Effect Of Inhibition Of Extracelluar-Signal Regulated Protein Kinase On Arsenic Trioxide-induced Apoptosis In K562/A02 Cells

Objectives: To investigate the apoptosis of multidrug resistant K562/A02 cells induced by AS₂O₃ and its signal transduction mechanisms.Methods: MTT assay, light microscopy, confocallaser scanning microscopy, PI/Annexinâ…¤and cell cycle analysis were used to detect apoptosis in K562/A02 cells. The expression of mdrl, Survivin and caspase-3 mRNA was examined with reverse transcription polymerase chain reaction (RT-PCR). The expression of ERK1/2, p-ERK1/2 and Survivin protein was examined with western-blot.Resμlts: AS₂O₃ coμld inhibit growth of K562/A02 cells in a manner depending on both concentration and time (R24h=0.81,R48h=0.86,R72h=0.90,P<0.01). Morphological changes typical of apoptosis induced by AS₂O₃ were observed through microscopy. Cell cycl analysis indicated the increased Sub-G1, proportion (P<0.01) as well as the apparent G2/M phase arrest (P<0.05) in AS₂O₃ treated cells. We noted that the expression of caspase-3 mRNA significantly increased after application of 2μmol/L and 5μmol/L. AS₂O₃ for 48 hours (P<0.01), and a 48 hour treatment of 5u mol/L AS₂O₃ coμld down-regμlate the expression of mdr1 mRNA. It was shown that the level of survivin decreased after administration of 5μmol/L. AS₂O₃ for 48 hours. The results of FCM indicated that in cells treated with 2μmol/L and 5μmol/L AS₂O₃ for 48 hours, AS₂O₃ coμld induce the apoptosis of K562/A02 cells in a manner depending on both concentration and time. We observed that effect of apoptosis in the cells treated w ith combination of AS₂O₃ and the selective inhibitor of extracellular-signal regulated kinase (U0126) was enhanced markedly.Enzymicactivity assay revealedth at caspase-3 coμld be gradually activated when action of 2μmol/L and 5 umol/L AS₂O₃ had lasted for 48 hours. Remarkable decrease of enzymic activity was detected 48 hours after 5μmol/L AS₂O₃ was applied. The result of RT-PCR confirmed that caspase-3 mRNA coμld also be up regμlated by 5μmol/L AS₂O₃ 48 hours after the agent was given.We found that 5μmol/L AS₂O₃ treatment for 48 hours coμld reduce content of mdrl mRNA in K562/A02 cells, and there was a marked fall in the expression of Survivin when the cells had been treated with 5μmol/L AS₂O₃ for 48 hours.Conclusions: We observed that effect of apoptosis in the cells treated w ith combination of AS₂O₃ and the selective inhibitor of extracellular-signal regulated kinase (U0126) was enhanced markedly. AS₂O₃ induces apoptosis in K562/A02 cells by activating caspase-3 pathway, and suppresses the expression of mdrl and Survivin gene by down regμlation of mRNA transcription and protein synthesis, U0126 could specifically and effectively inhibit the activity of ERK to the basal level or even lower. The selective Inhibition of extracellular-signal regulated Kinase can increase the sensitivity of arsenic trioxide iduced apoptosis in K562/A02 cells.