MZ3 can induce G2/M-phase arrest and apoptosis in human leukemia cells.
OBJECTIVE
4-(4-Bromopheny1)-2,3-dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-2-oxoidmi-dazole-1-carboxamide (MZ3) is one of the novel synthesized Combretastatin A-4 analogs. In previous research, we found that MZ3 is a potent and specific compound against leukemia cell lines both in vitro and in vivo. In this paper, our purpose is to investigate the mechanisms of MZ3 induced cell cycle arrest and apoptosis in K562 cells.


METHODS
Cytotoxicity was measured by MTT method; apoptosis and cell cycle arrest were measured by flow cytometry. DNA fragmentation was tested by agarose gel electrophoresis. Protein expression was analyzed by western blotting. The polymerization of microtubules in cell was detected through immunofluorescence.


RESULTS
MZ3 increases cyclin B1 levels and decreases the expression of cdc2, cdc25C and activation of Wee1. The changes in cdc2, cdc25C and Wee1 coincide with the appearance of phosphoepitopes recognized by a marker of mitosis, MPM-2. MZ3 induces apoptosis in K562 cells, proved by condensed DNA (DAPI stain) and DNA ladder. This apoptosis is related with the activation of caspase-9, caspase-3 and PARP cleavage, both of which are at the downstream of mitochondria. The changes of protein expression of Bcl-2 and Bax confirm the causal relationship between MZ3 and mitochondrial pathway, and the downregulation of AKT, p-AKT and XIAP indicate that the AKT pathway may participate in regulating this apoptosis. Moreover, MZ3 can reduce the soluble tubulin in K562 cells and inhibit microtubule assembly.


CONCLUSIONS
MZ3 is a promising antileukemia compound with antimitotic and apoptotic activity that has potential for management of various leukemias.
Fang
Liang
L
Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, #388 Yuhangtang Road, Hangzhou, Zhejiang 310058, China.
Shen
Li
L
Fang
Yanfen
Y
Hu
Yongzhou
Y
He
Qiaojun
Q
Yang
Bo
B
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D016203:CDC2 Protein Kinase; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects*; D024161:Collagen Type XI / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D018844:Cyclin-Dependent Kinases; D019008:Drug Resistance, Neoplasm; D005455:Fluorescent Antibody Technique; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors / Q000378:metabolism; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D020014:K562 Cells; D007938:Leukemia / Q000378:metabolism / Q000473:pathology*; D008938:Mitosis / Q000187:drug effects; D015394:Molecular Structure; D009687:Nuclear Proteins / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000378:metabolism
C517487:4-(4-bromophenyl)-2,3-dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-2-oxoidmi-dazole-1-carboxamide; C494826:BAX protein, human; C499319:COL11A2 protein, human; D018797:Cell Cycle Proteins; D024161:Collagen Type XI; D019926:Cyclin B; C107585:FOXM1 protein, human; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors; D007093:Imidazoles; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014404:Tubulin; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D051028:bcl-2-Associated X Protein; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; D051057:Proto-Oncogene Proteins c-akt; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases; D053148:Caspase 3; D053453:Caspase 9