The Effects Of Ubiquitin-proteasome Pathway In TRAIL-induced Apoptotic Resistance On Lymphoma

Objective:The incidence of malignant lymphoma is the eighth in China.The death of median age is 49.9 years,so it is risk for child and youth.At present, chemotherapy is the key method for malignant lymphoma.Now,the important thing is we must look for effective chemotherapeutics to improve the sensitivity and overcome drug resistance.TRAIL(TNF-related apoptosis-inducing ligand) is the tumor necrosis factor (TNF) family members,which can selectively induce apoptosis on cancer cells,transformant and infection viral cells,but it has no toxicity for normal cells.In type I cells,the activated procaspase-8 or-10 through caspase dependent signaling pathway induce apoptosis. In type II cells, mitochondrial apoptotic pathway is the important method of apoptosis.Now, researches have realized that TRAIL can induce more tumor cells apoptosis,but it has no toxicity for normal cells,so TRAIL is considered as a promising drug for malignant tumors.However,most of tomor cells act as primary or secondary drug resistance.It is very important to expound the drug resistant mechanism for effective applying TRAIL.It can result drug resistance because of the dysfunction of any site in apoptotic signaling pathway.As we all known,the process of apoptosis is complicated in cells,which include death receptor pathway and mitochondria pathway.They can enlargement the death signal each other.In mitochondria,SMAC,cytochrome c play a key role in the process of apoptosis.So we think it can reverse drug resistance by enlarging the mitochondria parhway.The stability of mitochondria is associated with Bcl-2 family proteins,the family include pro-/anti-apoptotic proteins;Bax,Bak,Bid,Bad et al are pro-apoptosis proteins;Bcl-2,Bcl-XL et al are anti-apoptotic proteins;The rate of pro-/anti-apoptotic proteins influence the stability of mitochondria,so it can trigger or inhibit the process of apoptosis.Bax is an important pro-apoptotic protein.The post-translational modification is a crucial factor for the level of protein.In all cells,there are more degradative pathway of protein,including:lysosomal pathway,calpain system,ubiquitin-proteasome pathway and independent-ubiquitin et al. Polyubiquitinated protein can be degraded by ubiquitin-proteasome pathway.In all,through pre-work we find the decrease of Bax protein is a frequent event in tumor therapy,so Bax may be related to drug resistance.Our objective is to explore the regulated mechanism of ubiquitin-proteasome pathway on Bax protein,to expound the change of level,conformation and location on Bax at regulation of ubiquitin and to explain the regulated effect of ubiquitin-proteasome pathway on crucial protein. So it can give a new therapeutic target.Methods:一,To explore the apoptotic effect of TRAIL on malignant lymphoma cells.1,Cell culture and treatment:choose malignant lymphoma cells:Raji,U937 and the immortalization normal lymphocytes cell:Hmy2.ciR as object. They were maintained in RPMI 1640 with 10% heat-inactivated fetal bovine serum. All cells were cultured at 37℃in a humidified incubator containing 5% CO2.The concentration of TRAIL is 500ng/ml,and then it treat the cells at different time points2,Measure cell apoptosis:after cells were treated by TRAIL(500ng/ml) at 0,6,24 hours,PI and AnnexinV-FIFC to dye, flow cytometry to measure cell apoptosis3,Measure the activity of caspase-8,9:after cells were treated by TRAIL(500ng/ml) at 0,6,24 hours,collect and centrifugate the cells,add the fluorescence object,at last using TD-700 instrument measute the fluorescence at 400/505nm4,MTT assay the the activity of cells:after cells were treated by TRAIL(500ng/ml) at 0,2,4,6,8,24 hours,add MTT for another 4 hours,then add DMSO,at last detect the absorbance (A),make the curve of cell growth二,To evaluate the level of Bax in TRAIL-induced apoptosis on malignant lymphoma cells1,The level and stability of Bax in cells:collect and lysis the cells,western-blot evaluate the level of Bax2,Western-blot measure the protein level of Bax after TRAIL treated 3,qRT-PCR measure the mRNA level of Bax after TRAIL treated4,Cell metastructure extraction and western-blot assay the location of Bax after TRAIL treated三,To assay the regulation of ubiquitin-proteasome pathway on the protein level of Bax1,Regulation of ubiquitin-proteasome pathway on Bax in the process of TRAIL,using IP and western-blot to assay the combination between ubiquitin and Bax2,Analogy the regulation of ubiquitin-proteasome pathway on Bax in vitro四,The regulation of proteasome inhibitor on malignant lymphoma cells1,To evaluate the synergistic apoptosis-induced reaction between proteasome inhibitor and TRAIL2,The regulation of proteasome inhibitor on BaxResults:TRAIL-treated malignant lymphoma cells:Raji,U937,after 24 hours the apoptotic index was was lower than the control cell:Hmy2.ciR. The Bax protein in Raji and U937 is degraded, but the Bax mRNA expression level does not change significantly.When proteasome inhibitor and TRAIL were synergisted,it can greatly reverse the drug resistance of malignant lymphoma cells:Raji,U937 on TRAIL,meanwhile the level of Bax also increased by the time of extend. Cell metastructure extraction and western-blot assay show that the level of Bax decrease significantly in cytoplasm and mitochondrion of lymphoma cells:Raji,U937 after TRAIL-treated,however,nucleus's Bax protein don't change greatly.When PS-341and TRAIL were synergisted,it can increase Bax protein in cytoplasm and mitochondrion but not in nucleus.Using IP can find the level of polyubiquitinated Bax significantly improved.In vitro using ubiquitin (2μg/ml) result in the decrease of Bax protein in patient's lymphoma tissues,but not in the adjacent tissues.Conclusion:Malignant lymphoma cells are drug resistance on TRAIL-induced apoptosis,and the level of Bax also decresed greatly,but the mechanism is still unknown; Ubiquitin-proteasome pathway regulate the level of Bax; When proteasome inhibitor and TRAIL were synergisted,it can reverse the drug resistances increase the expression of Bax protein and induce apoptosis of lymphoma cells,therefore it can give a new therapic target.