Azolato-Bridged Dinuclear Platinum(II) Complexes Exhibit Androgen Receptor-Mediated Anti-Prostate Cancer Activity

Abstract

Prostate cancer is an androgen-dependent malignancy that presents a marked treatment challenge, particularly after progression to the castration-resistant stage. Traditional treatments such as androgen deprivation therapy often lead to resistance, necessitating novel therapeutic approaches. Previous studies have indicated that some of the azolato-bridged dinuclear platinum(II) complexes (general formula: [{cis-Pt(NH₃)₂}₂(μ-OH)(μ-azolato)]X₂, where azolato = pyrazolato, 1,2,3-triazolato, or tetrazolato and X = nitrate or perchlorate) inhibit androgen receptor (AR) signaling. Therefore, here we investigated the potential of 14 such complexes as agents for the treatment of prostate cancer by examining their antiproliferative activity in the human prostate adenocarcinoma cell line LNCaP. Several of the complexes, particularly 5-H-Y ([{cis-Pt(NH₃)₂}₂(μ-OH)(μ-tetrazolato-N2,N3)](ClO₄)₂), effectively inhibited LNCaP cell growth, even at low concentrations, by direct modulation of AR signaling, and by binding to DNA and inducing apoptosis, which is a common mechanism of action of Pt-based drugs such as cisplatin (cis-diamminedichloridoplatinum(II)). Comparative analysis with cisplatin revealed superior inhibitory effects of these complexes. Further investigation revealed that 5-H-Y suppressed mRNA expression of genes downstream from AR and induced apoptosis, particularly in cells overexpressing AR, highlighting its potential as an AR antagonist. Thus, we provide here insights into the mechanisms underlying the antiproliferative effects of azolato-bridged complexes in prostate cancer.

Figure 1

Molecular structures of three major Pt-drugs (cisplatin, carboplatin and oxaliplatin), a series of azolato-bridged dinuclear Pt(II) complexes (AMPZ, AMTA, 5-H-X, 5-H-Y), 5-H-Y derivatives with the general formula [{cis-Pt(NH₃)₂}₂(μ-OH)(μ-5-R-tetrazolato-N2,N3)](NO₃)₂ (complexes 1–10), and the androgen antagonist KW-365.

Figure 2

Inhibition of prostate cancer cell proliferation by 14 azolato-bridged dinuclear Pt(II) complexes. Cells of the androgen receptor (AR)–positive prostate cancer cell line LNCaP were treated with the indicated azolato-bridged complexes in the presence or absence of the androgen dihydrotestosterone (DHT). (A) Concentration-dependent inhibition of cancer cell proliferation by Pt complexes on LNCaP cells in the presence of 10 nM DHT. Dimethyl sulfoxide (DMSO) was used as a control, and the DMSO content in any medium was kept below 0.1%. (B) Analysis of the inhibitory effect of DHT on cell proliferation of LNCaP cells: comparison of the proliferative effect of 10 nM DHT-treated cells ((A) right) against cells not treated with DHT ((A) left). (C) Effect of KW-365 and cisplatin, 5-H-Y on DHT concentration-dependent cell viability. The structures of the compounds are shown in Figure 1. *p < 0.05, **p < 0.01, ***p < 0.001, N.S.; not significant compared with vehicle.

Figure 3

5-H-Y suppresses androgen receptor (AR) signaling and inhibits prostate cancer cell proliferation. (A, B) RNA expression levels of the AR-responsive genes encoding prostate-specific antigen (PSA) and transmembrane protease, serine 2 (TMPRSS2) in LNCaP cells after treatment with 5-H-Y. Conditions: dihydrotestosterone (DHT), 0.13 nM; KW365, 2 μM; 5-H-Y, 50 μM. ***p < 0.001, N.S.; not significant. (C) Western blots confirming that the small interfering RNA targeting AR (siAR) decreased endogenous AR expression, and that the AR expression plasmid (pcDNA3.1-AR) increased endogenous AR expression, in LNCaP cells. (D–F) Effects of siAR-induced knockdown of AR expression on the inhibition of cell viability by cisplatin and 5-H-Y in LNCaP cells. (G–I) Effects of pcDNA3.1-AR-induced overexpression of AR on the inhibition of cell viability by cisplatin and 5-H-Y in LNCaP cells.

Figure 4

5-H-Y induces apoptosis in prostate cancer cells through androgen receptor (AR) signaling. (A) Immunofluorescence images showing the induction of apoptosis in LNCaP cells treated with 5-H-Y. Arrows indicate areas where apoptosis occurred. DMSO, dimethyl sulfoxide. (B) Effect of concentration on the degree of apoptosis induced by 5-H-Y in LNCaP cells overexpressing AR (pcDNA3.1-AR) or not (pcDNA3.1). *p < 0.05, **p < 0.01, N.S.; not significant. (C) Effects of cisplatin and 5-H-Y on cell cycle in LNCaP cells. (D) Effects of cisplatin and 5-H-Y on cell cycle in LNCaP cells overexpressing AR or not.

Figure 5

5-H-Y binds directly to androgen receptor (AR). (A) Coomassie Brilliant Blue stain confirming that bovine serum albumin forms aggregates in the presence of 5-H-Y. (B) Western blot confirming that AR forms aggregates in the presence of 5-H-Y or cisplatin. (C–E) Cellular thermal shift assay results for AR after treatment with dihydrotestosterone (DHT), KW-365, or 5-H-Y. DMSO, dimethyl sulfoxide (vehicle).

Figure 6

Androgen receptor (AR) binds to 5-H-Y and is transported to the nucleus. (A) Fluorescence immunostaining showing the time-dependent changes in AR localization following treatment with dihydrotestosterone (DHT), cisplatin, or 5-H-Y. (B) Fluorescence immunostaining showing the time-dependent nuclear export of AR at 36 h after 5-H-Y treatment. (C) Changes in nuclear Pt accumulation following administration of cisplatin or 5-H-Y under conditions of AR overexpression. *p < 0.05, N.S.; not significant. DMSO, dimethyl sulfoxide (vehicle).