Oligonucleotide microarray analysis of apoptosis induced by 15-methoxypinusolidic acid in microglial BV2 cells.
OBJECTIVE
We conducted a genome wide gene expression analysis to explore the biological aspects of 15-methoxypinusolidic acid (15-MPA) isolated from Biota orientalis and tried to confirm the suitability of 15-MPA as a therapeutic candidate for CNS injuries focusing on microglia.


METHODS
Murine microglial BV2 cells were treated with 15-MPA, and their transcriptome was analysed by using oligonucleotide microarrays. Genes differentially expressed upon 15-MPA treatment were selected for RT-PCR (reverse transcription-polymerase chain reaction) analysis to confirm the gene expression. Inhibition of cell proliferation and induction of apoptosis by 15-MPA were examined by bromodeoxyuridine assay, Western blot analysis of poly-ADP-ribose polymerase and flow cytometry.


RESULTS
A total of 514 genes were differentially expressed by 15-MPA treatment. Biological pathway analysis revealed that 15-MPA induced significant changes in expression of genes in the cell cycle pathway. Genes involved in growth arrest and DNA damage [gadd45alpha, gadd45gamma and ddit3 (DNA damage-inducible transcript 3)] and cyclin-dependent kinase inhibitor (cdkn2b) were up-regulated, whereas genes involved in cell cycle progression (ccnd1, ccnd3 and ccne1), DNA replication (mcm4, orc1l and cdc6) and cell proliferation (fos and jun) were down-regulated. RT-PCR analysis for representative genes confirmed the expression levels. 15-MPA significantly reduced bromodeoxyuridine incorporation, increased poly-ADP-ribose polymerase cleavage and the number of apoptotic cells, indicating that 15-MPA induces apoptosis in BV2 cells.


CONCLUSIONS
15-MPA induced apoptosis in murine microglial cells, presumably via inhibition of the cell cycle progression. As microglial activation is detrimental in CNS injuries, these data suggest a strong therapeutic potential of 15-MPA.
Choi
Y
Y
College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Korea.
Lim
S Y
SY
Jeong
H S
HS
Koo
K A
KA
Sung
S H
SH
Kim
Y C
YC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D002460:Cell Line; D004224:Diterpenes / Q000302:isolation & purification / Q000633:toxicity*; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010936:Plant Extracts / Q000302:isolation & purification / Q000633:toxicity; D029783:Thuja
C464724:15-methoxypinusolidic acid; D004224:Diterpenes; D010936:Plant Extracts