| Background: Metastasis is one of the basic characteristics of malignant tumors.Lymphatic metastasis is the most common phenomenon for carcinoma which is closelyrelated to the poor prognosis because effective treatments are still lacking. Therefore itis essential to investigate the molecular mechanism of lymphatic metastasis and seek thepotentially therapeutic target.Hca-P and Hca-F is a pair of synogenetic mouse hepatocellular carcinoma ascitescell lines established in our laboratory possessing a specific potential of lymphaticmetastasis. When inoculated subcutaneously in615mice, they metastasized only to thelymph nodes.Hca-P cells showed a low metastatic potential (lymphatic metastasisrate<30%), whereas Hca-F cells showed a high metastatic potential (lymphaticmetastasis rate>70%). In our previous study, we applied the suppressive subtractedhybridization technique, gene chip and quantitative proteomics technique to identifydifferentially expressed genes and proteins for lymphatic metastasis between the twocell lines. Among them, Jnk1, Gsn and Rack1were the highly expressed proteins inHca-F cells.C-Jun N-terminal kinase1(Jnk1) is an important member of the mitogen-activatedprotein kinase (MAPK) superfamily which play key roles in the cell proliferation anddifferentiation. Gelsolin (Gsn), an actin binding protein, regulates actin-severing/depolymerizing in a calcium dependent manner which causes the deformation of thecells. Gsn and Jnk1were confirmed to be associated with the enhanced capabilities ofcell proliferation, migration and invasion of the Hca-F cells in vitro and lymphaticmetastasis in vivo by our previous studies. Receptor of activated C-kinase1(Rack1,GNB2L1), a36-kD intracellular scaffold protein with WD-repeat structures, ishomologous to the G protein β subunit and widely expressed in vital organs of human and animals. Since it could recruit and bind a variety of molecules such as integrins, Src,PKC impacting on multiple important signaling transduction pathways, it is known tobe involved in many physiological cellular processes. However, the influence of Rack1on the lymphatic metastasis and how these proteins interact with the signalingmolecules and affect the biological behaviors of the lymphatic metastasis cell lines areunclear yet.Leaving from the primary foci and migrating to the distant is the first step fortumor matastasis. FAK pathway is closely related to the cell adhesion, migration andproliferation, therefore it plays an important role on tumor matastasis. Rack1is not onlyinvovled in the formation of focal adhesion plaque and activates focal adhesion kinase(FAK) which is a key signaling molecules in the FAK pathway, but also interacts withother signaling molecules such as PI3K and PKC so that it can cross-talk betweenRas-MAPK pathway, PI3K-C-myc pathway and JAK-STAT pathway. Jnk1is known tobe activated by MAPK pathway. Gsn is reported to be activated by Rac1which islocated at the downstream of the PI3K signaling pathway. Therefore, we surmised thatRack1may serve as a scafford protein and interact with the signaling molecules such asFAK, PI3K which play roles on the lymphatic metastasis by regulating Jnk1and Gsn.In present study, we transfected Rack1into Hca-P cells with low lymphaticmetastasis protential to assess the effect of Rack1on the cell proliferation, migrationand invasion of Hca-P cells and the expression of Jnk1, Gsn, Rac1and PI3K proteins.Moreover, the influence of PI3K inhibitors on Hca-P cells transfected with Rack1wasinvestigated further.Methods:1The eukaryotic expression vectors pCDNA3.1(+)-Rack1wereconstructed and confirmed by DNA sequence analysis. Hca-P cells were transferred intoa6-well plate with8×105cells per well and transfected with2μg ofpCDNA3.1(+)-Rack1, pCDNA3.1(+) and pCDNA3.1(+)-GFP plasmid respectively bySofastTMtransfection reagent (Sunma Corp., China) according to the manufacture’sprotocal. The stably transfected cells were selected in culture medium plus neomycin(400μg/ml) for2weeks and maintained in medium containing200μg/ml neomycin.The relative mRNA and protein levels of Rack1were determined by Real-timeqRT-PCR and Western blot analysis, respectively. pCDNA3.1(+)-Rack1-Hca-P cellswere chosen for further experiments.2. Cells were divided into three groups:(a)pCDNA3.1(+)-Rack1-Hca-P cells;(b) control pCDNA3.1(+)-Hca-P cells;(c)unmanipulated Hca-P cells. Cell proliferation was assessed by Cell counting kit-8 (CCK8) assay according to the protocol of the manufacturer. The Boyden-transwellassay (8-μm pore size) was performed to analyze the influence of over-expressed Rack1on Hca-P cell migration and invasion.3.30μg protein from each group were loaded andseparated by12%sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Next, the protein bands were transferred to the polyvinylidene fluoride(PVDF) membranes (Millipore, USA). After blocked in the5%dried milk for1h atroom tempratur, these membranes were incubated with a primary antibody againstRack1(BD, USA;1:2500), Jnk1(Bioworld,USA,1:400), Gsn (Proteintech, USA,1:800),PI3K(Proteintech, USA,1:500), Rac1(Proteintech, USA,1:500) and GAPDH (1:7500)overnight at4℃,respectively, and then incubated with horseradish peroxidase-linkedsecond antibodies (1:5000dilution) for1h at room temperature. Enhancedchemiluminescence (ECL; GE Healthcare, USA) was performed to visualize the proteinbands.4. pCDNA3.1(+)-Rack1-Hca-P cells were intervened with various doses ofLY294002(PI3K inhibitors). Cell growth inhibition was evaluated by CCK8assay. Theeffects of LY294002(40μM) on the migration and invasion ofpCDNA3.1(+)-Rack1-Hca-P cells in vitro were evaluated by the Boyden-transwellassay.5. The expression of Jnk1, Gsn and Rac1of pCDNA3.1(+)-Rack1-Hca-P cellswere detected by WB after pretreated with various dose of LY294002.6. Theexpression of Gsn of pCDNA3.1(+)-Rack1-Hca-P cells were detected by WB afterpretreated with NSC33766(Rac1inhibitor,50μM).Resμlts:1. The recombinant plasmids of pCDNA3.1(+)-Rack1were constructedsuccessfully by DNA sequence analysis. The expression of Rack1was increased by1.9fold at mRNA levels and2.8fold at protein levels in pCDNA3.1(+)-Rack1-Hca-P cellscompared to controls.2. The growth numbers of three groups of cells were almost thesame at24h. Then, the pCDNA3.1(+)-Rack1-Hca-P cells grew faster than the controlpCDNA3.1(+)-Hca-P cells and unmanipulated Hca-P cells after48h. The number ofpCDNA3.1(+)-Rack1-Hca-P cells migrated through the filter was more than that ofcontrol pCDNA3.1(+)-Hca-P cells (p<0.05) and unmanipulated Hca-P cells (p<0.05);The number of pCDNA3.1(+)-Rack1-Hca-P cells invaded through the filter was muchmore than that of control pCDNA3.1(+)-Hca-P cells (p<0.05) and unmanipulated Hca-Pcells (p<0.05)3. Tow bands of PI3K proteins were detected by Westen Blot. One was at110KD, the other was at85KD. The relative protein expression level of PI3K was notincreased in pCDNA3.1(+)-Rack1-Hca-P cells compared to control pCDNA3.1(+)-Hca-P cells and Hca-P cells. But the expression of Jnk1, Gsn and Rac1in pCDNA3.1(+)-Rack1-Hca-P cells were higher than those in controlpCDNA3.1(+)-Hca-P cells (P<0.05).4. The pCDNA3.1(+)-Rack1-Hca-P cells with5μM LY294002pretreatment showed keeping growth within72h and then falling down,the amount of cell proliferation was significantly lower than that of thepCDNA3.1(+)-Rack1-Hca-P cells without LY294002pretreatment (P<0.05). While, thepCDNA3.1(+)-Rack1-Hca-P cells with10μM or more LY294002pretreatment showedcontinued cell growth inhibition. The difference among groups were statisticallysignificant (P<0.05).5. The expression level of Gsn and Rac1were decreased with theincreasing concentration of LY294002(P<0.05). The expression level of Jnk1wasdecreased significantly when LY294002was at low concentration (<10μM), but it wasincreased when LY294002was at high concentration (>20μM).6. When thepCDNA3.1(+)-Rack1-Hca-P cells were pretreated with NSC33766at the concentrationof50μM, the relative protein expression level of Gsn was decreased.Conclusion: The pCDNA3.1(+)-Rack1-Hca-P cells with over-expressed Rack1were successfully established. Over-expressed Rack1protein significantly enhanced theproliferative capability, migration and invasion potential of thepCDNA3.1(+)-Rack1-Hca-P cells. Over-expressed Rack1increased the expression ofJnk1, Gsn and Rac1in the pCDNA3.1(+)-Rack1-Hca-P cells. The proliferation,migration and invasion capability of Hca-P cells enhanced by over-expressed Rack1could be attenuated by LY294002. LY294002can inhibite the expression of Gsn andRac1dose-dependently in the pCDNA3.1(+)-Rack1-Hca-P cells; the expression of Jnk1can be inhibited by LY294002at a low concentration, while it can be improved byLY294002at a high concentration.The expression of Gsn can be inhibited byNSC33766(an inhibitor of Rac1).Taken together, Rack1/PI3K/Rac1signaling pathway may play a crucial role inmalignant biological behaviors of mouse hepatocarcinoma cells with lymphaticmetastasis potential by regulating Jnk1and Gsn. It may be a potential target for therapyof cancer lymphatic metastasis. |
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