Abstract
We investigated the signaling pathways associated with microtubule interaction and apoptosis in U937 cells in vitro and in the U937 xenograft model in vivo by using 6-pyrrolidinyl-2-(2-hydroxyphenyl)-4-quinazolinone (MJ-29). MJ-29 induced growth inhibition and cell death of leukemia cell lines (U937, HL-60, K562, and KG-1) in a dose- and time-dependent manner but did not obviously impair the viability of normal cells (peripheral blood mononuclear cells and human umbilical vein endothelial cells). MJ-29 interacted with α- and β-tubulin, inhibited tubulin polymerization both in vitro and in vivo, and disrupted microtubule organization. MJ-29 caused mitotic arrest by activating cyclin-dependent kinase 1 (CDK1)/cyclin B complex activity. MJ-29-induced growth inhibition and activation of CDK1 activity were significantly attenuated by roscovitine (CDK inhibitor) and CDK1 small interfering RNA (siRNA). Furthermore, MJ-29-induced Bcl-2 phosphorylation was also significantly attenuated by CDK1 siRNA. MJ-29 caused an increase in the protein levels of cytosolic cytochrome c, apoptotic protease-activating factor-1, procaspase-9, and apoptosis-inducing factor. MJ-29-promoted activation of caspase-9 and caspase-3 during apoptosis was significantly attenuated by caspase-9 and caspase-3 inhibitors. It is noteworthy that in BALB/cnu/nu mice bearing U937 xenograft tumors MJ-29 inhibited tumor growth in vivo. The terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling-positive apoptotic cells of tumor sections significantly increased in MJ-29-treated mice compared with the control group. In conclusion, our results suggest that MJ-29 induces mitotic arrest and apoptosis in U937 cells via CDK1-mediated Bcl-2 phosphorylation and inhibits the in vivo tumor growth of U937 xenograft mice.
Footnotes
This work was supported by the National Science Council of Taiwan [Grants NSC 94-2320-B-264-002, NSC94-2320-B-039-013] and China Medical University, Taichung, Taiwan [Grant CMU98-S-12].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.165415.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
-
ABBREVIATIONS:
- MJ-29
- 6-pyrrolidinyl-2-(2-hydroxyphenyl)-4-quinazolinone
- AIF
- apoptosis-inducing factor
- Apaf-1
- apoptotic protease-activating factor-1
- CDK
- cyclin-dependent kinase
- CAK
- CDK-activating kinase
- DCFH-DA
- 2′-7′-dichlorfluorescein-diacetate
- DiOC6
- 3,3′-dihexyloxacarbocyanine iodide
- DMSO
- dimethyl sulfoxide
- ECL
- enzyme chemiluminescence
- FADD
- Fas-associated protein with death domain
- FasL
- Fas ligand
- HRP
- horseradish peroxidase
- MTA
- microtubule-targeting agent
- NAC
- N-acetyl-cysteine
- PBS
- phosphate-buffered saline
- PI
- propidium iodide
- PMSF
- phenylmethane sulfonyl fluoride
- HUVEC
- human umbilical vein endothelial cell
- PBMC
- peripheral blood mononuclear cell
- TNF
- tumor necrosis factor
- TNF-R1
- TNF receptor 1
- TRAIL-R1
- TNF-related apoptosis-inducing ligand receptor 1
- TRAIL-R2
- TNF-related apoptosis-inducing ligand receptor 2
- TUNEL
- terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling
- siRNA
- small interfering RNA
- RT-PCR
- reverse transcription-polymerase chain reaction
- GAPDH
- glyceraldehyde 3-phosphate dehydrogenase
- DTT
- dithiothreitol
- ANOVA
- analysis of variance
- ΔΨm
- mitochondrial membrane potential
- ROS
- reactive oxygen species.
- Received December 31, 2009.
- Accepted May 11, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|