20(S)-protopanaxadiol-aglycone downregulation of the full-length and splice variants of androgen receptor

Abstract

As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer.

Fig. 1

A. Structures of PPD and DHT. B & C. PPD inhibition of the growth of LNCaP cells (B), as well as LAPC-4, C4-2 and 22Rv1 cells (C). Cells were treated with different doses of PPD as indicated (B) or 20 μg/ml PPD (C) for 24, 48, or 72 hr, and growth response determined by the SRB assay. *, P < 0.01 from vehicle control. D. PPD suppression of AR transactivation. LNCaP cells transfected with the ARE-luciferase construct were treated with 20 μg/ml PPD for indicated time in the presence or absence of 1 nM DHT, and luciferase activity analyzed. * and **, P < 0.01 from control or DHT-treated sample, respectively.

Fig. 2

A. PPD downregulation of PSA mRNA in LNCaP cells. Cells were treated with 20 μg/ml PPD for indicated time in the presence or absence of 1 nM DHT, and PSA mRNA determined by qRT-PCR. * and **, P < 0.01 from control or DHT-treated sample, respectively. B. PPD downregulation of PSA protein in LNCaP cells. Cells were treated with 20 μg/ml PPD for indicated time, and PSA protein detected by Western blotting. C. PPD downregulation of PSA mRNA in LAPC-4, C4-2, and 22Rv1 cells. Cells were treated with 20 μg/ml PPD for 12 hr, and PSA mRNA determined. *, P < 0.01 from vehicle control. D. No weakening of PPD effect on PSA expression by increasing concentrations of DHT. LNCaP cells were treated with 20 μg/ml PPD and indicated concentration of DHT for 6 hr, and PSA mRNA determined by qRT-PCR. *, P < 0.01 from respective DHT-treated sample.

Fig. 3

A. PPD downregulation of AR protein. Western blot analysis of AR protein in cells treated with 20 μg/ml PPD. B. Specific knockdown of the full-length AR does not affect PPD downregulation of PSA mRNA. 22Rv1 cells infected with lentivirus encoding the control shRNA or a full-length-AR-specific shRNA were treated with 20 μg/ml PPD in androgen-depleted condition, and the cell lysates subjected to qRT-PCR analysis of PSA mRNA or Western blot analysis of AR proteins. * and **, P < 0.01 from untreated control-shRNA-transfected or AR-shRNA-transfected cells, respectively. C. PPD downregulation of AR protein in the presence or absence of DHT. Western blot analysis of AR protein in LNCaP or 22Rv1 cells treated with 20 μg/ml PPD in the presence or absence of 1 nM DHT for 6 or 12 hr. The numbers in the tables denote relative normalized intensities of the AR protein bands compared to the untreated control value of 100.

Fig. 4

A & B. PPD downregulation of AR transcripts. LNCaP, LAPC-4, C4-2 cells (A) or 22Rv1 cells (B) were treated with 20 μg/ml PPD, and subjected to qRT-PCR analysis of the full-length (AR) and truncated isoforms (AR3, AR4, and AR5) of AR mRNA. *, P < 0.01 from control. C. PPD suppression of AR transcription. LNCaP cells transfected with the AR-promoter-luciferase construct, pGL4-ARpro8 or pGL4-ARpro1.7, were treated with 20 μg/ml PPD for 12 hr, and luciferase activity analyzed. *, P < 0.01 from untreated control. D. PPD induction of proteasome-mediated AR degradation. Western blot analysis of AR or AR3 protein in LNCaP or 22Rv1 cells, respectively. Cells were pretreated with 20 μg/ml PPD for 6 hr prior to the combined treatment with PPD and 10 μM MG132 for an additional 6 hr. The bar graph represents densitometry analysis of the Western blot data from three independent experiments. * and Δ, P < 0.01 from control or cells treated with PPD alone for 12 hr, respectively. E. No effect of PPD on Hsp90-AR association. LNCaP cells cultured in hormone-deprived condition were treated with 20 μg/ml PPD for 2 or 4 hr, and immunoprecipitation was conducted with anti-AR antibody or mouse IgG. Two top panels are Hsp90 or AR Western analysis of the immunoprecipitates. Two bottom panels are Hsp90 or AR Western analysis of the input samples. F. PPD disruption of AR N-C interaction. AR N-C interaction assay in PC-3 cells transfected with a mammalian two-hybrid system. Cells were treated with 1 nM DHT ± 20 μg/ml PPD. * and **, P < 0.01 from control or DHT-treated sample, respectively.

Fig. 5. PPD inhibition of the growth of LNCaP xenograft tumors and PSA secretion

A. Mean tumor volumes in each group (n=14 for the control group; n=13 for the PPD group). *, P < 0.05 from the control group. B. Mean body weights of the mice in each group. C. Weights of individual tumors in each group at the conclusion of the experiment. *, P < 0.01 from the control group. D. Mean serum PSA levels, normalized by tumor weights, in each group at the conclusion of the study. *,P < 0.05 from the control group.

Fig. 6. PPD downregulation of AR in LNCaP xenograft tumors

A. AR immunohistochemical staining of sections of LNCaP xenograft tumors. Upper panel: Quantitation of the results, which is presented as % of pixels in each image field with different AR intensity. The numbers next to the distribution curves represent the mean pixel intensity of the respective group ± SEM. *, P < 0.01 from the control group. The vertical dashed lines segment the three tertiles of immunostaining intensity. Lower panels: Representative images from control and PPD groups (magnification, 200×). B. AR staining of sections of mouse dorsolateral prostates. C & D. H&E staining of sections of LNCaP xenograft tumors or mouse dorsolateral prostates, respectively.