Effect of Monoamine oxidase A (MAOA) inhibitors on androgen-sensitive and castration-resistant prostate cancer cells

Abstract

Background: Monoamine oxidase A (MAOA) is best known for its role in neuro-transmitter regulation. Monoamine oxidase inhibitors are used to treat atypical depression. MAOA is highly expressed in high grade prostate cancer and modulates tumorigenesis and progression in prostate cancer. Here, we investigated the potential role of MAOA inhibitors (MAOAIs) in relation to the androgen receptor (AR) pathway and resistance to antiandrogen treatment in prostate cancer.

Methods: We examined MAOA expression and the effect of MAOI treatment in relation to AR-targeted treatments using the LNCaP, C4-2B, and 22Rv1 human prostate cancer cell lines. MAOA, AR-full length (AR-FL), AR splice variant 7 (AR-V7), and PSA expression was evaluated in the presence of MAOAIs (clorgyline, phenelzine), androgenic ligand (R1881), and antiandrogen (enzalutamide) treatments. An enzalutamide resistance cell line was generated to test the effect of MAOAI treatment in this model.

Results: We observed that MAOAIs, particularly clorgyline and phenelzine, were effective at decreasing MAOA activity in human prostate cancer cells. MAOAIs significantly decreased growth of LNCaP, C4-2B, and 22Rv1 cells and produced additive growth inhibitory effects when combined with enzalutamide. Clorgyline decreased expression of AR-FL and AR-V7 in 22Rv1 cells and was effective at decreasing growth of an enzalutamide-resistant C4-2B cell line with increased AR-V7 expression.

Conclusions: MAOAIs decrease growth and proliferation of androgen-sensitive and castration-resistant prostate cancer cells. Clorgyline, in particular, decreases expression of AR-FL and AR-V7 expression and decreases growth of an enzalutamide-resistant cell line. These findings provide preclinical validation of MAOA inhibitors either alone or in combination with antiandrogens for therapeutic intent in patients with advanced forms of prostate cancer.

Keywords: androgen receptor splice variant; clorgyline; enzalutamide; phenelzine; prostate cancer.

FIGURE 1

The potency of MAO inhibitors on MAOA activity in prostate cancer cell lines. Effect of various doses of clorgyline, moclobemide, deprenyl, tranylcypromine, and phenelzine on MAOA catalytic activity on LNCaP (A) and C4–2B (B) prostate cancer cell lines was determined. Data is expressed as % MAOA catalytic activity measured in untreated control cells and log (MAOA inhibitor concentration in M). Data is summarized in inset (C). MAOA catalytic activity of LNCaP, C4–2B, and 22Rv1 cells determined by radioassay using serotonin as substrate is summarized (D)

FIGURE 2

Clorgyline decreases proliferation of androgen-dependent and androgen-independent prostate cancer cells determined by MTS assay. A total of 2–5 × 10³ LNCaP (A), C4–2B (B), and 22Rv1 (C) prostate cancer cells were seeded into 96-well plates. The cells were serum starved for 24 h before the addition of indicated concentrations of R1881 and clorgyline for 72 h. The viability for each cell line was assessed with the MTS assay. Cell number in untreated control was set at 100%. Each data set represents the mean ± SD of three independent populations (n = 3). *P < 0.05

FIGURE 3

Clorgyline blocks ligand-induced androgen signaling in prostate cancer cells. LNCaP cells were treated with vehicle (Ctr), 10nM R1881 (R1881), 10μM clorgyline (Clg) or both (R1881 + Clg) for 24 h in fresh medium after serum starvation for 24 h. The cells were collected and processed for (A) RT-PCR or (B) Western blot analysis. Each experiment was repeated at least three times and shows a representative blot. (B) The bar diagram (mean ± SEM, n = 3)) showing the fold change in the intensity of the band with respect to GAPDH and relative to untreated group (Ctr), (C) 20 000 cells were plated into each well of 6-well plate. The cells were starved for 24 h before the addition of indicated concentrations of clorgyline and absence or presence of 1nM R1881. The medium was collected after 24 h and change in PSA secretions were determined in cell free medium using prostatic-specific antigen ELISA kit. Each data set represents the mean ± SD of three independent populations (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001

FIGURE 4

Clorgyline and phenelzine enhances anti-proliferative effects of enzalutamide in LNCaP and 22Rv1 cells. 5000 cells from each prostate cancer cell line were seeded into each well of 96-well plates. Indicated concentrations of clorgyline (Clg) in the presence or absence of 2 μM enzalutamide (Enz) were added for 72 h and cell proliferation rate was assessed using MTS assay in (A) LNCaP and (B) 22Rv1 cells. The same experimental setup was used to study effects of phenelzine (Phen) at the indicated concentrations in companion with 2 μM enzalutamide (C and D). Each data point is calculated as % of untreated control and represents mean ± SEM of three independent experiments (n = 3). *P < 0.05 for clorgyline vs clorgyline with enzalutamide

FIGURE 5

Clorgyline decreases androgen receptor-full length (AR-FL) and AR-variant 7 (AR-V7) expression in 22Rv1 cells. 22Rv1 cells were treated with vehicle (Ctr), 2 μM enzalutamide (Enz), 10 μM clorgyline (Clg), or enzalutamide and clorgyline (Enz + Clg) for 24 h in fresh medium for 24 h. The cells were collected and processed for protein (A) and mRNA (B) expression analysis. Each experiment was repeated at least three times and average results are plotted. (A) Representative Western blot (upper panel) and semi-quantiative expression of AR-FL and AR-V7 (lower panel) in 22Rv1 cells following indicated treatments is shown with GAPDH present as a loading control. Bar diagram (mean ± SEM, n = 3) showing the fold change in the intensity of the band with respect to GAPDH and relative to untreated group (Ctr), (B) 22Rv1 cells were treated with vehicle (Ctr), 10 μM clorgyline, (Clg) 2 μM enzalutamide (Enz) or enzalutamide and clorgyline (Enz + Clg) for24 h. RNA was isolated and mRNA expressions for AR-FL, AR-V7, PSA and UBE2C were measured and normalized to 18S housekeeping gene. The histogram showing the mean ± SEM of three independent experiments, n = 3. *P < 0.05

FIGURE 6

C4–2B cells chronically treated with enzalutamide (C4–2B-ER) express AR-V7, increase expression of MAOA and are sensitive to clorgyline: (A) C4–2B and C4–2B-ER cells (5000) were plated in 96-well plate and treated with increasing concentration of enzalutamide for 72 h followed by MTS assay. Dose response curves were generated. Cell number in untreated control (Ctr) was set at 100%. N = 3, (B) Expression of AR-FL, AR-V7, PSA, and MAOA by western blot analysis in C4–2B and C4–2B-ER cells. (C) MAOA catalytic activity measured in C4–2B and C4–2B-ER cells. (D) Histogram showing mRNA expression of AR-FL, AR-V7, and MAOA measure by RT-PCR and (E) comparison of dose response curves for C4–2B and C4–2B-ER cells treated with either increasing concentrations of clorgyline or enzalutamide for three independent populations, *P < 0.05