Cyperenoic acid, a sesquiterpene derivative from Croton crassifolius, inhibits tumor growth through anti-angiogenesis by attenuating VEGFR2 signal pathway in breast cancer.

BACKGROUND: Cyperenoic acid, one of the main chemical constituents of the root of Croton crassifolius, exhibited potent anti-angiogenic property on the zebrafish embryo model with little cytotoxicity. Nevertheless, its anti-angiogenic mechanism and anti-tumor effect have not been investigated. PURPOSE: To investigate the anti-angiogenic mechanisms of cyperenoic acid and evaluate it whether could exert anti-tumor effect by inhibiting angiogenesis. STUDY DESIGN: Targeting vascular endothelial growth factor receptor-2 (VEGFR2) pathway to inhibit tumor angiogenesis is a significant strategy for cancer treatment. Initially, the anti-angiogenic effect of cyperenoic acid as well as the mechanisms of the action was studied using both in-vitro and in-vivo methodologies. Then, its anti-tumor effect through anti-angiogenesis by attenuating VEGFR2 signaling pathway was evaluated. METHODS: The in-vitro inhibitory effect of cyperenoic acid on the vascular endothelial growth factor (VEGF)-induced angiogenesis was evaluated using human umbilical vein endothelial cells (HUVECs) model. Moreover, its ex-vivo and in-vivo effects were evaluated using the aortic ring assay and the matrigel plug assay. The influence of the cyperenoic acid on tyrosine phosphorylation of VEGFR2 was studied by western blotting assay and the influence on downstream signaling pathway of VEGFR2 also be detected. Computer-docking simulations were carried out to study the interaction between cyperenoic acid and VEGFR2. Finally, its inhibitory effect on tumor growth was studied using breast cancer xenograft model. RESULTS: Cyperenoic acid possessed little toxicity to HUVECs, but it significantly inhibited VEGF-induced proliferation, invasion, migration and tube formation of HUVECs. Moreover, it inhibited VEGF-induced sprout formation ex vivo and vessel formation in vivo. Further mechanistic study showed that cyperenoic acid could suppress VEGFR2 tyrosine kinase activity and alter its downstream signaling pathways in VEGF-induced HUVECs. In addition, it could form two hydrogen bonds with the ATP binding pocket of the VEGFR2 kinase domain by docking. For breast cancer xenograft model, cyperenoic acid suppressed tumor growth, but no obvious toxic pathologic changes were observed in mice. Besides, it suppressed the phosphorylation of VEGFR2 in tumor, demonstrating its anti-angiogenic ability in vivo partly targeting the VEGFR2. CONLUSION: Cyperenoic acid could exert anti-tumor effect in breast cancer by inhibiting angiogenesis via VEGFR2 signaling pathway.

Penduliflaworosin, a Diterpenoid from Croton crassifolius, Exerts Anti-Angiogenic Effect via VEGF Receptor-2 Signaling Pathway.

Anti-angiogenesis targeting vascular endothelial growth factor receptor-2 (VEGFR-2) has been considered as an important strategy for cancer therapy. Penduliflaworosin is a diterpenoid isolated from the plant Croton crassifolius. Our previous study showed that this diterpenoid possesses strong anti-angiogenic activity by inhibiting vessel formation in zebrafish. This study was conducted to further investigate the anti-angiogenic activity and mechanism of penduliflaworosin. Results revealed that penduliflaworosin significantly inhibited VEGF-induced angiogenesis processes including proliferation, invasion, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Moreover, it notably inhibited VEGF-induced sprout formation of aortic rings and blocked VEGF-induced vessel formation in mice. Western blotting studies showed that penduliflaworosin inhibited phosphorylation of the VEGF receptor-2 and its downstream signaling mediators in HUVECs, suggesting that the anti-angiogenic activity was due to an interference with the VEGF/VEGF receptor-2 pathway. In addition, molecular docking simulation indicated that penduliflaworosin could form hydrogen bonds within the ATP-binding region of the VEGF receptor-2 kinase unit. Finally, cytotoxicity assay showed that penduliflaworosin possessed little toxicity toward both cancer and normal cells. Taken together, our findings demonstrate that penduliflaworosin exerts its anti-angiogenic effect via the VEGF receptor-2 signaling pathway. The anti-angiogenic property and low cytotoxicity of penduliflaworosin suggest that it may be useful in cancer treatments.

Pyran-2-one Derivatives from the Roots of Croton crassifolius.

One new pyran-2-one derivative [crotonpyrone C (1)] and two known ones (2-3) were isolated from the supercritical fluid extract (SFE) of the roots of Croton crassifolius. Their structures were elucidated using spectroscopic (IR, UV, 1D and 2D NMR) and HRESIMS methods. The isolated compounds were evaluated for their anti-angiogenic activity using a zebrafish model, but showed little activity.

Anti-angiogenic activity and mechanism of kaurane diterpenoids from Wedelia chinensis.

BACKGROUND: Wedelia chinensis is a traditional medicinal herb used in Asia and it has been reported to possess various bioactivities including anti-inflammatory and anticancer effects. However, its anti-angiogenic activity has never been reported. PURPOSE: To determine the most potent anti-angiogenic component in W. chinensis and its molecular mechanism of action. STUDY DESIGN: Initially, the active fraction of the plant was studied. Then, we determined the active components of the fraction and explored the mechanism of the most active compound. METHODS: The ethanol extract of W. chinensis and its four fractions with different polarities were evaluated for their anti-angiogenic activity in the Zebrafish model using quantitative endogenous alkaline phosphatase (EAP) assay. The molecular mechanism of the most active compound from the active fraction was studied using the real-time polymerase chain reaction (PCR) assay on Zebrafish embryos. The inhibitory effect of the most active compound on the proliferation, invasion and tube formation steps of angiogenesis was evaluated using the vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs) model, and the influences of the active compound on tyrosine phosphorylation of VEGF receptor (VEGFR-2) and its downstream signal pathway were evaluated by western blotting assay. Moreover, its anti-angiogenic effect was further evaluated by the VEGF-induced sprouts formation on aortic ring assay and the VEGF-induced vessel formation of mice on matrigel plug assay, respectively. RESULTS: Petroleum ether (PE) fraction of the plant displayed potent anti-angiogenic activity. Twelve kaurane diterpenoids (1-12) isolated from this fraction showed quite different effects. Compounds 9-12 could dose-dependently inhibit vessel formation in the Zebrafish embryos while the others showed little inhibitory effect. Among the active diterpenoids, compound 10, 3α-cinnamoyloxy-9ß-hydroxy-ent-kaura-16-en-19-oic acid (CHKA), possessed the strongest effect, and it affected multiple molecular targets related to angiogenesis including VEGF and angiopoietin in Zerbrafish. Moreover, CHKA significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, invasion, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR-2 tyrosine kinase activity and its downstream signaling pathways in HUVECs. CHKA also obviously inhibited sprouts formation of aortic ring, and block vessel formation in mice. CONCLUSION: Our findings demonstrate that kaurane diterpenoids is one of anti-angiogenic components in W. chinensis, and CHKA may become a promising candidate for the development of anti-angiogenic agent.

Potent anti-angiogenic component in Croton crassifolius and its mechanism of action.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Croton crassifolius Geisel is traditionally used in China for the treatment of snake bites, stomach ache, sternalgia, joint pain, pharyngitis, jaundice and rheumatoid arthritis, while in Thailand, it has been used as an anticancer herbal medicine by the indigenous people. Yet, its pharmacological studies are still limited, especially towards its anticancer property. Anti-angiogenesis is a promising therapeutic strategy in the anti-cancer treatment. Previous studies have shown strong anti-angiogenic activity in the low polar fraction of the herb. Nevertheless, the potent compound which is responsible for the anti-angiogenesis, and its molecular mechanism have never been reported. AIM OF THE STUDY: To determine the potent anti-angiogenic component in C. crassifolius and its molecular mechanism of action. MATERIALS AND METHODS: C. crassifolius was extracted using supercritical fluid extraction and steam distillation. The anti-angiogenic activities of the two extracts were evaluated in the zebrafish model by quantitative endogenous alkaline phosphatase assay. The chemical compounds in the active extract were isolated using chromatographic methods, and their structures were elucidated using different spectroscopic techniques. The content/quantity of the active compounds in this extract was determined with HPLC analysis. The molecular mechanism of the most active compound was further studied using the real-time PCR assay. Besides, its cytotoxicity on various cancer and normal cell lines was evaluated using the cell-counting kit. RESULTS: Supercritical fluid extract (SFE) of C. crassifolius showed better anti-angiogenic activity than that of steam distillation extract (SDE). Three sesquiterpenes, namely, cyperenoic acid, 8-hydroxy-α-guaiene and (+)-guaia-l(10),ll-dien-9-one, were isolated and identified in the SFE. Among them, cyperenoic acid displayed the strongest anti-angiogenic activity by 51.7% of the control at 10µM, while the others showed little effect. HPLC results showed that cyperenoic acid was the major component in the SFE with 9.97% (w/w). Results of the real-time PCR assay suggested that the cyperenoic acid affected multiple molecular targets related to angiogenesis including vascular endothelial growth factor (Vegfa), angpiopoietin (Angpt), and their receptors. Cytotoxicity assay showed cyperenoic acid possessed little toxicity toward cancer and normal cells. CONCLUSIONS: Cyperenoic acid is an important anti-angiogenic component present in C. crassifolius and serve as a potent inhibitor in the angiogenesis in the zebrafish embryo model. The anti-angiogenic property, but not the cytotoxicity, of C. crassifolius provides a scientific basis for its traditional use in cancer treatment.