Evaluation of apoptosis and necrosis induced by statins using fluorescence-enhanced flow cytometry.
The purpose of this study was to evaluate the apoptosis and necrosis induced by five kinds of statins in IM-9 human lymphoblasts with fluorescence-enhanced flow cytometry using avidin-biotin complex. IM-9 human lymphoblasts (2 x 10(4) cells/cm2) were seeded into tissue culture plates and incubated with five kinds of statins. Statin-treated cells were first incubated with biotin-annexin V, followed by addition of avidin-FITC and propidium iodide, and then subjected to flow cytometry. The fluorescence intensity was enhanced using an avidin-biotin complex system, resulting in successful separate determination of the statin-induced apoptosis and necrosis by flow cytometry, which enabled us to quantitatively evaluate the statin-induced cell damage. Flow cytometric analysis results in the intensity of statin-induced apoptosis in IM-9 cells as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. The intensity of statin-induced necrosis in IM-9 cells was expressed as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. The total damage of IM-9 cells induced by five kinds of statins were expressed as the sum of both percentages of apoptosis and necrosis as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. Our studies show that fluorescence enhancement with avidin-biotin complex is useful for the identification and quantitation of annexin-positive apoptosis cells and thus, the fluorescence-enhanced flow cytometry was shown to be applicable for screening of statins as new anti-leukemia agents.
Yasuda
Noriko
N
School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien, Kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan.
Matzno
Sumio
S
Iwano
Chihiro
C
Nishikata
Mayumi
M
Matsuyama
Kenji
K
D017209:Apoptosis; D000069059:Atorvastatin; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D004345:Drug Industry; D004354:Drug Screening Assays, Antitumor; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D000077340:Fluvastatin; D006538:Heptanoic Acids / Q000494:pharmacology; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000032:analysis* / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007938:Leukemia / Q000188:drug therapy; D008856:Microscopy, Fluorescence / Q000379:methods*; D009336:Necrosis; D004364:Pharmaceutical Preparations / Q000032:analysis; D017035:Pravastatin / Q000494:pharmacology; D011419:Propidium / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D019821:Simvastatin / Q000494:pharmacology; D013997:Time Factors
D005229:Fatty Acids, Monounsaturated; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007211:Indoles; D004364:Pharmaceutical Preparations; D011725:Pyridines; D011758:Pyrroles; D001360:Avidin; D011419:Propidium; D000077340:Fluvastatin; D001710:Biotin; D000069059:Atorvastatin; D019821:Simvastatin; C086276:cerivastatin; D016650:Fluorescein-5-isothiocyanate; D017035:Pravastatin