Mechanism Of The Cell Surface GRP78Facilitates The Invasion Of Hepatocellular Carcinoma Cells

ObjectiveIn order to study the role of cell surface GRP78on the invasion ofhepatocellular carcinoma cells, this experiment used Mahlavu andSMMC7721as the models, proving the role and possible mechanismof the cell surface GRP78on facilitated the invasive ability ofhepatocellular carcinoma cells.Methods1. Effects of endogenous cell surface GRP78on the invasivepotential of hepatocellular carcinoma cells. In Cell Western andinvasion assay explored the expression of cell surface GRP78and theinvasion ability of different HCC cells (PLC, SMMC7721andMahlavu). Two hepatocellular cells Mahlavu and SMMC7721whichexpressed higher cell surface GRP78were chosed for next experiment.Using GRP78(N20) neutralized cell surface GRP78, and treatedwith IgG antibody as control. Invasion and adhesion assay to detectthe invasion and adhesion abilities of hepatocellular carcinoma cellswith different expression of cell surface GRP78; Gelatin zymographassays to test the activity of exocrine metalloproteinases MMP-2andMMP-9; Western blot to reveal the secretion level of MMP-2,MMP-9, and the expression level of E-cadherin, N-cadherin.2. Effects of exogenous cell surface GRP78on the invasive potential of hepatocellular carcinoma cells. Using SMMC7721whichtransfected with GRP78KDEL motif deleted mutant (ΔKDEL) to stablyoverexpressing GRP78on the cell surface, transfected withpcDNA3.1(+) empty vector as control. In cell Western and flowcytometry was used to detect the expression level of cell surfaceGRP78; invasion and adhesion assay to detect the invasion andadhesion ability; Gelatin zymography assay to test the secretion levelof exocrine metalloproteinases MMP-2and MMP-9; Western blot toreveal the secretion level of MMP-2,MMP-9,and the expression levelsof E-cadherin, N-cadherin.Results1. In-cell western analysis revealed that Mahlavu cells showedthe highest cell surface GRP78level, while PLC cells showed thelowest cell surface GRP78level. Transwell analysis revealed that theinvasive potential of Mahlavu cells was highest, whereas PLC cellswas the lowest. We neutralized the cell surface GRP78using the N20antibody in SMMC7721and Mahlavu cells and cells treated with thegoat isotype IgG as control. Transwell assay showed the N20antibodydecreased the invasive potential of cancer cells to～50%in Mahlavucells and to～58.5%in SMMC7721cells as compared with isotypegoat IgG treated cells (P<0.01). Cell adhesion assay revealed thatthe N-20antibody reduced the adhesion of cancer cells to FN to～51%in Mahlavu cells,～60%in SMMC7721cells as compared withcontrol cells (P <0.01). Gelatin Zymography analysis revealed that theN-20antibody significantly inhibited the activity of MMP-2in bothMahlavu and SMMC7721cells, while it did not affect the activity ofMMP-9. Western blot analysis revealed that the N20antibodysignificantly decreased the content MMP-2in the condition medium and increased E-Cadherin protein expression level while decreasedN-Cadherin protein expression level in Mahlavu cells. Similar resultswere obtained in SMMC7721cells.2. We constructed GRP78ΔKDEL mutant and transfectedSMMC7721cells, the cells stably overexpressing GRP78on the cellsurface were selected by G418(400μg/mL). The cells transfected withempty pcDNA3.1(+) vector served as the mock transfectants. In-cellwestern showed that the level of the cell surface GRP78in the ΔKDELtransfectants appeared to be～2-fold higher than that on the cellsurface of mock transfectants.Flow cytometry showed that thepercentage of the cell surface GRP78positive cells increasedsignificantly in the ΔKDEL transfectants as compared with the mocktransfectants (56.3%versus95.4%, P <0.01). Cell adhesion assayrevealed that the ΔKDEL transfectants exhibited increased celladhesion to FN-coated culture dishes compared with the mocktransfectant. Transwell assay revealed that the invasive potential ofΔKDEL transfectants increased significantly compared with the mocktransfectants (P<0.05). Gelatin zymography analysis revealed theΔKDEL transfectants exhibited markedly increased the activity ofMMP-2. Western blot analysis revealed the ΔKDEL transfectantsexhibited decreased the content of MMP-2in the condition mediumand decreased E-Cadherin protein level while increased N-Cadherinprotein level.Conclusion1. GRP78is expressed on the cell surface of hepatocellularcarcinoma cells.2. The cell surface GRP78could promote the invasive ability ofHCC. 3. The cell surface GRP78induced the invasion of cancer cells byenhancing the activity of MMP-2and regulating EMT process.