Human castration resistant prostate cancer rather prefer to decreased 5α-reductase activity - PubMed (original) (raw)

Human castration resistant prostate cancer rather prefer to decreased 5α-reductase activity

Takeo Kosaka et al. Sci Rep. 2013.

Abstract

Physiologically relevant steroid 5α-reductase (SRD5A) activity that is essential for dihydrotestosterone (DHT) biosynthesis in human castration-resistant prostate cancer (CRPC) has not been fully characterized yet. In this study to ascertain the potential SRD5A activity, we cultured two human CRPC cell lines, C4-2 and C4-2AT6, with the steroid precursor: ¹³C-[2,3,4]-androstenedione (13C-Adione), and analyzed the sequential biosynthesis of ¹³C-[2,3,4]-testosterone (13C-T) and ¹³C-[2,3,4]-DHT (13C-DHT) by liquid chromatography/mass spectrometry (LC/MS/MS). The 13C-DHT/13C-T concentration ratio detected by LC/MS/MS in C4-2AT6 cells appeared to reflect the SRD5A activity. The ratio in C4-2AT6 was significantly lower than that in C4-2. An increased concentration of DHT did not have a positive effect on cell proliferation, rather it exhibited inhibitory effects. 5α-reductase inhibitors did not have any inhibitory effect at clinically achievable concentrations. These results indicate that CRPC cells may have an unknown regulation system to protect themselves from an androgenic suppressive effect mediated by SRD5A activity.

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Figures

Figure 1

Figure 1. Detection of T and DHT in CRPC cells by LC/MS/MS analysis.

(A) T and DHT concentrations in the supernatant of C4-2 incubated for 6 hrs *** p < 0.001. (B) T and DHT concentrations in the supernatant of C4-2AT6 incubated for 6 hrs *** p < 0.001. (C) The mRNA expression of SRD5A1 and SRD5A2 in C4-2 and C4-2AT6 cells. ** p < 0.01

Figure 2

Figure 2

(A) Simplified schematic representation of the co-culture system with 13C steroid precursors. (B) Typical selected ion recordings of the 13C-T and 13C-DHT extracted from cultured medium. (C) 13C-T and 13C-DHT concentrations in the supernatant of C4-2 and C4-2AT6 cells. *** p < 0.001. (D) DHT/T ratio appeared to reflect 5AR activity in cancer cells. C4-2AT6 cells exhibited significantly reduced 5AR activity, compared to C4-2.

Figure 3

Figure 3. At high concentrations DHT exhibited inhibitory effects on C4-2AT6 cells.

(A) mRNA expression of AR in C4-2 and C4-2AT6 cells. *** p < 0.001. (B) Western blot analysis of AR expression in the nucleus in C4-2 and C4-2AT6 cells. (C) mRNA expression of PSA in C4-2 cells increased in a dose-dependent manner. (D) mRNA expression of PSA in C4-2AT6 cells increased in a dose-dependent manner. *p < 0.05, *** p < 0.001. (E) WST cell viability assay exhibited no significant proliferative response to DHT at 10−14 M to 10−12 M DHT concentration in C4-2 cells. DHT treatment at a concentration of 10−8 M resulted in significant decreased cell viability. *p < 0.05. (F) When treated with 10−10 M DHT or 10−8 M DHT, C4-2AT6 cells showed significant decreased cell viability compared with that at lower DHT concentrations. *p < 0.01. (G) Response to DHT in C4-2 and C4-2AT6 cells detected by phase contrast field.

Figure 4

Figure 4. Efficacy of 5ARIs assessed using PCA cell lines.

The efficacy of 5ARIs in prostate cancer cells was assayed by incubation with dutasteride (A, C) or finasteride (B, D) at various concentrations for 96 h. In LNCaP cells, dutasteride and finasteride exhibited significant inhibitory actions within the clinically achievable 5ARI concentration of 10 nM. In C4-2 and C4-2AT6 cells, neither dutasteride nor finasteride inhibited cell viability at the same concentrations. *p < 0.05, **p < 0.01. (C, D) qPCR analysis of PSA, Nkx3.1, TMPRSS2 in C4-2 (C) and C4-2AT6 (D) after treated with 5ARIs. Dutasteride and finasteride did not show a significant effect on these AR target genes. (D) Dutasteride, (F) Finasteride.

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