The Effects Of Autophagy On Bortezomib-induced Apoptosis In Leukemia Cells

Objective:Acute lymphocytic leukemia is the most common malignancy in children.Although the remission rate of the patients has improved a lot in recent years,relapse and drugresistance are still the main problems in the treatment.And the exploration on new drugs has never stopped.Bortezomib is a kind of widespread used new anti-cancer drug.Its possible mechanism is to inhibit the activity of proteasome which can influence multiple cell signaling pathways and leads to the cell apoptosis.Several studies have proved that bortezomib has a great potential in the treatment of leukemia.Autophagylysosome system and ubiquitin-proteasome system are the two protein degradation pathways in eukaryocytes,and the interactions between them may have influence on the efficacy of bortezomib by autophagy regulation when used alone.In this study,we used bortezomib to inhibit proteasome activity and explore the effects of bortezomib on Jurkat T lymphocytic leukemia cells.Besides,we used autophagy regulators 3-methyladenine or rapamycin to inhibit or induce autophagy to observe the effects of autophagy on bortezomib-induced apoptosis in Jurkat cells.Then,we also observed the influence of chemotherapy on autophagy through the detection of autophagy-related protein in bone marrow cells before and after treatment of acute lymphocytic leukemia patients.Methods:Jurkat cells were cultured until they reached the logarithm growth period,then they were treated with bortezomib with or without autophagy inhibitor 3-methyladenine or autophagy inducer rapamycin for 24 h.Cell viability assay was detected by a CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit,and the results were analyzed by Graphpad Prism 5.0.After the protein of Jurkat cells was extracted,we used BCA kit to measure the concentration of protein.Then we used western blot to identify apoptosis-related and autophagy-related proteins,and the results were analyzed by the Quantity One software.Bone marrow smears from acute lymphocytic leukemia patients before and after treatment and immune thrombocytopenia patients were collected and the autophagy-related protein,p62 was detected by immunohistochemistry stain.The positive cells were observed by microscope,then statistical analysis was carried out by Graphpad Prism 5.0.Results:Cell viability of Jurkat cells was reduced by bortezomib in a dose-dependent way,and the level of autophagy-related protein LC-3B in Jurkat cells treated by bortezomib improved in a dose-dependent manner,too.Cell viability of Jurkat cells was reduced more when cells were treated by 3-methyladenine combined with bortezomib than bortezomib alone,and the level of LC-3B was reduced than treated with bortezomib alone.Cell viability of Jurkat cells was reduced more when cells were treated by rapamycin combined with bortezomib than bortezomib alone,and it was lower than treated by 3-methyladenine combined with bortezomib,and the level of LC-3B elevated in Jurkat cells treated by rapamycin combined with bortezomib than bortezomib alone.The level of autophagy-related protein p62 decreased in bone marrow cells from acute lymphocytic leukemia patients when compared to the control group(immune thrombocytopenia patients),and it improved after chemotherapy,even higher than the control group.Conclusions: 1.Bortezomib can induce both apoptosis and autophagy in a dose-dependent way in Jurkat cells.2.Both inhibition and induction of autophagy could enhance apoptosis induced by bortezomib in Jurkat cells,and induction of autophagy was more effectively.3.The level of autophagy improved in acute lymphocytic leukemia patients’ bone marrow cells,and chemotherapy could reduce it.