The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 cells.
Bufalin, an active principle of Chinese medicine, chan'su, induced typical apoptosis in human leukemia U937 cells. When U937 cells were treated with 10(-8) M bufalin in the absence of serum, mitogen-activated protein (MAP) kinase activity was markedly increased 6 h after the start of treatment and elevated so for 12 h. Prior to the activation of MAP kinase, increased activities of Ras, Raf-1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf-1, and MAP kinase kinase to MAP kinase. In association with this signal transduction, the concentration of cAMP in the cells decreased markedly, suggesting that Raf-1 was also activated by a decrease in the extent of phosphorylation by protein kinase A. In fact, pretreatment of U937 cells with forskolin and 3-isobutyl-1-methylxanthine, which are known to increase the concentration of cAMP in the cells, and subsequent treatment with bufalin resulted in a decrease in both Raf-1 activity and DNA fragmentation. To confirm the participation of MAP kinase in the apoptotic process, antisense cDNA for MAP kinase kinase 1 was expressed in U937 cells. The transformants were significantly resistant to both DNA fragmentation and cell death in response to bufalin. Our findings suggest that a pathway with the persistent activation of MAP kinase in U937 cells in response to bufalin is at least one of the signal transduction pathways involved in the induction of apoptosis.
Watabe
M
M
Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan.
Masuda
Y
Y
Nakajo
S
S
Yoshida
T
T
Kuroiwa
Y
Y
Nakaya
K
K
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002018:Bufanolides / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D016373:DNA, Antisense / Q000096:biosynthesis; D004273:DNA, Neoplasm / Q000187:drug effects; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D007938:Leukemia; D020929:Mitogen-Activated Protein Kinase Kinases; D011494:Protein Kinases / Q000096:biosynthesis / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000378:metabolism
D000970:Antineoplastic Agents; D002018:Bufanolides; D016895:Culture Media, Serum-Free; D016373:DNA, Antisense; D004273:DNA, Neoplasm; D011518:Proto-Oncogene Proteins; D005576:Colforsin; D000242:Cyclic AMP; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins; D015056:1-Methyl-3-isobutylxanthine; C022777:bufalin