Inhibition of 5alpha-reductase enhances testosterone-induced expression of U19/Eaf2 tumor suppressor during the regrowth of LNCaP xenograft tumor in nude mice

Abstract

Background: Intermittent androgen deprivation therapy (IADT) was developed to improve the quality of life and retard prostate cancer progression to castration resistance. IADT involves regrowth of the tumor during the off cycle upon testosterone recovery. Our previous studies showed that testosterone is more potent than dihydrotestosterone (DHT) in the induction of a subset of androgen-responsive genes during rat prostate regrowth. However, it is not clear if the same phenomenon would occur during androgen-induced regrowth of prostate tumors. Understanding the differences between testosterone and DHT in inducing androgen-responsive genes during prostate tumor regrowth may provide new insight for improving IADT.

Methods: Nude mice bearing androgen-sensitive LNCaP xenograft were castrated and followed up for 7-10 days before being randomized into various androgen manipulations, consisting of continuous castration (C) or testosterone replacement (T) in the absence or presence of dutasteride (D), a 5alpha-reductase inhibitor that blocks the conversion of testosterone to DHT. Testes-intact animals in the absence or presence of D were used as controls. The expression of five androgen-responsive genes, including the tumor suppressor U19/Eaf2, was determined using real-time RT-PCR, 3 days after randomization.

Results: In LNCaP tumors, the expression of U19/Eaf2 was higher in the T+D group as compared with T alone (2.87-fold, P < 0.05). In contrast, dutasteride treatment in testes-intact animals inhibited the expression of U19/Eaf2.

Conclusions: Inhibition of 5alpha-reductase during LNCaP tumor regrowth enhanced the expression of U19/Eaf2, an androgen-regulated tumor suppressor. This finding suggests that off cycle 5alpha-reductase inhibition may enhance the efficacy of IADT.

Fig. 1

Effect of finasteride on the expression of U19/Eaf2 and PSA during LNCaP tumor regrowth. Tumor bearing mice were castrated when the tumor sizes were 0.5 cm in diameter. After 2 weeks, they were randomized into three groups: castrated with no intervention (C), with testosterone-replacement (T), and with testosterone-replacement plus finasteride (T+F). Drug pellets were implanted for 3 days and tumors harvested for gene expression analysis. The expression of U19/Eaf2 was enhanced 1.8-fold in the T+F group (n = 9) versus the T group (n = 12). *P < 0.05, independent samples t-test. Error bars depict SEM.

Fig. 2

Flowchart of experimental design. Tumor bearing mice were castrated and followed up for 7–10 days before been randomized to receive testosterone (T), testosterone + dutasteride (T+D), dutasteride (D) or no intervention (C).T implantation mimicked intermittent androgen deprivation therapy (IADT),while T+D implantation mimicked IADT + OFF cycle 5α-reductase inhibition. Testes-intactmice, with or without dutasteride implantation, were kept as controls (TIC, TIC+D).

Fig. 3

LNCaP tumor response to androgen manipulation. a: Effect of castration on tumor volume. Tumor bearing mice were castrated, or were followed up without intervention. Tumors in both groups showed a significant increase in volume from 5 days before castration until castration day (116.34mm³ vs. 82.26mm³ in the castrated group; 131.87mm³ vs. 88.21mm³ in testes intact group, **P < 0.01 for both).Castration led to an arrest of tumor growth, and after 1 week, the mean tumor volume (104.6mm³)was modestly lower than that at castration day (116.34mm³, *P < 0.05).Over the same period, testes intact controls showed a significant increase in tumor volume, from 131.87 to 177.58mm³ (**P < 0.01). Error bars depict SEM, and the paired t test was used for p value calculation. b: Effect of androgen replacement on serum PSA. Tumor bearing mice castrated for 7–10 days were implanted with testosterone (T)or testosterone + dutasteride(T+D)pellets. Three days after pellet implantation, both groups showed increases in serum PSA levels, from 14.54 to 73.99 ng/ml in the T group (*P < 0.05) and from 18.68 to 68.01 ng/ml in the T+D group (**P < 0.01).Error bars depict SEM, and the paired t test was used for p value calculation. c: Serum PSA levels at sacrifice. Serum PSA was significantly different across the groups (P = 0.002). Serum PSA was lower in the castrated (C) group versus the testes intact control (TIC) group (18.89ng/ml vs. 59.82ng/ml, *P < 0.05), the T group (18.89 ng/ml vs. 72.63 ng/ml, **P < 0.01) and the T+D group (18.89 ng/ml vs.62.31 ng/ml, **P < 0.01). Error bars indicate SEM. One-way ANOVA with Tukey’s post-hoc test was used for statistical analyses.

Fig. 4

Effect of dutasteride on the expression of indicated androgen-responsive genes. Tumor bearing mice were castrated when the tumors reached a volume of 100 mm³.After castration, they were randomized to C,C+D,T, or T+D.TIC and TIC+D mice were followed up concurrently. Transcript levels for the assayed genes were normalized to GAPDH using the ΔCP method. Each dot represents a single sample, and the horizontal line depicts the median. PSA (**P < 0.01) and U19/Eaf2 (*P < 0.05) expression was significantly decreased in castrated mice when compared to TIC. Expression of PSA, U19/Eaf2, Calreticulin and ADI1 was significantly decreased, **P < 0.01, in TIC mice treated with D. Expression of U19/Eaf2 and calreticulin was enhanced in the T+D group, **P < 0.01. Unpaired t-test was used for P-value calculation.

Fig. 5

DHT levels in tumor tissues the DHT levels were significantly different across the groups (P = 0.0085, one-way ANOVA). The testes intact mice had a mean tumor DHT concentration of 296.2 pg/ml, and in castrated mice it was 124.27 pg/ml. Testosterone pellet implantation (T) restored the intra tumor DHT levels to a mean of 370 pg/ml and the addition of dutasteride to the off-cycle (T+D) caused a modest decline to 278.78 pg/ml. Error bars depict SEM.