Requirement of reactive oxygen species generation in apoptosis of leukemia cells induced by 2-methoxyestradiol.
OBJECTIVE
To investigate the effects of 2-methoxyestradiol (2-ME) on 2 myeloid leukemia cell lines HL-60 and U937, and to explore its mechanisms.


METHODS
Human myeloid leukemia cells HL-60 and U937 were used. Measurement of mitochondrial membrane potential (Dym) was performed using 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide ( JC-1). Apoptosis and cellular nitric oxide (NO) were detected by flow cytometry using Annexin V and NO sensor dye. Superoxide anion was measured with a fluorescent plate reader by dihydroethidium (DHE). Cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay.


RESULTS
2-ME resulted in viability decrease in a dose-dependent manner. 2-ME treatment also generated reactive oxygen species (ROS), including NO and superoxide anions, which resulted in mitochondria damage. 2-ME-induced apoptosis was correlated with an increase in ROS. The quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from 2-ME cytotoxicity and prevented apoptosis induction by 2-ME. Furthermore, the addition of manumycin, a farnesyltransferase inhibitor, significantly enhanced apoptosis induced by 2-ME.


CONCLUSIONS
Cellular ROS generation plays an important role in the cytotoxic effect of 2-ME. It is possible to use ROS generation agents, such as manumycin, to enhance the antileukemic effect. The combination strategy needs further in vivo justification and may have potential clinical application.
She
Miao-rong
MR
Department of Hematology, Guangdong Provincial Peopleos Hospital, Guangzhou 510080, China.
Li
Jing-gao
JG
Guo
Kun-yuan
KY
Lin
Wei
W
Du
Xin
X
Niu
Xin-qing
XQ
D000077584:2-Methoxyestradiol; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism; D011090:Polyenes / Q000494:pharmacology; D053284:Polyunsaturated Alkamides / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology; D020298:U937 Cells
D011090:Polyenes; D053284:Polyunsaturated Alkamides; D017382:Reactive Oxygen Species; D050257:Tubulin Modulators; D013481:Superoxides; D009569:Nitric Oxide; D004958:Estradiol; D000077584:2-Methoxyestradiol; C054474:manumycin