Limits...
Tylophorine, a phenanthraindolizidine alkaloid isolated from Tylophora indica exerts antiangiogenic and antitumor activity by targeting vascular endothelial growth factor receptor 2-mediated angiogenesis.

Saraswati S, Kanaujia PK, Kumar S, Kumar R, Alhaider AA - Mol. Cancer (2013)

Bottom Line: Therefore, we examined its anti-angiogenic effects and mechanisms in vitro and in vivo.Tylophorine significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells.Molecular docking simulation indicated that tylophorine could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit.

View Article: PubMed Central - HTML - PubMed

Affiliation: Camel Biomedical Research Unit, College of Pharmacy and Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia. saritasaraswati@gmail.com

ABSTRACT

Background: Anti-angiogenesis targeting VEGFR2 has been considered as an important strategy for cancer therapy. Tylophorine is known to possess anti-inflammatory and antitumor activity, but its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is still unknown. Therefore, we examined its anti-angiogenic effects and mechanisms in vitro and in vivo.

Methods: We used tylophorine and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVEC) in vitro and Ehrlich ascites carcinoma (EAC) tumor in vivo.

Results: Tylophorine significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR2 tyrosine kinase activity and its downstream signaling pathways including Akt, Erk and ROS in endothelial cells. Using HUVECs we demonstrated that tylophorine inhibited VEGF-stimulated inflammatory responses including IL-6, IL-8, TNF-α, IFN-γ, MMP-2 and NO secretion. Tylophorine significantly inhibited neovascularization in sponge implant angiogenesis assay and also inhibited tumor angiogenesis and tumor growth in vivo. Molecular docking simulation indicated that tylophorine could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit.

Conclusion: Tylophorine exerts anti-angiogenesis effects via VEGFR2 signaling pathway thus, may be a viable drug candidate in anti-angiogenesis and anti-cancer therapies.

Show MeSH

Related in: MedlinePlus

Western blot analysis of the effect of tylophorine on VEGFR2-mediated downstream signaling. (A) HUVECs were pre-treated with tylophorine followed by the stimulation with 50 ng/mL of VEGF for 20 min. Data were presented as means ± SEM, n = 6. (B). Effect of tylophorine on VEGF-induced MMP-2 secretion from HUVECs after 20 h examined by zymography. Data were presented as means ± SEM, n = 6. (C). Effect of tylophorine on HUVECs intracellular ROS level as detected by DCFH-DA staining assay. Data were presented as means ± SEM, n = 6. ##p < 0.01 VEGF-treated group versus no VEGF-treated group; **p < 0.01; ***p < 0.001 versus VEGF-stimulated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3733984&req=5

Figure 4: Western blot analysis of the effect of tylophorine on VEGFR2-mediated downstream signaling. (A) HUVECs were pre-treated with tylophorine followed by the stimulation with 50 ng/mL of VEGF for 20 min. Data were presented as means ± SEM, n = 6. (B). Effect of tylophorine on VEGF-induced MMP-2 secretion from HUVECs after 20 h examined by zymography. Data were presented as means ± SEM, n = 6. (C). Effect of tylophorine on HUVECs intracellular ROS level as detected by DCFH-DA staining assay. Data were presented as means ± SEM, n = 6. ##p < 0.01 VEGF-treated group versus no VEGF-treated group; **p < 0.01; ***p < 0.001 versus VEGF-stimulated group.

Mentions: Binding of VEGFR2 with VEGF led to the activation of various downstream signaling molecules responsible for endothelial cell migration, proliferation and survival [35]. To further delineate the mechanism that underlies the anti-angiogenic effects of tylophorine, we screened some key kinases involved in VEGFR2-mediated signaling pathway. VEGF induces survival of endothelial cells (ECs) mainly via the activation of AKT [37], whereas activation of ERK1/2 MAPKs is thought to be essential for VEGF-induced proliferation [38]. To assess the effect of tylophorine on these pathways, serum-starved HUVECs were treated with VEGF for 20 minutes in the presence or absence of tylophorine and cell lysates were subjected to immunodetection using antibodies against either P-AKT (Ser473) or P-ERK1/2. The result showed that P-ERK1/2 is enhanced by VEGF treatment while the expression level of ERK1/2 remains unchanged. Tylophorine was found to inhibit the phosphorylation of ERK1/2 at the concentration of 20 μM without affecting total ERK1/2 expression level (Figure 4A). A recent study suggests that the AKT/mTOR pathways and Hsp90, which are critical for angiogenesis, are phosphorylated or activated by VEGFR2 activation in the endothelial cells [39]. As shown in Figure 4A, expression levels of P-AKT and p-mTOR increases by VEGF treatment. Pretreatment of the HUVECs with tylophorine significantly inhibited the phosphorylation of AKT and mTOR, while the total amount of AKT and mTOR remains unchanged. Further, the action of tylophorine on the phosphorylation of FAK and Src were determined. The result showed that tylophorine inhibited VEGF-induced phosphorylation of FAK at the dose of 10 and 20 μM and Src at the concentration of 20 μM respectively (Figure 4A). Tylophorine could evidently inhibit VEGF-stimulated eNOS expression. In addition, both the MMP-9 and MMP-2 activities were suppressed with tylophorine treatment (Figure 4B). ROS is known to be downstream signaling after VEGFR2 activation [40], therefore, we detected the ROS levels by DCFH-DA probe. The results showed that the intracellular ROS levels were significantly reduced after tylophorine administration (Figure 4C). Taken together, our result revealed that tylophorine inhibited in vitro angiogenesis by directly targeting VEGFR2 on the surface of endothelial cells, and further downregulating VEGFR2-mediated signaling pathway.


Tylophorine, a phenanthraindolizidine alkaloid isolated from Tylophora indica exerts antiangiogenic and antitumor activity by targeting vascular endothelial growth factor receptor 2-mediated angiogenesis.

Saraswati S, Kanaujia PK, Kumar S, Kumar R, Alhaider AA - Mol. Cancer (2013)

Western blot analysis of the effect of tylophorine on VEGFR2-mediated downstream signaling. (A) HUVECs were pre-treated with tylophorine followed by the stimulation with 50 ng/mL of VEGF for 20 min. Data were presented as means ± SEM, n = 6. (B). Effect of tylophorine on VEGF-induced MMP-2 secretion from HUVECs after 20 h examined by zymography. Data were presented as means ± SEM, n = 6. (C). Effect of tylophorine on HUVECs intracellular ROS level as detected by DCFH-DA staining assay. Data were presented as means ± SEM, n = 6. ##p < 0.01 VEGF-treated group versus no VEGF-treated group; **p < 0.01; ***p < 0.001 versus VEGF-stimulated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733984&req=5

Figure 4: Western blot analysis of the effect of tylophorine on VEGFR2-mediated downstream signaling. (A) HUVECs were pre-treated with tylophorine followed by the stimulation with 50 ng/mL of VEGF for 20 min. Data were presented as means ± SEM, n = 6. (B). Effect of tylophorine on VEGF-induced MMP-2 secretion from HUVECs after 20 h examined by zymography. Data were presented as means ± SEM, n = 6. (C). Effect of tylophorine on HUVECs intracellular ROS level as detected by DCFH-DA staining assay. Data were presented as means ± SEM, n = 6. ##p < 0.01 VEGF-treated group versus no VEGF-treated group; **p < 0.01; ***p < 0.001 versus VEGF-stimulated group.
Mentions: Binding of VEGFR2 with VEGF led to the activation of various downstream signaling molecules responsible for endothelial cell migration, proliferation and survival [35]. To further delineate the mechanism that underlies the anti-angiogenic effects of tylophorine, we screened some key kinases involved in VEGFR2-mediated signaling pathway. VEGF induces survival of endothelial cells (ECs) mainly via the activation of AKT [37], whereas activation of ERK1/2 MAPKs is thought to be essential for VEGF-induced proliferation [38]. To assess the effect of tylophorine on these pathways, serum-starved HUVECs were treated with VEGF for 20 minutes in the presence or absence of tylophorine and cell lysates were subjected to immunodetection using antibodies against either P-AKT (Ser473) or P-ERK1/2. The result showed that P-ERK1/2 is enhanced by VEGF treatment while the expression level of ERK1/2 remains unchanged. Tylophorine was found to inhibit the phosphorylation of ERK1/2 at the concentration of 20 μM without affecting total ERK1/2 expression level (Figure 4A). A recent study suggests that the AKT/mTOR pathways and Hsp90, which are critical for angiogenesis, are phosphorylated or activated by VEGFR2 activation in the endothelial cells [39]. As shown in Figure 4A, expression levels of P-AKT and p-mTOR increases by VEGF treatment. Pretreatment of the HUVECs with tylophorine significantly inhibited the phosphorylation of AKT and mTOR, while the total amount of AKT and mTOR remains unchanged. Further, the action of tylophorine on the phosphorylation of FAK and Src were determined. The result showed that tylophorine inhibited VEGF-induced phosphorylation of FAK at the dose of 10 and 20 μM and Src at the concentration of 20 μM respectively (Figure 4A). Tylophorine could evidently inhibit VEGF-stimulated eNOS expression. In addition, both the MMP-9 and MMP-2 activities were suppressed with tylophorine treatment (Figure 4B). ROS is known to be downstream signaling after VEGFR2 activation [40], therefore, we detected the ROS levels by DCFH-DA probe. The results showed that the intracellular ROS levels were significantly reduced after tylophorine administration (Figure 4C). Taken together, our result revealed that tylophorine inhibited in vitro angiogenesis by directly targeting VEGFR2 on the surface of endothelial cells, and further downregulating VEGFR2-mediated signaling pathway.

Bottom Line: Therefore, we examined its anti-angiogenic effects and mechanisms in vitro and in vivo.Tylophorine significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells.Molecular docking simulation indicated that tylophorine could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit.

View Article: PubMed Central - HTML - PubMed

Affiliation: Camel Biomedical Research Unit, College of Pharmacy and Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia. saritasaraswati@gmail.com

ABSTRACT

Background: Anti-angiogenesis targeting VEGFR2 has been considered as an important strategy for cancer therapy. Tylophorine is known to possess anti-inflammatory and antitumor activity, but its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is still unknown. Therefore, we examined its anti-angiogenic effects and mechanisms in vitro and in vivo.

Methods: We used tylophorine and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVEC) in vitro and Ehrlich ascites carcinoma (EAC) tumor in vivo.

Results: Tylophorine significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR2 tyrosine kinase activity and its downstream signaling pathways including Akt, Erk and ROS in endothelial cells. Using HUVECs we demonstrated that tylophorine inhibited VEGF-stimulated inflammatory responses including IL-6, IL-8, TNF-α, IFN-γ, MMP-2 and NO secretion. Tylophorine significantly inhibited neovascularization in sponge implant angiogenesis assay and also inhibited tumor angiogenesis and tumor growth in vivo. Molecular docking simulation indicated that tylophorine could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit.

Conclusion: Tylophorine exerts anti-angiogenesis effects via VEGFR2 signaling pathway thus, may be a viable drug candidate in anti-angiogenesis and anti-cancer therapies.

Show MeSH
Related in: MedlinePlus