Experimental Study Of Effect To Proliferation Of Glioma By Inhibiting Proteasome Activity

Glioma is the malignant tumor in central nervous system with infiltrative growth, accounting for 30% of primary brain tumor in adult. Glioblastoma, a representative type of glioma, is the most malignant one with high recurrence rate and poor prognosis. Chemotherapy is a main treatment for glioma. However glioma is resistant to many kinds of drug to some extent, it gradually becomes to be the focus to search for the new and effective drugs in treating the glioma. Ubiquitin-proteasome pathway is the main pathway of intracellular protein degradation. Many short-lifed regulative protiens involving in cell proliferation, differentiation, apoptosis and so on are degraded in proteasome. Proteasome inhibitor can bind the active sites of the proteasome, which suppress the target protien degradation. Many studies revealed that proteasome inhibitor can suppress the many kinds of tumor cells proliferation, but the mechanism is unclear. In our experiments, we investigate the effects of proteasome inhibitor Lactacystin on rat C6 glioma cell in vitro and vivo to explore the effect and mechanism on inhibiting proteasome activity of glioma.Lactacystin, a natural and irreversible proteasome inhibitor, can specially bind the active sites of proteasome in a covalence manner. Rat C6 glioma cells have the same pathology features with the human glioblastoma cells. And Rat C6 glioma cells infiltratively grow too.Our experements include two parts:the vitro experment and the vivo experiment. MTT assay, Flow cytometry (FCM), RT-PCR, Western Blotting and Immunohistochemistry were used. The cell death has three types, apoptosis, autophagy and necrosis. Apoptosis is a suicide cell death with typical morphorlogical features and is a Caspace-dependent multistep reaction process. Caspace-3 is the main effective protease in apoptosis. Upregulation of the Caspace-3 expression could promote cell apoptosis. In our experiments, RT-PCR detected Caspace-3 mRNA expression and Western Blotting detected Caspace-3 protien expression. Apoptosis have three pathways: Mitochondrial pathway, Death receptor-mediated pathway and Extrinsic pathway. Cytochrome c release from the mitochondrion into the cytoplasm and mitochondrial membrane potential collapse are the characteristics of the Mitochondrial pathway. In our experiments, we detected the mitochondrial membrane potential by FCM to identify the type of apoptosis. Bcl-2 family protiens are important regulative protiens of apoptosis, including proapoptotic protiens and antiapoptotic protiens with locating in mitochondrial membrane. Bax and bcl-2 are two key members of the family. Bax can promote the apoptosis while bcl-2 can inhibit the apoptosis. The Bax/ bcl-2 ratio control the direction of the apoptosis. In our experiments, Bax and bcl-2 mRNA expression was detected by RT-PCR and their protien expression was detected by Western Blotting. Sequently Bax/ bcl-2 mRNA ratio and ther protien ratio are calculated. Nuclear factorκB (NF-κB) lies in the cytoplasm in the inactive state in most types of cells. When the cell is triggered, NF-κB is activated and translocates to the nucleus and upregulates the transcriptions of the target genes. The target genes involve in apoptosis, cell cycle control, migation and so on. NF-κB becomes constitutively activated in many kinds of tumor cells. In our experiments. P65 protien, a NF-κB protien, was detected by Western Blotting to explore the affect of Lactacystin on NF-κB of rat C6 glioma cell.Methods:(1) In vitro experiment, according to the different concentration of Lactacystin, the cultured C6 glioma cells were divised for the control group, Lactacystin 2.5μmol.L-1Group, Lactacystin 5.0μmol.L-1 Group and Lactacystin 10.0μmol.L-1Group. Proliferation rate of C6 glioma cells in each group was detected by MTT assay. FCM detected apoptosis and mitochondrial membrane potential of C6 glioma cells in each group. RT-PCR detected Bax and bcl-2 mRNA expression in each group. Bax, bcl-2, NF-κB(P65) protein expression was also detected by Western Blotting in C6 glioma cells of each group.(2) In vivo experiment, the cultured C6 glioma cells in the logarithmic growth phase were selceted and inoculated subcutaneously in the back of the nude mice. Then the nude mice were administered intraperitoneally with Lactacystin for 7 days. The nude mice were divided for model group (Model), Lactacystin 0.5μg/20g group, Lactacystin 1μg/20g Group, and Lactacystin 5μg/20g group with each group including three mice. The general condition of nude mice such as food, coat color and activities was observed every day.The measurement and calculation of tumor volume at different time points was got. RT-PCR detected Bax, bcl-2 and Caspase-3 mRNA expression in the glioma cells of each group. Bax, bcl-2, And Caspase-3 protein expression was also detected by Western Blotting and immuno- histochemistry.Results:(1) The results of vitro experiment: Compared with the Con group, in Lactacystin 2.5μmol.L-1Group, Lactacystin 5.0μmol.L-1 Group and Lactacystin 10.0μmol.L-1Group, the proliferation rate of cells were decreased(P<0.05); Compared with the Con group, in Lactacystin 5μmol.L-1Group and Lactacystin 10μmol.L-1 Group, mitochondrial membrane potential and NF-κB(P65)protein expression were decreased(P<0.05), while the apoptosis rate ,Bax/bcl-2 mRNA and protein ratio were increased(P<0.05).(2) The results of vivo experiment: Compared with the Model group, in Lactacystin 0.5μg/20g Group, Lactacystin 1μg/20g Group and Lactacystin 5μg/20g Group,the tumor volume was significantly reduced( P<0.05), Bax/bcl-2 mRNA and protein ratio was increased (P<0.05), the Caspase-3 mRNA expression was up-regulated ( P<0.05). Compared with the Model group, in Lactacystin 5μg/20g Group , the Caspase-3 protien expression was up-regulated ( P<0.05). Immunohistochemistry revealed that the tumor cells with positive Bax, bcl-2 and Caspase-3 proteins were markedly increased.Conclusions:(1) Proteasome inhibitor Lactacystin showed the antiproliferative activity on rat C6 glioma cell in vitro and vivo.(2) Proteasome inhibitor Lactacystin showed the induction of the apoptosis on rat C6 glioma cell in vitro and vivo.(3) Proteasome inhibitor Lactacystin definitely induces the apoptosis of the rat C6 glioma cell, which may be caused through the mitochondrial pathway. Addtionally,Lactacystin perhaps upregulates the ratio of Bax/bcl-2 and Caspase-3 expression to promote the apoptosis of the C6 glioma cell.(4) Proteasome inhibitor Lactacystin suppresses NF-κB signal pathway, which may result in the antiproliferative activity and the induction of apoptosis on rat C6 glioma cell.(5) Suppressing proteasome activity can obviously inhibit the proliferation and induce the apoptosis in glioma cell.Both vitro and vivo experiments revealed that Suppressing proteasome activity can obviously inhibit the proliferation and induce the apoptosis in glioma cell and futheremore the mechanisms were initially analyzed. It supplied the experimental and theoretical basis for the Proteasome inhibitor in treating glioma in clinic in future.