Figure 1.
Chemical structures of stilbenes.
Resveratrol (Res), trans-3,5,4′-trihydroxystilbene; Pterostilbene (PTER), trans-3,5-dimethoxystilbene; Trimethoxy-Resveratrol (3M-Res), trans-3,5,4′-trimethoxystilbene; Piceatannol (PIC), trans-3,5,3′4′-tetrahydroxystilbene; Dimethoxystyrylaniline (DMSA), trans-4-(3,5-dimethoxystyryl)aniline; Diacetyl-Resveratrol (2Ac-Res), trans-3,5-diacetylstilbene; Triacetyl-Resveratrol (3Ac-Res), trans-3,5,4′-triacetylstilbene.
Figure 2.
Differential expression of MTA1 in PCa cells analyzed by Western blot.
A. Cell lines represent different stages of PCa progression: RWPE-1, “normal” immortalized prostate epithelial cells; LNCaP, androgen-responsive cells; Du145, androgen-resistant cells; PC3M, aggressive metastatic cells. Cells were grown in RPMI-1640 media containing 10% FBS and antibiotics. 75 µg of protein was separated on 10% gel, transferred to membrane and probed with MTA1 antibodies. β-actin was a loading control. Pterostilbene has the highest potency in inhibiting MTA1. B. Dose-dependent effects of Res/analogues on MTA1 protein levels in LNCaP; in Du145 (C ) and in PC3M (D) cells. (i) cells were treated with 5–100 µM of Res/analogues for 24 hr and analyzed by immunoblotting. (ii) graphical representation of results. The Ctrl is set to 1 and MTA1 level changes are expressed as a percentage of Ctrl. The means±SE of four independent experiments are shown. •p<0.05, #p<0.01, *p<0.001. (iii) effective doses (ED50) were calculated by Chou-Martin method.
Figure 3.
Pterostilbene increases MTA1-mediated p53 acetylation in Du145 cells.
Du145-EV and Du145-MTA1shRNA cells (Fig. S1) were treated with 50 µM of Res or PTER for 24 hr and analyzed for MTA1, p53, Ac-p53 by Western blot as described in “Materials and methods”. A representative blot is shown. Quantitation of Ac-p53/p53 ratio was conducted by Image J software and data shown as mean±SEM from three independent experiments.
Figure 4.
MTA1-mediated therapeutic activity of resveratrol and pterostilbene in orthotopic PCa xenografts.
Male nude mice were injected orthotopically with Du145-EV-Luc (EV) or Du145-MTA1shRNA-Luc (MTA1shRNA) cells and treated with vehicle (Ctrl), Res or PTER, 50 mg/kg/day, every day, i.p. A. Normalized representative BL images of mice from each group are shown. B, left, Quantitative analysis of tumor light emission in Total Flux (photons/sec/cm2/sr) is plotted against time. The means ± SE are shown (n = 6 at the start), *p = 0.05. Right, log trends for each group are shown. Significant growth inhibition was detected in EV- vs. MTA1shRNA-tumors as groups, **p<0.01 and between EV-Ctrl vs. MTA1shRNA-Res and MTA1shRNA-PTER, ***p<0.001.
Figure 5.
Increased p53 acetylation and apoptosis and decreased angiogenesis by resveratrol and pterostilbene are linked to MTA1 inhibition.
Left, representative IHC images of A. Ki-67 (proliferation); B. Ac-p53/p53 (MTA1 signaling); C. M30 (apoptosis); and D. CD31 (angiogenesis) in EV- and MTA1shRNA-tumors are shown. Right, quantification of positively stained cells as percentage of total cells. Data are box plots of randomly chosen five fields analyzed independently by two investigators. Pairwise comparisons, *p<0.05; **p<0.01; ***p<0.001 vs. EV-Ctrl; #p<0.05 and ##p<0.001 vs. MTA1shRNA-Ctrl; +p<0.05 and ++p<0.001, of compounds between EV and MTA1shRNA groups.
Figure 6.
Effects of resveratrol and pterostilbene on spontaneous metastasis: involvement of MTA1.
A. BL images of metastasis are shown. Signals detected after prostate removal consisted of metastatic and non-specific signals. Removal of skin and muscles eliminated non-specificity. B. Top, ex vivo images of metastatic organs. Bottom, Validation of metastatic lesions (T) in kidneys (K), liver (Li) and lung/heart (L/H) by H&E staining. C, quantitative analysis of total metastatic Luc signals in Total Flux (photons/sec/cm2/sr). Open circles represent outliers. *p<0.05; **p<0.01; ***p<0.001 are pairwise comparisons vs. EV-Ctrl. D, quantitative analysis of organ-specific metastasis calculated by luciferase signals as Total Flux (photons/sec/cm2/sr). Color-coded histograms of signals for each group are shown. PTER was more effective in inhibiting metastasis in all organs compared to Res in EV-group. In MTA1-knockdown group, Res exhibited more effects by eliminating kidney metastasis.