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 × 104 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.