| Angiogenesis, the arising of new blood vessels from preexisting ones, is a complex multi-step process, including the destabilization of established vessel, endothelial cell proliferation, migration, tube formation and remodeling of vascular basement membrane. Tumor angiogenesis is the expansion of a network of blood vessels penetrating into the cancerous growths to supply nutrients and oxygen and to remove metabolic waste from tumors. Tumor growth and metastasis depend on angiogenesis, and inhibiting angiogenesis is effective strategies for cancer treatment.There are many inhibitory factors and inducing factors, which regulate angiogenesis. And Vascular Endothelial Growth Factor (VEGF) is the crucial one of inducing factors, which acts on endothelial cells as both a chemotactic and mitogenic factor via endothelial cell-specific receptors:VEGFR-1(Flt-1), VEGFR-2(Flk-1/KDR), and VEGFR-3(Flt-4), among which vascular endothelial growth factor receptor2(VEGFR2) is the major mediator of pro-angiogenic effects induced by VEGF. In endothelial cells, VEGFR2signaling activates a number of downstream mediators, including Janus kinase2(Jak2)/signal transducer and activator of transcription-3(STAT3) and members of mitogen-activated protein kinase (MAPK) pathway. In previous studies, protein/peptide and small-molecule inhibitors of VEGFR2, including Rastuzumab, Bevacizumab, Sorafenib and Temsirolimus, have been shown to inhibit tumor angiogenesis. Many inhibitors of angiogenesis, such as sorafenib and sunitinib, were FDA approval granted for the treatment of patients with tumors.Here, we tested herbal monomer our abundant Chinese herbal medicine resources and novel compounds modified from natural small molecules with experiments in vitro and in vivo. We found some small molecule compounds could significantly inhibit tumor growth and metastasis, and revealed the novel molecular mechanism of cancer, provided potential drugs, and theoretical basis. Moreover, we found these compounds could inhibit tumor growth and metastasis, and explicated the related molecular mechanism. Detailed contents are follows.1. Cucurbitacin E (CuE, a-elaterin), a tetracyclic triterpenes compound from folk traditional Chinese medicine plants, has been shown to inhibit cancer cell growth, inflammatory response and bind bilirubin-albumin. However, the effects of Cucurbitacin E on tumor angiogenesis and its potential molecular mechanism are still unknown. Here we demonstrated that Cucurbitacin E significantly inhibited human umbilical vascular endothelial cells (HUVECs) proliferation, migration, and tubulogenesis in vitro and blocked angiogenesis in chick embryo chorioallantoic membrane assay and mouse corneal angiogenesis model in vivo. Furthermore, we found that Cucurbitacin E remarkably induced HUVECs apoptosis, inhibited tumor angiogenesis, and suppressed human prostate tumor growth in xenograft tumor model. Finally, we showed that Cucurbitacin E blocked VEGFR2mediated JaK2/STAT3signaling pathway in endothelial cells and suppressed the downstream protein kinases, such as extracellular signal-regulated kinase and p38mitogen-activated protein kinases. Therefore, our studies provided the first evidence that Cucurbitacin E inhibited tumor angiogenesis by inhibiting VEGFR2-mediated JaK/STAT3and MAPK signaling pathways and Cucurbitacin E is a potential candidate in angiogenesis related disease therapy. 2. While significant progress have been made in understanding the effect of Oridonin on anti-inflammation and anti-proliferation of tumor cells, little is known whether Oridonin could affect tumor angiogenesis and metastasis. Here, we showed that a small synthetic molecule extracted from Chinese herb Oridonin suppressed angiogenesis and metastasis in vivo. Importantly, Oridonin could significantly inhibit VEGF-induced the breast cancer cell line in mice,4T1transendothelial migration. In addition, we reported that Oridonin inhibits vascular endothelial growth factors (VEGF) induced cascade process through blocking Jagged2, the ligand of Notch1that triggered Notch signal pathway activation and promoted capillary-like tube formation in human umbilical vein endothelial cells (HUVECs). Oridonin suppressed significantly Neovascularization induced by VEGF in jagged1-expressing HUVECs in vitro. Moreover, the activity of Notch1was significantly down-regulated by Oridonin, and the same results were shown in the direct target genes of Notch1, including Hes-1and HESR1which were significantly correlated with blood vessel content, tube formation and associated with tumor development. Our results first elucidated a novel mechanism of Oridonin inhibits tumor growth and metastasis through regulating VEGF-induced Notch signaling pathway, and further provided evidence for therapy tumor by modulating Notch signal pathway.3. Pathologic retinal neovascularization occurs in various ocular disorders including proliferative diabetic retinopathy, retinopathy of prematurity and secondary neovascular glaucoma, which often result in angiogenesis and then blindness. However, Oridonin whether could inhibit retinal neovascularization has not been studied yet. We have demonstrated that Oridonin has anti-angiogenic activity in vitro and in mouse tumor previously. In this study, we assessed the efficacy of Oridonin both in vitro and in vivo and evaluated its anti-neovascularization mechanisms. We found that Oridonin significantly suppressed mice retinal neovascularization in vivo through inhibiting retinal angiogenesis. Moreover, we found the proteins in FAK/Paxillin pathway were down regulated significantly which extracted from the mice retina through study. Because the presence of the blood-ocular barrier and blood-retinal barrier, which make drugs in the eye when systemic administration is difficult to achieve an effective concentration, so most effective drugs for treatment of eye disease was Local Administration. Oridonin may be a candidate for treatment of angiogenesis in retinal Neovascularization diseases.4. Fatostatin is a small molecular compound, which binding to activator of SREBPs, which could significantly downregulate the activity of SREBPs. In addition, SREBPs are critical to angiogenesis. In this study, we design a series of small molecular compounds from Fatostatin. We found WB339has stronger inhibitory effect and lower cytotoxicity on angiogenesis among all the modified compounds. Further in vivo study found WB339could significantly inhibit tumor growth, angiogenesis, and tumor metastasis. In addition, the chemical structure of WB339is similar to Lysophospholipids acid (LPA) analogues. Previous studies reported LPA analogues inhibited prostate cancer cells proliferation through antagonizing LPA activate G protein coupling receptor (GPCRs). LPA is a simple natural phospholipids secreted by a variety of cells to the cell surface, which activating its receptor, namely the specific GPCRs, activate the downstream signal pathways. GPCRs are widely distributed in multiple types of cells, which play an important role in the development and execution of physiological function. Designing small molecules to adjust the corresponding signal pathways make in normal activation range is a promising drug development strategy.. In addition, WB339inhibited the activity of focal adhesion kinase (FAK) and its downstream protein expression, including the phosphorylated Paxillin and the changes of arranged F-actin, which revealed again that WB339inhibits tumor growth and metastasis through regulating FAK signal pathway. To sum up, WB339is probably an efficient antitumor growth and metastasis drug candidate, with low-toxic potential.Over all, we found three small molecular compounds, Cucurbitacin E, Oridonin, and WB339could effectively suppress tumor growth and metastasis. Cucurbitacin E inhibited tumor growth through inhibiting VEGFR2mediated JAK/STAT3signaling pathway. Oridonin regulated Notch signaling pathway to adjust the angiogenesis in pathological conditions. In addition, we found that Oridonin significantly inhibited proliferative retinopathy of angiogenesis by regulating FAK signal pathway. At the same time, we designed a series of small molecular compounds from Fatostatin, and found out a novel small molecule, WB339, which could inhibit tumor growth and tumor metastasis through regulating FAK signal pathway. We established and improved a series of studies of tumor angiogenesis models, including in vitro, in vivo, the molecular mechanisms and disease model. Moreover, providing an example for the study of tumor angiogenesis, as well as supplying anti-angiogenic drug candidate for treatment of tumors. |
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