Preliminary Research On Roles Of Special Nuclear Matrix Proteins During Apoptosis Of Human Esophageal Cancer EC9706 Cells

In this study,30μmol/L curcumin was used to induce the apoptosis of human esophageal cancer EC9706 cells.On this base,by the methods of subcellular proteomics,immunocytochemistry and cellular molecular biology,we studied systematically the changes of nuclear matrix proteins during apoptosis of EC9706 cells induced by curcumin,analysed their co-localizational relationship with the products of related oncogenes and tumor suppressor genes,and speculated their functions to induce apoptosis of EC9706 cells.It's useful for us to find out the roles of differential expressed nuclear matrix proteins on EC9706 cells and explore the molecular mechanisms of carcinogenesis and apoptosis in a relatively systematical level.We found that the proliferation of EC9706 cells were inhibited effectively after being treated with 30μmol/L curcumin,and the inhibitory rate was 83.88%.The cell cycle was arrested in G₀/G₁ phase.The malignant morphological characteristics of EC9706 cells were changed after treatment with curcumin by means of light microscope and electron microscope,with the results that the cells shrinked,cell nucleus concerntrated and chromatin agglutinated.Immunocytochemistry results revealed that the expression level of Bax,P53,Fas were significantly upregulated while the expression level of Bcl-2 was significantly downregulated in the cells treated with curcumin.Agarose gel electrophoresis revealed that cell DNA fragment exhibited characteristic DNA ladder.We also found that cell nucleus concentrated and appeared granular fluorescence by Hoechst33258 staining.Meanwhile,29 nuclear matrix proteins(NMPs) changed significantly during EC9706 cells apoptosis,12 of which were identified by MALDI-TOF-MS analysis,including the up-regulated proteins,such as Transcription elongation factor A(SII)-like 4(TCEAL4),CCT8 protein,Tara,and the down-regulated proteins,such as Prohibitin,Spindling-2B,Nucleophosmin,Translation initiation factor eIF-2B subunit beta.In addition,we confirmed the changes of Nucleophosmin,Prohibitin,HSP70,Spindling-2B, Vimentin by western blot.Four specific NMPs,Nucleophosmin,Prohibitin, Spindling-2B and HSP70 were co-localized with anti-apoptosis protein Bcl-2 and pro-apoptosis protein Bax.Their expression level and location were changed during the apoptosis.Our study showed that the human esophageal cancer EC9706 cells were effectively induced into apoptosis after treatment with curcumin,with the expression of anti-apoptosis gene bcl-2 downregulated and pro-apoptosis gene bax,p53,fas upregulated.Nuclear matrix proteins(NMPs) have changed in expression level during the apoptosis,3 of which including TCEAL4,CCT8 protein,Tara upregulated and 9 of which including Prohibit,Spindling-2B,Nucleophosmin downregulated.The co-localization of Nucleophosmin,Prohibitin,Spindling-2B and HSP70 with the products of anti-apoptosis gene bcl-2 and pro-apoptosis gene bax also changed.In conclusion,the special NMPs exist in EC9706 cells and they play an important role in the apoptosis of EC9706 cells.Our results revealed that the apoptosis of EC9706 cells induced by curcumin was closely related with its roles in alterring the expression of NMPs which further regulate the activities of apoptosis-related genes.Our findings will help to explore the mechanisms of induced apoptosis of tumor cells,elucidate the signaling pathways and mechanisms of carcinogenesis and apoptosis,and provide a new way for clinical therapies targeting esophageal caner.