Effects of 5 alpha reductase inhibitors on androgen-dependent human prostatic carcinoma cells

Abstract

Purpose: To investigate the effects of MK906, a selective 5 alpha reductase (5alphaR) type 2 (5alphaR2) inhibitor, and of MK386, a specific 5alphaR1 inhibitor, on the cellular proliferation of androgen-dependent human prostatic cancer (PCa) cells in cultures of cells derived from bioptic and surgical tissues.

Methods: In this study we tested the effects of MK906 and MK386 in 30 cultures derived from PCa, 6 from PIN and 10 from benign prostatic hyperplasia specimens.

Results: Prostate primary cultures under short-term conditions (with <4 subcultures) represent a mixture of epithelial and stromal cells. Epithelial cells require testosterone (T) for optimal growth, but were not able to grow in the presence of T under long-term conditions even if DHT was able to induce cellular proliferation to a similar extent in both conditions, suggesting that 5alphaR can be lost in long-term cultures. Therefore, our studies were performed under short-term conditions. Both 5alphaR inhibitors decreased cell proliferation significantly and dose-dependently in all the samples tested. MK906 was more efficient than MK386 in 7 out of 10 cultures derived from BPH tissues, in 4 out of 6 cultures derived from PIN and in 18 out of 30 cultures derived from PCa. In 3 out of 10 BPH, in 2 out of 6 PIN and in 5 out of 30 PCa-derived cultures, both inhibitors presented similar efficacy, whereas in 1 out of 10 BPH and 7 out of 30 PCa-derived cultures MK386 was more efficient than MK906. In addition, MK386 was more efficient than MK906 in 4 out of 15 non-metastatic PCa and 2 out of 7 metastatic PCa-derived cultures.

Conclusions: Considering that 5alphaR1 (responsible primarily for androgenic catabolism) is mostly expressed in epithelial cells and that 5alphaR2 (responsible for local DHT synthesis and release) is expressed in the stromal cells (which provides several paracrine growth factors and DHT itself to the epithelial cells), our experiments suggest that the inhibition of both 5alphaR1 and 5alphaR2 by MK386 and MK906, respectively, may have therapeutic potential in order to reduce the growth and progression of human prostatic cancers, through the inhibition of autocrine or paracrine mechanisms involving the stromal cell compartment. In addition, some effects of 5alphaR inhibitors could be mediated by estrogens, which are synthesized by the aromatase enzyme present in the epithelial cells. These aspects could be considered in order to improve the therapeutical management of PCa and for future clinical trials.

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. 1 Proliferative effects of DHT. 2×10⁵ cells were seeded in 60 mm petri dishes, left to attach overnight and to grow for 72 h in 5% charcoal-treated FCS DMEM plus DHT at different concentrations. Each series of samples was performed in triplicate. The results are averages (±SD, bars) from three independent experiments

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. 2 Proliferative effects of T and DHT in short-term and long-term cultures. 2×10⁵ cells were seeded in 60 mm petri dishes, left to attach overnight and grow for 72 h in 5% charcoal-treated FCS DMEM plus T and DHT. The figure shows higher effects of TSTR (10^–9 M) in short-term cultures compared to those observed in long-term cultures. DHT (10^–10 M) shows similar effects under both conditions. Each series of samples was performed in triplicate. The results are averages (±SD, bars) from three independent experiments

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. 3 Scheme for the stepwise characterization of prostate cells in primary cultures (Festuccia et al. 2005)

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. 4a-f Morphological changes in response to treatment with MK906 in the presence of a 20% stromal contamination. PU166 (G6 pT2N1M1a PCa-derived culture) cells were treated with or without 10 μM MK906 for up to 7 days. Panels A, B and C show untreated cells at 3, 5 and 7 days of culture whereas panels D, E and F represent MK906-treated cells at the same culture times. Initially cells became elongated with a morphology resembling that of neuron-like cells (panel D black arrows, original magnification ×100) compared to untreated cells at the same culture time (panel A original magnification ×200). Similar elongated neuron-like cells with elongated cytoplasm were also observed in untreated cells at about 5 days of culture when we observed a subconfluence condition (panel B black arrows, original magnification ×200) whereas no neuron-like cells were observed at confluence (panel C original magnification ×100). Prolonged MK906 treatment induced hypertrophy of cells (white arrow) and detachment of cells (black arrows) as shown in panels E and F

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. 5a-d Morphological changes in response to treatment with MK386. PU140 (G 7 pT3N0M0 PCa-derived culture) cells were treated with or without 10 μM MK906 for up to 7 days. Cytoplasmic vacuoles became more apparent in treated cells, C 3 days of culture, original magnification ×100, D 7 days of culture, original magnification ×400, compared to untreated cells (A×100, B×400)

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. 6a-d Proliferative effects of 10⁻¹⁰ M DHT, 10⁻⁹ M TSTR on LNCaP (A, B) and BPH-1 (C, D) cells alone (A, C) or in co-culture with PCa-derived fibroblasts (B, D). 2×10⁵ cells were seeded in 6-well plates, left to attach overnight and grow for 72 h in 5% charcoal-treated FCS DMEM plus TSTR and DHT. For co-culture experiments we used the transwell chambers in which the upper compartments were covered by a polycarbonate filter with 0.8 µm pores. In the upper compartment we seeded LNCaP and BPH1 cells, whereas in the lower compartment we seeded 2×10⁵ fibroblasts. Each series of samples was performed in triplicate and MK906 and MK386 were used at 1.0 μM. The results are averages (±SD, bars) from three independent experiments

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. 7a-c Effects of selected sex steroids and inhibitors on LNCaP cell proliferation. 2×10⁵ cells were seeded in 6-well plates, left to attach overnight and to grow for 72 h in 5% charcoal-treated FCS DMEM plus steroids. A Effects of single agents: 10⁻¹⁰ M of both 3α-adiol and 3β-adiol, 10⁻⁹ M E2 and 10⁻⁹ M T on LNCaP, 1.0 μM MK386 partially blocked T-mediated LNCaP cell proliferation. B Effects of T plus 1.0 μM of anastrazole (ANS), MK386 and BCLT. C Combination effects: 0.1 μM anastrazole, 0.1 μM MK386 and 0.05 μM anastrazole, MK386 and BCLT in combination completely block LNCaP cell proliferation, with doses which are at least 10 times lower compared to doses achieving inhibition with the same drugs used alone. Each series of samples was performed in triplicate. ΜΚ906 and ΜΚ386 were used at 1.0 μM. The results are averages (±SD, bars) from three independent experiments