Molecular and biochemical effects of a kola nut extract on androgen receptor-mediated pathways

Abstract

The low incidence of prostate cancer in Asians has been attributed to chemopreventative properties of certain chemicals found in their diet. This study characterized the androgenic and chemopreventative properties of the Jamaican bush tea "Bizzy," using androgen receptor positive and negative cell lines. Exposure of prostate cells to Biz-2 resulted in a growth inhibition (GI(50)) of 15 ppm in LNCaP cells and 3.6 ppm in DU145 cells. Biz-2 elicited a 2-fold increase in the mRNA of the anti-apoptotic gene Bcl2, with a 10-fold increase in that of the proapoptotic gene Bax. We observed a 2.4- to 7.5-fold change in apoptotic cells in both cell lines. Biz-2 at 10 ppm elicited a time- and dose-dependent stimulation of both the protein and mRNA levels of several androgen-regulated genes. Biz-2 caused a 36% decrease in PSA secretion and a significant increase in PSA mRNA. The relative binding affinity (IC(50)) of Biz-2 for AR was 2- to 5-fold lower than that of the synthetic androgen R1881. Biz-2 was found to be a specific ligand for the AR in that the natural ligand, DHT, and the anti-androgen, flutamide, displaced Biz-2 bound to AR and inhibited Biz-2-induced transcription and PSA secretion. This study provided evidence that Biz-2 extract possesses the ability to modulate prostate cancer cell biology in an AR-dependent manner.

Figure 1

Biz-2 modulates PSA secretion by LNCaP cells. LNCaP cells (1E⁷) were grown in serum-free medium for 24 hours, then induced with 100 ppm of Biz-2 for 0, 6, 12, and 24 hours. Equal aliquots (10 uL) of the condition medium were removed and analyzed by PSA ELISA (see Section 2). (a) Time-dependent secretion of PSA. (b) PSA secretion induced with 10 ppm Biz-2 for 24 hours in the presence and absence of 10 nM flutamide. The values are the mean ± SEM of three separate experiments performed in triplicate. CON: control; DHT: dihydrotestosterone; Biz-2: ether extract of Bizzy nut; RES: resveratrol.

Figure 2

Stimulation of PSA mRNA expression by Biz-2. LNCaP cells were induced with Biz-2 or DHT, and 5 μg of DNase-I-treated RNA isolated from induced or uninduced cells were subjected to two-step Taqman real-time RT-PCR (see Section 2). The relative expression of PSA mRNA expression was calculated by the 2^ΔΔ_Ct method. First, relative quantitation of PSA mRNA expression was performed by first normalizing the C_t values of PSA amplification against the C_t values of endogenous 18S rRNA, then the resulting C_t values were normalized using the C_t value of the vehicle control sample. (a) Relative amplification plot of mRNA expression (1–NTC. 2–18S rRNA at 24 hours, 3 and 4- CON at 6 and 24 hours, 5 and 6- Biz-2 at 6 and 24 hours). (b) PSA mRNA expression relative to time. (c) Dose-dependent response of PSA to Biz-2. The values are mean ± SEM of induction samples analyzed in triplicate.

Figure 3

Flutamide inhibition of Biz-2 stimulation of PSA mRNA expression. LNCaP cells were induced with Biz-2 in the presence or absence of flutamide, and RNA from induced or uninduced cells was subjected to two-step Taqman real-time RT-PCR. Relative quantitation of PSA mRNA expression was preformed as described in Figure 2. CON: vehicle control; FLU: flutamide; Biz-2: Bizzy nut extract-2; RES: resveratrol; Act.D: actinomycin-D; CHX: cycloheximide. The values are the mean ± SEM of three separate experiments performed in triplicate.

Figure 4

Biz-2 competes for AR binding in LNCaP cells. AR protein from LNCaP cells, cells transfected with pCMV-AR, or purified recombinant AR protein was incubated with 2 nM [³H] R1881 with or without increasing concentration of Biz-2 for 18 hours at 4°C. Bound and free [³H] R1881 were separated using the HAP assay (see Section 2). Each data point represents the mean ± SEM of a representative experiment performed in triplicate. The IC₅₀ was calculated from the competition curve using a one-site competition model. K_i was calculated by K_i = IC₅₀/(1 + [ligand]/K_d) in which K_d and ligand concentration [ligand] were set to 1 and 2 nM, respectively. (a) Single-point assay in the presence of 500X excess R1881 (number above the bar is % of total binding). (b) Displacement curve of [³H] R1881 by Biz-2 (insert is displacement curve of [³H] R1881 by inert R1881). N = 2 separate experiments performed in triplicate.

Figure 5

Modulation of prostate cell proliferation by Bizzy nut extracts. LNCaP or DU145 cells were grown in the presence of 0–1000 ppm Biz-2 for 24 hours, and cell growth was determined using (a) ³[H] thymidine incorporation, (b) MTT cell viability assay and tryphan blue dye staining, and (c) cytotoxicity of Biz-2 in LNCaP cells that was performed as described in Section 2. Growth inhibitory activity of the extracts was determined as [(A_490(control) − A_490(extract))/A_490(control)] × 100. Each value represents mean ± SEM for three experiments performed in eight wells of a 96-well plate. Values followed by ∗ P < .05 differed significantly from the controls.

Figure 6

Cellular and nuclear morphology indicative of apoptosis induced by Biz-2. LNCaP cells were grown on a microscope slide, then induced with 1 or 100 ppm of Biz-2 for 24 hours. Cells were stained with a solution of acridine orange and ethidium bromide for 5 minutes, then examined by light microscopy. (a) Confocal laser fluorescent microscope images taken with a 550 nM filter (Top panel: acridine orange, middle panel: ethidium bromide, bottom panel: both). (b) Apoptotic index was determined. We calculate the apoptotic index by averaging the number of apoptotic cells per field (70–60 cells), then dividing by the total number of cells perfield (120–140 cells) (total number of cells − total number of apoptotic cells)/(total number of cells).

Figure 7

Modulation of apoptotic protein levels in the presence of Biz-2. LNCaP cells were grown in a serum-free medium in the presence of increasing concentrations of Biz-2 for 24 hours. Whole-cell extracts were isolated with RIPA buffer, and 30 μg of protein extract were analyzed by immunoblotting for Bcl-2, Bax, AR, and GDPH expression. (a) Immunoblot of Biz-2-induced proteins. (b) Ratio of Bcl2/Bax expression. (c) Biz-2 induces increase in Caspase 3/7 activity.

Figure 8

Biz-2 increases Caspase 3/7 levels in LNCaP cells. LNCaP cells (1 × 10⁶) were grown in test medium and induced with 100 ppm Biz-2 for 24 hours, and the resulting Caspase 3/7 activity was determined using the Apo-One Homogeneous Caspase-3/7 assay as described in Section 2. (a) Western blot of Bcl2 and Bax proteins following induction of LNCaP with Biz-2 in the absence and presence of flutamide. (b) Caspase 3/7 activity was expressed as n-fold increase relative to untreated control (n = 8, mean ± SEM).