The Effects of Ginseng Pectins on Tumor Cell Migration and Their Mechanisms Yuying Fan 26 th, Nov, 2012

Outlines: 1st, Research Background 2nd, Research Strategies 3rd, Research Progress

Research Background- panax ginseng C.A.meyer Ginseng (Ginnsuu), belongs to the panax genus in the family Araliaceae. Ginseng American Ginseng (Panax quinquefolius) Asian (Japanese/Korea/China) Ginseng (Panax ginseng) Prince Ginseng (Pseudostellaria heterophylla) Siberian Ginseng (Eleutherococcus senticosus)

Research Background- classification of asian ginseng panax ginseng fresh ginseng: raw product white ginseng (WG): fresh ginseng which has been dried red ginseng (RG): heated by steaming or sun-drying sun ginseng (SG): steaming at a higher temperature than RG

Research Background- structures of ginseng polysaccharides  chemical compounds: ginseng polysaccharides, ginsenosides, ginseng peptides and other small molecules.  Ginseng polysaccharides=neutral polysaccharides (starch-like glucans)+acidic substances (ginseng pectin) flower, fruit stipe leaf stalk radicula rootlet taproot root tip

 It has been shown that ginseng polysaccharides have many bioactivities, including immunomodulation, anti-tumor, anti- fatigue, hpyerglycemic, anti-atheroscloresis, anti-radiation… ginseng polysacch arides immunomodulation anti- tumor hpyerglycemic anti- fatigue tumor cell proliferation tumor cell apoptosis tumor cell metastasis Research Background- activities of ginseng polysaccharides

Research Background-tumor metastasis

Research Background- cell migration Cell polarization refers to the tendency of migrating cells to have a distinct, stable front and rear. The polarity is reinforced and often even arises from environments that provide a directional cue. These directional cues can be chemotactic, (induced by chemoattractants or morphogens), haptotactic (caused by varying concentrations of substrate), mechanotactic (breakdown of cell-cell contacts, as in wound healing), electrotactic (induced by electric fields), and 'durotactic', (due to differences in pliability), or combinations of any of these. Polarization

The result is a defined cell front and a rear. The leading edge is usually characterized by intense actin polymerization that generates a protrusive structure, and by adhesion to the substratum. The trailing edge is characterized by stable bundles and the release and disassembly of adhesions. The central part of the cell usually contains the nucleus and microtubules (which exhibit different degrees of polarization depending on the cell type).

Major mechanisms that control nuclear and microtubule positioning during cell migration. The small GTPase Cdc42 acts through MRCK and myosin II to control the distance and position of the nucleus with respect to the centrosome (MTOC). Cdc42 also controls the Par3/Par6/aPKC module to control microtubule dynamics through the dynein/dynactin complex.

Protrusion Protrusion is the de novo formation of membrane extensions, or protrusions, in the direction of migration, i.e. the leading edge. It has three major components: the expansion of the plasma membrane, the formation of an underlying backbone that supports membrane extension, and the establishment of contacts with the substratum, which provides traction for the movement of the rest of the cell body and signals that regulate actin polymerization.

The protrusion is produced by local actin polymerization. One kind of protrusion is flat and fan-like, the edge of which is often called the lamellipodium and within which actin is polymerizing and often branched. Spike-like filopodia are another kind of protrusion; these structures comprise polymerized actin filaments that are arranged into long parallel bundles. These two forms of protrusion are thought to serve different roles: filopodia act as mechanosensory, exploratory devices, whereas lamellipodia provide wide surfaces that generate traction for forward movement.

Actin polymerization results from the nucleation of new filaments and the availability and addition of new monomers. The Arp2/3 complex is a molecular heptamer that attaches itself to the side of a pre-existing actin filament and nucleates de novo actin polymerization at a fixed angle. This is essential for branching in lamellipodia, and also important in the formation of the base of filopodia. Monomers are provided by severing of older filaments and their dissolution into actin monomers. This is mediated by an actin-filament severing protein, cofilin.

Scheme depicts the major mechanisms and molecules implicated in regulating and enabling actin polymerization/depolymerization and organization. The Arp2/3 complex induces branching by attaching to the side of a pre-existing filament and promoting de novo polymerization. Formins such as mDia1 and mDia2 induce processive actin polymerization by attaching themselves to the barbed end of the filament. The formins compete with actin capping proteins, such as gelsolin and capZ (not depicted). Ena/VASP serves a dual role as an adaptor protein and a 'leaky' capper, which enables actin polymerization through other molecules that are part of a complex (not shown). Myosin II (in green, between filaments) cross links antiparallel actin mediates contraction due to its ATPase-dependent motor domain, and its cross-linking, ATPase-independent activities. Cofilin (grey arrowhead) severs actin filaments, promoting the formation of new barbed ends. It also generates actin monomers to be incorporated to the filaments via profilin/formin (treadmilling). Another small protein, thymosin β4, sequesters actin monomers and maintains the pool of available monomers. Some of the signaling proteins that control these molecules are also shown. They include: Rho-kinase (ROCK), which is under the control of RhoA and activates LIMK to activate cofilin, blocks the myosin phosphatase, promoting myosin activation, and phosphorylates and activates myosin directly; PAK also activates LIMK but it inhibits MLCK, inactivating myosin II; the WAVE/Scar-Abi complex and WASP, which activate the Arp2/3 complex, are under the control of Rac and Cdc42, respectively; finally, formins are also activated by small GTPases directly (mDia1 by RhoA; mDia2 by Cdc42).

adhesion Cell adhesion (the physical interaction of a cell with another cell or with the extracellular matrix (ECM)) is essential for cell migration and tissue integrity. Cell-cell adhesion maintains epithelial tissues, supports functional contacts between specialized cells, and can facilitate directed migration (for example, radial glia can guide neuronal progenitor cells to specific layers of the brain). Cell- matrix adhesion is the best-studied form of adhesion that mediates cell migration, and is the focus of this outline.

Scheme depicts the process of adhesion maturation. Nascent adhesions (on the right) are connected to the actin cytoskeleton by an actin linkage that includes talin, vinculin and α-actinin; other signaling adaptors are also recruited to these complexes, including FAK and paxillin. These adhesions generate signals that activate Rac, promoting actin polymerization and preventing myosin II engagement in the lamellipodium. These signals are also required for the dissolution of some adhesions (turnover) as the cell advances. Some adhesions mature into focal adhesions; this process is characterized by an enlargement and elongation of the complexes as well as the appearance of new players, like tensin. Some components have conformations that are tension sensitive (like talin, vinculin, and p130Cas, not shown here). Rho activation is thought to generate focal adhesions and actin bundling due to increased myosin II activity. Other events that occur at adhesions are internalization of the integrins, which is mediated by dynamin; and microtubule targeting, which may contribute to adhesion disassembly.

For a cell to migrate, protrusion of the front and translocation of the cell body must be followed by retraction of the rear of the cell. Cell body translocation immediately follows protrusion and is independent of actin polymerization. In keratocytes, the cell body 'rolls' behind the front protrusion. This movement is propelled by a coordinated contraction of the actomyosin cytoskeleton, and thus depends on myosin II. Myosin-mediated contraction and microtubule motors (e.g. dynein) also control the translocation of the nucleus. On the other hand, rear retraction requires the coordinated contraction of the actin cytoskeleton and disassembly of the adhesions at the trailing edge. Several mechanisms converge to promote adhesion disassembly: actomyosin contraction, microtubule-induced adhesion relaxation, endocytosis of adhesion receptors, and proteolytic cleavage of focal adhesion proteins. Cell body translocation and rear retraction

At the rear of the cell, adhesions disassemble; this process is mediated by at least four major mechanisms: 1) actomyosin contraction, controlled by RhoA - Rho- kinase - Myosin II; 2) protein dephosphorylation and inactivation, mediated by phosphatases, e.g. calcineurin; 3) proteolysis of adhesion components, mediated by calpain; 4) microtubule targeting; the identity of the disassembly signal is not known.

Research Background ？ relationship of structure and activity mechanism development and application of ginseng better

fractions with different structures （ fine structure ） ginseng pectin ion exchange/molecular gel filtration chromatograph structure-activity relationship and mechanism ginseng pectin （ pectins mixture ） ginseng root amylase treat tumor cell migration scratch/ transwell scratch / transwell / cell adhesion / cell spreading / cell morphology / cytoskeleton / phosphorylation of signaling molecular STEP:1STEP:2 Research Strategies

Ginseng pectin inhibited tumor cell migration (specificity and selective) Mechanisms(HG and RG-I pectins have synergy effect on migration, cell morphology, cytoskeleton, focal adhesion, phosphorylation of signaling molecules→cell adhesion and spreading→cell migration, without galectin-3 ） Research Progress