AA as a receptor-specific androgen antagonist that inhibits PSA expression
Previously, we have described the isolation of AA from a P. africanum methylene chloride extract . To investigate the potential anti-androgenic activity of AA, we performed transient transfection assays in CV1 cells with cotransfecting human AR with the mouse mammary tumour virus (MMTV)-luc reporter gene, a well-known AR responsive promoter . CV1 cells were used because they lack functional AR, GR or PR that are able to bind to the same response element and thus interfere with the functional assays. Also, CV1 cells lack functional thyroid hormone and oestrogen receptors. Initial experiments revealed that AA effectively inhibited the AR-mediated transaction at 10–5 M (supplementary data). Cells were treated without or with increasing concentrations of the agonists DHT (Fig. 1A) or methyltrieneolone (R1881; Fig. 1B). The latter is a synthetic AR-specific agonist that is not metabolized, whereas DHT can be degraded and may serve as ligands for other nuclear receptors . Co-treatment of androgens with AA leads to the inhibition of the ligand-activated human AR whereas treatment of AA alone did not affect the reporter activity. Thus, the data reveal that AA inhibits the ligand-induced transactivation of AR and suggest that AA has an anti-androgenic activity. Concentration series of the natural androgen DHT were performed to determine the efficacy to inhibit DHT-activated AR (Fig. 1A). At 10 nM DHT, AA (10 μM) was able to strongly inhibit AR mediated transactivation. As control, treatment of cells with the solvent itself did not affect the reporter. AA also potently inhibited AR-mediated transactivation in a dose-dependent manner using the synthetic AR-ligand R1881 (Fig. 1B). This provides an indication that AA competes with DHT or R1881 for binding to AR.
Figure 1. Inhibition of transactivation function of ligand-activated human AR by AA isolated from P. africanum bark extracts. CV1 cells, lacking functional endogenous AR were transfected with the expression vector for human AR and the androgen responsive reporter MMTV-luciferase treated with the indicated concentration series of the androgen (white bars) DHT (A) or with the synthetic AR ligand R1881 (B). In addition cells were treated with 10 μM AA (black bars), dissolved in ethanol/DMSO (1:1) or as control with this solvent alone. Luciferase values obtained were normalized to the cotransfected internal control and indicated as relative light units. The error bars indicate the variation of the mean.
Taken together, the data suggest that AA is able to repress the transactivation function mediated by agonist-induced human AR.
To analyse the receptor specificity of AA, various members of the nuclear hormone receptor superfamily were used for reporter assays (Fig. 2). The closely related members are GR and PR, for which the MMTV reporter is also a suitable responsive reporter. Ten and 100 μM of AA did not affect the glucocorticoid-induced transactivation of human GR significantly. In case of human PR, both isoforms were tested, the PR-B isoform with a more potent transactivation function and the PR-A isoform, which is generated by an internal translation start site. The progesterone-induced transactivation of both PR forms were unaffected by co-treatment with 10 μM AA, whereas the transactivation mediated predominately by the PR-B isoform was strongly inhibited at 10-fold higher AA concentrations (100 μM; Fig. 2A). Using TR with a TR responsive reporter, p(DR4)2-tk-luc, the addition of AA did not affect significantly the thyroid hormone (T3)-induced transactivation of TR (Fig. 2B). Furthermore, AA was tested for its influence on the oestrogen receptors ER-α- and ER-β-mediated transactivation. In contrast to AR and GR, the ER is localized predominantly in the nucleus in the absence of ligand [38–40]. Interestingly, the hormone-induced transactivation of both ERs was not affected by AA. However, in the absence of oestrogens both ERs were only weakly induced by 10 μM and more efficiently by 100 μM AA in a dose- and receptor-dependent manner (Fig. 2C and not shown). This suggests that AA at higher concentrations serves as ER agonist. Taking together, this indicates that AA is at lower concentrations specific for AR and affects only at higher concentrations PR and ER significantly.
Figure 2. AA at lower concentrations inhibits specifically ligand-activated human AR. Closely related members of the nuclear hormone receptor superfamily were selected to test for their inhibition by AA. The human glucocorticoid receptor (hGR), both human progesterone receptors (PR-A and -B forms), the human thyroid hormone receptor β (human TR-β), and both human oestrogen receptors (ER-α and ER-β) were tested with the appropriate reporters (MMTV-luciferase for hGR and both PR forms (A), p(DR4)2-tk-luc for TR (B), and p3ERE-TATA-luc for the ERs (C), respectively) in CV1 cells, lacking functional endogenous GR, PR, ER or TR. Indicated concentrations of AA were tested for inhibition of glucocorticoid (dexamethasone) induced hGR, progesterone-induced PR-A and -B isoforms, estradiol for both ERs, and of thyroid hormone for TRβ. (D) The mutant AR T877A, which is endogenously expressed in LNCaP cells was compared to the wild-type AR for inhibition by AA. (E) AA inhibits the transactivation mediated by the OH-F treated mutant AR T877A.
This suggests that at lower concentrations of 10 μM AA has a strong preference for the AR and is an AR-specific antagonist.
LNCaP cells are used as a model cell line for analysing human PCa. For that purpose we used the human LNCaP PCa cell line that is known to grow androgen dependently. In addition, we employed the C4–2 PCa cell line that also expresses functional endogenous AR and exhibits an androgen-independent growth  making them an interesting model representing the transition of the initial androgen-dependent disease to an androgen-independent state. Both cell lines express the mutant AR, AR T877A, which can be activated by the complete androgen antagonist OH-F . The activation of this AR mutant by the complete antagonist OH-F represents a mechanism by which the cancer becomes resistant to hormone therapy treatment. Because AA (for structure see supplemental data) has more structural similarities to OH-F compared to other AR antagonists used in PCa therapy, we focused on comparing AA with OH-F activity.
Therefore, first pre-tests were performed to test whether AA is able to inhibit the transactivation of the mutant AR T877A. The effect of AA with 10 mM AA with or without androgen agonist on wild-type and the mutant AR T877A was compared (Fig. 2D). In contrast, OH-F activated the mutant AR effectively in the absence of agonist and poorly inhibited AR transactivation in the presence of R1881 (supplementary data). As expected, AA inhibited also the OH-F-induced transactivation of the mutant AR T877A (supplementary data). Taken together, the obtained data reveal that AA inhibits the agonist-induced transactivation of both wild-type and mutant AR T877A.
To analyse whether AA represses an endogenous AR-regulated gene, the well-known AR target gene PSA was analysed. Androgens induce the expression PSA through the AR that binds to androgen response elements in the promoter and enhancer region of PSA. Therefore, to confirm the AR antagonism of AA also for an endogenous AR target gene, the endogenous PSA expression of both LNCaP and C4–2 cells were analysed by treatment with or without DHT or R1881 in the absence or presence of AA at 10−4 or 10−5 M. The RNA was extracted and analysed by quantitative RT-PCR for PSA expression levels (Fig. 3). Treatment of both cell types with either agonist induced the PSA mRNA expression. For LNCaP cells, AA inhibited the expression of PSA at 10−5 M. The inhibition was more pronounced using 10−4 M AA (Fig. 3A). Treatment of C4–2 cells with 10−4 M AA inhibited the expression of PSA also in the absence of agonist (Fig. 3B), which might reflect the notion that AR is partially active in these cells . At 10−4 M AA, both DHT- and R1881-induced PSA expression was potently inhibited. Notably, this was also observed using the lower 10−5 M AA concentration. Thus, these findings suggest that AA inhibits the expression of the PSA gene in both androgen-dependent and androgen-independent PCa cells.
Figure 3. AA inhibits the endogenous PSA gene expression of androgen-dependent and androgen-independent PCa cells. The androgen-dependent LNCaP (A) or the androgen-independent C4–2 cells (B) were treated with the indicated androgens DHT or R1881 (methyltrienolone) with and without AA. Quantitative real-time RT-PCR was performed for PSA mRNA and β-actin mRNA for normalization.
Compared to OH-F, which has among the AR antagonists used in PCa therapy structurally the most similarities to AA and is an agonist of the AR mutant T877A, the obtained data using AA suggest that AA appears to act distinct from OH-F.
Taken together, AA can inhibit the AR-mediated transactivation and endogenous androgen-induced gene expression. Thus, these findings suggest that AA is a novel AR antagonist.
Growth inhibition of human prostate cancer cells by AA
The growth of prostate cells and PCa cells is initially dependent on androgens [11, 13]. To analyse whether the androgen antagonism of AA affects cell growth, LNCaP and C4–2 cells were used. Although AA represents a novel structural platform for AR antagonists (for structure see supplementary data), AA has some structural similarities to OH-F. Therefore, we compared the effect of AA with that of OH-F on cell growth using the human PCa cell lines LNCaP and C4–2, expressing endogenously the human AR, with the PC3 as well as with CV1 kidney cell lines, that lack endogenously expressed AR. Because growth of LNCaP cells in long-term cultures is impaired with charcoal-stripped serum to remove hormones, we used normal serum that is known to contain androgens naturally and represents a similar situation as in patients with PCa. For that purpose an equal number of cells were seeded out in 5% untreated serum together with or without addition of OH-F or AA. As expected, treatment of LNCaP and C4–2 cells with OH-F revealed no significant inhibition of LNCaP cell growth (Fig. 4A, 4B). Interestingly, treatment of LNCaP cells with AA inhibited potently LNCaP proliferation. Growth inhibition was observed after 4 to 6 days of treatment with AA. We did not observe enhanced de-attachment or an increase in the apoptosis rate by AA. The growth inhibitory effect was more prominent after day 10 of treatment. This strongly indicates that AA is mechanistically different to OH-F for mediating inactivation of AR.
Treatment of the androgen-independent PCa cell line C4–2 also led to a growth reduction by AA and not by OH-F (Fig. 4B) without observing toxic effects. Even after treatment with 10−4 M AA no detectable cell toxicity was observed. In contrast to C4–2 cells, using CV1 cells that lack endogenously expressed AR, no significant change in growth was observed by treatment with either OH-F or AA (Fig. 4C). Similarly, the growth rate of the AR-deficient PC3 PCa cell line was not affected by AA (supplementary data). The lack of influence on the growth of both AR negative CV1 and PC3 cells suggests that AA has no significant toxicity and implies that AA inhibits the AR.
Taken together, these data suggest that the androgen antagonism of AA is able to inhibit growth of human PCa cells.
AA inhibits cell invasion through ECM
The AR agonist R1881, besides promoting PCa cell proliferation, also enhances cell invasion through ECM, thought to be an important step towards cancer metastasis . In line with previous observations, R1881 enhances cellular invasion of LNCaP cells through ECM (Fig. 5). Interestingly, addition of AA at 10 μM abrogated this promoting effect. Further, using 100 μM AA cell invasion was further inhibited. AA itself had no significant effect on cell invasion (supplementary data). These findings suggest that AA is an AR antagonist also at the level of cellular invasion.
Figure 5. AA inhibits cellular invasion of LNCaP cells through extracellular matrix (ECM). Cell invasion through ECM was analysed using LNCaP cells. The cells were seeded out with 0.1% charcoal-treated FCS with R1881 (3 × 10−11 M), with and without AA (10−4 or 10−5 M) and incubated for 48 or 72 hrs at 37°C under 5% CO2 atmosphere. Migrated cells through ECM were counted with a microscope.
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Thus, these observations suggest that AA inhibits cellular invasion through ECM.
AA inhibits ligand-induced nuclear localization
To get insights into the molecular mechanisms of AA-mediated inhibition of AR, we first analysed whether the LBD of AR is target of inhibition by AA. As expected, deletion of the LBD (AR –ΔC) leads to a hormone-independent transactivation (Fig. 6). However, compared with the wild-type AR, the AR –ΔC activity was unaffected by AA (Fig. 6). This suggests that the LBD is the target of AA-mediated repression. The N-terminal deletion of AR (AR –ΔC) exhibited no significant transactivation function and was not affected by AA, indicating that AA treatment has no significant influence on this mutant and does not unspecifically influence the promoter activity. Further, because the SUMO-mutant of AR, that was shown to lack binding of the corepressor SMRT  and Alien , was also inhibited by AA (Fig. 6A), it appears that corepressor binding to AR is not involved as a molecular mechanism of repression of AR by AA. Lack of interaction of various corepressors with AR in mammalian cells treated with of AA suggests this notion (supplementary data).
Figure 6. The ligand-binding domain of AR is target for AA-mediated inhibition. (A) The indicated AR deletion mutants were tested for their ability to be repressed by AA. AR –ΔN, N-terminal deletion of AR (aa 1–504 are deleted); AR –ΔC, C-terminal deletion (aa 628–919 are deleted); AR K385E and K518E represent amino acid exchanges at these positions. (B) Competitive whole cell binding assays were performed using pSG5-flag-wtAR transfected COS-7 cells incubated with 1 nM (3H)-mibolerone in the absence and presence of increasing concentrations of either unlabelled mibolerone (circle) or AA (rectangles) for 90 min. Competition for binding is illustrated by the percentage of [3H] mibolerone specifically bound to the AR. Results are averages of three independent experiments with each condition in triplicate (±S.E.M.).
To further investigate the molecular mechanism of AA-mediated inhibition of AR, corepressor recruitment and AR stability were analysed. It is known that androgen antagonists recruit corepressors, such as NCoR, SMRT or Alien to inactivate the AR [27, 32, 36, 43–46]. However, using our previously established modified two-hybrid test system we were unable to detect corepressor interaction with AR in the presence of AA (supplementary data).
Because the deletion analysis suggests that AA acts through the hormone-binding domain of AR, competitive hormone binding assays were performed to detect whether AA is able to compete for androgen binding to AR (Fig. 6B). Using increasing amounts of AA, competition of the androgen mibolerone was observed at the concentration of 10 μM that was effective in both inhibition of transactivation and growth proliferation. Thus, these data suggest that AA binds to the LBD of AR.
Another potential possibility to inhibit AR-mediated transactivation is to decrease AR protein levels by AA. Pre-tests of control experiments of AR levels in transfected CV1 cells used for the reporter assays suggested that AA did not reveal a change of AR protein level (supplemental data). To investigate the protein levels of endogenously expressed AR, LNCaP cells were stimulated with or without AA in the presence of 5% FCS prior preparation of whole cell extracts. Subsequent Western analyses did also not reveal an AA-induced decrease of AR protein levels (Fig. 7A).
To analyse the possibility that AA inhibits the ligand-induced translocation of AR to the nucleus LNCaP cells were treated with or without androgen and AA prior cell fractionation to yield a cytosolic and nuclear fraction (Fig. 7B and C). Ligand-induced nuclear localization of AR was observed with methyltrienolone treatment decreasing the amount of cytosolic AR and increasing the nuclear AR (Fig. 7B, compare lane 1 with lane 4). As controls for loading the detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Fig. 7B) and for cytosolic and nuclear fractionation that of actin and nucleophosmin were used (Fig. 7C). Interestingly, addition of AA abrogated the methyltrienolone-induced nuclear translocation of AR (Fig. 7B and D, lane 5 and 6) at the effective concentrations that lead to inhibition of AR-mediated transactivation and cell growth. To confirm the inhibition of the AR translocation a GFP-AR fusion was employed. Treatment of cells with R1881 resulted in predominantly nuclear staining of living cells whereas AA alone or AA in combination with R1881 resulted in predominant cytosolic staining (Fig. 7). These data confirm the observed inhibition of agonist-induced nuclear translocation of AR.
Thus, these data suggest that AA competes with androgens for the binding to AR and inhibits the nuclear translocation of AR.