Figure 1.
Structural formula of compound AZA1.
(N*2*,N*4*-Bis-(2-methyl-1H-indol-5-yl)-pyrimidine-2,4-diamine).
Figure 2.
Compound AZA1 inhibits Rac1 and Cdc42 activation.
A, Rac1 B, Cdc42 and C, RhoA activation in 22Rv1, DU145 and PC3 prostate cancer cells after incubation (60 min) with different concentrations of compound AZA1 and stimulation with 50 ng/ml EGF. Means of three independent experiments are shown. *, significantly different from EGF.
Figure 3.
Effects of Rac1 and Cdc42 inhibition by AZA1 on cell proliferation in 22Rv1 prostate cancer cells.
A, Relative density of cancer cells up to 72 h following treatment with 2, 5, and 10 µM compound AZA1 in unstimulated (left panel) or EGF-stimulated (right panel) cancer cells was measured using the WST-1 cell proliferation assay. AZA1 suppresses 22Rv1 prostate cancer cell proliferation in both unstimulated and EGF-stimulated cancer cells in a dose-dependent manner. Means of three independent experiments are shown. *, significantly different from control (left panel) and from control and EGF-stimulated cells (right panel); +, significantly different from control (right panel);. B, Representative flow cytometry histograms showing cell populations in sub- G0/G1, G0/G1, S and G2/M phases. 22Rv1 cells were incubated with 10 µM AZA1 for 24 h. Control cells received no treatment. Cellular DNA content was analyzed by flow cytometry after staining with propidium iodide. C, Cyclin D1 expression. Representative flow cytometry analysis and quantification of fluorescence intensity in 22Rv1 cells treated with 10 µM AZA1 for 60 min (red histogram) compared to untreated cells (bold line) and isotype controls (thin line). Compound treatment reduced Cyclin D1 levels. *, significantly different vs. control.
Figure 4.
Rac1 and Cdc42 blockade reduces prostate cancer cell migration and affects cytoskeletal dynamics.
A, Representative images of migrated prostate cancer cells from an in vitro migration assay are shown. 22Rv1 prostate cancer cells were stimulated with 50 ng/ml EGF and treated with 2, 5 or 10 µM AZA1 for 24h and migrated cancer cells quantified subsequently in in vitro migration assays. Data were collected from five individual consecutive fields of view (40x) from three replicate Boyden chambers. *, significantly different from control; +, significantly different from control and EGF-stimulated cells. B, Effect of AZA1 treatment on lamellipodia and filopodia formation. 22Rv1 prostate cancer cells were plated on cell culture chambers, stimulated with 50 ng/ml EGF and incubated with 5 and 10 µm AZA1 for 24 h. Paraformaldehyde fixed cells were stained with Atto-488 phalloidin (F-actin, green) to detect polymerized actin cytoskeleton, filopodia and lamellipodia and counterstained with DAPI (blue) and photographed (magnification, x1000). Arrow head indicates filopodia, arrow indicates lamellipodia. The numbers of filopodia and lamellipodia per cell were calculated from 25 cells in each group. AZA1 leads to changes in cellular morphology and suppresses filopodia and lamellipodia formation. *, significantly different from controls; +, significantly different from controls and EGF-stimulated cells; ‡, significantly different from EGF-stimulated cells. Co, control. C, Effect of AZA1 on actin dynamics. Expressions of F-actin and G-actin in 22Rv1, DU 145 and PC-3 cells analyzed by immunoblotting (F-actin/G-actin ratio). Bars represent the F/G actin mean value ±SD. *, significantly different from controls of the respective cell line; +, significantly different from controls and EGF-stimulated cells of the respective cell line.
Figure 5.
AZA1 down-regulates PAK signaling.
Analysis of PAK, AKT and BAD-phosphorylation in EGF-stimulated 22Rv1 prostate cancer cells following AZA1 treatment. Representative Western blot images and quantification of immunoblots stained with phospho-PAK1/2 (pPAK), phospho-AKT (pAKT) and phospho-BAD (pBAD) antibodies before and after treatment with 2, 5 and 10 µM AZA1 for 24 h. Rac1/Cdc42 blockade reduces phosphorylation of PAK1, AKT and BAD in 22Rv1 prostate cancer cells compared to controls (means of 3 independent experiments). *, significantly different from unstimulated and untreated controls; +, significantly different from EGF-stimulated control; ‡, significantly different from unstimulated, untreated control and EGF-stimulated control. SP, specific protein; LC, loading control.
Figure 6.
Rac1 and Cdc42 blockade suppresses tumor growth and prolongs animal survival.
A, Tumor volume curves in 22Rv1 tumor xenograft bearing mice treated with AZA1 or solvent only (Control). *, significantly different from control. B, representative images of human prostate tumor xenografts on day 24 from mice treated with solvent (Control) or 100 µg/day AZA1 (AZA1 d24) (bar = 1 cm). C, AZA1 significantly suppresses tumor weight of human prostate xenografts in mice. Data are shown as mean tumor weights on day 24. *, significantly different from control on day 24. D, left panel: Representative immunohistochemistry images of tumor tissue sections from mice treated with solvent (Control) or 100 µg/day AZA1 on day 24 stained with Ki-67 antibody (bar = 200 µm). Cellular proliferation is reduced following Rac1 and Cdc42 blockade. Right panel: Quantitative histomorphometric analysis of Ki-67-positive, proliferating tumor cells. *, significantly different from control. E, effect of AZA1 treatment on animal survival in 22Rv1 prostate cancer bearing mice. AZA1 significantly prolongs animal survival vs. untreated controls.
Figure 7.
Proposed AZA1 regulated pathways downstream of Rac1 and Cdc42 in prostate cancer.
AZA1 inhibits activation of Rac1 and Cdc42 GTPases, shifting the balance towards cell growth inhibition and apoptosis. These effects are exerted by different mechanisms. AZA1 inhibits the PAK pathway via the cell cycle regulator Cyclin D1 and suppresses PAK and AKT activation leading to reduced BAD phosphorylation to induce pro-apoptotic activity. In addition, suppression of Rac1 and Cdc42 suppresses cancer cell migration and invasion by affecting actin polymerization and subsequent inhibition of lamellipodia and filopodia formation.