Dong-Soon Im

Effects of ginsenosides Rg3 and Rh2 on the proliferation of prostate cancer cells

Ginseng has an anti-cancer effect in several cancer models. This study was to characterize active constituents of ginseng and their effects on proliferation of prostate cancer cell lines, LNCaP and PC3. Cell proliferation was measured by [3H]thymidine incorporation, the intracellular calcium concentration by a dual-wavelength spectrophotometer system, effects on mito-gen-activated protein (MAP) kinases by Western blotting, and cell attachment and morphologic changes were observed under a microscope. Among 11 ginsenosides tested, ginsenosides Rg3 and Rh2 inhibited the proliferation of prostate cancer cells. EC50s of Rg3 and Rh2 on PC3 cells were 8.4 μM and 5.5 μM, respectively, and 14.1 μM and 4.4 μM on LNCaP cells, respectively. Both ginsenosides induced cell detachment and modulated three modules of MAP kinases activities differently in LNCaP and PC3 cells. These results suggest that ginsenosides Rg3 and Rh2-induced cell detachment and inhibition of the proliferation of prostate cancer cells may be associated with modulation of three modules of MAP kinases.

Lysophosphatidic Acid-induced Mitogenesis Is Regulated by Lipid Phosphate Phosphatases and Is Edg-receptor Independent

Lysophosphatidic acid (LPA) is an extracellular signaling mediator with a broad range of cellular responses. Three G-protein-coupled receptors (Edg-2, -4, and -7) have been identified as receptors for LPA. In this study, the ectophosphatase lipid phosphate phosphatase 1 (LPP1) has been shown to down-regulate LPA-mediated mitogenesis. Furthermore, using degradation-resistant phosphonate analogs of LPA and stereoselective agonists of the Edg receptors we have demonstrated that the mitogenic and platelet aggregation responses to LPA are independent of Edg-2, -4, and -7. Specifically, we found that LPA degradation is insufficient to account for the decrease in LPA potency in mitogenic assays, and the stereoselectivity observed at the Edg receptors is not reflected in mitogenesis. Additionally, RH7777 cells, which are devoid of Edg-2, -4, and -7 receptor mRNA, have a mitogenic response to LPA and LPA analogs. Finally, we have determined that the ligand selectivity of the platelet aggregation response is consistent with that of mitogenesis, but not with Edg-2, -4, and -7.

Cell-surface residence of sphingosine 1-phosphate receptor 1 on lymphocytes determines lymphocyte egress kinetics

The sphingosine 1-phosphate receptor 1 (S1P1) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein–coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P1 internalization is necessary for this effect. We characterize a knockin mouse (S1p1rS5A/S5A) in which the C-terminal serine-rich S1P1 motif, which is important for S1P1 internalization but dispensable for S1P1 signaling, is mutated. T cells expressing the mutant S1P1 showed delayed S1P1 internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P1 agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P1 expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P1 on T cells is a primary determinant of lymphocyte egress kinetics in vivo.

Inhibitory Role of Sphingosine 1-Phosphate Receptor 2 in Macrophage Recruitment during Inflammation

Macrophage recruitment to sites of inflammation is an essential step in host defense. However, the mechanisms preventing excessive accumulation of macrophages remain relatively unknown. The lysophospholipid sphingosine 1-phosphate (S1P) promotes T and B cell egress from lymphoid organs by acting on S1P receptor 1 (S1P1R). More recently, S1P5R was shown to regulate NK cell mobilization during inflammation, raising the possibility that S1P regulates the trafficking of other leukocyte lineages. In this study, we show that S1P2R inhibits macrophage migration in vitro and that S1P2R-deficient mice have enhanced macrophage recruitment during thioglycollate peritonitis. We identify the signaling mechanisms used by S1P2R in macrophages, involving the second messenger cAMP and inhibition of Akt phosphorylation. In addition, we show that the phosphoinositide phosphatase and tensin homolog deleted on chromosome 10, which has been suggested to mediate S1P2R effects in other cell types, does not mediate S1P2R inhibition in macrophages. Our results suggest that S1P serves as a negative regulator of macrophage recruitment by inhibiting migration in these cells and identify an additional facet to the regulation of leukocyte trafficking by S1P.

Life on the edg

The identification of cloned receptors that mediate lysolipid phosphate signalling has greatly advanced the study of these important mediators. The amino acid sequence similarity that exists between these receptors reflects the structural similarity of LPA and S1P, and suggests that an underlying commonality exists between these two fields that have developed somewhat in parallel. Lipid phosphate mediators, particularly LPA, have been implicated in a variety of pathophysiological conditions including blood clotting27xSchumacher, K.A., Classen, H.G., and Spth, M. Thromb. Haemost. 1979; 42: 631–640PubMedSee all References27, corneal wounding28xLiliom, K. et al. Am. J. Physiol. 1998; 274: C1065–C1074PubMedSee all References28, subarachinoid haemorrhage29xTigyi, G. et al. Am. J. Physiol. 1995; 268: H2048–H2055PubMedSee all References29, inflammation30xFourcade, O. et al. Cell. 1995; 80: 919–927Abstract | Full Text PDF | PubMed | Scopus (423)See all References30 and colitis31xSturm, A., Sudermann, T., Schulte, K.M., Goebell, H., and Dignass, A.U. Gastroenterology. 1999; 117: 368–377Abstract | Full Text | Full Text PDF | PubMed | Scopus (78)See all References31, and have been shown to accumulate in malignant ascites32xXu, Y. et al. Clin. Cancer Res. 1995; 1: 1223–1232PubMedSee all References32. Furthermore, LPA is reported to be an anti-apoptotic factor in several cell types including kidney proximal tubule cells33xLevine, J.S., Koh, J.S., Triaca, V., and Lieberthal, W. Am. J. Physiol. 1997; 273: F575–F585PubMedSee all References33 and Schwann cells34xWeiner, J.A. and Chun, J. Proc. Natl. Acad. Sci. U. S. A. 1998; 96: 5233–5238Crossref | Scopus (193)See all References34, and it also promotes skin thickening35xPiazza, G.A., Ritter, J.L., and Baracka, C.A. Exp. Cell Res. 1995; 216: 51–64Crossref | PubMed | Scopus (68)See all References35. The availability of recombinant receptor subtypes will help to identify selective compounds to explore the biology of these fascinating molecules.

Omega-3 fatty acids in anti-inflammation (pro-resolution) and GPCRs

Omega-3 fatty acids, such as, DHA and EPA, have well established beneficial effects on human health, but their action mechanisms remain unknown. Recent pharmacological studies have suggested several molecular targets for the anti-inflammatory effects of omega-3 fatty acids, namely, nuclear receptor PPARγ and the G protein-coupled receptor GPR120. Furthermore, the conversions of omega-3 fatty acids to anti-inflammatory and pro-resolving resolvins and protectins and the identifications of putative target GPCRs, ChemR23, BLT₁, ALX/FPR2, and GPR32, have drawn great attention. In addition, the pharmacology of omega-3 fatty acids is now under scrutiny. However, questions remain to be answered regarding the in vivo effects of omega-3 fatty acids at the molecular level. In this review, anti-inflammatory effects of omega-3 fatty acids are discussed from the viewpoint of molecular pharmacology, particularly with respect to the above-mentioned GPCRs.

Cloning and characterization of additional members of the G protein-coupled receptor family

A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.

Pharmacological tools for lysophospholipid GPCRs: development of agonists and antagonists for LPA and S1P receptors

AbstractPrevious studies on lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) using various approaches have shown that both the molecules can act as intercellular signaling molecules. The discovery of the Edg subfamily of G-protein-coupled receptors (GPCRs) (later renamed LPA1–3 and S1P1–5) for these molecules has opened up a new avenue for pathophysiological research on lysophospholipids. Genetic and molecular studies on lysophospholipid GPCRs have elucidated pathophysiological impacts and roles in cellular signaling pathways. Recently, lysophospholipid GPCR genes have been used to develop receptor subtype-selective agonists and antagonists. The discovery of FTY720, a novel immune modulator, along with other chemical tools, has provided a means of elucidating the functions of each lysophospholipid GPCR on an organ and the whole body level. This communication attempts to retrospectively review the development of agonists and antagonists for lysophospholipid GPCRs, provide integrated information on pharmacological tools for lysophospholipid GPCR signaling, and speculate on future drug development.

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor.

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK‐OV3 ovarian cancer cells and OVCAR‐3 ovarian cancer cells were responsive to LPE. LPE‐stimulated intracellular calcium concentration ([Ca2+]i) increase was inhibited by U‐73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca2+]i increase by LPE, indicating the involvement of PTX‐sensitive G‐proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK‐OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE‐stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B‐driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK‐OV3 ovarian cancer cells.

Linking Chinese medicine and G-protein-coupled receptors

Following the purification of the immunosuppressant ISP-1 from a Chinese medicine, Japanese scientists have developed a more potent immune modulator, FTY720, that induces T-cell homing. FTY720, a promising immunosuppressant for use in patients with tissue transplants and autoimmune diseases, is currently in clinical trials. Two recent studies have elucidated that the mechanism of action of FTY720 is via a subset of G-protein-coupled receptors for the lysophospholipid mediator sphingosine-1-phosphate.