Atorvastatin and celecoxib in combination inhibits the progression of androgen-dependent LNCaP xenograft prostate tumors to androgen independence

Abstract

Epidemiology studies suggest that statins and nonsteroidal anti-inflammatory drugs reduce the risk of prostate cancer. In the present study, LNCaP cells were cultured in regular medium containing fetal bovine serum or in medium supplemented with charcoal-stripped fetal bovine serum to mimic androgen deprivation treatment. We found that atorvastatin (Lipitor) or celecoxib (Celebrex) treatment of LNCaP cells cultured in regular or androgen-depleted medium inhibited growth and stimulated apoptosis. A combination of atorvastatin and celecoxib was more effective than either agent alone. In animal studies, severe combined immunodeficient mice were injected s.c. with LNCaP cells in Matrigel. After 4 to 6 weeks, mice with LNCaP tumors (about 0.6 cm wide and 0.6 cm long) were surgically castrated and received daily i.p. injections of vehicle, atorvastatin (10 microg/g body weight/d), celecoxib (10 microg/g/d), or a combination of atorvastatin (5 microg/g/d) and celecoxib (5 microg/g/d) for 42 days. In all groups, the androgen-dependent LNCaP tumors regressed initially in response to castration, but the tumors eventually progressed to androgen independence and started to grow. Treatment of the mice with atorvastatin or celecoxib alone suppressed the regrowth of LNCaP tumors after castration. A combination of low doses of atorvastatin and celecoxib had a more potent effect in inhibiting the growth and progression of LNCaP tumors to androgen independence than a higher dose of either agent alone. Our results indicate that administration of a combination of atorvastatin and celecoxib may be an effective strategy for the prevention of prostate cancer progression from androgen dependence to androgen independence.

Figure 1

Schematic illustration of in vivo androgen-deprivation treatment by surgical castration and effects of atorvastatin or celecoxib alone or in combination on the development and growth of androgen-independent LNCaP tumors in SCID mice.

Figure 2

Effect of atorvastatin or celecoxib alone or in combination on the growth and activation of Akt, Erk1/2 and NF-κB in cultured LNCaP cells. (A) LNCaP cells were seeded at a density of 0.5×10⁵ cells/ml in cell culture dishes and incubated in regular or androgen-depleted medium. The cells were treated with atorvastatin (10 μM) or celecoxib (10 μM) alone or in combination for 96 h. The number of viable cells was determined by using a trypan blue exclusion assay. (B) LNCaP cells were seeded at a density of 1×10⁵ cells/ml of regular RPMI medium and incubated for 24 h. The regular medium was then changed to androgen-depleted medium and the cells were treated with atorvastatin (10 μM) or celecoxib (10 μM) alone or in combination for 24 h. Activated Akt and Erk1/2 were determined by using Western blotting with anti-phosphorylated-Akt (#9275, Cell Signaling Technology, MA) and anti-phosphorylated Erk1/2 (#4376, Cell Signaling Technology). (C) LNCaP cells were seeded at a density of 0.2×10⁶ cells/ml of medium in 60 mm culture dishes (6 ml/dish) and incubated for 24 h. The cells were then transfected with a NF-κB-luciferase construct using Lipofectamine^™ 2000 (LF2000, Invitrogen Life Technology). The cells were treated with atorvastatin (10 μM) or celecoxib (10 μM) alone or in combination for 24 h. The luciferase activity was determined by using a luciferase assay kits (E1500, Promega Madison). For immunostaining of NF-κB in LNCaP cells, cytospin slides were stained with anti-NF-κB antibody (sc-114; Santa Cruz Biotechnology Inc). Photomicrographs were taken using a light microscope (Nikon Optiphot-2, Japan) linked to an Image System (Media Cybernetics, Silver Spring, MD). Representative photomicrographs of NF-κB staining in cells treated with DMSO, atorvastatin, celecoxib or atorvastatin + celecoxib are shown.

Figure 3

Plasma concentration of celecoxib or atorvastatin at various times after an i.p. injection of celecoxib (10 mg/kg) or atorvastatin (10 mg/kg) in male SCID mice. Each value is the mean ± S.E. (N=3).

Figure 4

Effect of i.p. injections of atorvastatin or celecoxib alone or in combination on the progression and growth of androgen-dependent LNCaP xenograft prostate tumors to androgen-independence. Male SCID mice were injected subcutaneously with LNCaP cells in 50% Matrigel (2.0 × 10⁶ cells/0.1 ml). After 4–6 weeks, mice with LNCaP tumors (0.6–1.0 cm wide and 0.6–1.0 cm long) were surgically castrated. Castrated mice were injected i.p with atorvastatin (10 μg/g body weight/day), celecoxib (10 μg/g body weight/day) or a combination of atorvastatin (5 μg/g body weight/day) and celecoxib (5 μg/g body weight/day) for 42 days. Tumor size (length × width) and body weight were measured once every three days and expressed as percent of initial tumor size and percent of initial body weight, respectively. (A). Growth curve of LNCaP tumors in each group. Each value represents the mean ± S.E. from 8 mice (B). Individual body weight of mice after treatment for 42 days.