The Effect Of Truncated4E-BPs And Crocin On C6Glioma Cells

Background:The morbidity of malignant glioma is about5/100,000, and approximately14,000and55,000new cases were diagnosed in America and China separately every year, and thetrend of new cases is also gradually rising in recent years. Glioblastoma accounts forapproximately60-70%of all malignant gliomas. The mortality of malignant glioma isstill keeping high in spite of progressing treatment means in tumors, so it is impending toexplore and search new intervening measures. The eukaryotic translation initiation factors(eIFs) is a class of necessary protein molecule in initial translation of eukaryotic cells, andthe eIF-4E has a huge potential for targeting treatment of malignant neoplasms.Translation of mRNA into protein is a fundamental process and requires tight regulation.Primary control occurs at the initiation step. For eukaryotic cells, eIF-4F compoundsinvolving eIF-4E eIF-4A and eIF-4G activates the translation of5’ hat structure mRNA,and its accurate assembly is ruled via eIF-4E binding proteins (4E-BPs) whichphosphorylation level decides the binding with eIF-4E. PTEN/PI3K/AKt/mTOR signaltransduction pathway plays a leading roole in phosphorylation of4E-BPs, and mTORdiscerns RAIP gene sequence of amino terminal and TOS gene sequence of carboxylterminal that RAIP gene sequence recognizes the phosphorylation sites of the amino terminal and carboxyl terminal of4E-BPs and TOS gene sequence discerns thephosphorylation sensitive sites of s65, T70and sirolimus. Therefore the outcome of theelimination of RAIP and TOS gene sequence may avoid the phosphorylation of4E-BPs,prevent the assembly of eIF-4F compound, then inhibit the growth of tumor cells andpromote their apoptosis.Traditional Chinese medicine is not only valuable treasure of our motherlandmedicine, but also a significant domain of the treatment and prevention of tumor.Literatures show that Crocin, the effective constituent of Crocus sativus L,Safforn ofChinese herbal medicine, will be a new-type native anticarcinogen that shows distinctantitumor activity to multiple tumor cells and has nearly no negative effect onphysiological function of normal tissue cells[56,57]. The mechanism research ofanti-glioma activity of Crocin is only reported individually in domestic, but not inforeign[50].Objective:We hope to build eukaryotic expression vector via deleting the coding sequence ofRAIP and TOS of4E-BPs cDNA, and explore the proliferation, pro-apoptosis and relatingproteins expression of C6glioma cells of truncate4E-BPs in molecular biology.Methods and Results:(1) The eukaryotic expression vector of truncate4E-BPs was builded via RT-PCR andmolecular cloning technique, and authenticated by restriction enzyme digestion andgene sequencing. The results showed that the insertion sequence accorded with thedesign sequence.(2) The proliferation ability of cells was detected by MTT and PI labeling flow cytometry.The results demonstrated that the proliferation of C6glioma cells was inhibited bytruncated4E-BPs.(3) The changes of C6cells morphology were observed under phase contrast microscopeand using Hoechst fluorescent staining. The results demonstrated that Corcin caninhibit obviously the growth of C6cells in vitro. (4) The apoptotic level of cells was tested via Annexin V-PI double labeling flowcytometry. The results revealed the pro-apoptotic effect of C6glioma cells of truncated4E-BPs.(5)The relating proteins expression was determined via western blot. The results showedthat the expression of the tumor-related proteins of C6glioma cells was inhibited by thetruncated4E-BPs; the expression of the proteins Servivin and Livin was down regulationby Crocin.