Anti-Androgen Abiraterone Acetate Improves the Therapeutic Efficacy of Statins on Castration-Resistant Prostate Cancer Cells

Abstract

The treatment of castration-resistant (CR) prostate cancer (PCa) is limited. A sub-population of CR PCa tumors can synthesize androgens for intracrine androgen receptor (AR) activation, thus targeting androgen biosynthesis could be an effective therapeutic option for these patients. We determined that androgen biosynthesis inhibitors simvastatin, atorvastatin, and ketoconazole directly inhibit growth, migration, and colony formation of LNCaP C-81 cells, which exhibit de novo androgen biosynthesis, with simvastatin being the most effective. Importantly, in combination treatments, statins specifically enhanced growth suppression with added effects by anti-androgen abiraterone acetate on the CR PCa cells. Thus, statins can be used in conjunction with abiraterone acetate to enhance anti-androgen therapy for CR PCa.

Keywords: Anti-Androgens; Castration-Resistant Prostate Cancer; Combination Treatments; Prostate Cancer; Statins.

Figure 1(A-D):

Effects of Statins on LNCaP C-81 PCa Cell Growth in SR Conditions. LNCaP C-33 (A) and LNCaP C-81 (C) cells were plated in regular steroid-containing medium at 3 × 10⁴ and 2×10⁴ cells per well, respectively, in 6-well plates. After 72 h, cells were conditioned to SR medium for 48 h, and treated with simvastatin, atorvastatin, and ketoconazole ranged from 0–20 μM concentrations in SR medium for 72 h. Control cells were treated with DMSO alone. Attached cells were harvested via trypsinization and living, shiny cells were counted with trypan blue dye. After counting, cells were lysed, and total cell lysate was analyzed for PCNA (B and D). β-actin was used as a loading control. Protein levels (B and D) were quantified with ImageJ software. Results presented are mean ± SE. n=3×3. *p<0.05; ^**p<0.005; ^***p<0.0005.

Figure 2(A-B):

Effects of Steroid Biosynthesis Inhibitors on LNCaP C-81 Cell Tumorigenicity. (A) Cell migration via transwell assay. LNCaP C-81 cells were plated in the upper chamber of inserts at 5×10⁴ cells per well in regular steroid-containing medium. Both upper and lower chambers contained medium with 10 μM of simvastatin, atorvastatin, or ketoconazole. Control cells were treated with DMSO alone. After 24 h, cells in the lower chamber were stained, while cells remaining in the upper chamber were removed via cotton swab. Images shown are at 40x magnification. Results presented are mean ± SE. n=2×3. (B) Clonogenic assay. LNCaP C-81 cells were plated in 6-well plates at 3000 cells per well in regular steroid-containing medium for 72 h. Cells were then treated with 10 μM of simvastatin, atorvastatin, or ketoconazole for 9 days, with fresh medium containing the specified treatment every 3 days. Control cells were treated with DMSO alone. On day 12, cells were stained, and the number of colonies was counted. Representative images are at 40x magnification. Results presented are mean ± SE. n=3×3. *p<0.05; ^**p<0.005; ^***p<0.0005.

Figure 3(A-E):

Effects of Combination Treatments of Simvastatin with Casodex, Abiraterone Acetate, and Enzalutamide. Combination treatments of statins and anti-androgens on LNCaP C-81 and VCaP-AI cells. LNCaP C-81 cells were plated in 6-well plates at 2×10⁴ cells per well in regular steroid-containing RPMI 1640 medium, while VCaP-AI cells were plated in regular steroid-containing DMEM medium at 2×10⁵ cells per well. After 72 h, cells were conditioned to SR medium for 48 h before treatment in SR conditions with 10 μM of statin compounds and/or 10 μM of anti-androgens: Casodex (Cas), Abiraterone Acetate (AA), or Enzalutamide (Enz) for 72 h. Control cells were treated with DMSO alone. Cells were harvested via trypsinization and counted using trypan blue exclusion dye. (A) Simvastatin combination treatments on LNCaP C-81 cells. (B) LNCaP C-81 cells were treated with 10 μM simvastatin and 1 μM, 5 μM, or 10 μM abiraterone acetate. (C) LNCaP C-81 cells were treated with 5 μM simvastatin and 10 μM abiraterone acetate. (D) Simvastatin combination treatment on VCaP-AI cells. (E) Atorvastatin combination treatment on LNCaP C-81 cells. Results presented are mean ± SE. n=3×3. *p<0.05; ^**p<0.005; ^***p<0.0005.

Figure 4(A-D):

Western Blot Analysis of Simvastatin and Abiraterone Acetate Combination Treated LNCaP C-81 Cells. LNCaP C-81 cells (A) were plated in T75 flasks at 1.5 × 10⁴ cells per flask in regular steroid-containing medium for 72 h, and then steroid starved for 48 h. VCaP-AI cells (C) were plated at 5 × 10⁴ cells per T75 in regular steroid-containing DMEM medium for 72 h, then adjusted to SR conditions for 48 h. Cells were treated with 10 μM of simvastatin, abiraterone acetate, or both simvastatin and abiraterone acetate for 72 h under SR conditions. Control cells were treated with DMSO alone. Cells were harvested via scrapping and lysed. Total cell lysates were analyzed for phosphorylated AKT by site-specific Ser473 phospho-antibodies as well as total AR, PCNA, AKT, Survivin, Bcl_XL, BAX, PARP, Caspase 3 protein levels. β-actin protein level was used as a loading control. Protein density was determined using ImageJ Software (B and D). Results presented are mean ± SE. n=3. *p<0.05; ^**p<0.005; ^***p<0.0005.

Figure 5:

Proposed Mechanism of Action of Simvastatin and Abiraterone Acetate. Simvastatin inhibits HMG-CoA reductase (HMGCR), the rate-limiting enzyme of cholesterol biosynthesis. Simvastatin has been demonstrated to reduce AKT activity upon reduction of cellular cholesterol levels. AR levels are reduced in the presence of simvastatin; thus, simvastatin can potentially inhibit AR activity. Inhibition of these proteins results in the inhibition of PCa survival and growth via reduced Survivin protein levels, as well as the cleavage of Caspase 3 and PARP via Bax. Abiraterone acetate inhibits Cytochrome P450 side chain cleavage enzyme (CYP17A1) to reduce cellular DHT levels. This has the potential to result in the inhibition of AR and AKT activity, leading to inhibition of PCa growth and survival.