Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS-/- mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor NG-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca2+ levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca2+ levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol-treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca2+ chelator, calmodulin antagonist, and CaMKKbeta siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca2+-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca2+/CaMKKbeta-dependent eNOS phosphorylation and Ca2+-dependent eNOS dimerization.

Colchicine-derived compound CT20126 promotes skin allograft survival by regulating the balance of Th1 and Th2 cytokine production

Colchicine has been shown to regulate the expression of inflammatory gene, but this compound possesses much weaker anti-inflammatory activity. In this study, we synthesized a new colchicine derivative CT20126 and examined its immunomodulatory property. CT20126 was found to have immunosuppressive effects by inhibiting lymphocyte proliferation without cytotoxicity and effectively inhibit the transcriptional expression of the inflammatory genes, iNOS, TNF-alpha, and IL-1beta, in macrophages stimulated by LPS. This effect was nearly comparable to that of cyclosporine A. This compound also significantly suppressed the production of nitric oxide and Th1-related pro-inflammatory cytokines, IL-1beta, TNF-alpha, and IL-2, with minimal suppression of Th2-related anti-inflammatory cytokines IL-4 and IL-10 in the sponge matrix allograft model. Moreover, administration of CT20126 prolonged the survival of allograft skins from BALB/c mice (H-2d) to the dorsum of C57BL/6 (H-2b) mice. The in vivo immune suppressive effects of CT20126 were similar to that of cyclosporine A. These results indicate that this compound may have potential therapeutic value for transplantation rejection and other inflammatory diseases.

SIRT1 promotes DNA repair activity and deacetylation of Ku70

Human SIRT1 controls various physiological responses including cell fate, stress, and aging, through deacetylation of its specific substrate protein. In processing DNA damage signaling, SIRT1 attenuates a cellular apoptotic response by deacetylation of p53 tumor suppressor. The present study shows that, upon exposure to radiation, SIRT1 could enhance DNA repair capacity and deacetylation of repair protein Ku70. Ectopically over-expressed SIRT1 resulted in the increase of repair of DNA strand breakages produced by radiation. On the other hand, repression of endogenous SIRT1 expression by SIRT1 siRNA led to the decrease of this repair activity, indicating that SIRT1 can regulate DNA repair capacity of cells with DNA strand breaks. In addition, we found that SIRT1 physically complexed with repair protein Ku70, leading to subsequent deacetylation. The dominant-negative SIRT1, a catalytically inactive form, did not induce deacetylation of Ku70 protein as well as increase of DNA repair capacity. These observations suggest that SIRT1 modulates DNA repair activity, which could be regulated by the acetylation status of repair protein Ku70 following DNA damage.

beta-Carotene inhibits inflammatory gene expression in lipopolysaccharide-stimulated macro phages by suppressing redox-based NF-kappaB activation

beta-Carotene has shown antioxidant and antiinflammatory activities; however, its molecular mechanism has not been clearly defined. We examined in vitro and in vivo regulatory function of beta-carotene on the production of nitric oxide (NO) and PGE2 as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, TNF-alpha, and IL-1beta. beta-Carotene inhibited the expression and production of these inflammatory mediators in both LPSstimulated RAW264.7 cells and primary macrophages in a dose-dependent fashion as well as in LPS-administrated mice. Furthermore, this compound suppressed NF-kappaB activation and iNOS promoter activity in RAW264.7 cells stimulated with LPS. beta-Carotene blocked nuclear translocation of NF-kappaB p65 subunit, which correlated with its inhibitory effect on IkappaBalpha phosphorylation and degradation. This compound directly blocked the intracellular accumulation of reactive oxygen species in RAW264.7 cells stimulated with LPS as both the NADPH oxidase inhibitor diphenylene iodonium and antioxidant pyrrolidine dithiocarbamate did. The inhibition of NADPH oxidase also inhibited NO production, iNOS expression, and iNOS promoter activity. These results suggest that beta-carotene possesses anti-inflammatory activity by functioning as a potential inhibitor for redox-based NF-kappaB activation, probably due to its antioxidant activity.

Prostaglandin E2 stimulates angiogenesis by activating the nitric oxide/cGMP pathway in human umbilical vein endothelial cells

Prostaglandin E2(PGE2), a major product of cyclooxygenase, has been implicated in modulating angiogenesis, vascular function, and inflammatory processes, but the underlying mechanism is not clearly elucidated. We here investigated the molecular mechanism by which PGE 2 regulates angiogenesis. Treatment of human umbilical vein endothelial cells (HUVEC) with PGE 2 increased angiogenesis. PGE 2 increased phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), eNOS activity, and nitric oxide (NO) production by the activation of cAMP-dependent protein kinase (PKA) and phosphatidylinositol 3-kinase (PI3K). Dibutyryl cAMP (DB-cAMP) mimicked the role of PGE 2 in angiogenesis and the signaling pathway, suggesting that cAMP is a down-stream mediator of PGE 2. Furthermore, PGE 2 increased endothelial cell sprouting from normal murine aortic segments, but not from eNOS-deficient ones, on Matrigel. The angiogenic effects of PGE 2 were inhibited by the inhibitors of PKA, PI3K, eNOS, and soluble guanylate cyclase, but not by phospholipase C inhibitor. These results clearly show that PGE 2 increased angiogenesis by activating the NO/cGMP signaling pathway through PKA/PI3K/Akt-dependent increase in eNOS activity.

Tetraspanin CD9 induces MMP-2 expression by activating p38 MAPK, JNK and c-Jun pathways in human melanoma cells

Expression of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), which correlates with tumor invasion and metastasis, has been known to be regulated by several intracellular signaling pathways. Since the CD9 membrane protein has been implicated in signal transduction and malignant progression of cancer cells, we examined the functional involvement of CD9 in the regulation of MMP-2 and MMP-9 expression by using stable CD9 transfectant clones of MelJuso human melanoma cells. The CD9 cDNA-transfected cells with elevated CD9 expression displayed increased MMP-2 and decreased MMP-9 expression when compared with the mock transfectant cells. Among several signal pathway inhibitors tested, SB203580 and SP600125, which inhibit p38 MAPK and JNK respectively, completely blocked the CD9-stimulated MMP-2 expression. Phosphorylation levels of p38 MAPK and c-Jun in MelJuso cells were also significantly increased by CD9 transfection. In addition, the down-regulation of p38 MAPK and JNK by siRNA transfection resulted in a decrease in MMP-2 expression by MelJuso cells. Promoter analysis and gel shift assay showed that the CD9-induced MMP-2 expression is mediated by a functional AP-1 site through interactions with AP-1 transcription factors including c-Jun. These results suggest that CD9 induces MMP-2 expression by activating c- Jun through p38 MAPK and JNK signaling pathways in human melanoma cells.

Metastasis-suppressor KAI1/CD82 induces homotypic aggregation of human prostate cancer cells through Src-dependent pathway

To investigate the functional role of KAI1/CD82, a metastasis suppressor for human prostate cancer, in the regulation of homotypic cell adhesion, we transfected KAI1 cDNA into DU 145 human prostate cancer cells and established stable transfectant clones with high KAI1/CD82 expression. The KAI1 transfectant cells exhibited significantly increased homotypic cell aggregation in comparison with the control transfectant cells. This aggregation of the KAI1 transfectants was further enhanced upon exposure to anti-CD82 antibody, suggesting that KAI1/CD82 may be involved in the intracellular signaling for the cell adhesion. Among several signal pathway inhibitors tested, PP1, an inhibitor of Src family kinases, significantly suppressed homotypic aggregation of the KAI1 transfectant cells. Ligation of KAI1/CD82 with anti-CD82 antibody increased endogenous Src kinase activity of the KAI1 transfectant cells. When different types of src expression constructs were retransfected into the KAI1-transfected DU 145 cells, kinase-negative mutant src transfectant cells exhibited much lower homotypic aggregation than the mock cells transfected with an empty vector. Moreover, homotypic aggregation of the mutant src transfectant cells was not enhanced by KAI1/CD82 ligation with anti- CD82 antibody. These results suggest that Src mediates the intracellular signaling pathway of KAI1/CD82 for the induction of homotypic adhesion of human prostate cancer cells.

A decrease in the expression of CD63 tetraspanin protein elevates invasive potential of human melanoma cells

CD63, which belongs to the tetraspanin membrane proteins, has been proposed to play an important role in inhibiting melanoma metastasis. To determine whether reduction of CD63 expression, which frequently occurs in the malignant progression of human melanoma, is responsible for metastasis promotion, we transfected the antisense CD63 cDNA into MelJuso melanoma cells having endogenous CD63 expression. The antisense CD63 transfectant clones showing decreased CD63 expression displayed increased cell motility, matrix-degrading activity, and invasiveness in vitro when compared with the control transfectant cells. The antisense CD63 cDNA-transfected cells also exhibited altered adhesiveness to extracellular matrix. The results suggest that reduced CD63 expression contributes to the invasive and metastatic ability of human melanoma cells.

Immunoreactivity of CD99 in Stomach Cancer

CD99 is characteristically expressed in Ewing's sarcoma/primitive neuroectodermal tumor. Recently its immunoreactivity has also been reported in other tumors. However, the significance of CD99 isoforms expressed in these tumors has not been elucidated. In this study, we evaluated the expression of CD99 isoforms and its relationship with histopathologic parameters in gastric adenocarcinomas. Paraffin sections of 46 gastric adenocarcinomas were stained with an anti-CD99 monoclonal antibody, YG32. Twelve (26.1%) cases of 46 gastric adenocarcinomas showed immunoreactivity to YG32. The CD99 expression was also seen both in non-neoplastic foveolar epithelial cells and infiltrating lymphocytes. In addition, Western blot and RT-PCR analyses revealed that the type I is the predominant isoform of CD99 in non-neoplastic and neoplastic gastric tissues. The CD99 expression was usually seen in the intestinal type adenocarcinoma, while rarely in the diffuse type. The CD99 immunoreactivity decreased in MMP-2-overexpressing adenocarcinomas (p=0.028). Our results suggest that the type I is the major isoform of CD99 expressed in non-neoplastic gastric mucosa and gastric adenocarcinomas and its downregulation in gastric adenocarcinoma may be associated with cellular dedifferentiation and/or MMP-2 overexpression.