title: 4-Chlorobenzoyl berbamine, a novel berbamine derivative, induces apoptosis in multiple myeloma cells through the IL-6 signal transduction pathway and increases FOXO3a-Bim expression.
abstract: Multiple myeloma (MM) is an incurable hematopoietic malignancy, although many novel therapeutic agents have been explored. In the present study, we showed that 4-chlorobenzoyl berbamine (BBD9), a novel derivative of berbamine, inhibited the growth of 4 MM cell lines (U266, RPMI 8226, MM1.R and MM1.S). After a 24-h treatment with BBD9, the half maximal inhibitory concentration (IC(50)) values were 1.8, 2.3, 1.5 and 2.4 µg/ml, respectively, using MTT assays. In BBD9-treated U266 and RPMI 8226 cells, Annexin V (AV)-propidium iodide (PI) staining and FACS analysis demonstrated that apoptosis was involved in this inhibition. This was confirmed by western blot analysis indicating activation and cleavage of caspase-3, -8, -9 and PARP. BBD9 also induced G2/M phase cell cycle arrest in these cells. To investigate the mechanisms responsible for BBD9-induced apoptosis, U266 cells were incubated with 0, 1 or 2 µg/ml of BBD9 combined with 0 or 150 ng/ml of interleukin (IL)-6. MTT assays showed that IL-6 partially abrogated the BBD9-induced cell growth inhibition. Furthermore, BBD9 inhibited autocrine IL-6 production, and downregulated membrane IL-6 receptor (IL-6R) expression. Crucial proteins downstream of the IL-6 signaling pathway, including AKT and STAT3, were inactivated in BBD9-treated U266 cells, although exogenous IL-6 did not abrogate this effect. Forkhead transcription factor class 3a (FOXO3a), a nuclear transcription factor downstream from AKT, was upregulated in the nuclei of BBD9-treated U266 cells. Bim, the target gene of FOXO3a, was upregulated at both the protein and mRNA levels, as shown by western blot analysis and quantitative PCR. These results suggest that BBD9 induces apoptosis in MM cells through the inhibition of the IL-6 signaling pathway, leading to FOXO3a activation and upregulation of pro-apoptotic Bim.
authors.affiliation: Department of Hematology, Institute of Hematology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China.
authors.forename: Jia-Kun
authors.initials: JK
authors.lastname: Shen
authors.forename: Hua-Ping
authors.initials: HP
authors.lastname: Du
authors.forename: Qiuling
authors.initials: Q
authors.lastname: Ma
authors.forename: Min
authors.initials: M
authors.lastname: Yang
authors.forename: Yun-Gui
authors.initials: YG
authors.lastname: Wang
authors.forename: Jie
authors.initials: J
authors.lastname: Jin
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis; D000072224:Bcl-2-Like Protein 11; D044182:Benzylisoquinolines / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000096:biosynthesis*; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis; D009101:Multiple Myeloma / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019947:Receptors, Interleukin-6 / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C548958:4-chlorobenzoylberbamine; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D044182:Benzylisoquinolines; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D015850:Interleukin-6; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019947:Receptors, Interleukin-6; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9