Relationship between phosphatase activity and cytotoxic effect of two protein phosphatase inhibitors, okadaic acid and pervanadate, on human myeloid leukemia cell line.
Protein phosphatases are signalling molecules that regulate a variety of fundamental cellular processes including cell growth, metabolism and apoptosis. The aim of this work was to correlate the cytotoxicity of pervanadate and okadaic acid on HL60 cells and their effect on the phosphatase obtained from these cells. The cytotoxicity of these protein phosphatase inhibitors was evaluated on HL60 cells using phosphatase activity, protein quantification and MTT reduction as indices. The major phosphatase presents in the cellular extract showed high activity (80%) and affinity (Km = 0.08 mM) to tyrosine phosphate in relation to p-nitrophenyl phosphate (pNPP)-(Km = 0.51 mM). Total phosphatase (pNPP) was inhibited in the presence of 10 mM vanadate (98%), 200 microM pervanadate (95%) and 100 microM p-chloromercuribenzoate (80%) but okadaic acid caused a slight increase in enzyme activity (25%). When the HL60 cells were treated with the phosphatase inhibitors (pervanadate and okadaic acid) for 24hours, only 20% residual activity was observed in presence of 200 microM pervanadate, whereas in the presence of okadaic acid this inhibitory effect was not observed. However, in respect to mitochondrial function, cell viability decreased about 80% in the presence of 100 nM okadaic acid. The total protein content was decreased 25% when the cells were treated with 100 nM okadaic acid in combination with 200 microM pervanadate. Our results suggest that both phosphatase inhibitors presented different mechanisms of action on HL60 cells. However, their effect on the cell redox status have to be considered.
Freire
Ana Claudia Galvão
AC
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, 13083-970 Campinas, São Paulo, Brasil.
Aoyama
Hiroshi
H
Haun
Marcela
M
Ferreira
Carmen Veríssima
CV
D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000633:toxicity*; D018922:HL-60 Cells; D006801:Humans; D015394:Molecular Structure; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology* / Q000633:toxicity*; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology* / Q000633:toxicity*
D004791:Enzyme Inhibitors; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C076715:pervanadate; D019319:Okadaic Acid; C008644:nitrophenylphosphate; D014638:Vanadates; D010749:Phosphoprotein Phosphatases