MAZ51 Blocks the Tumor Growth of Prostate Cancer by Inhibiting Vascular Endothelial Growth Factor Receptor 3

Abstract

Vascular endothelial growth factor (VEGF) signaling plays a critical role in the carcinogenesis and tumor development of several cancer types. However, its pathological significance in prostate cancer, one of the most frequent and lethal malignancies in men, remains unclear. In the present study, we focused on a pathological role of the VEGF receptors (VEGFRs), and examined their expression and effects of MAZ51 (an inhibitor of the tyrosine kinase of VEGFR-3) on cell proliferation, migration, and tumor growth in human prostate cancer cells. The expression level of VEGFR-3 was higher in androgen-independent and highly metastatic prostate cancer PC-3 cells than in other prostate PrEC, LNCaP, and DU145 cells. In PC-3 cells, VEGFR-3 and Akt were phosphorylated following a stimulation with 50 ng/ml VEGF-C, and these phosphorylations were blocked by 3 μM MAZ51. Interestingly, PC-3 cells themselves secreted VEGF-C, which was markedly larger amount compared with PrEC, LNCaP, and DU145 cells. MAZ51 reduced the expression of VEGFR-3 but not VEGFR-1 and VEGFR-2. The proliferation of PC-3 cells was inhibited by MAZ51 (IC₅₀ = 2.7 μM) and VEGFR-3 siRNA, and partly decreased by 100 nM GSK690693 (an Akt inhibitor) and 300 nM VEGFR2 Kinase Inhibitor I. MAZ51 and VEGFR-3 siRNA also attenuated the VEGF-C-induced migration of PC-3 cells. Moreover, MAZ51 blocked the tumor growth of PC-3 cells in a xenograft mouse model. These results suggest that VEGFR-3 signaling contributes to the cell proliferation, migration, and tumor growth of androgen-independent/highly metastatic prostate cancer. Therefore, the inhibition of VEGFR-3 has potential as a novel therapeutic target for the treatment for prostate cancer.

Keywords: MAZ51; PC-3; VEGF-C; VEGFR-3; prostate cancer; vascular endothelial growth factor.

FIGURE 1

Up-regulation of VEGFR-3 in human prostate cancer PC-3 cells. The protein expression of VEGFRs was examined in human prostate epithelial PrEC cells and prostate cancer LNCaP (androgen-dependent/weakly metastatic), PC-3 (androgen-independent/highly bone metastatic), and DU145 (androgen-independent/moderate brain metastatic) cells by Western blotting. (A) Representative blots of VEGFR-1, VEGFR-2, and VEGFR-3 in PrEC, LNCaP, PC-3, and DU145 cells. β-actin was used as an endogenous marker. (B) Expression levels of VEGFR-1, VEGFR-2, and VEGFR-3 in PrEC, LNCaP, PC-3, and DU145 cells (n = 4–11). The expression levels of VEGFRs were normalized by that of β-actin. Note that the expression level of VEGFR-3 was higher in PC-3 cells than in PrEC, LNCaP, and DU145 cells. Data are presented as means ± S.D.

FIGURE 2

Abundant expression of VEGFR-3 in PC-3 cells. The expression of VEGFR-3 protein was examined in PrEC, LNCaP, PC-3, and DU145 cells by Western blotting using three different antibodies for VEGFR-3. (A) Representative blots of VEGFR-3 in PrEC, LNCaP, PC-3, and DU145 cells using three different VEGFR-3 antibodies: #79-633 (ProSci), sc-28297, and sc-321 (Santa Cruz Biotechnology). (B) Expression level of VEGFR-3 in PrEC, LNCaP, PC-3, and DU145 cells (n = 4). The expression level of VEGFR-3 were normalized by that of β-actin. (C) Representative blots of VEGFR-3 in PC-3 cells treated with 50 nM control or VEGFR-3 siRNA for 48 h. (D) Expression level of VEGFR-3 in PC-3 cells treated with control or VEGFR-3 siRNA (n = 6). Data are presented as means ± S.D. **p < 0.01 vs. control siRNA (unpaired two-tailed t-test).

FIGURE 3

Inhibition of the VEGF-C-induced phosphorylation of VEGFR-3 by MAZ51. The phosphorylation of VEGFRs after a stimulation with VEGF-C (a ligand for VEGFR-2 and VEGFR-3) in the absence and presence of MAZ51 in human prostate cancer PC-3 cells using Western blotting and co-immunoprecipitation methods. (A) Representative blots of phosphorylation of VEGFR-3 stimulated by 50 ng/ml VEGF-C in the absence and presence of 3 μM MAZ51 for 4 h in PC-3 cells. (B) The phosphorylation level of VEGFR-3 by the VEGF-C stimulation in the absence and presence of MAZ51 in PC-3 cells (n = 4). (C) Representative blots of phosphorylation of VEGFR-1 stimulated by VEGF-C in the absence and presence of MAZ51 in PC-3 cells. (D) The phosphorylation level of VEGFR-1 by the VEGF-C stimulation in the absence and presence of MAZ51 in PC-3 cells (n = 5). (E) Representative blots of phosphorylation of VEGFR-2 stimulated by VEGF-C in the absence and presence of MAZ51 in PC-3 cells. (F) The phosphorylation level of VEGFR-2 by the VEGF-C stimulation in the absence and presence of MAZ51 in PC-3 cells (n = 5). Data are presented as means ± S.D. *p < 0.05, **p < 0.01 vs. 0 min; ^## p < 0.01 vs. control (unpaired two-tailed t-test).

FIGURE 4

Phosphorylation pathway following VEGF-C-induced VEGFR-3 activation. The phosphorylation signaling after VEGF-C-induced VEGFR-3 activation was assayed in the absence and presence of MAZ51 in human prostate cancer PC-3 cells by Western blotting. (A) Representative blots of phosphorylation of Akt (Ser473), ERK1/2, and p38 after 50 ng/ml VEGF-C stimulation in the absence and presence of 3 μM MAZ51 for 4 h in PC-3 cells. (B–D) The phosphorylation level of Akt (B), ERK1/2 (C), and p38 (D) by the VEGF-C stimulation in the absence and presence of MAZ51 in PC-3 cells (n = 4). Data are presented as means ± S.D. *p < 0.05 vs. 0 min; ^# p < 0.05, ^## p < 0.01 vs. control (unpaired two-tailed t-test).

FIGURE 5

VEGF-C levels in human prostate cancer cells. The levels of VEGF-C in the culture media of human prostate epithelial PrEC cells and prostate cancer LNCaP, PC-3, and DU145 cells were measured using the human VEGF-C Quantikine ELISA kit. Note that larger amounts of VEGF-C were secreted from PC-3 cells than from PrEC, LNCaP, and DU145 cells (n = 10). Data are presented as means ± S.D. **p < 0.01 vs. PrEC, LNCaP, or DU145 cells (Scheffé’s test following ANOVA).

FIGURE 6

Down-regulation of VEGFR-3 expression and p-Akt levels by MAZ51. The effects of MAZ51 on VEGFR expression and its downstream signal pathway, Akt, were examined in human prostate cancer PC-3 cells by Western blotting. (A) Representative blots of VEGFR-3, p-Akt (Ser473), and total-Akt in the absence and presence of 1 and 3 μM MAZ51 for 48 h in PC-3 cells. (B–D) The expression levels of VEGFR-3 (B), p-Akt (C), and total-Akt (D) in the absence and presence of MAZ51 in PC-3 cells (n = 8). (E,F) Representative blots (E) and the expression levels (F) of VEGFR-1 in the absence and presence of MAZ51 in PC-3 cells (n = 4). (G,H) Representative blots (G) and the expression levels (H) of VEGFR-2 in the absence and presence of MAZ51 in PC-3 cells (n = 4). These expression levels were normalized by that of β-actin. Data are presented as means ± S.D. *p < 0.05, **p < 0.01 vs. 0 μM MAZ51 (Scheffé’s test following ANOVA).

FIGURE 7

Inhibitory effects of MAZ51 on the proliferation of human prostate cancer cells. The effects of MAZ51 on the cell viability and proliferation of human prostate epithelial PrEC cells and prostate cancer LNCaP, PC-3, and DU145 cells using MTT and BrdU assays, respectively. (A) Time-dependent cell growth of PrEC, LNCaP, PC-3, and DU145 cells (n = 9–16). (B) The effects of MAZ51 for 48 h on the viability of PrEC, LNCaP, PC-3, and DU145 cells (n = 8–34). The IC₅₀ values of MAZ51 on the viabilities of PrEC, LNCaP, PC-3, and DU145 cells were 7.0, 6.0, 2.7, and 3.8 μM, respectively. (C) The effects of 100 nM GSK690693 (an Akt inhibitor) for 48 h on the viability of PrEC, LNCaP, PC-3, and DU145 cells (n = 12–18). (D) The effects of VEGFR2 Kinase Inhibitor I for 48 h on the viability of PrEC, LNCaP, PC-3, and DU145 cells (n = 12–24). (E) The effects of MAZ51 for 48 h on the proliferation of PC-3 cells (n = 12). (F) The effects of 50 and 100 nM VEGFR-3 siRNA for 48 h on the proliferation of PC-3 cells (n = 16–24). Data are presented as means ± S.D. *p < 0.05, **p < 0.01 vs. 6 h, 0 μM drug, or control siRNA; ^# p < 0.05, ^## p < 0.01 vs. PrEC cells (Scheffé’s test following ANOVA).

FIGURE 8

Inhibitory effects of MAZ51 and VEGFR-3 siRNA on the migration of human prostate cancer PC-3 cells. The effects of VEGF-C on the migration of human prostate cancer PC-3 cells in the absence and presence of MAZ51 or VEGFR-3 siRNA were examined using the Transwell kit. (A) Representative images in the absence and presence of 50 ng/ml VEGF-C and 3 μM MAZ51 for 18 h in PC-3 cells. (B) The effects of VEGF-C and MAZ51 on the migration of PC-3 cells (n = 4). (C) Representative images in the absence and presence of VEGF-C for 18 h in PC-3 cells treated with 50 nM VEGFR-3 siRNA for 48 h. (D) The effects of VEGF-C and VEGFR-3 siRNA on the migration of PC-3 cells (n = 3). Data are presented as means ± S.D. *p < 0.05, **p < 0.01 vs. control or control siRNA/control; ^## p < 0.01 vs. VEGF-C alone or control siRNA/VEGF-C (Scheffé’s test following ANOVA).

FIGURE 9

Inhibition of the tumor growth of PC-3 cells by MAZ51. The effects of MAZ51 on the tumor growth of human prostate cancer PC-3 cells were examined in a xenograft mouse model. (A) Representative tumors after the treatment with vehicle (0.1% DMSO), 1, or 3 μM MAZ51 for 1–4 weeks. (B,C) Tumor volumes (B) and weights (C) after the treatment with vehicle or MAZ51 for 1–4 weeks (n = 5–6). Note that the VEGFR-3 kinase inhibition by MAZ51 effectively blocked the tumor growth of prostate cancer in ex vivo model. Data are presented as means ± S.D. *p < 0.05, **p < 0.01 vs. vehicle control (non-parametric Mann-Whitney U-test).