title: Autophagy counteracts apoptosis in human multiple myeloma cells exposed to oridonin in vitro via regulating intracellular ROS and SIRT1.
abstract: OBJECTIVE
  To explore the mechanisms underlying the oridonin-induced apoptosis and autophagy in human multiple myeloma cells in vitro.
  
  
  METHODS
  Human multiple myeloma RPMI8266 cells were used. The cell viability was assessed using MTT assay. Morphological changes of apoptosis and autophagy were observed under transmission electron microscope. TUNEL and annexin V-FITC/PI dual staining assays were used to measure apoptosis. Autophagy was analyzed using Western blot analysis and immunofluorescence staining with a QDs(605 nm)-Anti-LC3 fluorescent probe. Intracellular ROS was estimated with flow cytometry using DCFH-DA fluorescent probe. Protein levels of active caspase 3, Beclin 1 and SIRT1 were determined with Western blot analysis.
  
  
  RESULTS
  Exposure to oridonin (1-64 μmol/L) inhibited the proliferation of RPMI8266 cells in a concentration-dependent manner with an IC(50) value of 6.74 μmol/L. Exposure to oridonin (7 μmol/L) simultaneously induced caspase 3-mediated apoptosis and Beclin 1-dependent autophagy of RPMI8266 cells. Both the apoptosis and autophagy were time-dependent, and apoptosis was the main effector pathway of cell death. Exposure to oridonin (7 μmol/L) increased intracellular ROS and reduced SIRT1 nuclear protein in a time-dependent manner. The blockade of intracellular generation of ROS by NAC (5 mmol/L) abrogated apoptosis, autophagy and the decrease of SIRT1 in the cells exposed to oridonin (7 μmol/L). The inhibition of autophagy by 3-MA (5 mmol/L) sensitized the cells to oridonin-induced apoptosis, which was accompanied by increased intracellular ROS and decreased SIRT1.
  
  
  CONCLUSIONS
  Oridonin simultaneously induces apoptosis and autophagy of human multiple myeloma RPMI8266 cells via regulation of intracellular ROS generation and SIRT1 nuclear protein. The cytotoxicity of oridonin is mainly mediated through the apoptotic pathway, whereas the autophagy protects the cells from apoptosis.
authors.affiliation: Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Zeng
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Shuai
authors.initials: S
authors.lastname: Zhao
authors.forename: Guo-hui
authors.initials: GH
authors.lastname: Cui
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D045744:Cell Line, Tumor / Q000187:drug effects / Q000648:ultrastructure; D049109:Cell Proliferation / Q000187:drug effects; D045786:Diterpenes, Kaurane / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009101:Multiple Myeloma / Q000188:drug therapy / Q000473:pathology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D056564:Sirtuin 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D045786:Diterpenes, Kaurane; D008565:Membrane Proteins; D017382:Reactive Oxygen Species; C011959:oridonin; C447939:SIRT1 protein, human; D056564:Sirtuin 1