The anti-angiogenic potential of (±) gossypol in comparison to suramin

Abstract

Cotton, a staple fiber that grows around the seeds of the cotton plants (Gossypium), is produced throughout the world, and its by products, such as cotton fibers, cotton-seed oil, and cottonseed proteins, have a variety of applications. Cotton-seed contains gossypol, a natural phenol compound. (±)-Gossypol is a yellowish polyphenol that is derived from different parts of the cotton plant and contains potent anticancer properties. Tumor growth and metastasis are mainly related to angiogenesis; therefore, anti-angiogenic therapy targets the new blood vessels that provide oxygen and nutrients to actively proliferating tumor cells. The aim of the present study was to evaluate the anti-angiogenic potential of (±)-gossypol in vitro. (±)-Gossypol has anti-proliferative effects on cancer cell lines; however, its anti-angiogenic effects on normal cells have not been studied. Anti-proliferative activities of gossypol assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, anti-angiogenic activities using tube formation assay, and cell migration inhibition capability using a wound-healing assay on human umbilical vein endothelial cells (HUVECs) were revealed. (±)-Gossypol displayed the following potent anti-angiogenic activities in vitro: it inhibited the cell viability of HUVECs, it inhibited the migration of HUVECs, and disrupted endothelial tube formation in a dose-dependent manner. In addition, the anti-angiogenic effects of (±)-gossypol were investigated in ovo in a model using a chick chorioallantoic membrane (CAM). Decreases in capillary density were assessed and scored. (±)-Gossypol showed dose-dependent anti-angiogenic effects on CAM. These findings suggest that (±)-gossypol can be used as a new anti-angiogenic agent.

Keywords: (±)-Gossypol; Anti-angiogenesis; Human umbilical vein endothelial cell (HUVEC); Suramin.

Fig. 1

Inhibitory effect of gossypol (A) and suramin (B) on cell viability. Cells were incubated with various concentrations of gossypol and suramin for 24, 48, and 72 h, and cell viability was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. The results are expressed as the mean  ± SD. *Indicates significant difference from the control group using the Tukey test (P < 0.05)

Fig. 2

Inhibitory effect of gossypol and suramin on human umbilical vein endothelial cell (HUVEC) tube formation. A1 gossypol control cells, A2 suramin control cells, B1 dimethyl sulfoxide (DMSO) solvent control cells, B2 DMSO solvent control cells, C1 HUVECs were treated with 0.5 µM gossypol, C2 0.5 µM suramin, D1 2.5 µM gossypol, D2 2.5 µM suramin, E1 5 µM gossypol, E2 5 µM suramin, F1 10 µM gossypol, F2 10 µM suramin, G1 20 µM gossypol, G2 20 µM suramin, H1 30 µM gossypol, and H2 30 µM suramin. Images are representative of independent triplicate assays. Scale bar: 500 µm

Fig. 2

Inhibitory effect of gossypol and suramin on human umbilical vein endothelial cell (HUVEC) tube formation. A1 gossypol control cells, A2 suramin control cells, B1 dimethyl sulfoxide (DMSO) solvent control cells, B2 DMSO solvent control cells, C1 HUVECs were treated with 0.5 µM gossypol, C2 0.5 µM suramin, D1 2.5 µM gossypol, D2 2.5 µM suramin, E1 5 µM gossypol, E2 5 µM suramin, F1 10 µM gossypol, F2 10 µM suramin, G1 20 µM gossypol, G2 20 µM suramin, H1 30 µM gossypol, and H2 30 µM suramin. Images are representative of independent triplicate assays. Scale bar: 500 µm

Fig. 3

Effect of gossypol and suramin on cell migration. A1 Gossypol control cells, A2 suramin control cells, B1 dimethyl sulfoxide (DMSO) solvent control cells, B2 DMSO solvent control cells, C1 human umbilical vein endothelial cells (HUVECs) were treated with 0.5 µM gossypol, C2 0.5 µM suramin, D1 2.5 µM gossypol, D2 2.5 µM suramin, E1 5 µM gossypol, E2 5 µM suramin, F1 10 µM gossypol, F2 10 µM suramin, G1 20 µM gossypol, G2 20 µM suramin, H1 30 µM gossypol, and H2 30 µM suramin. Images are representative of independent triplicate assays. The black lines show the wound area created in the cell layer and the cell migration in the wound area after application of gosspol and suramin. Scale bar: 500 µm

Fig. 3

Effect of gossypol and suramin on cell migration. A1 Gossypol control cells, A2 suramin control cells, B1 dimethyl sulfoxide (DMSO) solvent control cells, B2 DMSO solvent control cells, C1 human umbilical vein endothelial cells (HUVECs) were treated with 0.5 µM gossypol, C2 0.5 µM suramin, D1 2.5 µM gossypol, D2 2.5 µM suramin, E1 5 µM gossypol, E2 5 µM suramin, F1 10 µM gossypol, F2 10 µM suramin, G1 20 µM gossypol, G2 20 µM suramin, H1 30 µM gossypol, and H2 30 µM suramin. Images are representative of independent triplicate assays. The black lines show the wound area created in the cell layer and the cell migration in the wound area after application of gosspol and suramin. Scale bar: 500 µm

Fig. 4

Effect of gossypol on chick chorioallantoic membrane (CAM). A1 Untreated CAM before, A2 untreated CAM after 24 h, B1 negative control (dimethyl sulfoxide; DMSO) before, B2 negative control (DMSO) after 24 h, C1 15 mM gossypol before, C2 15 mM gossypol, after 24 h, (the arrow indicates vascular development after treatment of gossypol) D1 15 mM suramin before, D2 15 mM suramin after 24 h, (the arrow indicates vascular development after treatment of suramin) E1, 30 mM gossypol before E2 30 mM gossypol after 24 h, (the arrow indicates vascular development after treatment of gossypol) F1 30 mM suramin before, F2 30 mM suramin after 24 h, (the arrow indicates vascular development after treatment of suramin) G1 60 mM gossypol before, G2 60 mM gossypol after 24 h, (the arrow indicates vascular development after treatment of gossypol) H1 60 mM suramin before, H2 60 mM suramin after 24 h, (the arrow indicates vascular development after treatment of suramin) I1 120 mM gossypol before, I2 120 mM gossypol after 24 h, (the arrow indicates vascular development after treatment of gossypol) J1 120 mM suramin before, J2 120 mM suramin after 24 h. (the arrow indicates vascular development after treatment of suramin). CAMs were photographed using 8x magnification

Fig. 5

Anti-angiogenic scores of gossypol (A) and suramin (B) at 15, 30, 60, and 120 mM. The data are expressed as the median (min–max) in scatter plots. P < 0.05

Fig. 6

Average antiangiogenic scores of gossypol (A) and suramin (B) at 15, 30, 60, and 120 mM. P < 0.05