figure 5B). Similar effects of DHT (100 nM) on AT-101-mediated apoptosis in VCaP cells placed in androgen-depleted media was observed when caspase-3 activation was assessed by immunoblot analysis (Figure 5C), although DHT did not inhibit the AT-101-induced decrease in Bcl-2 or Mcl-1 expression in VCaP or PC-3 cells (Figure 5D).

Figure 5.

Figure 5

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DHT rescues AT-101-induced apoptosis in VCaP cells in vitro. VCaP (A) and PC-3 (B) cells were grown in androgen-depleted media and treated with AT-101 [5 µM] for 72 hours in the presence or absence of DHT (100 nM). Apoptosis was measured by ELISA and data are presented as the mean ± SD from three independent experiments (*P < .001). (C) The effects of DHT (100 nm) on AT-101 [5 µM] for 72 hours on caspase-3 activation was assessed by immunoblot analysis. 1, DMSO vehicle control; 2, AT-101 (5 µM, 72 hours); 3, DHT (100 nM, 72 hours); 4, AT-101 (5 µM) + DHT (100 nM). (D) The effects of DHT (100 nm) on AT-101 [5 µM] for 72 hours on Bcl-2, Mcl-1, Bcl-xL, Bak, Bad, and Bax was assessed by immunoblot analysis. 1, DMSO vehicle control; 2,-AT-101 (5 µM, 72 hours); 3, DHT (100 nM, 72 hours); 4, AT-101 (5 µM) + DHT (100 nM).

Kinetics of Bcl-2 Expression in VCaP Prostate Cancer Cells Following Surgical Castration

The kinetics of castration on Bcl-2 expression was assessed by immunoblot analysis. Animals harboring a VCaP subcutaneous tumor underwent surgical castration as described above. Tumors were collected at various time points (0, 4, and 12 hours and 1, 2, 3, 5, 7, 10, and 14 days postcastration). Bcl-2 expression was significantly upregulated as early as 4 hours postcastration and remained significantly elevated (Figure 6).

Figure 6.

Figure 6

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Bcl-2 protein expression in VCaP cells is significantly elevated following surgical castration. Male SCID mice implanted with VCaP subcutaneous tumors were castrated when tumors reached 200 mm3 by caliper measurement. Tumors were harvested immediately after castration (0 hour) and at 4, 12, 24, 48, 72, 120, 168, 240, and 336 hours postcastration. Bcl-2 protein expression was determined by immunoblot analysis with β-actin as a control for each sample. (A) Representative immunoblot of Bcl-2 expression and (B) densitometric analysis of Bcl-2 protein expression from two independent experiments. Data are graphed as mean ± SD.

Discussion

Bcl-2 family proteins are overexpressed in many cancers and have been proposed as therapeutic targets in non-Hodgkin's lymphoma (Bcl-2), myeloma (Mcl-1), chronic lymphocytic leukemia (Bcl-2 and Mcl-1), prostate cancer (Bcl-xL and Bcl-2), melanoma (Bcl-2), and other cancers. Prosurvival proteins products of Bcl-2, Bcl-xL, Mcl-1, and Bcl-W may contribute to cancer cell pathogenesis and resistance to cytotoxic therapy by binding to BH3 domains of proapoptotic family members, sequestering them and inhibiting their ability to promote cell death. Previously, we have demonstrated the upregulation of Bcl-2 expression in VCaP prostate cancer cells in vivo following surgical castration as a mechanism of progression to androgen independence [10]. In this study, we demonstrate that administration of AT-101, a small molecule inhibitor of proapoptotic members of the Bcl-2 family, delays the onset of androgen-independent growth of VCaP prostate cancer xenografts in vivo. Administration of AT-101 attenuated the upregulation of Bcl-2 on castration and prevented the enhanced prosurvival advantage of elevated Bcl-2 levels. In vitro AT-101-induced apoptosis through the activation of caspase-9, which is consistent with the activation of the mitochondrial death pathway. The mechanism of Bcl-2-mediated antiapoptosis is through binding to proapoptotic proteins (Bax, Bak, and Bad) and inhibiting their activation of the mitochondrial death pathway by compromising the mitochondrial membrane integrity releasing cytochrome C and thus activating caspase-9 cleavage.

The transition to androgen-independent prostate cancer is a tremendous hurdle to overcome for the treatment of prostate cancer. Multiple mechanisms have been identified that contribute to the transition to the androgen-independent phenotype [13–17]. Receptor tyrosine kinases become activated and the AR is phosphorylated producing a ligandindependent AR [18]. Other growth factors and hormones have been shown to induce AR activation and translocation to the nucleus [18,19]. In this study we demonstrate that upregulation of Bcl-2 is an important mechanism by which VCaP prostate cancer cells transition to androgen independence in vivo following surgical castration. Interestingly, Bcl-2 expression was significantly upregulated within 4 hours following surgical castration and maintained an elevated expression status up to 14 days postcastration. These data suggest that administration of AT-101 may be more appropriately administered before castration (hormonal manipulation) to achieve a greater efficacy in preventing upregulation of Bcl-2 and thereby further delaying the onset of androgen-independent disease. Recently, AT-101 was tested in 23 men with hormone refractory prostate cancer who progressed following hormonal therapy but who had not yet received systemic chemotherapy [20]. Preliminary evidence of AT-101 activity in this population was demonstrated, as three (15%) patients met the criteria for a prostate-specific antigen (PSA) response (e.g., ≥ 50% decline in PSA), with two (10%) patients having a confirmed response. Of 12 (47.8%) patients who had measurable disease, 53.8% had radiographically stable disease following AT-101 monotherapy. These data indicate that AT-101 has biologic activity as a single agent in hormone refractory prostate cancer as demonstrated by declines in PSA slope and PSA partial responses per the Bubley criteria. The activity of AT-101 in this population of patients is being explored further in an ongoing Phase 1/2 trial of AT-101 in combination with docetaxel and prednisone. An additional trial of AT-101 in combination with anti-androgen therapy in men with untreated D2 prostate cancer is also planned. In conclusion, in this study, AT-101-mediated inhibition of Bcl-2 and Mcl-1 has proven to be efficacious in delaying the onset of androgen-independent prostate cancer using the VCaP prostate cancer model as previously described [10]. Further studies are needed to understand the mechanisms involved in AT-101 downregulation of Bcl-2 and Mcl-1 as well as to identify the most effective time of AT-101 delivery around hormonal therapy.

Acknowledgements

We thank Ascenta Therapeutics, Inc. for kindly providing AT-101 and Karen Giles for assisting with manuscript preparation.

Abbreviations

ADT

androgen deprivation therapy

AR

androgen receptor

DHT

dihydrotestosterone

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

p.o.

per os

PARP

poly(ADP-ribose) polymerase

PCR

polymerase chain reaction

PSA

prostate-specific antigen

QPCR

quantitative PCR

SCID

severe combined immunodeficiency disease

Footnotes

1

Grant Information: Dr. Loberg and Dr. Pienta are supported by the National Institutes of Health (NIH) (PO1 A093900), NIH Specialized Program of Research Excellence 1 (P50 CA69568), the Southwest Oncology Group, and the Prostate Cancer Foundation.

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