Zhengfang Yi

Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways

Thymoquinone, a component derived from the medial plant Nigella sativa, has been used for medical purposes for more than 2,000 years. Recent studies reported that thymoquinone exhibited inhibitory effects on cell proliferation of many cancer cell lines and hormone-refractory prostate cancer by suppressing androgen receptor and E2F-1. Whether thymoquinone inhibits tumor angiogenesis, the critical step of tumor growth and metastasis, is still unknown. In this study, we found that thymoquinone effectively inhibited human umbilical vein endothelial cell migration, invasion, and tube formation. Thymoquinone inhibited cell proliferation and suppressed the activation of AKT and extracellular signal-regulated kinase. Thymoquinone blocked angiogenesis in vitro and in vivo, prevented tumor angiogenesis in a xenograft human prostate cancer (PC3) model in mouse, and inhibited human prostate tumor growth at low dosage with almost no chemotoxic side effects. Furthermore, we observed that endothelial cells were more sensitive to thymoquinone-induced cell apoptosis, cell proliferation, and migration inhibition compared with PC3 cancer cells. Thymoquinone inhibited vascular endothelial growth factor–induced extracellular signal-regulated kinase activation but showed no inhibitory effects on vascular endothelial growth factor receptor 2 activation. Overall, our results indicate that thymoquinone inhibits tumor angiogenesis and tumor growth and could be used as a potential drug candidate for cancer therapy. [Mol Cancer Ther 2008;7(7):1789–96]

Gambogic Acid Inhibits Angiogenesis and Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2 Signaling

Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been previously reported to activate apoptosis in many types of cancer cell lines by targeting transferrin receptor and modulating nuclear factor-kappaB signaling pathway. Whether GA inhibits angiogenesis, which is crucial for cancer and other human diseases, remains unknown. Here, we found that GA significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, migration, invasion, tube formation, and microvessel growth at nanomolar concentration. In a xenograft prostate tumor model, we found that GA effectively inhibited tumor angiogenesis and suppressed tumor growth with low side effects using metronomic chemotherapy with GA. GA was more effective in activating apoptosis and inhibiting proliferation and migration in HUVECs than in human prostate cancer cells (PC3), suggesting GA might be a potential drug candidate in cancer therapy through angioprevention with low chemotoxicity. Furthermore, we showed that GA inhibited the activations of vascular endothelial growth factor receptor 2 and its downstream protein kinases, such as c-Src, focal adhesion kinase, and AKT. Together, these data suggest that GA inhibits angiogenesis and may be a viable drug candidate in antiangiogenesis and anticancer therapies.

Acetyl-11-Keto-β-Boswellic Acid Inhibits Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2-Mediated Angiogenesis

The role of angiogenesis in tumor growth and metastasis is well established. Identification of a small molecule that blocks tumor angiogenesis and is safe and affordable has been a challenge in drug development. In this study, we showed that acetyl-11-keto-beta-boswellic acid (AKBA), an active component from an Ayurvedic medicinal plant (Boswellia serrata), could strongly inhibit tumor angiogenesis. AKBA suppressed tumor growth in the human prostate tumor xenograft mice treated daily (10 mg/kg AKBA) after solid tumors reached approximately 100 mm(3) (n = 5). The inhibitory effect of AKBA on tumor growth was well correlated with suppression of angiogenesis. When examined for the molecular mechanism, we found that AKBA significantly inhibited blood vessel formation in the Matrigel plug assay in mice and effectively suppressed vascular endothelial growth factor (VEGF)-induced microvessel sprouting in rat aortic ring assay ex vivo. Furthermore, AKBA inhibited VEGF-induced cell proliferation, chemotactic motility, and the formation of capillary-like structures from primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Western blot analysis and in vitro kinase assay revealed that AKBA suppressed VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) kinase (KDR/Flk-1) with IC(50) of 1.68 micromol/L. Specifically, AKBA suppressed the downstream protein kinases of VEGFR2, including Src family kinase, focal adhesion kinase, extracellular signal-related kinase, AKT, mammalian target of rapamycin, and ribosomal protein S6 kinase. Our findings suggest that AKBA potently inhibits human prostate tumor growth through inhibition of angiogenesis induced by VEGFR2 signaling pathways.

Regulation of bone formation and remodeling by G-protein-coupled receptor 48

G-protein-coupled receptor (GPCR) 48 (Gpr48; Lgr4), a newly discovered member of the glycoprotein hormone receptor subfamily of GPCRs, is an orphan GPCR of unknown function. Using a knockout mouse model, we have characterized the essential roles of Gpr48 in bone formation and remodeling. Deletion of Gpr48 in mice results in a dramatic delay in osteoblast differentiation and mineralization, but not in chondrocyte proliferation and maturation, during embryonic bone formation. Postnatal bone remodeling is also significantly affected in Gpr48-/- mice, including the kinetic indices of bone formation rate, bone mineral density and osteoid formation, whereas the activity and number of osteoclasts are increased as assessed by tartrate-resistant acid phosphatase staining. Examination of the molecular mechanism of Gpr48 action in bone formation revealed that Gpr48 can activate the cAMP-PKA-CREB signaling pathway to regulate the expression level of Atf4 in osteoblasts. Furthermore, we show that Gpr48 significantly downregulates the expression levels of Atf4 target genes/proteins, such as osteocalcin (Ocn; Bglap2), bone sialoprotein (Bsp; Ibsp) and collagen. Together, our data demonstrate that Gpr48 regulates bone formation and remodeling through the cAMP-PKA-Atf4 signaling pathway.

Morelloflavone, a Biflavonoid, Inhibits Tumor Angiogenesis by Targeting Rho GTPases and Extracellular Signal-Regulated Kinase Signaling Pathways

Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown antioxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We showed that morelloflavone could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the mouse aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we showed that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway kinases without affecting VEGF receptor 2 activity. Together, our results indicate that morelloflavone exerts antiangiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action.

Cucurbitacin E, a tetracyclic triterpenes compound from Chinese medicine, inhibits tumor angiogenesis through VEGFR2-mediated Jak2-STAT3 signaling pathway.

Cucurbitacin E (CuE, α-elaterin), a tetracyclic triterpenes compound from folk traditional Chinese medicine plants, has been shown to inhibit cancer cell growth, inflammatory response and bilirubin-albumin binding. However, the effects of CuE on tumor angiogenesis and its potential molecular mechanism are still unknown. Here, we demonstrated that CuE significantly inhibited human umbilical vascular endothelial cell (HUVEC) 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 CuE remarkably induced HUVEC apoptosis, inhibited tumor angiogenesis and suppressed human prostate tumor growth in xenograft tumor model. Finally, we showed that CuE blocked vascular endothelial growth factor receptor (VEGFR) 2-mediated Janus kinase (Jak) 2-signal transducer and activator of transcription (STAT) 3 signaling pathway in endothelial cells and suppressed the downstream protein kinases, such as extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Therefore, our studies provided the first evidence that CuE inhibited tumor angiogenesis by inhibiting VEGFR2-mediated Jak-STAT3 and mitogen-activated protein kinases signaling pathways and CuE is a potential candidate in angiogenesis-related disease therapy.

Kisspeptin-10, a KISS1-Derived Decapeptide, Inhibits Tumor Angiogenesis by Suppressing Sp1-Mediated VEGF Expression and FAK/Rho GTPase Activation

Kisspeptin-10 (Kp-10), a decapeptide derived from the primary translation product of KISS1 gene, has been reported previously to be a key hormone for puberty and an inhibitor for tumor metastasis via the activation of G protein-coupled receptor 54. However, whether Kp-10 inhibits angiogenesis, which is critical for tumor growth and metastasis and other human diseases, is still unknown. Here we show that Kp-10 significantly inhibits human umbilical vein endothelial cell (HUVEC) migration, invasion, and tube formation, key processes in angiogenesis. Using chicken chorioallantoic membrane assay and vascular endothelial growth factor (VEGF)-induced mouse corneal micropocket assay, we show that Kp-10 inhibits angiogenesis in vivo. Furthermore, Kp-10 inhibits tumor growth in severe combined immunodeficient mice xenografted with human prostate cancer cells (PC-3) through inhibiting tumor angiogenesis, whereas Kp-10 has little effect on the proliferation of HUVECs and human prostate cancer cells. In deciphering the underlying molecular mechanisms, we show that Kp-10 suppresses VEGF expression by inhibiting the binding of specificity protein 1 to VEGF promoter and by blocking the activation of c-Src/focal adhesion kinase and Rac/Cdc42 signaling pathways in HUVECs, leading to the inhibition of tumor angiogenesis.

Hispidulin, a small flavonoid molecule, suppresses the angiogenesis and growth of human pancreatic cancer by targeting vascular endothelial growth factor receptor 2‐mediated PI3K/Akt/mTOR signaling pathway

Hispidulin, an active component from Artemisia vestita, a traditional Tibetan medicinal plant, has been shown to possess anti‐inflammatory and anti‐oxidative activities. However, the functional role of hispidulin on tumor growth and angiogenesis has not been elucidated. We found that hispidulin significantly inhibited human pancreatic tumor growth in xenograft mice when s.c. treated at a dosage of 20 mg/kg daily, and this effect was accompanied with a potent inhibition on angiogenesis. When examining the cytotoxicity of hispidulin on HUVECs and pancreatic cancer cells in vitro, we found that HUVECs were more susceptible to the treatment, suggesting angiogenesis might be the primary target of hispidulin. Our results further showed that hispidulin inhibited vascular endothelial growth factor (VEGF)‐induced cell migration, invasion, and capillary‐like structure formation of HUVECs in a dose‐dependent manner. In ex vivo and in vivo angiogenesis assays, we showed that hispidulin suppressed VEGF‐induced microvessel sprouting of rat aortic rings and corneal neovascularization in C57/BL6 mice. To understand the underlying molecular basis, we next examined the effects of hispidulin on different molecular components in treated HUVECs, and found that hispidulin suppressed the VEGF‐triggered activation of VEGF receptor 2, PI3K, Akt, mTOR, and ribosomal protein S6 kinase, but had little effect on focal adhesion kinase or extracellular signal‐regulated kinase at an effective concentration. Taken together, our results indicate that hispidulin targets the VEGF receptor 2‐mediated PI3K/Akt/mTOR signaling pathway in endothelial cells, leading to the suppression of pancreatic tumor growth and angiogenesis. (Cancer Sci 2011; 102: 219–225)

Maslinic acid potentiates the anti-tumor activity of tumor necrosis factor α by inhibiting NF-κB signaling pathway

BackgroundTumor necrosis factor alpha (TNFα) has been used to treat certain tumors in clinic trials. However, the curative effect of TNFα has been undermined by the induced-NF-κB activation in many types of tumor. Maslinic acid (MA), a pharmacological safe natural product, has been known for its important effects as anti-oxidant, anti-inflammatory, and anti-viral activities. The aim of this study was to determine whether MA potentiates the anti-tumor activity of TNFα though the regulation of NF-κB activation.ResultsIn this study, we demonstrate that MA significantly enhanced TNFα-induced inhibition of pancreatic cancer cell proliferation, invasion, and potentiated TNFα-induced cell apoptosis by suppressing TNFα-induced NF-κB activation in a dose- and time-dependent manner. Addition of MA inhibited TNFα-induced IκBα degradation, p65 phosphorylation, and nuclear translocation. Furthermore, MA decreased the expression levels of NF-κB-regulated genes, including genes involved in tumor cell proliferation (Cyclin D1, COX-2 and c-Myc), apoptosis (Survivin, Bcl-2, Bcl-xl, XIAP, IAP-1), invasion (MMP-9 and ICAM-1), and angiogenesis (VEGF). In athymic nu/nu mouse model, we further demonstrated that MA significantly suppressed pancreatic tumor growth, induced tumor apoptosis, and inhibited NF-κB-regulated anti-apoptotic gene expression, such as Survivin and Bcl-xl.ConclusionsOur data demonstrate that MA can potentiate the anti-tumor activities of TNFα and inhibit pancreatic tumor growth and invasion by activating caspase-dependent apoptotic pathway and by suppressing NF-κB activation and its downstream gene expression. Therefore, MA together with TNFα could be new promising agents in the treatment of pancreatic cancer.

Inhibition of STAT3 Signaling Pathway by Nitidine Chloride Suppressed the Angiogenesis and Growth of Human Gastric Cancer

STAT3 has been strongly implicated in human malignancies, and constitutive activation of STAT3 serves a crucial role in cell survival, angiogenesis, immune evasion, and inflammation. In this study, we showed that nitidine chloride, a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb) DC, exerts potent anticancer activity through STAT3 signaling cascade. Nitidine chloride dose dependently suppressed VEGF-induced endothelial cell proliferation, migration, and tubular structure formation in vitro and dramatically reduced VEGF-triggered neovascularization in mouse cornea and Matrigel plugs in vivo. This angiogenesis inhibition mediated by nitidine chloride was well interpreted by the suppression of Janus kinase 2/STAT3 signaling and STAT3 DNA-binding activity in endothelial cells. Furthermore, nitidine chloride suppressed the constitutively activated STAT3 protein, its DNA-binding activity, and the expression of STAT3-dependent target genes, including cyclin D1, Bcl-xL, and VEGF in human gastric cancer cells. Consistent with the earlier findings, nitidine chloride inhibited gastric tumor cell growth and induced tumor cell apoptosis in vitro and effectively suppressed the volume, weight, and microvessel density of human SGC-7901 gastric solid tumors (n = 8) at a dosage of 7 mg/kg/d (intraperitoneal injection). Immunohistochemistry and Western blot analysis further revealed that the expression of STAT3, CD31, and VEGF protein in xenografts was remarkably decreased by the alkaloid. Taken together, we propose that nitidine chloride is a promising anticancer drug candidate as a potent STAT3 signaling inhibitor. Mol Cancer Ther; 11(2); 277–87. ©2011 AACR.