Phosphodiesterase inhibitors control A172 human glioblastoma cell death through cAMP-mediated activation of protein kinase A and Epac1/Rap1 pathways.
OBJECTIVE
We investigated whether cAMP-mediated protein kinase A(PKA) and Epac1/Rap1 pathways differentially affect brain tumor cell death using 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone(rolipram), specific phosphodiesterase type IV(PDE IV) inhibitor.


METHODS
A172 and U87MG human glioblastoma cells were used. Percentage of cell survival was determined by MTT assay. PKA and Epac1/Rap1 activation was determined by western blotting and pull-down assay, respectively. Cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis.


RESULTS
Non-specific PDE inhibitors, isobutylmethylxanthine(IBMX) and theophylline reduce survival percentage of A172 and U87MG cells. The expression of PDE4A and PDE4B was detected in A172 and U87MG cells. Rolipram-treated A172 or U87MG cell survival was lower in the presence of forskolin, adenylate cyclase activator, than that in its absence. Co-treatment with rolipram and forskolin also enhanced CREB phosphorylation on serine 133 that was inhibited by H-89, PKA inhibitor and cAMP-responsive guanine nucleotide exchange factor 1(Epac1), a Rap GDP exchange factor-mediated Rap1 activity in A172 cells. When A172 cells were treated with cell-permeable dibutyryl-cAMP(dbcAMP), PKA activator or 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate(CPT), Epac1 activator, basal level of cell death was increased and cell cycle was arrested at the phase of G2/M. Rolipram-induced A172 cell death was also increased by the co-treatment with dbcAMP or CPT, but it was inhibited by the pre-treatment with H-89.


CONCLUSIONS
These findings demonstrate that PKA and Epac1/Rap1 pathways could cooperatively play a role in rolipram-induced brain tumor cell death. It suggests that rolipram might regulate glioblastoma cell density through dual pathways of PKA- and Epac1/Rap1-mediated cell death and cell cycle arrest.
Department of Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea. eunyimoon@sejong.ac.kr
Eun-Yi
EY
Moon
Geun-Hee
GH
Lee
Myung-Shik
MS
Lee
Hwan-Mook
HM
Kim
Jae-Wook
JW
Lee
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001932:Brain Neoplasms / Q000201:enzymology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000032:analysis / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005909:Glioblastoma / Q000201:enzymology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020889:Rolipram / Q000494:pharmacology*; D000089804:Shelterin Complex; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology; D034501:Telomere-Binding Proteins / Q000378:metabolism*; D013806:Theophylline / Q000494:pharmacology
D020662:Guanine Nucleotide Exchange Factors; D007546:Isoquinolines; D010726:Phosphodiesterase Inhibitors; D047428:Protein Kinase Inhibitors; C116122:RAPGEF3 protein, human; D000089804:Shelterin Complex; D013449:Sulfonamides; C440809:TERF2IP protein, human; D034501:Telomere-Binding Proteins; D005576:Colforsin; D013806:Theophylline; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D015056:1-Methyl-3-isobutylxanthine