title: Leptomycin B-induced apoptosis is mediated through caspase activation and down-regulation of Mcl-1 and XIAP expression, but not through the generation of ROS in U937 leukemia cells.
abstract: Leptomycin B (LMB), which is originally isolated from Streptomyces, possesses anti-tumor properties in vivo and in vitro. Though it was previously reported that LMB induces cell cycle arrest and p53-mediated apoptosis in certain cancer cells, however, the mechanism by which LMB induces apoptosis remains poorly understood. Here, we investigated the mechanisms of apoptosis induced by LMB in U937 cells. Treatment with LMB concentration-dependently induced cytotoxicity and apoptosis in U937 cells that correlated temporally with activation of caspases and down-regulation of Mcl-1 and XIAP. LMB did not change the expressions of Bcl-2 or Bax. A broad spectrum caspase inhibitor, z-VAD-fmk, blocked caspase-3 activation and elevated the survival in LMB-treated U937 cells, suggesting that caspase-3 activation is critical for LMB-induced apoptosis. Interestingly, Bcl-2 overexpression that blocked cytochrome c release by LMB effectively attenuated the apoptotic response to LMB, suggesting that LMB-induced apoptosis is mediated through the mitochondrial pathway. Antioxidants or antioxidant enzymes had no effects on LMB-induced apoptosis. Data of flow cytometry analysis using 2',7'-dichlorofluorescein-diacetate further revealed no reactive oxygen species (ROS) generation by LMB, indicating that apoptosis induced by LMB is ROS-independent. However, the apoptotic response to LMB was not shown in U937 cells pretreated with the sulfhydryl group-containing antioxidant N-acetylcysteine (NAC). Further analysis suggested that NAC directly binds LMB and abolishes the apoptotic effects of LMB. Collectively, these findings suggest that LMB potently induces apoptosis in U937 cells, and LMB-induced apoptosis in U937 cells is related with cytochrome c release, activation of caspases, and selective down-regulation of Mcl-1 and XIAP.
authors.affiliation: Chronic Disease Research Center and Institute for Medical Science, Keimyung University School of Medicine, Jung-gu, Daegu 700-712, South Korea. jangbc12@kmu.ac.kr
authors.forename: Byeong-Churl
authors.initials: BC
authors.lastname: Jang
authors.forename: Ji-Hye
authors.initials: JH
authors.lastname: Paik
authors.forename: Hye-Yun
authors.initials: HY
authors.lastname: Jeong
authors.forename: Hyun-Ji
authors.initials: HJ
authors.lastname: Oh
authors.forename: Jong-Wook
authors.initials: JW
authors.lastname: Park
authors.forename: Taeg Kyu
authors.initials: TK
authors.lastname: Kwon
authors.forename: Dae-Kyu
authors.initials: DK
authors.lastname: Song
authors.forename: Jong-Gu
authors.initials: JG
authors.lastname: Park
authors.forename: Sang-Pyo
authors.initials: SP
authors.lastname: Kim
authors.forename: Jae-Hoon
authors.initials: JH
authors.lastname: Bae
authors.forename: Kyo-Chul
authors.initials: KC
authors.lastname: Mun
authors.forename: Min-Ho
authors.initials: MH
authors.lastname: Suh
authors.forename: Minoru
authors.initials: M
authors.lastname: Yoshida
authors.forename: Seong-Il
authors.initials: SI
authors.lastname: Suh
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism* / Q000502:physiology; D002470:Cell Survival; D015536:Down-Regulation; D004789:Enzyme Activation; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D007938:Leukemia / Q000473:pathology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020298:U937 Cells; D051636:X-Linked Inhibitor of Apoptosis Protein
chemical_list: D000903:Antibiotics, Antineoplastic; D005231:Fatty Acids, Unsaturated; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C030193:immunosuppressive acidic protein; D020169:Caspases; C038753:leptomycin B