Sodium salicylate-triggered apoptosis in HL-60 cells depends on caspase-8 activation.
For investigation of the killing and proapoptotic effects of sodium salicylate (Na-Sal) on HL-60 cells, the cytotoxic activity of Na-Sal was measured by means of MTT assay. Apoptosis was identified and analyzed with the help of transmission electron microscopy, annexin V staining, and DNA gel electrophoresis, and the association of caspase-8 activation with apoptosis was determined with the specific protease inhibitor IETD-fmk. After exposure of HL-60 cells to increasing concentrations of Na-Sal (0.5, 1, 3, 5, and 7 mmol/L) for 24 hours, the mean cell viability gradually dropped to 92%, 83%, 68%, 50%, and 42%. With treatment of target cells with 5-mmol/L (IC50) Na-Sal for 6, 12, 24, or 36 hours, the mean cell survival tapered to 91%, 81%, 48% (P <.05 versus control), and 14% (P <.05 versus control). Again incubated with 5-mmol/L Na-Sal for 12 or 24 hours, HL-60 cells displayed clear early or late signs of apoptosis, including (1) notable enhancement of phosphatidylserine externalization, (2) cell shrinkage, membrane blebbing, and eventual disintegration into numerous apoptotic bodies, and (3) formation of ladder DNA. The viability of HL-60 cells increased significantly during 24 or 36 hours of coculture with 100-micromol/L IETD-fmk in combination with 5-mmol/L Na-Sal compared with the viability when 5-mmol/L Na-Sal was used alone (P < .05). Moreover, the target cells showed a considerable decrease in phosphatidylserine exposure and DNA fragmentation after coincubation for 12 or 24 hours performed as described above. The findings presented herein strongly suggest that Na-Sal can exert potent killing and proapoptotic activity against HL-60 cells, and this effect appears to depend on caspase-8 activation.
Chen
Xiequn
X
Department of Hematology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China. xiequnchen@sina.com
Wan
Youfeng
Y
Bai
Qingxian
Q
Zhang
Weiping
W
Zhu
Huafeng
H
D017304:Annexin A5 / Q000032:analysis; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism* / Q000502:physiology; D002462:Cell Membrane / Q000648:ultrastructure; D053938:DNA Fragmentation; D018922:HL-60 Cells / Q000187:drug effects; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism; D012980:Sodium Salicylate / Q000494:pharmacology*
D017304:Annexin A5; D000894:Anti-Inflammatory Agents, Non-Steroidal; D010718:Phosphatidylserines; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D012980:Sodium Salicylate