Tissue ACE phenotyping in prostate cancer

Figures

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  Figure 1. ACE activity in homogenates of prostate tissues (I cohort). Homogenates (1:9 weight/volume ratio) were prepared from surgically removed prostate tissues of 4 patients with PC, from TURP-treated prostate tissues of 6 patients with BPH and from tissues of 2 unrelated patients (died due to accident) used as controls. (A–B) ACE activity was quantified using a spectrofluorometric assay with ZPHL (2 mM, A) and HHL (5 mM, B) as substrates calculated as mU/mg of protein and expressed as a % from mean value for control samples. (C) ZPHL/HHL ratio. Data for ACE activity were expressed as ratios of the rates of the hydrolysis of Z-Phe-His-Leu to rates of the hydrolysis of Hip-His-Leu for each sample and presented as a % from mean value for control samples. Values increased more than 20% were highlighted with orange; more than 50% with brown. Bars highlighted with yellow - values decreased more than 20%, with blue - more than 50%. Bars were highlighted if values were statistically significant (* p < 0.05). Data presented as a mean of at least 2 independent experiments in duplicates (with intra-assay standard deviations - SD <10%).

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  Figure 2. ACE activity in prostate tissues taken by biopsy (III cohort). (A–B) Prostate tissue homogenates were prepared from needle biopsies separately from 7 patients with BPH and 2 patients with PC. Homogenates from postmortem prostate tissue from 2 unrelated individuals (died due to accidents) served as controls. ACE activity was quantified as in Figure 1 legend and expressed as a % from mean values for control samples. (C) ZPHL/HHL ratio. Data presented as a mean of at least 2 independent experiments in du-or triplicates (with intra-assay standard deviations - SD <10%). Coloring was as in Figure 1, and individual values increased more than 100% were highlighted with red. Bars were highlighted if values were statistically significant (* p < 0.05) from control values. Values in ACE activity for PC (as a group) were significantly lower than for BPH group (p = 0.058 for ZPHL and p = 0.034 for HHL).

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  Figure 3. ACE conformational fingerprinting in prostate tissues (III cohort). ACE activity was precipitated by 4 different mAbs from homogenates from individual prostate biopsies from 2 PC patient, 7 BPH patients and 2 from postmortem counterparts. Data were presented as ratio of ACE activity precipitated by one of the tested mAbs to that precipitated by strongest mAb, 9B9. (A) 1G12/9B9: (B) 4E3/9B9, (C) 3F10/9B9. Data presented as a mean of at least 2 independent experiments in du-triplicates. Bars were highlighted if values were statistically significant (* p < 0.05). Coloring is as in Figure 1.

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  Figure 4. ACE conformational fingerprinting in biopsies from prostate tissues. ACE activity was precipitated by 3 different mAbs from homogenates from individual prostate biopsies: 6 of prostate cancer tissues, 18prostate tissues from patients with BPH and 6 from postmortem counterparts. Immunoprecipitated ACE activity is presented as ratio of ACE activity precipitated by one of the tested mAbs mAbs to that precipitated by mAb 9B9. (A) 3F10/9B9: (B) 4E3/9B9. (C) The structure of the C domain of ACE with marked potential glycosylation sites (by green) and epitopes for mAbs (by circles). Human C domain structure was based on PDB 1O86. The epitopes for mAbs were marked according to [42]. The positions of the epitopes are shown by circles. The potential sites of N-glycosylation are marked by green. Some amino acid residues are shown by numbers according to [11] for orientation. Data presented as a mean of at least 2 independent experiments. Bars were highlighted if values were statistically significant (* p < 0.05). Coloring – as in Figure 1.

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  Figure 5. ACE activity in biopsies of prostate tissues. Data on ACE activity in individual samples were presented for all biopsies that were analysed separately (cohorts III, IV and V): 6 samples of prostate cancer tissues, 18 prostate tissues from patients with BPH and 6 from postmortem counterparts. ACE activity was expressed as a % from mean value for control samples. (A) with ZPHL as a substrate; (B) with HHL as a substrate; (C) as ZPHL/HHL ratio. (D) the level of PSA in individual homogenates. Coloring as in Figure 1. Bars were highlighted if values were statistically significant (* p < 0.05). Data presented as a mean of at least 2 independent experiments in du-or triplicates (with intra-assay standard deviations - SD <10%). Values in ACE activity for PC (as a group) were significantly lower than for BPH group (p = 0.0000058 for ZPHL and p = 0.0000078 for HHL).

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  Figure 6. Prostate ACE phenotype in BPH and PC. Individual data on ACE phenotype characteristics were presented for all biopsies that were analysed separately as in Figure 5 and expressed as a % from mean value for control samples. (A) ACE activity with ZPHL (the same as Figure 5A). (B) amount of immunoreactive ACE protein (determined by precipitation with mAb 9B9. Values in ACE activity precipitation by mAb 9B9 for PC (as a group) were significantly lower than for BPH group (p = 0.000193). (C) the ratio of immunoreactive ACE protein to ACE activity. (D) PSA in the blood. Coloring as in Figure 1. Data presented as a mean of at least 2 independent experiments in du-or triplicates (with intra-assay standard deviations - SD <10%).