(-)-Gossypol suppresses the growth of human prostate cancer xenografts via modulating VEGF signaling-mediated angiogenesis

Abstract

(-)-Gossypol, a natural BH3-mimetic and small-molecule Bcl-2 inhibitor, shows promise in ongoing phase II clinical trials for human cancers. However, whether (-)-gossypol plays functional roles in tumor angiogenesis has not been directly elucidated yet. In this study, we showed that (-)-gossypol dose dependently inhibited the expression of VEGF, Bcl-2, and Bcl-xL in human prostate cancer cells (PC-3 and DU 145) and primary cultured human umbilical vascular endothelial cells (HUVEC) in vitro. Notably, the growth of human prostate tumor PC-3 xenografts in mice was significantly suppressed by (-)-gossypol at a dosage of 15 mg/kg/d. This inhibitory action of (-)-gossypol in vivo was largely dependent on suppression of angiogenesis in the solid tumors, where VEGF expression and microvessel density were remarkably decreased. Furthermore, (-)-gossypol inhibited VEGF-induced chemotactic motility and tubulogenesis in HUVECs and human microvascular endothelial cells and suppressed microvessel sprouting from rat aortic rings ex vivo. When examined for the mechanism, we found that (-)-gossypol blocked the activation of VEGF receptor 2 kinase with the half maximal inhibitory concentration of 2.38 μmol/L in endothelial cells. Consequently, the phosphorylation of key intracellular proangiogenic kinases induced by VEGF was all suppressed by the treatment, such as Src family kinase, focal adhesion kinase, extracellular signal-related kinase, and AKT kinase. Taken together, the present study shows that (-)-gossypol potently inhibits human prostate tumor growth through modulating VEGF signaling pathway, which further validates its great potential in clinical practice.

Figure 1. (−)-Gossypol decreases cell viability via apoptosis induction and inhibits Bcl-2/Bcl-xL/VEGF signaling in prostate cancer cells and endothelial cells

A, the chemical structure of (−)-gossypol. B, (−)-gossypol inhibited prostate cancer cell viability in a dose-dependent manner. PC-3 and DU 145 cells (5~6×10³ cells) were directly treated with or without various concentrations of (−)-gossypol for 48 h. Cell viability was quantified by MTS assay. Columns, mean; bars, standard error; **, P < 0.01 vs. untreated group. C, (−)-gossypol induced potent apoptosis in prostate cancer cells. PC-3 and DU 145 cells were treated with (−)-gossypol for 24 h. The whole cell protein was applied to western blotting analysis. The full length of PARP was cleaved into 89 KD form as indicated. D, (−)-gossypol suppressed the expression of VEGF, Bcl-2 and Bcl-xL in human prostate cancer cells and endothelial cells. PC-3, DU 145 and HUVECs were incubated with (−)-gossypol for 24 h. The whole cell protein was harvested and probed with specific antibodies.

Figure 2. (−)-Gossypol suppresses tumor growth and angiogenesis of human prostate tumor xenografts

PC-3 cells were injected into 5- to 6-week-old BALB/cA nude mice (5×10⁶ cells per mouse). After solid tumors established, the mice were subcutaneously treated with or without (−)-gossypol at dosage of 15 mg/kg daily. A, (−)-gossypol inhibited tumor growth as measured by tumor volume. B, the weight of solid tumors in the (−)-gossypol-treated mice was significantly lower than that of the control group. C, anti-CD 31, anti-VEGF and anti-VEGFR2 immunohistochemistry revealed that (−)-gossypol inhibited neovascularization and VEGF expression in solid tumors. D, microvessel density was analyzed by Image-Pro Plus 6.0 software. Columns, mean; bars, standard error; *, P < 0.05 vs. the control group.

Figure 3. (−)-Gossypol inhibits VEGF-induced viability, chemotactic motility and capillary-like structure formation of endothelial cells

A, (−)-gossypol dose-dependently inhibited VEGF-induced cell viability. HUVECs (6~7×10³ cells/well) were starved with serum-free medium and then treated with or without VEGF (50 ng/mL) and various concentrations of (−)-gossypol for 48 h. Cell viability was quantified by MTS assay. Columns, mean; bars, standard error; **, P < 0.01 vs. VEGF alone group. B, (−)-gossypol inhibited cell migration in HUVECs and HMEC-1. Endothelial cells were seeded in the upper chamber of Transwells and treated with different concentrations of (−)-gossypol. The bottom chamber was filled with medium supplemented with 30 ng/mL VEGF. Cells that migrated through the membrane were photographed (magnification, 160×). C, (−)-gossypol inhibited the capillary-like structure formation in endothelial cells. Pretreated HUVECs and HMEC-1 were placed in 24-well plates coated with Matrigel. After 4~6 h, cells were fixed, and tubular structures were photographed (original magnification, 100×).

Figure 4. (−)-Gossypol inhibits VEGF-induced microvessel sprouting ex vivo

Aortic segments isolated from Sprague-Dawley rats were placed in Matrigel-covered wells and treated with VEGF in the presence or absence of (−)-gossypol for 6 d. A, representative photographs of endothelial cell sprouts from aortic rings. B, sprouts were scored from 0 (least positive) to 5 (most positive) in a double-blinded manner. Columns, mean; bars, standard error; **, P < 0.01 vs. VEGF alone group.

Figure 5. (−)-Gossypol is a VEGFR2 kinase inhibitor

A, (−)-gossypol suppressed the activation of VEGFR2 triggered by VEGF in endothelial cell. HUVECs were starved in serum-free medium for 4~6 h, pretreated with (−)-gossypol for 30 min, and then stimulated with 50 ng/mL VEGF for 2 min. The activation of VEGFR2 from different treatments was analyzed by western blotting and probed with anti-phospho-VEGFR2 antibody at Tyr 1175 and Tyr 996 sites. B, the relative optical density was qualified by Image J software. C, (−)-gossypol inhibited VEGFR2 kinase activity in vitro. Dots, mean; Bars, standard error.

Figure 6. (−)-Gossypol inhibits activation of key proangiogenic molecules involved in VEGF signaling

A, (−)-gossypol inhibited VEGF-induced activation of Src, FAK, AKT and ERK kinase in endothelial cells. Several key signaling molecules that mediate angiogenesis were analyzed by western blotting assay. HUVECs were starved in serum-free medium for 4~6 h, pretreated with (−)-gossypol for 30 min, and then stimulated with 50 ng/mL VEGF for 5~20 min. Protein was harvested for the analysis. B, schematic model depicted the (−)-gossypol-mediated antiangiogenic signaling pathway. (−)-Gossypol potentially affected the production of VEGF released from tumor cells and endothelial cells by inhibition of Bcl-2 family protein, and further it blocked the activation of VEGFR2 and intracellular kinases in vascular endothelial cells.