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. 2013 May;12(5):621-31.
doi: 10.1158/1535-7163.MCT-12-0978. Epub 2013 Feb 26.

Spongian Diterpenoids Inhibit Androgen Receptor Activity

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Free PMC article

Spongian Diterpenoids Inhibit Androgen Receptor Activity

Yu Chi Yang et al. Mol Cancer Ther. .
Free PMC article

Abstract

Androgen receptor is a ligand-activated transcription factor and a validated drug target for all stages of prostate cancer. Antiandrogens compete with physiologic ligands for androgen receptor ligand-binding domain (LBD). High-throughput screening of a marine natural product library for small molecules that inhibit androgen receptor transcriptional activity yielded the furanoditerpenoid spongia-13(16),-14-dien-19-oic acid, designated terpene 1 (T1). Characterization of T1 and the structurally related semisynthetic analogues (T2 and T3) revealed that these diterpenoids have antiandrogen properties that include inhibition of both androgen-dependent proliferation and androgen receptor transcriptional activity by a mechanism that involved competing with androgen for androgen receptor LBD and blocking essential N/C interactions required for androgen-induced androgen receptor transcriptional activity. Structure-activity relationship analyses revealed some chemical features of T1 that are associated with activity and yielded T3 as the most potent analogue. In vivo, T3 significantly reduced the weight of seminal vesicles, which are an androgen-dependent tissue, thereby confirming the on-target activity of T3. The ability to create analogues of diterpenoids that have varying antiandrogen activity represents a novel class of chemical compounds for the analysis of androgen receptor ligand-binding properties and therapeutic development.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest.

Figures

Figure 1
Figure 1. Inhibition of AR-dependent proliferation by diterpenoids
(A) Chemical structures of diterpenoids (T1, T2, and T3), DHT, synthetic androgen R1881, and BIC. (B) Proliferation of LNCaP cells after 4 days of treatment, and (C) PC3 cells after 3 days of treatment. Proliferation assays employed BrdU incorporation. Both cell lines were pre-treated for 1 hour with 10 µM of BIC, or T1, or T2, or T3, or vehicle (VEH), prior to the addition of 0.1 nM R1881 (black bars) or ethanol control (white bars). Error bars represent the mean ± SEM, n = 4 separate experiments. Student’s t test: *p < 0.05; **p < 0.01; ***p < 0.001. (D) Morphology of LNCaP cells four days after treament of 10 uM of indicated compounds with ethanol (ETOH) vehicle or 0.1 nM R1881.
Figure 2
Figure 2. Inhibition of AR transcriptional activity
(A) LNCaP cells were transfected with ARR3-LUC and treated with 10 µM of BIC, or T1, or T2, or T3, or vehicle (VEH) in the absence or presence of 1 nM R1881 (-- or +) for 48 hours. (B) LNCaP cells transfected with ARR3-LUC were treated with diterpenoids at 10 µM, 1 µM, 0.1 µM, and 0 µM in the presence of 1 nM R1881 for 48 hours. (C) CV1 cells were cotransfected with pSV-AR0, the expression vector for wild-type AR, and ARR3-LUC, prior to treatment with 10 µM of BIC, or T1, or T2, or T3, or VEH in the absence or presence of 1 nM R1881 (-- or +) for 24 hours. (D) CV1 cells transiently cotransfected with pSV-AR0 (wild-type AR) and ARR3-LUC were treated with diterpenoids at 10 µM, 5 µM, 2.5 µM, 1 µM, and 0 µM in the presence of 1 nM R1881 for 24 hours. Error bars represent the mean ± SEM, n = 3 independent experiments. Student’s t test: *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 3
Figure 3. Reduction of endogenous expression of androgen-regulated genes
(A) Levels of mRNA of four androgen-regulated genes (PSA, KLK2, FKBP5, and TMPRSS2) in LNCaP cells were measured by qRT-PCR. LNCaP cells were pre-treated for 1 hour with 10 µM of BIC, or T1, or T2, or T3, or vehicle (VEH), prior to the addition of 1 nM R1881 (black bars) or ethanol control (white bars) for 48 hr. Endogenous expression of AR in LNCaP cells under the same treatment conditions as mentioned above were detected by (B) qRT-PCR for AR mRNA levels and (C) Western blot analysis for AR protein levels. (D) Levels of PSA, FKBP5, and TMPRSS2 mRNAs were also measured in VCaP cells. Levels of expression of each gene were normalized to levels of GAPDH mRNA. Error bars represent the mean ± SEM, n = 3 separate experiments. Student’s t test: *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4
Figure 4. Binding affinities for AR and inhibition of AR N/C interaction
(A) Recombinant AR LBD was tested for the binding affinity of the diterpenoids by measuring fluorescence polarization (mP) with an excitation wavelength of 470 nm and emission wavelength of 535 nm. Serial dilution was performed for the test compounds using synthetic androgen R1881 as a positive control. A representative plot from at least 3 independent assays is shown, and error bars represent the mean ± SEM. (B) CV1 cells transfected with 5XGAL4Luc reporter vector, VP16-AR-NTD, and GAL4DBD-AR-LBD (wild-type) were pre-treated for 1 hour with 10 µM of BIC, or T1, or T2, or T3, or vehicle (VEH), prior to the addition of 1 nM synthetic androgen R1881 for 24 hours. Error bars represent the mean ± SEM, n=5 independent experiments. Student’s t test: *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5
Figure 5. Effects of diterpenoids on transcriptional activity of related steroid receptors
(A) LNCaP cells were transiently cotransfected with an expression plasmid for full-length human progesterone receptor-beta (PRβ) and a PR-driven (PRE) luciferase reporter. Cells were pre-treated for 1 hour with 10 µM of BIC, or RU486, or each diterpenoid, or vehicle (VEH), prior to the addition of 10 nM 4-pregnene-3,20 dione (Preg) as indicated by black bars (+), or ethanol control (white bars and --). (B) LNCaP cells were transiently cotransfected with an expression plasmid for full-length human glucocorticoid receptor (GR) and GR-driven (GRE) reporter and pre-treated with 10 µM of BIC, or diterpenoids, or VEH, prior to the addition of 10 nM dexamethasone (Dex) as indicated by black bars (+), or ethanol control (white bars and --). 48 hours after treatment, luciferase assay was performed and relative luminescent unit (RLU) per minute was measured and normalized to protein concentration. Error bars represent the mean ± SEM, n ≥ 3 independent experiments. Student’s t test: *p < 0.05; **p < 0.01; ***p < 0.001. (C) Recombinant human PR LBD and (D) recombinant human full-length ERα were tested for the binding affinity of the diterpenoids by measuring fluorescence polarization (mP) with an excitation wavelength of 470 nm and emission wavelength of 535 nm. Serial dilution was performed for the testing compounds. A representative plot from at least 3 independent assays is shown for each receptor.
Figure 6
Figure 6. Efficacy of diterpenoids in vivo
Male NOD-SCID intact mice were treated with DMSO, or BIC, or T3 at 10 mg/kg body weight for two weeks. Tissues were collected, and their weights were measured. The weight of seminal vesicle (A) and testes (B) were plotted as Whisker plots. Body weight (C) was plotted as the percentage change between initial and final weights. Error bars represent the mean ± SEM, n = 8 to 10 as indicated. Student’s t test: *p < 0.05; **p < 0.01; ***p < 0.001.

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