Figure 1.
Effect of constitutive Rap1 expression (cRap1) and/or 8CPT treatment on PC3 xenograft growth.
(A and B) Left Panel: Tumor volume (expressed in mm3) of human PC3 and PC3-cRap1 xenografts treated as indicated was measured as described in Methods. Day 0 represents the first day of treatment; *p<0.05, n = 7 for each group. Right Panel: Images of a representative tumor from each treatment group at the end of the experiment (Day 28). (C) Tumor volume (expressed in mm3) of human PC3 and PC3-cRap1 xenografts treated as indicated was measured as in A; *p<0.05, n = 7 for each group. (D) Tumors from panel C were weighed at the end of the experiment (Day 28) and expressed in grams; *p<0.05; (n = 7).
Figure 2.
Effect of 8CPT treatment on angiogenesis and tumor growth in PC3 and PC3-cRap1 xenografts.
(A and B) CD31 and VEGF immunoreactivity in PC3 and PC3-cRap1 tumors. Immunostaining was measured as described in Methods. Right Panels: Representative photomicrographs. Left Panels: Quantification of immunohistochemical staining of tumor slices; *p<0.05; n = 7; a.u. = arbitrary units. (C) Tumor volume (expressed in mm3) of human PC3-cRap1 xenografts. PC3-cRap1 cells were injected as xenografts at Day 0 and allowed to grow for 10 days before a osmotic minipump containing the treatments was implanted for constant infusion (Day 10, pump). Tumors treated as indicated were measured as described in Methods; *p<0.05; n = 8 for each group. (D) Tumor weight of human PC3-cRap1 xenografts. Tumors from panel C were weighed at the end of the experiment (Day 38) and expressed in grams; *p<0.05; (n = 8).
Figure 3.
Effect of 8CPT treatment on HIF-1α and VEGF in PC3 and PC3-cRap1 cells under hypoxic-like conditions.
(A) VEGF levels were measured by ELISA in PC3 and PC3-cRap1 cells under hypoxic-like conditions (CoCl2) as described in Methods; *p<0.05, (n = 3). (B) Immunoblot analysis of nuclear extracts from PC3 and PC3-cRap1 cells treated as indicated. HIF-1α protein levels were determined by immunoblotting with specific antibody; (C) Analysis of VEGF mRNA levels by qPCR in PC3 and PC3-cRap1 cells treated as indicated. Data were normalized to GAPDH levels; *p<0.05 (n = 3).
Figure 4.
Effect of SDF-1α on secreted VEGF in prostate cancer cells under hypoxic-like conditions.
Cells were treated as indicated and VEGF levels were measured by ELISA as described in Methods. (A and B) PC3 or PC3-cRap1 cells; *p<0.05, n = 3. (C and D) LNCaP or LNCaP-cRap1 cells; *p<0.05, n = 2.
Figure 5.
Effect of PKA inhibitor and PKA activator on secreted VEGF in PC3-cRap1 and LNCaP-Rap1 cells under hypoxic-like conditions.
Cells were treated as indicated, and VEGF levels were measured by ELISA as described in Methods. (A) Reversal of 8CPT effects by H-89 in PC3-cRap1 cells; *p<0.05, n = 3. (B) Reversal of 6BzcAMP (6Bn) effects by H-89 in PC3-cRap1 cells; *p<0.05, n = 3. (C) Reversal of 8CPT effects by H-89 in PC3-cRap1cells; *p<0.05, n = 3.
Figure 6.
Effect of PKA inhibitor on tumor growth and angiogenesis in PC3-cRap1 xenografts.
(A and B) The PKA inhibitor, H-89 (2.5 µmol), reversed tumor growth inhibition and tumor weight reduction mediated by 8CPT in PC3-cRap1 xenografts. PC3-cRap1 tumors from mice infused with PBS, 8CPT, or H-89 were measured and tumor volume determined as described in Methods; * p<0.05; n = 8 for each treatment group. (C) CD31 and VEGF immunoreactivity in PC3-cRap1 tumors treated as indicated. Immunostaining was measured as described in Methods. Upper Panels: Representative photomicrographs. Lower Panels: Quantification of immunohistochemical staining of tumor slices; *p<0.05; n = 8 for each treatment group; a.u. = arbitrary units.
Figure 7.
Schematic depicting interplay between Rap1 and PKA in the tumor microenvironment.