SFRPs Family Invovled In Growth Inhibition Of K562 Cells Induced By Artesunate

Objective:Studies have shown that abnormal activation of Wnt signaling pathway and is closely related to tumor genesis.Cell surface coil protein (Frizz -led,Fzd) is a specific receptor for Wnt pathway to activate its downstream sig -naling throughβ-catenin to stimulate the growth of a range of tumor cells.Sec -reted frizzled related proteins (SFRPs) is the main antagonist of Wnt pathway, and functions through competing with Fzd receptor to bind with Wnt proteins to inhibit Wnt pathway. Recent studies have found that SFRPs family member -es expressed at very low levels in a variety of tumor cells, which was closely related to the methylation of sfrps gene promoter.DNA methyltransferase (DN -MTs) are the key enzymes involved in the process of DNA methylation, whic -h can promote the methylation of tumor suppressor gene promoters and inhibi -t transcription of tumor suppressor genes,and subsequently induce tumor gene -sis. Artesunate(ART),a semi-synthetic derivative of artemisinin, is very effect -ive in antimalaria.Recent studies have shown that to ART can inhibit the grow -th of several kinds of tumor cells.To evaluate the growth inhibition effect of artesunate on K562 cells and investigate the potential mechanism,the expressi -on of sfrps family,dnmts family mRNA andβ-catenin protein were measured in the present study and revealed that SFRPs family were involved in the grow -th inhibition of K562 cells induced by artesunate.Methods:Cytotoxicity,apoptosis rate and cell cycle distribution of K562 cells induced by ART(0, 4, 10, 20,40μg/ml) were measured by MTT assay and by flow cytometry,respectively.Furthermore,protein expression levels ofβ-cate -nin in K562 cells were detected by western blot.The mRNA expression levels of sfrp1,sfrp2,sfrp4 and dnmt1,dnmt3a,dnmt3b in K562 cells were evaluated b -y reverse transcription-polymerase chain reaction(RT-PCR).Results: 1 Artesunate significantly inhibited proliferation and induced apoptosis o -f K562 cells.After treated with ART for 48h, the growth inhibition rate of K5 62 cells treated with 4μg/ml ART was 54.29%,and in K562 cells treated with 10μg/ml ART was 58.03%,which was statistically significant higher than in 4μg/ml ART group(P<0.05).The gowth inhibition rate was 69.33% in K562 cell -s treated with 20μg/ml ART and was significantly higher than in the former t -hree groups(P<0.05),and 77.98% in K562 cells treated with 40μg/ml ART,wh -ich was the highest one among the five groups(P<0.05).2 After K562 cells had been treated with ART at different concentrations for 48 hours,the apoptosis rate of K562 cells from 4μg/ml ART group was 15. 87±2.48%,increased compared with that in 0μg/ml ART group(P<0.05); the ap -optosis rate of K562 cells from 10μg/ml ART group was 21.90±2.29%,increa -sed compared with that in 4μg/ml ART group (P<0.05); the apoptosis rate of K562 cells was 49.47±5.08% in 40μg/ml ART group, which was the highest o -ne among the five goups( P<0.05).3 Results from flow cytometry analysis showed that after K562 cells bei -ng treated for 48 hours with ART at different concentrations,cell numbers in G2/M phase increased significantly (P<0.05), while the cell numbers in G0/Gl +S phase decreased significantly in a dose dependent manner,when compared with that of control group.4 Results from RT-PCR assay showed that treatment with ART at the fin -al concentrations of 0, 4, 10, 20 and 40μg/ml, the relative expression levels of sfrp1,sfrp2,sfrp4 mRNA in K562 cells increased significantly, while the expre -ssion levels of dnmtl, dnmt3a, dnmt3b mRNA decreased significantly compar -ed with the control group.Results from Western blot showed thatβ-catenin pr -otein levels decreased in a dose dependent manner, when compared with that of the control group (P<0.05).Conclusion:Artesunate increased the mRNA expression of sfrps and decre -ased the mRNA expression of dnmts in K562 cells, indicating that artesunate could inhibite the mRNA expression of DNMTs family and minimize the meth -ylation of sfrps gene promoter. Therefore, SFRPs could inhibit the function of Wnt pathway by competing with Fzd receptors to bind with Wnt proteins,and decrease the expression ofβ-catenin in K562 cells.It may be one of the mecha -nisms in inhibiting proliferation of K562 cells induced by artesunate.The resu -lts from this experiment provides a new theoretical basis for clinical aplicatio -n of artesunate in leukemia treatment.