Effects Of Sulfasalazine On Human Acute Myeloid Leukemia And MUTZ-1 Cells

Acute myeloid leukemia(AML)is a heterogenous clonal disorder of haemopoietic progenitor cells,which lose the ability to differentiate normally and to respond to normal regulators of proliferation.Myelodysplastic syndromes(MDS)are a group of disorders characterized by progressive cytopenias and transformation to acute leukemia.Although the recent development of new treatment strategies have made progress, new strategies are still urgently needed both in AML and in MDS.Sulfasalazine(SSZ)is a synthetic anti-inflammatory drug and routinely used for the treatment of inflammatory bowel disease and rheumatoid arthritis.Recently,SSZ demonstrated anti-cancer properties on several human tumor cell lines and in vivo studies also confirmed its anti-cancer properites in glioblastomas.In the present study,we investigated the effects and mechanisms of SSZ in human AML cells and MDS cell line MUTZ-1.Firstly,we examined the effect of SSZ on human AML cells.Using MTT assay,we examined the effects of different concentrations of SSZ on the growth of human AML cells.The results indicated that cell viability in the presence of SSZ decreased in a dose-dependent manner.The inhibitory rates of cell growth were positively correlated with SSZ concentrations.The growth-inhibitory IC₅₀values were 0.372mM,0.302mM,0.734mM,1.043mM,0.824mM in KASUMI-1,NB-4,K-562,U-937,KG-1 AML cells after the treatment of SSZ for 48 hours,respectively.SSZ also inhibited the growth of freshly isolated AML cells from 8 patients with an IC₅₀(at 48h)of 0.4-1.2mM.In contrast,the IC₅₀of SSZ was more than 2.4mM in normal cells.To explore the possible mechanism of the anti-leukemic activity of SSZ in AML cells,further investigation was carried out.In order to investigate whether apoptosis is associated with the anti-leukemic activity of SSZ in AML cells,we observed the cell morphology using Wright-Giemsa staining,evaluated the exposure of phosphatidylserine(PS)on KASUMI-1 and NB-4 cells after double staining with fluorescein isothiocyanate(FITC)-labeled annexin V and propidium iodide(PI).We found typical apoptotic morphology after KASUMI-1 and NB-4 cells were exposed to SSZ for 48hr.And Annexin V/PI staining also confirmed SSZ induced apoptosis in KASUMI-1 and NB-4 cells in a dose-dependent manner.In order to explore the possible mechanism and pathway of the signal conduction of the cell apoptosis induced by SSZ,we examined mitochondrial transmembrane potential and revealed that SSZ led to mitochondrial dysfunction in KASUMI-1 and NB-4 cells.Besides,western blot confirmed that Caspase-9,Caspase -3 and PARP were activated in KASUMI-1 cell following treatment with 0.5-1.5mM SSZ for 24h.These results indicate that mitochondrial pathway may play an important role in ssz induced apoptosis in KASUMI-1 cell.Meanwhile,the role of Bcl-2 family and IAPs family members were also investigated by western blot when 0.5-1.5mM SSZ was given for 24h in KASUMI-1 cell,the results showed that Bcl-2/Bax and Bcl-xl/Bax ratio were negatively correlated with SSZ concentrations,and the IAPs family members XIAP,cIAP-1,cIAP-2 protein levels were also negatively correlated with SSZ concentrations.These results indicate that both Bcl-2 family and IAPs family were involved in ssz induced apoptosis in KASUMI-1 cell.We also found an decrease of nuclear NF-kB(p65)expression in KASUMI-1 cell following treatment with 0.5-1.5mM SSZ for 24h by western blot which indirectly indicated that NF-kB inhibition may be an important mechanism for anti-leukemic activity of SSZ in KASUMI-1 cell. Meanwhile,we confirmed that AML1-ETO fusion protein which carried by KASUMI-1 cell was also down-regulated with 0.5-1.5mM SSZ for 24h by western blot which indicated that AML-ETO may be another important target for SSZ in KASUMI-1 cell.Secondly,we examined the effect of SSZ on human MDS cell line MUTZ-1.MTT assay revealed that MUTZ-1 cell viability in the presence of SSZ decreased in a dose-dependent manner.The inhibitory rates of cell growth were positively correlated with SSZ concentrations.The growth-inhibitory IC₅₀values was 0.672mM after the treatment of SSZ for 48h.Besides,Annexin V/PI staining confirmed SSZ induced apoptosis in MUTZ-1 cell in a dose dependent manner.Further more,JC-1 staining revealed mitochondrial dysfunction in MUTZ-1 cell after 1.5mM SSZ treatment for 24h.Western blot revealed activation of caspase-3 when 0.5-1.5mM SSZ was treated for 24h in MUTZ-1 cell.These results indicated that SSZ can inhibit the viability and induce apoptosis of MUTZ-1 cell,dissipation of mitochondrial transmembrane and activating of Caspase-3 were involved in this process.In conclusion,we found out that SSZ had potent anti-leukemic activity in AML and anti-MDS activity in MUTZ-1 cell in vitro,suggesting that further investigation of this agent is warranted.