Mei-Hong Li

Quercetin, a dietary-derived flavonoid, possesses antiangiogenic potential

Quercetin, a dietary-derived flavonoid, suppresses tumor growth in vitro and in vivo, and inhibits the activity of tyrosine kinase. The effects of quercetin on the angiogenic process were examined in this study. Quercetin was found to inhibit several important steps of angiogenesis including proliferation, migration, and tube formation of human microvascular dermal endothelial cells in a dose-dependent manner. Additionally, the effect of quercetin on endothelial cell proliferation was confirmed using human umbilical vein endothelial cells. The activity of quercetin on the proliferation of endothelial cells was stronger than that on A549, BEL-7402, MKN-45 tumor cells and NIH-3T3 fibroblast cells. The chicken chorioallantoic membrane assay revealed that addition of quercetin displayed an antiangiogenic effect in vivo. After exposure to quercetin, a decrease in the expression and activity of matrix metalloproteinase-2, which is involved in the angiogenic process of migration, invasion, and tube formation, was observed by reverse transcription-polymerase chain reaction (RT-PCR) and gelatin zymography. These findings suggest that quercetin has antiangiogenic potential and that this effect may be related to an influence on the expression and activity of matrix metalloproteinase-2.

Pseudolaric Acid B Inhibits Angiogenesis and Reduces Hypoxia-Inducible Factor 1α by Promoting Proteasome-Mediated Degradation

Purpose: Pseudolaric acid B (PAB), the naturally occurring diterpenoid isolated from the root bark of Pseudolarix kaempferi Gordon tree (Pinaceae), possesses potent antifungal and pregnancy-terminating effects that may be tightly associated with angiogenesis. This study was to examine its angiogenic inhibition, impact on vascular endothelial growth factor (VEGF) secretion from tumor cells and the possible mechanism of action. Experimental Design: Angiogenesis inhibition was assessed by the human umbilical vascular endothelial cell proliferation, migration, and tube-formation assays, as well as the chorioallantoic membrane assay. ELISA, reverse transcription-PCR, and Western blotting analyses were performed to examine VEGF protein secretion, mRNA expression, and the possible mechanism in hypoxic MDA-MB-468 cells. Results: PAB displayed potent in vitro antiangiogenic activity shown by inhibiting VEGF-stimulated proliferation and migration and fetal bovine serum-stimulated tube formation of human umbilical vascular endothelial cells in a concentration-dependent manner. Moreover, PAB (10 nmol per egg) significantly suppressed in vivo angiogenesis in the chorioallantoic membrane assay. On the other hand, PAB abrogated hypoxia-induced VEGF secretion from MDA-MB-468 cells via reducing HIF-1α protein. Additional analyses using LY294002 and U0126 indicated that the increase in hypoxia-inducible factor 1 (HIF-1)α protein level was highly dependent on phosphatidylinositol 3′-kinase and p42/p44 mitogen-activated protein kinase activities in hypoxic MDA-MB-468 cells. However, PAB treatment did not affect the active (phosphorylated) forms of Akt and Erk. Interestingly, the selective proteasome inhibitor MG-132 completely reversed the reduction of HIF-1α protein in the PAB-treated MDA-MB-468 cells. Conclusions: PAB displays the dual antiangiogenic activities of directly inhibiting endothelial cells and abrogating paracrine stimulation of VEGF from tumor cells due to reducing HIF-1α protein by promoting its proteasome-mediated degradation in MDA-MB-468 cells, which has potential clinical relevance.

Grateloupia longifolia polysaccharide inhibits angiogenesis by downregulating tissue factor expression in HMEC‐1 endothelial cells

1 The antiangiogenic and antitumor properties of Grateloupia longifolia polysaccharide (GLP), a new type of polysaccharide isolated from the marine alga, were investigated with several in vitro and in vivo models. Possible mechanisms underlying its antiangiogenic activity were also assessed. 2 GLP dose‐dependently inhibited proliferation of human microvascular endothelial cells (HMEC‐1) and human umbilical vein endothelial cells (HUVEC), with IC50 values of 0.86 and 0.64 mg ml−1, respectively. In tube formation and cell migration assays using HMEC‐1 cells, noncytotoxic doses of GLP significantly inhibited formation of intact tube networks and reduced the number of migratory cells. Inhibition by GLP was VEGF‐independent. 3 In the chick chorioallantoic membrane (CAM) assay, GLP (2.5 μg egg−1) reduced new vessel formation compared with the vehicle control. GLP (0.1 mg plug−1) also reduced the vessel density in Matrigel plugs implanted in mice. 4 The levels of pan and phosphorylated recptors for VEGF, VEGFR‐1 (flt‐1) and VEGFR‐2 (KDR) were not significantly altered by 5 mg ml−1 GLP treatment of HMEC‐1, although tissue factor (TF) showed significant decreases at both mRNA and protein levels following GLP treatment. 5 In mice bearing sarcoma‐180 cells, intravenous administration of GLP (200 mg kg−1) decreased tumor weight by 52% without obvious toxicity. Vascular density in sections of the tumor was reduced by 64% after GLP treatment. 6 Collectively, these results indicate that GLP has antitumor properties, associated at least, in part, with the antiangiogenesis induced by downregulation of TF.

c-Jun Protects Hypoxia-Inducible Factor-1α from Degradation via Its Oxygen-Dependent Degradation Domain in a Nontranscriptional Manner

Although hypoxia-inducible factor-1alpha (HIF-1alpha) has long been intensively investigated as a drug target by interfering with its expression or transcriptional function, the regulatory mechanisms of HIF-1alpha remain to be further clarified. We report here that c-Jun associates with HIF-1alpha via its oxygen-dependent degradation domain, masks the sites for ubiquitination, and thus protects HIF-1alpha from proteasome-executing degradation. All of these together resulted in the stabilization and accumulation of HIF-1alpha, consequently promoting the transcription of its target gene and driving angiogenesis-related events. The stabilization of HIF-1alpha was dependent on the domains of c-Jun for DNA binding and heterodimerization but independent of the Ser(63/73) phosphorylation that is critical for transcriptional function. These findings highlight a previously unrecognized nontranscriptional function of c-Jun on the one hand and a distinct regulatory mechanism of HIF-1alpha activity on the other, consequently offering profound mechanistic insights into multiple events simultaneously involving both c-Jun and HIF-1alpha in tumor progression.

Antiangiogenic potential of 10-hydroxycamptothecin.

To investigate the antiangiogenic potential of 10-hydroxycamptothecin (HCPT), the proliferation of human microvascular endothelial cells (HMEC) and seven human tumor cell lines were detected by SRB assay, and the endothelial cell migration and tube formation were assessed using two in vitro model systems. Also, inhibition of angiogenesis was determined with a modification of the chick embryo chorioallantoic membrane (CAM) assay in vivo. Morphological assessment of apoptosis was performed by fluorescence microscope. HCPT 0.313-5 micromol x L(-1) treatment resulted in a dose-dependent inhibition of proliferation, migration and tube formation in HMEC cells, and HCPT 6.25-25 nmol x egg(-1) inhibited angiogenesis in CAM assay. HCPT 1.25-5 micromol x L(-1) elicited typical morphological changes of apoptosis including condensed chromatin, nuclear fragmentation, and reduction in volume in HMEC cells. HCPT significantly inhibited angiogenesis both in vitro and in vivo at relatively low concentrations, and this effect was related with induction of apoptosis in HMEC cells. These results taken collectively suggest that HCPT may be a potent antiangiogenetic and cytotoxic drug and further investigation is warranted.

Antimetastatic effect of Salvicine on human breast Cancer MDA-MB-435 orthotopic xenograft is closely related to rho-dependent pathway

Purpose: Salvicine is a novel DNA topoisomerase II inhibitor with potent anticancer activity. In present study, the effect of salvicine against metastasis is evaluated using human breast carcinoma orthotopic metastasis model and its mechanism is further investigated both in animal and cellular levels. Experimental Design: The MDA-MB-435 orthotopic xenograft model was applied to detect the antimetastatic effect of salvicine. Potential target candidates were detected and analyzed by microarray technology. Candidates were verified and explored by reverse transcription-PCR and Western blot. Salvicine activities on stress fiber formation, invasion, and membrane translocation were further investigated by immunofluorescence, invasion, and ultracentrifugal assays. Results: Salvicine significantly reduced the lung metastatic foci of MDA-MB-435 orthotopic xenograft, without affecting primary tumor growth obviously. A comparison of gene expression profiles of primary tumors and lung metastatic focus between salvicine-treated and untreated groups using the CLOTECH Atlas human Cancer 1.2 cDNA microarray revealed that genes involved in tumor metastasis, particularly those closely related to cell adhesion and motility, were obviously down-regulated, including fibronectin, integrin α3, integrin β3, integrin β5, FAK, paxillin, and RhoC. Furthermore, salvicine significantly down-regulated RhoC at both mRNA and protein levels, greatly inhibited stress fiber formation and invasiveness of MDA-MB-435 cells, and markedly blocked translocation of both RhoA and RhoC from cytosol to membrane. Conclusion: The unique antimetastatic action of salvicine, particularly its specific modulation of cell motility in vivo and in vitro, is closely related to Rho-dependent signaling pathway.

Pseudolaric acid B-driven phosphorylation of c-Jun impairs its role in stabilizing HIF-1alpha: a novel function-converter model

We have recently discovered that c-Jun executes a non-transcriptional function to stabilize hypoxia inducible factor 1α (HIF-1α) and that pseudolaric acid B (PAB) accelerates HIF-1α degradation and phosphorylates c-Jun at Ser63/73. In this study, PAB was used as a probe to investigate whether and how the Ser63/73 phosphorylation of c-Jun regulates its functions. The PAB-induced reduction of HIF-1α protein was rescued through supplying additional non-phosphorylated c-Jun. However, c-Jun siRNA, which reduced both the PAB-driven phosphorylated c-Jun and the total c-Jun protein, did not prevent the PAB-induced decrease in HIF-1α. HIF-1α was revealed to be co-immunoprecipitated only with the non-phosphorylated c-Jun. PAB increased the phosphorylated c-Jun while reducing the non-phosphorylated c-Jun at Ser63/73, which impaired its function in stabilizing HIF-1α. Consequently, PAB led to the degradation of HIF-1α, thus resulting in the decreased HIF-1α-dependent expression of mdr-1 and VEGF. We accordingly propose a function-converter model of c-Jun: the Ser63/73 phosphorylation serves as a function converter to convert c-Jun from its non-transcriptional function to its transcriptional function.