Fig. 1. Photomicrograph of the PC-3 cells visualized (×40 magnification) using Nikon inverted phase contrast microscope. (a, e, i and m) Control cells treated with 0.1% BSA vehicle — 24, 48, 72 and 96 h, respectively; (b, f, j and n) 1 nM estradiol-treated cells — 24, 48, 72 and 96 h, respectively; (d, h, l and p) 100 nM estradiol-treated cells — 24, 48, 72 and 96 h, respectively. The dose and duration-dependent effect was observed: 10 and 100 nM estradiol induces in all time points than 1 nM estradiol.

Fig. 2. The assessment of mitochondrial function in PC-3 human prostate cancer cells incubated for 24, 48, 72 and 96 h with serum free medium alone or with increasing concentration of estradiol (1, 10 and 100 nM) were analyzed for the ability of mitochondria to MTT. Bars are mean ± SEM for three experiments; each with eight replicates, representing MTT reduction as OD indicates impaired mitochondrial function. The letter a denotes comparison between control and estradiol-treated cells at significant levels denoted (P < 0.05).

Fig. 3. Effects of estradiol on the secretion of IGFBP-3 in PC-3 cells in conditioned media for 24 and 48 h. Bars represent mean ± SEM for three experiments, the significance compared to the control: letter a denotes P < 0.05.

Fig. 4. Effects of estradiol on the secretion of IGFBP-4 in PC-3 cells in conditioned media for 24 and 48 h. Bars represent mean ± SEM for three experiments, the significance compared to the control: letter a denotes P < 0.05.

Fig. 5. After 48 h exposure of estradiol (10 and 100 nM), the cells were lysed for western analysis of (a) MMP-2, (b) MMP-9 and (c) IGF-IR in PC-3 cells. The equal loading of the sample confirmed by (d) β-actin as an internal control. The MMP-2 levels significantly decreased after the estradiol exposure as shown in the quantification graph (a: MMP-2, MMP-9 and IGF-IR significantly decreased after the estradiol exposure as shown in quantification graph Fig. 5a, b and c, respectively). —Denotes the significance (P < 0.05) compared to the control, a—indicates significance (P < 0.05) in between groups.

Fig. 6. Estradiol decreases MMP-2 and MMP-9 activity in PC-3 cells. Forty-eight hours after plating, human prostate cells were changed to serum-free media, treated with the indicated concentrations of estradiol (1, 10 and 100 nM), and allowed to condition the media for 48 h. The cells were lysed and used for zymography, the MMP activity measured as described in Materials and methods.

Fig. 7. Photomicrograph of ethidium bromide and acridine orange (Etbr/Ao) staining of the PC-3 cells incubated for 48 h and visualized and photographed at ×20 magnification. (a) Control cells treated with 0.1% BSA, viable cells showed in green color stain. (b) 10 nM estradiol-treated cells, proapoptotic (yellow color stained) and apoptotic (reddish orange stained) cells were observed. (c) 100 nM estradiol-treated cells, a tremendous number of apoptotic (reddish orange stained) cells were observed. (For the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Diagram of Annexin V binding and propidium iodide (PI) influx in PC-3 cells after incubation with media alone or with 100 nM estradiol for 48 h. The lower left quadrants of each panels show the viable cells, which exclude PI and are negative for FITC–Annexin V binding. The upper right quadrants (Rl) contain the nonviable, apoptotic cells, positive for FITC–Annexin V binding and for PI uptake. The lower right quadrants (R2) represent the proapoptotic cells, FITC–Annexin V positive and PI negative demonstrating cytoplasmic membrane integrity. The upper right quadrants (R3) contain the nonviable necrotic cells, positive for FITC–Annexin V and for PI uptake. One representative experiment out of three is shown. The annexin and propidium iodide binding are analyzed quantifically (a). The data represent mean ± SEM of two independent experiments and indicate the percentage of cells staining positively for annexin V and PI. Bars with letter (a) indicated significance (P < 0.05) between control and estradiol-treated groups.

Fig. 9. DNA fragmentation pattern of PC-3 cells after the treatment of estradiol for 48 h. Lane 1, 100–1000 bp marker; lane 2, control cells; lane 3, 1 nM estradiol treatment; lane 4, 10 nM estradiol treatment; and lane 5, 100 nM estradiol treatment.