The Role Of CLIC4 And 14-3-3 Protein In Autophagy Induced By Starvation In Glioma Cells

Malignant Gliomas account for over 50% of all brain tumors and are the most common primary brain tumors in adults. Despite the use of conventional treatments, including surgery, irradiation therapy, and chemotherapy, the average life expectancy of glioma patients after the initial diagnosis is usually less than 1 year. Although caspase mediated apoptosis is the best-defined cell death program counteracting tumor growth, glioma cells are resistant to the conventional proapoptotic cancer therapeutics. Therefore, effective treatment of malignant gliomas may rely on the development of novel strategies for inducing nonapoptotic cell death, such as autophagic cell death or cell death through mitotic catastrophe, which has been recently described as a new alternative death pathway.Autophagy, a regulated process of degradation and recycling of cellular constituents, also participates in organelle turnover and in the bioenergetic management of starvation. During autophagy, parts of the cytoplasm or entire organelles are sequestered into double-membraned vesicles called autophagic vacuoles or autophagosomes. These autophagosomes ultimately fuse with lysosomes to generate single-membraned autophago -lysosomes capable of degrading their contents. There are two main signal markers for the detection of autophagy, one is Beclin 1, a phylogenically conserved protein essential for autophagy, is a haplo insufficient tumor suppressor, activate and up-regulate in the autophagy through class III PI3K pathway. LC3 protein is another autophagy marker, cytosolic protein LC3-I covalently linked to phosphatidylethanolamine, then converts to LC3-II, associates with the formation of autophagosome. Although autophagy can serve as a protective mechanism against apoptosis and starvation by recycling macromolecules and removing damaged mitochondria and other organelles, it can also lead to growth arrest, reduction in cell number, and a nonapoptotic cell death associated with appearance of excessive autophagic vesicles. Therefore, high levels of autophagy can function as a cell death effector mechanism. Recently, induction of autophagy or autophagic cell death has been reported in several types of cancer cells in response to radiation or chemotherapy. But the molecular mechanisms of autophagy remain largely unknown, still need further investigations.The best characterized of the CLIC family members is CLIC4, essential for p53 and c-Myc mediated apoptosis and its promoter is a direct downstream target of these transcription factors. Cytoplasmic CLIC4 translocates to the nucleus under conditions of metabolic stress, growth arrest, apoptosis and DNA damage, this is mediated by a functional nuclear localization signal (NLS). CLIC4 has emerged as a crucial player in many physiological processes, including tubular morphogenesis during angiogenesis, transdifferentiation of mammary fibroblasts to myofibroblasts, and adipocyte differentiation. CLIC4 locate on the mitochondrial membrane, as the only member which is locate on the mitochondria, CLIC4 is important for the maintaince of the mitochondria founction and structure, to affect the founction of CLIC4 by genetic of biological techniques, maybe affect the normal mitochondria founction, and cell differentiation, generation and cell death urther. Mitochondria is regarded as the key of cell apoptosis and autophagy, so whether the CLIC4 protein which is relate to the cell apoptosis, is also relate to the autophagy, become a new subject that is great worth to discuss and research.14-3-3 proteins are a family of about 30kDa dimeric well conserved a-helical phosphoserine/threonine binding proteins. They are able to bind multiple protein ligands. 14-3-3 protein is involved in many different cellular processes, including mitogenesis, DNA damage, cell cycle control, and apoptosis, plays a significant role in the formation of malignant tumours. Latest research indicated that 14-3-3 protein isoform Tau regulated the level of Beclin1 and required for autophagy, but whether the other forms of 14-3-3 family are involved in autophagy process still not clear.In this study, we induced autophagy in human glioma U251 cell line by starvation, partially silenced chloride intracellular channel protein 4 (CLIC4) with small interfere RNA (siRNA), investigate the mechanism of CLIC4 and 14-3-3 protein in autophagy, and the relationship between autophagy and apotosis which are the two main forms of programmed cell death.Methods:(1) Human glioma U251 cell culture.(2) Base on the gene sequence and the RNAi desine principles, pSilencerTM3.1-H1 neo CLIC4 siRNA plasmid was constructed and sequencing. The level of CLIC4 mRNA and protein expression was analyzed by RT-PCR and Western Blot.(3) To detect cell vitality, MTT method was applied. Fluorescence staining of MDC,LC3, Hoechst to determine the change of cell autophagy. The levels of Bax,Bcl-2,cleaved caspase-3 and cytosolic cyt c protein were detected by Western blot, investigate the effect of the inhibition of CLIC4 on cell apoptosis.(4) Fluorescence staining of MDC,LC3,Hoechst and AO to determine the autophagy under starvation. The levels of apoptotic protein Bax,Bcl-2,cleaved caspase-3 and cytosolic cyt c were detected by Western blot. The cell apoptosis rate was detected with PI/Annexin-V FITC staining by flow cytometry.(5) To investigate the effect of the inhibition of CLIC4 on the autophagy induced by starvation, fluorescence staining of MDC,LC3,Hoechst and AO was applied for the autophagy detection, and Western Blot analysis to determine the LC3 protein level.(6) To investigate the effect of the inhibition of CLIC4 on the apoptosis induced by starvation. The levels of apoptotic protein Bax,Bcl-2,cleaved caspase-3 and cytosolic cyt c were detected by Western blot. The cell apoptosis rate was detected by flow cytometry.(7) To investigate the effect of the inhibition of CLIC4 on the co-location of 14-3-3 epsilon protein and CLIC4, fluorescence staining of Hoechst,14-3-3 epsilon,CLIC4 was used and Western Blot analysis to determine the expression level of Beclin 1 and 14-3-3 epsilon protein. Immunoprecipitation was applied to detect the binding of 14-3-3 protein and CLIC4 protein.(8) Mitochondrial membrane potential was measured with Rhodamine123 (Rho123) staining and Flow cytometry analysis. The effect of CLIC4 siRNA on the mitochondrial fusion genes Mfn1, Mfn2, Opa1 and fission genes Fis1, MTP18 were determined by RT-PCR.Results:(1) We constructed recombinant plasmid of pSilencerTM3.1-H1 neo CLIC4 siRNA successfully, confirmed by restrictive enzyme digestion and DNA sequencing. The level of CLIC4 mRNA and protern in U251 cells decreased significantly.(2) Compared with the control group, MTT results indicated that there is no obvious effect of cell vitality with CLIC4 siRNA transfection. The inhibition of CLIC4 has no significant effect on cell apoptosis and autophagy in U251 cells.(3) Compared with control group, the expression of autophagy-associated protein LC3 II was up-regulated significantly. No change of apoptosis related protein expression. Starvation of U251 cells could not lead to cell apoptosis.(4) The inhibition of CLIC4 enhanced cell autophagy, the accumulation of LC3 and cell autophagic vacuoles increased, LC3 II protein expression increased in U251 cells induced by starvation after 8 hours, compared with starvation group. Inhibition of CLIC4 triggered apoptosis in U251 cells under starvation.(5) The results of 14-3-3 epsilon and CLIC4 fluo-staining indicated that the co-location of 14-3-3 epsilon protein and ClIC4 increased significantly under starvation, showed that the regulation of CLIC4 on autophagy is relate to the 14-3-3 epsilon protein probably, and the inhibition of CLIC4 down-regulated the co-location of them. The results of Western Blot indicated that inhibition of CLIC4 caused the increasing of Beclin 1 and 14-3-3 protein expression. The co-precipitation of 14-3-3 epsilon protein and CLIC4 enhanced under starvation was decreased by inhibition of CLIC4. In a word, the inhibition of the interaction between 14-3-3 epsilon protein and CLIC4 by siRNA, and the following over activation of Beclin 1 signal pathway, maybe the reason of the inhibition of CLIC4 aggravated the autophagy under starvation.(6) The inhibition of CLIC4 decreased mitochondrial membrane potential, resulted mitochondrial injury and up-regulated mitochondrial fusion and fission genes,lead to mitochondria kinesics disorder, maybe the another reason of autophagy and apoptosis exacerbation.Conclusions:This research partially silenced mitochondria localized chloride intracellular channel protein 4 (CLIC4) with small interfere RNA (siRNA), induced human glioma U251 cell autophagy by starvation, to investigate the relation of autophagy and apoptosis, and the machenism of CLIC4 and 14-3-3 protein in this process. The results showed that inhibition of CLIC4 has no effect on cell autophagy and apoptosis. Under starvation, autophagy was detected in U251 cells but no apoptosis. Inhibition of CLIC4 enhanced the autophagy under starvation, on the same time, triggered caspase-dependent mitochondrial apoptosis. The mitochondrial dynamic related injury and decrease of the interaction between 14-3-3 protein and CLIC4, over activated Beclin 1 signaling pathway, maybe the reasons of these chain reactions.This study investigated the effect of choloride intracellular channel protein 4(CLIC4) which is located on mitochondria mainly and 14-3-3 protein in the autophagy induced by starvation for the first time. Further investigated the mechanism of cell autophagy in glioma cells, and the relationship between autophagy and apotosis which are the two main forms of programmed cell death, indicated that mitochondria is the key to connect autophagy and apoptosis. The further research on the relation of autophagy and apoptosis, introduce autophagy related methods into the therapic strategy, maybe the new route of cancer therapy.