The Relationship Between Protein Translocation And Apoptosis Induction And Their Signal Transduction Pathways In Gastric Cancer Cells

Protein kinase c (PKC) is a family member of cellular serinehareonine kinasesthat play a variety of regulatory roles in proliferaion, differenti,ation, apoptosis, geneexpression, membrane trynspohation, and signal tf8nsduction. 1there are at lease lldistince PKC isoforms. Athough only subtle differences have beel1 found in vivo amongvarious PKC isoforms with regard to their enZymatic propeml, ligand binding andsubstrate specificity, evidence from in vitro exPeriments further demonstraed that theseisoforms may also ethibit different tissue- and cell-tyPe-specificity in their exPressionpattems, suggestion that each PKC isoform may have itS unique, specific functionalcharact6riatics.In this study, we att6mpted to find out the relation batweerl translocation of PKCsand inducton of apoptOsis in gastric cancer cells. TPA cou1d significantly induceapoptosis in BGC-823 cells, Which was not due to changes in the protein levels of PKCa, PKC 0 I, PKC' and PKC A, since TPA treatInent could not change their proteinexpression levels. To determine whether TPA may cause translocation of PKCs in gastficcancer cells, the immunofluorescent localization of PKC isofoims, including PKC a,PKC 0 l, PKC (and PKC A, was conducted. The resultS illustraed that TPA couldinduce the translocation of PKC a from both mitochondria and Cytosol tO nucleus,while it had no effect on the redistributions of other PKC isoforms in BGC-823 cells. Wealso found that TPAinduced apoptOsis was repressed When the trgnslocation of PKC awas blocked by either Wortmannin (wort.) or PKC inhibitor pePtidere.I.P), both of whichI 8x.are PKC-specific inhibitors. In the case of 2hr pretreatment by wori f or P.I.P, followed byTPA treatment, the apoptOsis rates were only 7. l% and 6.4%. The suPpressions of Bcl-2and Bcl-xl exPression and the uP-regUlation of Bax, Caspase 3 and Caspase 9 exPressionsby TPA were not det6cted When cells were pretreated with woft. ()r P.I.P. Our findings,therefore, have drawn an inspiration that translocation of PKC c(may be one of theessential links involved in the mechanism of apoptosis induction by TPA in gastficcancer cell.Nur77 is an orphan recePtoI, Whose ligand is unknown. Just like many early-response genes, Nur77 exPression is induced raPidly by a variety of growth stimuli,including growth factors, phorbol esters, calcium ionorphores and cyclic AMP-dependentsynthesis pathways. Although Nur77 exerts itS effect on cell proliferation and apoptosisthrough its caPability of binding tO a variety to response elements and regUlating theirtrynsactivation acitivities, the intrinsic function of Nur77 has not been folly understood,especially because of itS regulation on apoptosis and proliferation is characterized as celltype- and collteat-dependent.In this study, we demonstrate that Nur77 regUlates aPoptosis via itS eXPression andtranslocation, rather than itS talsaCtivation aCtivity in gastric cancer cells. Nur77exPressed constitutively in BGC-823 cells, ThA tredrient not only resulted in up-regulation of Nur77 rnRNA level, but also led to translocation of Nur77 from nucleus tOmitochondria, in which it caused the release of cytochrome c. But we found that TPAcould not activatC the tr3nscriptional activity of Nur77. When CAT rePort gene, whichcontains a Nur-binding sequendce(Nume),was transiently transfected into BGC-823cells, the reporter gene tf8nscriPtional activity was not changed by tfCatmeni with TPA.The up-regulation of Nur77 mRNA was essential fOr TPA to induce apoptosis, since TPAcould not induce aPotOsis in BGC-823/aNur cells which were trarlsfected with antisense-Nur77. Furthermore, we found that TPAinduced apoptOsis and the release of cytochromec was inhibited in the presence of leptomycin B (LMB), whi(:h can non-sepeifically0.II !k0S@1ttX Abstrast 02-06-22block protein exPort6d form nucleus. These data demonstracted that translocation ofNur77 was associatOd with the initiati...