Figure 1.
Finasteride’s effects on MMP2 and MMP9 activities in the conditioned medium of DU145 cells.
a) Conditioned medium (CM) of untreated (Control) and finasteride-treated DU145 cells was collected, concentrated and analyzed for MMP2 and MMP9 activities using a gelatin zymography assay. (S) Standards, (P) Tissue extract of a gastric ulcer that was used as a positive control. These cells secreted high quantities of MMP2 and MMP9 into their CM, as shown by the bright bands on the dark background. b) High-dose finasteride (50 µM) downregulated MMP2 activity up to 43% after 72 hours of exposure, as quantified using the ImageJ™ software. Data are expressed as a fold-change graphic of the IOD values that were obtained from the bands in the (a) figure for finasteride-treated cells over control cells. (**) Statistically significant values with p<0.01. Experiments were carried out in triplicate.
Figure 2.
Finasteride’s effects on MMP2 and MMP9 activity and TIMP-1 and TIMP-2 expression in the conditioned medium of RWPE-1 cells.
a) Conditioned medium of untreated (Control) and finasteride-treated RWPE-1 cells were collected, concentrated and analyzed for MMP2 and MMP9 activities using their respective Biotrak® Activity Assays. Low-dose finasteride (10 µM) for 72 hours of exposure downregulated MMP2 and MMP9 activity by 25% and 30%, respectively, when compared to control values. High-dose finasteride (50 µM) downregulated MMP2 and MMP9 activities at all tested time points, up to 90% and 55% after 72 hours of exposure, respectively. b) Conditioned medium of untreated (Control) and finasteride-treated RWPE-1 cells were collected, concentrated and analyzed for TIMP-1 and TIMP-2 protein expression using their respective Biotrak® Assays. Finasteride exposure, at both doses, induced the upregulation of TIMP-2 expression at the 72 hour time point, up to 150% more expression than control levels. Data are expressed as a fold-change graphic of the IOD values that were obtained for finasteride-treated cells over those of the control cells. (*) Statistically significant values with p<0.05.
Figure 3.
Finasteride’s effects on MMP2 and MMP9 activity and TIMP-1 and TIMP-2 expression in the conditioned medium of PC3 cells.
a) Conditioned medium of untreated and finasteride-treated PC3 cells was collected, concentrated and analyzed for MMP2 and MMP9 activities using their respective Biotrak® Activity Assays. Low-dose finasteride exposure (10 µM) did not induce the downregulation of MMP2 or MMP9 activities, except at the 24 hour time point. High-dose finasteride induced the downregulation of MMP2 at the 48 hour and 72 hour time points and of MMP9 at all assessed time points, up to 70% reduction. b) Conditioned medium of untreated (Control) and finasteride-treated PC3 cells was collected, concentrated and analyzed for TIMP-1 and TIMP-2 protein expression using their respective Biotrak® Assays. Finasteride exposure did not induce any significant modulation of TIMP-1 and TIMP-2 expression, except for the 10 µM finasteride dose at 24 hours of exposure. Data are expressed as a fold-change of the IOD values obtained for finasteride treated cells over control cells. (*) Statistically significant values with p<0.05.
Figure 4.
Finasteride’s effects on the migration of the human prostate cell lines RWPE-1, LNCaP, PC3 and DU145.
The prostate cell lines were previously cultivated in 50 µM finasteride or control medium for 72 hours and individually cultivated in 200 µl of serum-free medium (with or without 50 µM finasteride) in the migration insert at a density of 1×105 total cells. After 22 hours, cells that migrated through the 8 µm porous membrane were fixed, stained and counted within five random fields using a light microscope. Experiments were performed in triplicate. Representative photomicrographs of untreated and finasteride-treated cells are shown on the left side. Human fibroblasts (WS1 cell line) were employed as migration-positive control cells, and a representative image is shown on the bottom-left side. Scale bar = 40 µm. Finasteride significantly inhibited the cell migration of the RWPE-1 and LNCaP cell lines but not of the PC3 and DU145 cell lines. Data are expressed as the mean ± SD of the migrating cells. (*) Statistically significant values with p<0.05. (**) Statistically significant values with p<0.01.
Figure 5.
Finasteride’s effects on the migration of the human prostate cell lines PC3 and DU145 in the wound closure migration assay.
Cells were cultivated in 6-well plates at 4×104 cells/cm2 until 100% confluence was achieved. The monolayers were scratched in a straight line, and the wounded monolayers were then given culture medium with or without 50 µM finasteride. The wound area was inspected after 24, 48, 72 and 96 hours using an inverted phase contrast microscope with a digital camera. a) Representative images showing the wound closure of finasteride-treated and control PC3 cells at 0 to 72 hours. Red highlighted areas represent the open-wound area. Scale bar = 50 µm. b) The initial red highlighted areas were measured using the ImageJ™ software, and the remaining areas were calculated as a percentage of the initial wound area. A wound-closure graphic was made by dividing the area values that were obtained at the indicated time points for the control and finasteride-treated cells. Finasteride slightly inhibited cell migration; however, this difference was not statistically significant (p>0.05). c) Representative images showing the wound closure of finasteride-treated and control DU145 cells at 0 to 72 hours. Red highlighted areas represent the open wound area. Scale bar = 50 µm. d) As observed for the PC3 cells, finasteride slightly inhibited cell migration (p>0.05).
Figure 6.
Finasteride’s effects on the invasion of the human prostate cell lines RWPE-1, LNCaP, PC3 and DU145.
The prostate cell lines were previously cultivated in 50 µM finasteride or control medium for 72 hours and individually cultivated in 200 µl of serum-free medium (with or without 50 µM finasteride) in the Matrigel® invasion insert at a density of 1×105 total cells. After 22 hours, cells that invaded through the matrigel matrix and porous membrane were fixed, stained and counted within five random fields using a light microscope. Experiments were performed in triplicate. Representative images of untreated and finasteride-treated cells are shown on the left side. Scale bar = 40 µm. Finasteride significantly inhibited the cell invasion of all tested cell lines. Data are expressed as the mean ± SD of the invading cells. (*) Statistically significant values, with p<0.05. (**) Statistically significant values, with p<0.01. (***) Statistically significant values, with p<0.001.
Figure 7.
Western blots of whole protein extracts from RWPE-1, LNCaP, PC3 and DU145 cell lines.
The prostate cell lines were cultivated in regular medium until 100% confluence and individually processed for protein extraction. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane and probed with an anti-androgen receptor (AR) antibody. After incubation with the secondary antibody, the bands were visualized with DAB. All cell lines employed in this investigation expressed AR except for the DU145 cell line. Experiments were carried out in triplicate. (MW) Molecular Weight Standard. The estimated MW of AR is approximately 110 kDa.