Procyanidin B2 3,3″-di-O-gallate inhibits endothelial cells growth and motility by targeting VEGFR2 and integrin signaling pathways

Abstract

Targeting angiogenesis, one of the hallmarks of carcinogenesis, using non-toxic phytochemicals has emerged as a translational opportunity for angioprevention and to control advanced stages of malignancy. Herein, we investigated the inhibitory effects and associated mechanism/s of action of Procyanidin B2-3,3″-di- O-gallate (B2G2), a major component of grape seed extract, on human umbilical vein endothelial cells (HUVECs) and human prostate microvascular endothelial cells (HPMECs). Our results showed that B2G2 (10-40 μM) inhibits growth and induces death in both HUVECs and HPMECs. Additional studies revealed that B2G2 causes a G1 arrest in cell cycle progression of HUVECs by down-regulating cyclins (D1 and A), CDKs (Cdk2 and Cdc2) and Cdc25c phosphatase and up-regulating CDK inhibitors (p21 and p27) expression. B2G2 also induced strong apoptotic death in HUVECs through increasing p53, Bax and Smac/Diablo expression while decreasing Bcl-2 and survivin levels. Additionally, B2G2 inhibited the growth factors-induced capillary tube formation in HUVECs and HPMECs. Interestingly, conditioned media (CCM) from prostate cancer (PCA) cells (LNCaP and PC3) grown under normoxic (~21% O2) and hypoxic (1% O2) conditions significantly enhanced the tube formation in HUVECs, which was compromised in presence of conditioned media from B2G2-treated PCA cells. B2G2 also inhibited the motility and invasiveness of both HUVECs and HPMECs. Mechanistic studies showed that B2G2 targets VEGFR2/PI3K/Akt and integrin signaling molecules which are important for endothelial cells survival, proliferation, tube formation and motility. Overall, we report that B2G2 inhibits several attributes of angiogenesis in cell culture; therefore, it warrants further investigation for efficacy for angioprevention and cancer control.

Fig. 1

Effect of B2G2 on endothelial cells growth and proliferation. (A) Chemical structure of Procyanidin B2 3,3″-di-O-gallate (B2G2). (B–C) HUVECs and HPMECs were grown in complete EGM-2 media with 2% FBS at the density of 5 × 10⁴ cell/well in six well plate. After 24 h of seeding, cells were treated with 10 to 40 μM concentrations of B2G2 for 6 h and 24 h. At the end of each time point, cells were harvested and counted as mentioned in ‘Materials and Methods’, and total cell number and percentage of dead cells are shown. Each value represents mean ± S.E. of three samples for each treatment. *p<0.05, significant with respect to control.

Fig. 2

Effect of B2G2 on cell cycle distribution and apoptosis in HUVECs. (A–B) HUVECs were grown in complete EGM-2 media with 2% FBS at the density of 4 × 10⁵ cell/60 mm plate. After 24 h of seeding, cells were treated with 10 to 40 μM concentrations of B2G2 for 24 h. At the end of experiment, cells were harvested and cell cycle distribution and apoptosis were analyzed by flow cytometry as described in ‘Materials and Methods’. Each value represents mean ± S.E. of three samples for each treatment. *p<0.05, significant with respect to control. (C–D) HUVECs were treated with B2G2 for 24 h as described above and total cell lysates were prepared and analyzed for cell cycle regulators (Cyclin D1, Cyclin A, Cdk2, Cdk4, Cdc2, Cdc25c, p21 and p27) and apoptosis-related molecules (cleaved PARP, p53, Bax, Smac/Diablo, Bcl-2, and Survivin) by immunoblotting. To check for protein loading, blots were stripped and re-probed with an antibody specific for β-actin.

Fig. 3

Effect of B2G2 on capillary tube formation by HUVECs and HPMECs. (A–B) Representative images depicting effect of B2G2 treatment (20–40 μM) on capillary tube formation by HUVECs and HPMECs on matrigel after 6 h and 8 h of cell seeding, respectively. Tubular structures were photographed at 100x magnification. (C) Tube length was measured as described in ‘Materials and Methods’. Tube length data is presented as mean ± standard error of three samples for each treatment. *p<0.05, significant with respect to BM treated group; #p<0.05, significant with respect to BM + GF treated group.

Fig. 4

Effect of B2G2 on Prostate cancer cells-induced capillary tube formation by HUVECs. (A) LNCaP and PC3 cells were treated with B2G2 (30 μM) and grown under normoxic (~21% O₂) and hypoxic (1% O₂) conditions and conditioned media was collected as described in ‘Materials and Methods’. HUVECs were seeded on matrigel along with 0.5% FBS containing media or conditioned media and EBM-2 (75:25 ratio) and capillary tube formation was analyzed after 6 h. Representative images depicting formation of capillary tubes are shown at 100x magnification. In this experiment, HUVECs incubated with 0.5% FBS containing LNCaP or PC-3 media alone served as a negative control. (B) Tube length was measured as described in ‘Materials and Methods’. Tube length data is presented as mean ± standard error of three samples for each treatment. *p<0.05, significant with respect to control group; #p<0.05, significant with respect to CCM treated group.

Fig. 5

Effect of B2G2 on invasion and migration of HUVECs and HPMECs. Representative images and graphical representation depicting the effect of B2G2 (20, 30 and 40 μM) on invasion (A and B) and migration (C and D) of HUVECs and HPMECs. Cells were allowed to invade and migrate for 10 h and 6 h, respectively, and non-invasive and non-migratory cells were removed by scraping with cotton swab. Invasive and migratory cells at the bottom of the membrane were fixed, stained and counted. Data are shown as percentage of cell invasion and migration compared to control (with control value as 100%). Each value represents mean ± S.E. of three samples for each treatment. All images are shown at 100x magnification. * p<0.05

Fig. 6

Effect of B2G2 on VEGFR2/PI3K/Akt and Integrin signaling pathways in HUVECs. HUVECs were treated with B2G2 (20–40 μM) for 6 h and total cell lysates were prepared and analyzed for mentioned signaling molecules by immunoblotting. To check for protein loading, blots were stripped and re-probed with an antibody specific for β-actin.