Jian Luo

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.

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.

Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2

The development of the anterior segment of the mammalian eye is critical for normal ocular function, whereas abnormal development can cause glaucoma, a leading cause of blindness in the world. We report that orphan G protein-coupled receptor 48 (Gpr48/LGR4) plays an important role in the development of the anterior segment structure. Disruption of Gpr48 causes a wide spectrum of anterior segment dysgenesis (ASD), including microphthalmia, iris hypoplasia, irdiocorneal angle malformation, cornea dysgenesis, and cataract. Detailed analyses reveal that defective iris myogenesis and ocular extracellular matrix homeostasis are detected at early postnatal stages of eye development, whereas ganglion cell loss, inner nuclear layer thinness, and early onset of glaucoma were detected in 6-month-old Gpr48−/− mice. To determine the molecular mechanism of ASD caused by the deletion of Gpr48, we performed gene expression analyses and revealed dramatic down-regulation of Pitx2 in homozygous knockout mice. In vitro studies with the constitutively active Gpr48 mutant receptor demonstrate that Pitx2 is a direct target of the Gpr48-mediated cAMP-CREB signaling pathway in regulating anterior segment development, suggesting a role of Gpr48 as a potential therapeutic target of ASD.

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.

Orphan G protein-coupled receptors (GPCRs): biological functions and potential drug targets

The superfamily of G protein-coupled receptors (GPCRs) includes at least 800 seven-transmembrane receptors that participate in diverse physiological and pathological functions. GPCRs are the most successful targets of modern medicine, and approximately 36% of marketed pharmaceuticals target human GPCRs. However, the endogenous ligands of more than 140 GPCRs remain unidentified, leaving the natural functions of those GPCRs in doubt. These are the so-called orphan GPCRs, a great source of drug targets. This review focuses on the signaling transduction pathways of the adhesion GPCR family, the LGR subfamily, and the PSGR subfamily, and their potential functions in immunology, development, and cancers. In this review, we present the current approaches and difficulties of orphan GPCR deorphanization and characterization.

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.

Ubiquitin-specific protease 4 (USP4) targets TRAF2 and TRAF6 for deubiquitination and inhibits TNFα-induced cancer cell migration

TRAF [TNF (tumour necrosis factor)-receptor-associated factor] 2 and 6 are essential adaptor proteins for the NF-κB (nuclear factor κB) signalling pathway, which play important roles in inflammation and immune response. Polyubiquitination of TRAF2 and TRAF6 is critical to their activities and functions in TNFα- and IL (interleukin)-1β-induced NF-κB activation. However, the regulation of TRAF2 and TRAF6 by deubiquitination remains incompletely understood. In the present study, we identified USP (ubiquitin-specific protease) 4 as a novel deubiquitinase targeting TRAF2 and TRAF6 for deubiquitination. We found that USP4 specifically interacts with TRAF2 and TRAF6, but not TRAF3. Moreover, USP4 associates with TRAF6 both in vitro and in vivo, independent of its deubiquitinase activity. The USP domain is responsible for USP4 to interact with TRAF6. Ectopic expression of USP4 inhibits the TRAF2- and TRAF6-stimulated NF-κB reporter gene and negatively regulates the TNFα-induced IκBα (inhibitor of NF-κBα) degradation and NF-κB activation. Knockdown of USP4 significantly increased TNFα-induced cytokine expression. Furthermore, we found that USP4 deubiquitinates both TRAF2 and TRAF6 in vivo and in vitro in a deubiquitinase activity-dependent manner. Importantly, the results of the present study showed that USP4 is a negative regulator of TNFα- and IL-1β-induced cancer cell migration. Taken together, the present study provides a novel insight into the regulation of the NF-κB signalling pathway and uncovers a previously unknown function of USP4 in cancer.

LGR4 is a receptor for RANKL and negatively regulates osteoclast differentiation and bone resorption

Tumor necrosis factor (TNF) superfamily member 11 (TNFSF11, also known as RANKL) regulates multiple physiological or pathological functions, including osteoclast differentiation and osteoporosis. TNFRSF11A (also called RANK) is considered to be the sole receptor for RANKL. Herein we report that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL. LGR4 competes with RANK to bind RANKL and suppresses canonical RANK signaling during osteoclast differentiation. RANKL binding to LGR4 activates the Gαq and GSK3-β signaling pathway, an action that suppresses the expression and activity of nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (NFATC1) during osteoclastogenesis. Both whole-body (Lgr4−/−) and monocyte conditional knockout mice of Lgr4 (Lgr4 CKO) exhibit osteoclast hyperactivation (including elevation of osteoclast number, surface area, and size) and increased bone erosion. The soluble LGR4 extracellular domain (ECD) binds RANKL and inhibits osteoclast differentiation in vivo. Moreover, LGR4-ECD therapeutically abrogated RANKL-induced bone loss in three mouse models of osteoporosis. Therefore, LGR4 acts as a second RANKL receptor that negatively regulates osteoclast differentiation and bone resorption.

Maslinic Acid Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Regulating RANKL-Mediated NF-κB and MAPK Signaling Pathways

Activation of NF‐κB and MAPK/activator protein 1 (AP‐1) signaling pathways by receptor activator NF‐κB ligand (RANKL) is essential for osteoclast activity. Targeting NF‐κB and MAPK/AP‐1 signaling to modulate osteoclast activity has been a promising strategy for osteoclast‐related diseases. In this study we examined the effects of maslinic acid (MA), a pentacyclic triterpene acid that is widely present in dietary plants, on RANKL‐induced osteoclastogenesis, osteoclast function, and signaling pathways by in vitro and in vivo assay systems. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, MA inhibited RANKL‐induced osteoclastogenesis in a dose‐dependent manner within nongrowth inhibitory concentration, and MA decreased osteoclastogenesis‐related marker gene expression, including TRACP, MMP9, c‐Src, CTR, and cathepsin K. Specifically, MA suppressed osteoclastogenesis and actin ring formation at early stage. In ovariectomized mice, administration of MA prevented ovariectomy‐induced bone loss by inhibiting osteoclast activity. At molecular levels, MA abrogated the phosphorylation of MAPKs and AP‐1 activity, inhibited the IκBα phosphorylation and degradation, blocked NF‐κB/p65 phosphorylation, nuclear translocation, and DNA‐binding activity by downregulating RANK expression and blocking RANK interaction with TRAF6. Together our data demonstrate that MA suppresses RANKL‐induced osteoclastogenesis through NF‐κB and MAPK/AP‐1 signaling pathways and that MA is a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis.