Selective induction of G2/M arrest and apoptosis in HL-60 by a potent anticancer agent, HMJ-38.
We previously reported that HMJ-38 was the most potent 2-phenyl-4-quinozolinone derivative in inhibiting tubulin polymerization and showed significant cytotoxicity against several human tumor cell lines. In this work, we studied its cytotoxic effect on HL-60 leukemia cells and the underlying mechanisms. We first investigated the effects of HMJ-38 on viability, cell cycle and induction of apoptosis in HL-60 and normal human peripheral blood mononuclear cells (PBMC). After 24-hour treatment with HMJ-38, a dose- and time-dependent decrease in the viability of HL-60 cells was observed and the approximate IC50 was 4.48 microM. The cytotoxic effect of HMJ-38 on PBMC was less significant than that on HL-60 cells, either with 24 or 48 hours of treatment. Cell cycle analysis showed that HMJ-38 induced significant G2/M arrest and apoptosis in HL-60 cells. The HMJ-38-induced G2/M arrest occurred before the onset of apoptosis. Within 24 hours of treatment, HMJ-38 influenced the CDK/cyclin B activity by increasing Chk1, Wee1 and p21 and decreasing Cdc25C protein levels. The HMJ-38-induced apoptosis was further confirmed by morphological assessment and DNA fragmentation assay. Induction of apoptosis in HMJ-38-treated HL-60 cells was accompanied by an apparent increase of cytosolic cytochrome c, down-regulation of Bcl-2, up-regulation of Bax and cleavage of pro-caspase-9, -3 and poly(ADP)ribosylpolymerase (PARP). The results of the significant reduction of caspase activities and apoptosis by caspase inhibitors indicated that the HMJ-38-induced apoptosis was mainly mediated by activation of caspases-9 and -3. HMJ-38 also activated ERK in HL-60 cells. Pre-incubating cells with ERK inhibitors (U0126 and PD98059) attenuated the HMJ-38-induced ERK activation and apoptosis. Nevertheless, cells remained arrested in G2/M. These results suggest that HMJ-38 is a potent anticancer drug and it shows a remarkable action on cell cycle before commitment for apoptosis is reached.
Yang
Jai-Sing
JS
Graduate Institute of Pharmaceutical Chemistry, School of Medicine, China Medical University, Taichung, Taiwan 404, Republic of China.
Hour
Mann-Jen
MJ
Kuo
Sheng-Chu
SC
Huang
Li-Jiau
LJ
Lee
Miau-Rong
MR
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D004789:Enzyme Activation / Q000187:drug effects; D016195:G2 Phase / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects; D020935:MAP Kinase Signaling System; D008938:Mitosis / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D011759:Pyrrolidines / Q000494:pharmacology*; D011799:Quinazolines / Q000494:pharmacology*; D052999:Quinazolinones; D015854:Up-Regulation / Q000187:drug effects; D051028:bcl-2-Associated X Protein
C416653:2-(3'-methoxyphenyl)-6-pyrrolidinyl-4-quinazolinone; D000970:Antineoplastic Agents; C494826:BAX protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011759:Pyrrolidines; D011799:Quinazolines; D052999:Quinazolinones; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases