| Glioma is the most common malignant brain tumor with infiltrative growth, accountingfor40%-50%of primary brain tumor in central nervous system. Glioblastoma, arepresentative type of glioma, is the most malignant one with high recurrence rate and poorprognosis,which average survival period is less than one year. Although it is difficult toremove the tumor completely, the standard treatment of glioma is still surgical resectioncombined with radiation therapy and chemotherapy. The majority of the tumor can be cut andverified the pathological types by operation. It is also benefit to choose the chemotherapydrugs. With the development of neurosurgery, in particular the application of neuronavigationand intraoperative MRI, our operations are more safe and more thorough than before, but wecan not solve the recurrence still. Chemotherapy is a main treatment for glioma. Howeverglioma is resistant to many kinds of drug to some extent, it gradually becomes to be the focusto search for the new and effective drugs in treating the glioma. Ubiquitin-proteasome systemis the main system of intracellular protein degradation. The proteasome is an evolutionarilyconserved protease complex with multiple catalytic activities and is mainly responsible forselective degradation of abnormal intracellular proteins and many cellular regulatory proteins.It is also involved in cellular differentiation, proliferation, apoptosis, antigen presentation andcell cycle modulation. Therefore, inhibition of proteasome activity has emerged as a newchemotherapy strategy for malignant tumors. Proteasome inhibitor can bind the active sites ofthe proteasome, which suppress the target protien degradation. Although studies have shownthat inhibition of proteasome activity could inhibit cellular proliferation in several tumor celllines including melanoma, prostate cancer and glioma cells, its underlying mechanism is notfully understood. MG-132is an aldehyde peptide compound that has shown potent inhibitoryeffects on proteasome chymotrypsin-like activity and is chemically distinct from bortezomib.Although previous studies in vitro showed that it could induce apoptosis in human gliomacells, its upstream events are largely unknown. In our experiments, we investigate the effectsof proteasome inhibitor MG-132on C6glioma cell in vitro to explore the effect and mechanism of proteasome inhibitor on the induction of apoptosis in glioma cell.Oxidative stress is a complex and dynamic situation characterized by overproduction ofROS that cannot be cleared from cells. Oxidative stress is a common damage in cells andtissues. Cellular antioxidant systems provide the primary defense mechanisms for cells tocope with oxidative stress via directly quenching reactive oxygen species. The UPP provides asecondary defense mechanism via degradation of abnormal proteins. Oxidative stress candamage macromolecules such as lipids, nucleic acids and proteins and trigger cell deaththrough the effects of ROS on signal transduction pathways. Therefore, oxidative stress couldcause cell death via apoptotic pathways. Apoptosis is a programmed cell death with typicalmorphorlogical features and is a caspase-dependent multistep reaction process. Severalstudies have shown that oxidative stress is closely associated with the proteasome, as theproteasome is involved in regulating anti-oxidants, degrading abnormal intracellular proteins.However, the relationship between proteasome dysfunction and oxidative stress is still amatter of dispute. In the central nervous system, proteasome dysfunction serves as animportant switch for the induction of oxidative stress. Therefore, in this study, we examinedthe effects of proteasome inhibition on rat C6glioma cells and investigated the relationshipbetween apoptosis and oxidative stress generated by inhibition of proteasome activity.MG-132is a synthetic tripeptide aldehyde proteasome inhibitor that exerts antitumoractivity by blocking the proteasome chymotrypsin-like activity. C6glioma cell has the samepathology features with the human glioblastoma cells. So MG-132and C6glioma cell wereused. MTT assay was used to analyze cell proliferation. Proteasome activity was assayedusing Suc-LLVY-AMC, and intracellular ROS level was evaluated with the redox-sensitivedye DCFH-DA. Apoptosis was detected using fluorescence and transmission electronmicroscopy as well as flow cytometry. The expression of apoptosis-related proteins wasinvestigated using Western blot analysis. Tiron, a well-known and widely used antioxidant,was used in this study to attenuate the oxidative stress that proteasome inhibition mightinduce. Results: MG-132inhibited C6glioma cell proliferation in a time-and dose-dependentmanner. MG-132(18.5umol/L) suppressed the proteasome activity by about70%at3h. Itinduced apoptosis via down-regulation of anti-apoptotic proteins Bcl-2and XIAP, up-regulation of pro-apoptotic protein Bax and caspase-3, and production of cleaved PARP. Itcaused a more than5-fold increase of reactive oxygen species. MG-132induces oxidativestress.Tiron (1mmol/L) effectively blocked oxidative stress induced by MG-132(18.5umol/L),attenuated proliferation inhibition and apoptosis in C6glioma cells, and reversed theexpression pattern of apoptosis-related proteins. Conclusion: MG-132induced apoptosis ofC6glioma cells via the oxidative stress.In summary, we showed that inhibition of proteasome activity by MG-132is an effectiveway to suppress the proliferation of C6glioma cells via the induction of apoptosis. Tiron notonly blocked the oxidative stress caused by MG-132but also suppressed MG-132-inducedapoptosis by decreasing the expression of Bax and caspase-3, rescuing the expression of Bcl-2and XIAP and attenuating PARP cleavage. These results suggest that there is a cause andeffect relationship between oxidative stress and apoptosis induced by MG-132. In one word,MG-132induced apoptosis of C6glioma cells via the oxidative stress.
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