Study Of Mechanism Of CD82/kAI1and Gangliosides Synergistically Inhibiting Cancer Metastasis

CD82/KAI1is one of the tetraspanin superfamily proteins. The major biologicalrole of CD82/KAI1is regulating the cell motility and migration. CD82is widelyexpressed in the normal tissue cells, but is not expressed or at a low level in the cancercells. CD82/KAI1was first identified as a specific metastasis suppressor of prostaticcarcinoma. Subsequent experiments had shown that CD82/KAI1can inhibit a variety ofdifferent tumors metastasis. CD82can inhibit the tumor cell movement and migrationin vitro, and metastasis in animal model. Clinically, the malignant degree andmetastasis ability of various tumors (especially the lymph node metastasis) werenegative correlation with the expression levels of CD82. Detection levels of CD82expression can be used as an important indicator to judge whether the tumor metastasisand prognosis. Thus, CD82/KAI1is considered as a wide-spectrum invasion-andmetastasis–suppressor.Since the tumor metastasis inhibition ability of CD82/KAI1was reported for thefirst time in1995, many of investigations have been performed to explore themechanism by which CD82/KAI1inhibits the tumor metastasis. CD82/KAI1inhibitsthe metastasis of tumor cells mainly through two ways: One is that CD82/KAI1directlyinteracts with some cell surface adhesion molecules such as integrins, membrane-boundimmunoglobulin (mIgG), thereby affect the invasion and migration ability of cell. Theother is that CD82/KAI1direct or indirectly acts on some cell membrane receptors suchas epidermal growth factor receptor, thereby regulating the intracellular signaltransduction pathways that control cell migration and movement..Gangliosides are sialylated glycosphingolipids that are characteristic componentsof mammalian cell membranes. Gangliosides are mainly located on the outer leaflet ofthe lipid bilayer of plasma membrane by inserting their hydrophobic portion (theceramide) into the membrane layer with the oligosaccharide moieties being exposed onthe cell surface. Gangliosides belongs to the trace constituent in plasma membrane ofthe cell, but it can interacts with other cell membrane components such as adhesionmolecule, receptor, participate in cell recognition, adhesion, regulate receptor mediatedsignal transmembrane conduction and cell differentiation and movement ability. The recent study had shown that the metastatic inhibition effect of CD82had been involvedin the gangliosides, especially GM2and GM3. A lot of studies had shown thatgangliosides with CD82can synergistically inhibiting metastasis in different tumorcells. For example, GM3and CD9can play a synergistic inhibition on tumor metastasis.GM2and CD82can play a synergistic action to inhibit tumor metastasis. At present,the mechanism of CD82and gangliosides synergistically inhibiting tumor metastasis isstill unknown. On the cell surface, CD82with gangliosides or other tetraspaninsuperfamily proteins are usually clustered and distributed in specific membranemicrodomains, which recruit some membrane molecules such as growth factorreceptors, integrins, phosphotydylinositol-3-kinase, PKC to form a membrane proteincomplex (Tetraspanin Web). The function of CD82inhibiting tumor metastasis dependson the formation of tetraspanin web. Inhibition of the formation of tetraspanin web,CD82would lost the fuction of metastatic inhibition. Therefore, it is very important tomake clear the mechanism of synergistic effect of CD82with ganglioside, and thetetraspanin web formation mechanism for thoroughly clarifing the molecularmechanism of CD82inhibiting tumor metastasis. To solve this problem, we must firstunderstand the molecular structure basis for the recognition, interaction between CD82and gangliosides and other molecule in tetraspanin web.CD82is a transmembrane glycoprotein. There are three N-glycosylation sites(Asn129, Asn157and Asn198) in its extracellular region. When these glycosylationsites were changed, the tumor metastasis inhibiting effect of CD82will decreased ordisappeared. This suggested that the N-sugar chain of CD82is required for itsfunctions of inhibiting tumor metastasis. It is known that the major role of carbohydrateis participating in recognition, adhesion between protein and protein, or protein andglycosphingolipids. Therefore, the N-sugar chain may be the important structure forthe interaction between CD82and gangliosides and other molecules.In the present work, in order to explore the mechanisms of gangliosides and CD82synergistically inhibiting the tumor metastasis, the studied had been performed as thefollowing1. Effects of ganglioside GM2/GM3on tumor cells in vitro and movement abilityand Research on the action mechanism. Two related sublines derived from murineascites hepatoma cell1ines Hca-F25, which were selected for their markedly differentmetastatic potential to lymph nodes，were found to be differ in their ganglioside patterns．The low metastatic cell line (HcaP) contained a major ganglioside GM3(about80%of total gangliosides), whereas the high metastatic cell line (HcaF) contained amajor ganglioside GM2(about80%of total gangliosides). These results suggest thatthe differences in expression of gangliosides of two cell lines may attributed to thedifferences in lymphatic metastasis potential between these two cell lines. Therefore,we further investigated the molecular mechanism of gangliosides affecting lymphnodes metastasis in two cell lines. Using the specific ganglioside synthesis inhibitor(PPPP) for treatment of low lymphatic metastasis potential Hca F cells, downregulatingthe content of GM3in Hca F cell; exogenous GM3for treatment of high lymphaticmetastasis potential Hca P cells, upgulating the content of GM3in Hca P cells, and theninvestigated the effect of GM3on phosphorylation of epidermal growth factor receptor(EGFR), activity of intracellular PI3K/AKT signal transduction pathway and in vitrocells migration by the Boyden cell culture and western bloting technology.Results:(1).Suppression of ganglioside synthesis by treatment of the cells with P4,an inhibitor of glucosylceramide synthesis, enhanced the mobility and migration of thelow metastatic HcaP cells in vitro, but did not affect the motility and migration of thehigh metastatic HcaF cells. It is suggested that that is GM3rather than GM2can affectthe cell movement and migration, the role of GM3is to inhibit cell movement andmigration.(2) Suppression of GM3synthesis by P4in low metastatic HcaP cellspromoted EGFR phosphorylation at the Tyr1173and PKB/Akt phosphorylation atSer473and Thr308. In contrast, increase in GM3content in high metastatic HcaF cellsby addition of exogenous GM3into the culture medium suppressed phosphorylation ofEGFR and PKB/Akt at the same residues. It is indicated that GM3can inhibit theactivity of EGFR, downregulate the activation of PI3K signal transduction pathway.This may be one of the mechanisms of GM3to inhibit cell movement and migration.(3)The motility and migration of Hca F cells in vitro could be suppressed by blocking thePI3K/Akt signaling pathway with LY294002, whereas the enhanced motility andmigration of HcaP cells caused by P4-suppressed ganglioside synthesis could also bereversed by blocking the PI3K/Akt signaling pathway with LY294002. these resultssuggested that the mechanism of GM3-suppressed cell motility and migration mayinvolve the inhibition of phosphorylation of epidermal Growth Factor Receptor and theactivity PI-3K/AKT signaling pathway.Conclusion:(1) GM3is one of the important factors affecting cell lymph node metastasis ability of the two cell lines, and its role is to inhibit the metastasis of lymphnode.(2) One of the possible molecular mechanisms of GM3suppressing lymph nodemetastasis is inhibition of EGFR phosphorylation and activation, down regulation ofPI3K/AKT signaling transduction pathway in HcaP and HcaF cells.2. Study of molecular mechanism of ganglioside and CD82synergistically inhibitthe tumor metastasis. Using human colon carcinoma SW620with high lymph nodemetastatic potential as a cell model, we investigated the molecular mechanism ofgangliosides and CD82synergistically inhibiting the tumor metastasis. SW620celllittle expresses CD82and contains GM2as a major ganglioside component. We alteredthe contents of ganglioside and CD82by the treating cells with P4(a specific inhibitorof glucosylceramide synthesis) or exogenous gangliosides, and/or by the transfection ofcells with CD82cDNA and then investigated the effect of GM3and CD82on Cellmigration in vitro, epidermal growth factor receptor (EGFR) activation, intracellularsignal transduction pathway such asPI3K/AKT, MAPK and PLC γ signal transductionpathway by the Boyden cell culture and western bloting technology.Results:(1) GM3suppresses the EGF-stimulated SW620cell motility andmigration and inhibits the EGF-stimulated phosphorylation of EGFR at the Tyr1173residue and the EGF-stimulated phosphorylation of Akt at Ser473. But GM2did not.(2)The overexpression of CD82inhibits the EGF-stimulated cell motility and migration,the EGF-stimulated phosphorylation of EGFR at Tyr1045, and the EGF-stimulatedphosphorylation of ERK.(3) Both GM3and GM2can enhance the inhibitory effect ofCD82on cell migration, but the combination of GM3with CD82synergisticallysuppresses the EGF-stimulated phosphorylation of EGFR at Tyr1173and theEGF-stimulated phosphorylation of Akt, whereas GM2with CD82synergisticallysuppress the EGF-stimulated phosphorylation of EGFR at Tyr1045and theEGF-stimulated phosphorylation of ERK.(4) The mixture of GM3and GM2withCD82was found to markedly suppress the EGF-stimulated SW620cell motility andmigration and to inhibit the EGF-stimulated phosphorylation of EGFR at both Tyr1173and Tyr1045and the EGF-stimulated phosphorylation of Akt and ERK. These resultssuggest that the mixture of GM3and GM2with CD82can exert the strongestsynergistic inhibitory effect on cell mobility and migration by inhibiting theEGF-stimulated phosphorylation of EGFR at Tyr1173and Tyr1o45, anddownregulating the PI-3K/AKT and the ERK signaling pathway. Conclusion:(1) GM3alone can inhibit the cell motility and migration and thephosphorylation of EGFR at the Tyr1173residue and the phosphorylation of Akt atSer473. But GM2did not.(2) CD82alone can inhibit the cell motility and migrationand the phosphorylation of EGFR at Tyr1045, and the phosphorylation of ERK.(3)Both GM3and GM2with CD82can exert synergistic effect, but through differentmechanisms. The combination of GM3with CD82synergistically suppresses thephosphorylation of EGFR at Tyr1173and the phosphorylation of Akt, whereas GM2with CD82synergistically suppress the phosphorylation of EGFR at Tyr1045and thephosphorylation of ERK.(4) The mixture of GM3and GM2with CD82can exert thestrongest synergistic inhibitory effect on cell mobility and migration by inhibiting theEGF-stimulated phosphorylation of EGFR at Tyr1173and Tyr1o45, anddownregulating the PI-3K/AKT and the ERK signaling pathway.3. Study on CD82N-sugar chain in gangliosides and CD82synergistic inhibitormetastasis and molecular mechanism It is known that one of the important roles ofcell surface glycolipid and glycoprotein glycan sugar chain is participating in therecognition, adhesion between protein-protein, protein–glycosphingolipids. In order toclarify the roles of CD82extracellular N-sugar chain in CD82and gangliosidessynergistic inhibiting tumor metastasis, we used gene recombination technique toconstruct the recombinant plasmid pcDNA4.1-CD82-V5carrying CD82cDNA. Usingthe point mutation technology, we constructed the recombinant plasmid of CD82withdifferent N-glycosylation site mutation, transfected the recombinant plasmids intoSW620cells and observed the distribution in cells of expression product of differentCD82N-glycosylation site mutation and the influence on cell movement and migrationability. We also screened several cell lines stably expressed CD82with N-glycosylation site mutation. The distribution of partial CD82mutants in cell surfacelipid rafts was studied.Results:(1) Construction of the recombinant plasmid pcDNA4.1-V5carryingCD82cDNA and the recombinant plasmid of CD82with different N-glycosylation sitemutants, including single point mutant: Asn129, Asn157, Asn198, double points mutant:Asn129/Asn157, Asn129/Asn198, Asn157/Asn198and three points mutantAsn129/Asn157/Asn198. Gene sequencing results proved that the construction ofCD82mutant was successful.(2) Histochemistry experimental results showed that allthe mutants with N-glycosylation site mutation were successfully expressed in SW620 cells. Besides three points mutant Asn157/Asn198and Asn129/Asn157/Asn198expressed inside cells, all the other mutants were expressed at the cell surface.(3)Asn129/Asn157, Asn157/Asn198and Asn129/Asn157/Asn198mutation can affect theinhibitory effect of CD82on cell migration in vitro.(4) Asn129/Asn157/Asn198mutation can affect the distribution of CD82in lipid raft.Conclution:(1) The recombinant plasmids of CD82with different N-glycosylation site mutation were constructed successfully.(2) All the CD82mutantswith different N-glycosylation site mutation can be successfully expressed inSW620.(3) Asn129/Asn157, Asn157/Asn198and Asn129/Asn157/Asn198N-glycosylation sites may be related to mechanism of CD82inhibition tumor metastasis.(4) Asn129/Asn157/Asn198N-glycosylation sites may affect the CD82metastasissuppressor function through affecting the distribution of CD82in the cell membranelipid rafts.