DETERMINATION OF APOPTOTIC EFFECT OF ASPIRIN AND ITS ANALOGUES ON AN ADHERENT CELL LINE USING FLOW CYTOMETRY: PROBLEMS AND POSSIBLE SOLUTIONS

Abstract

Aim: To determine the apoptotic effect of aspirin and its analogues on an adherent cell line using Flow 
cytometry and whether a change in the isomeric positions of the acetyl group to the benzene ring in 
these compounds might be responsible for any change in behaviour. Also, to observe the problems 
encountered together with possible solutions  
Study design: Qualitative and Quantitative. 
Place and Duration of Study: This study was conducted in the Research Institute in Healthcare 
Sciences, University of Wolverhampton, UK.  
Methodology: Flow cytometry and immunocytochemistry (ICC) were used to determine the apoptotic 
effects of aspirin and its analogues on SW480 CRC cell line. Annexin-V dye was used to detect 
apoptotic cells and PI to detect necrotic cells in flow cytometry, while YO-PRO®-1 and propidium 
iodide (PI) dyes were employed to detect apoptosis and necrosis respectively in ICC, using confocal 
microscopy to view cells. 
Results: Using flow cytometry, 308.9% of the cell population were necrotic after 16 h and this did 
not change even after 40 h of treatment with meta- (PN548) and para- (PN549) aspirin. On the other 
hand, the percentage of cell population treated with ortho-thioaspirin (PN590) were significantly 
necrotic with a small population apoptotic. But it’s meta- (PN591) and para- (PN592) isomers did not 
significantly cause apoptosis or necrosis with most of the cell population live/healthy even after 40 h 
of treatment. It was observed that flow cytometry showed more “necrotic” cells than ICC when treated 
with staurosporine (apoptotic control), H2O2 (necrotic control) and aspirin analogues. 
Conclusion: It was concluded that aspirin and its analogues with the exception of PN591 and PN592 
had some apoptotic effect after 16 h. However, a high necrotic effect was observed after 40 h, which 
was not observed using ICC. In order to obtain unambiguous and reliable results, it was concluded that 
flow cytometry, a popular method in the detection of apoptosis in various cell lines, is less suitable for 
adherent cell lines, unless additional measures are taken. Unfortunately, these additional measures will 
result in additional costs. Further research using ICC with the inclusion of DAPI (4’,6-diamidino-2
phenylindole), a blue fluorescence dye that stains the nucleus is required for accurate quantitative 
results.