Figure 1.
Casodex inhibits the EGF-triggered DNA synthesis in human fibrosarcoma HT1080 cells and reduces the growth of HT1080 xenografts.
Human fibrosarcoma HT1080 cells were used. In A, quiescent cells on coverslips were left un-stimulated or stimulated for 18 h with the indicated compounds. EGF (Roche) was used at 100 ng/ml; the synthetic androgen R1881 and DHT (both from Sigma) were used at 10 nM; Casodex (Sigma) was used at 10 μM. After in vivo pulse with BrdU (100 μM; Sigma), BrdU incorporation was analyzed by IF and expressed as % of total nuclei. Several independent experiments were performed in duplicate and the results were derived from at least 400 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. (**) p value< 0,001. Inset in A, shows the Western blot of HT1080 cell lysates with the antibodies against the indicated proteins. AR was revealed using two different antibodies raised against the AR amino (N-20) or the carboxyl (C-19)-terminal domains of the receptor. Tubulin (tub) was revealed by immunoblot, as a loading control. In B, xenografts were established in nude male mice as described in Methods. On alternate days, animals were intra-peritoneally injected with vehicle alone (vehicle), or the pure androgen antagonist Casodex (Cx: 0.1 μg/mice; 4µg/Kg body weight), or DHT (DHT: 0,1 μg/mice; 3 μg/Kg body weight) alone. Volumes of HT1080 cell xenografts were measured at the indicated times in two dimensions by a caliper and expressed as tumor mass (mm3). Mean and SEM are shown. n represents the number of experiments. (*) p value < 0,05.
Figure 2.
Inhibition by the S1 peptide of AR/Src complex, Src activation and DNA synthesis triggered by EGF in HT1080 cells.
Quiescent HT1080 cells were used. Cells were left un-stimulated or stimulated for 10 min with EGF (at 100 ng/ml) in the absence or presence of S1 or Ss peptide (both at 1 nM). Casodex (at 10 μM) was used for comparison with the S1 peptide. Upper section in A, Western blot of HT1080 cell lysates with anti-EGFR antibody. Tubulin (tubulin) was revealed by immunoblot, as a loading control. Lower section in A, lysates were immune-precipitated with anti-EGFR Ab and proteins in immune complexes were detected using antibodies against the indicated proteins. In B, lysates were immune-precipitated with the anti-Src MAb and Src activity in immune complexes was assayed using acidified enolase, as a substrate. In C, cells on coverslips were left untreated or treated for 18 h with the indicated compounds. After in vivo pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total nuclei. Several independent experiments were performed in duplicate and the results were obtained from at least 500 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. (*) p value < 0,001; (°) p value< 0,005.
Figure 3.
S1 peptide inhibits EGF-stimulated MMP-9 secretion, transmigration and wound closure in HT1080 cells.
Quiescent HT1080 cells were used. In A, the cells were left untreated or treated for 5 min with EGF (at 100 ng/ml), in the absence or presence of Casodex (Cx, at 10 μM), S1 or SS peptides (both peptides were used at 10 nM). MMP-9 protease activity was assayed in concentrated conditioned cell medium, as detailed in Methods. In B and C, the cells were left untreated or treated for 6 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex (Cx) was used at 10 μM; both S1 and SS peptides were used at 10 nM. In B, cells were allowed to migrate in collagen pre-coated Trans-well filters. Migrated cells were stained and counted as reported in Methods In A and B, results were derived from several independent experiments, each performed in duplicate. Data are expressed as relative increase. Mean and SEM are shown. n represents the number of experiments.
In A and B, (*) p value < 0.005; (**) p value < 0.001.
In C, contrast phase images from wounded cells were captured and shown. They are representative of 3 different experiments, each performed in duplicate.
Figure 4.
Casodex and S1 peptide inhibit EGF-stimulated BrdU incorporation and migration in LNCaP and MCF-7 cells.
Quiescent human prostate cancer-derived LNCaP cells were used. In A, quiescent cells on coverslips were left unstimulated or stimulated for 18 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex was used at 10 μM; S1 and Ss peptides were used at 10 nM. After in vivo pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total cells. Several independent experiments were performed in duplicate and the results were derived from at least 600 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. In B, the cells were left untreated or treated for 6 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex (Cx) was used at 10 μM; both S1 and SS peptides were used at 1 nM. Cells were allowed to migrate in collagen pre-coated Trans-well filters. Migrated cells were stained and counted as reported in Methods. Results were derived from several independent experiments, each performed in duplicate. Data are expressed as relative increase. Mean and SEM are shown. n represents the number of experiments.
Quiescent human breast cancer-derived MCF-7 cells were used. In C, quiescent cells on coverslips were left un-stimulated or stimulated for 18 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex was used at 10 μM; S1 and Ss peptides were used at 1nM. After in vivo pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total cells. Several independent experiments were performed in duplicate and the results were derived from at least 500 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. In D, the cells were left untreated or treated for 6 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex (Cx, at 10 μM); both S1 and SS peptides were used at 10 nM. Cells were allowed to migrate in collagen-pre-coated Trans-well filters. Migrated cells were stained and counted as reported in Methods. Results were derived from several independent experiments, each performed in duplicate. Data are expressed as relative increase. Mean and SEM are shown. n represents the number of experiments.
In A, B, C and D, (*) p value < 0.001; p value < 0,005 (**).
Panel E shows the Western blot of LNCaP or MCF-7 cell lysates with the antibodies against the indicated proteins: tubulin, epidermal growth factor receptor (EGFR) and androgen receptor (AR).
Figure 5.
Casodex and S1 peptide inhibit EGF-stimulated BrdU incorporation and migration in HCT116 and KP-2 cells.
Quiescent human colon cancer-derived HCT116 cells were used. In A, cells on coverslips were left un-stimulated or stimulated for 18 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex was used at 10 μM; S1 and Ss peptides were used at 10 nM. After in vivo pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total cells. Several independent experiments were performed in duplicate and the results were derived from at least 400 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. In B, the cells were left untreated or treated for 6 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex (Cx) was used at 10 μM; both S1 and SS peptides were used at 10 nM. Cells were allowed to migrate in collagen-pre-coated Trans-well filters. Migrated cells were stained and counted as reported in Methods. Results were derived from several independent experiments, each performed in duplicate. Data are expressed as relative increase. Mean and SEM are shown. n represents the number of experiments.
Quiescent human pancreatic cancer-derived KP-2 cells were used. In C, cells on coverslips were left unstimulated or stimulated for 18 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex was used at 10 μM; S1 and Ss peptides were used at 10 nM. After in vivo pulse with BrdU (100 μM), BrdU incorporation was analyzed by IF and expressed as % of total cells. Several independent experiments were performed in duplicate and the results were derived from at least 400 scored cells for each coverslip. Mean and SEM are shown. n represents the number of experiments. In D, the cells were left untreated or treated for 6 h with the indicated compounds. EGF was used at 100 ng/ml; Casodex (Cx) was used at 10 μM; both S1 and SS peptides were used at 10 nM. Cells were allowed to migrate in collagen-pre-coated Trans-well filters. Migrated cells were stained and counted as reported in Methods. Results were derived from several independent experiments, each performed in duplicate. Data are expressed as relative increase. Mean and SEM are shown. n represents the number of experiments.
In A, B, C and D, (*) p value < 0.001; (**) p value < 0,005.
Panel E shows the Western blot of HCT116 or KP-2 cell lysates with the antibodies against the indicated proteins: tubulin, epidermal growth factor receptor (EGFR) and androgen receptor (AR).