Sfrps Family Invovled in Growth Inhibition of K562 Cells Induced by Artesunate

Studies have shown that abnormal activation of wnt signaling pathway is closely related to tumor genesis. Cell surface coil protein (frizzled, fzd) is a specific receptor for wnt pathway to activate its downstream signaling through β-catenin to stimulate the growth of a range of tumor cells. Secreted frizzled related proteins (sfrps) are the main antagonists of wnt pathway. sfrps can inhibit the function of wnt pathway via competing with fzd receptor. Recent studies have found that sfrps family memberes expressed at very low levels in a variety of tumor cells,which was closely related to the methylation of sfrps gene promoters. DNA methyltransferase (dnmts) are the key enzymes involved in DNA methylation, which can promote the methylation of tumor suppressor genes' promoters and inhibit transcription of these genes, and induce tumor genesis. Artesunate, a semi-synthetic derivative of artemisinin, is very effective in antimalaria. Recent studies have shown that artesunate has anti-tumor functions. However, the effect of artesunate on the expression of sfrps- and dnmts- mRNAs and β-catenin protein are not clear. To evaluate the growth inhibition effect of Artesunate (at final concentrations of 0μg/ml, 4μg/ml, 10μg/ml, 20μg/ml and 40μg/ml) on K562 cells and to investigate its potential mechanism by detecting the expression levels of sfrps- and dnmts- mRNAs and β-catenin protein in K562 cells treated with artesunate.

Cell growth inhibition rate and cell cycle distribution of K562 cells induced by artesunate treatment were measured by MTT assay and flow cytometry, respectively. The mRNA expression levels of sfrp1, sfrp2, sfrp4 and dnmt1, dnmt3a, dnmt3b in K562 cells which had been treated with or without artesunate for 48 hours were evaluated with reverse transcription-polymerase chain reaction (RT-PCR). Protein expression level of β-catenin in K562 cells were detected by Western blot.

Artesunate treatment significantly induced growth inhibition of K562 cells in a concentration-dependent manner after the cells were treated with artesunate for 48 hours(p < 0.05). The inhibition rate of 4,10,20 and 40(μg/ml)artesunate exposure were 54.29%, 58.03%, 69.33% and 77.98% respectively.

Flow cytometry analysis showed that K562 cells were arrested at G₀/G_l and G₂/M phase in concentration-dependent manner after 48 hours exposure of artesunate (p < 0.05).

After treated with artesunate at the final concentrations of 0, 4, 10, 20 and 40μg/ml, the relative expression levels of sfrp1, sfrp2 and sfrp4 mRNA in K562 cells increased, while the expression levels of dnmtl, dnmt3a, dnmt3b mRNA decreased significantly compared with the control group.

Results from Western blot showed that β-catenin protein levels decreased in a concentration-dependent manner, when compared with that of the control group (P<0.05). Conclusion: Results from the present study indicated that artesunate could inhibite the mRNA expression of dnmts family and minimize the methylation of sfrps gene promoter. Therefore, sfrps could inhibit the function of wnt pathway through competing with fzd receptors. Meanwhile, artesunate could decrease the expression of β-catenin protein in K562 cells, and could further inhibit the function of wnt pathway. Therefore, data from the present experiment provides a new theoretical basis for clinical application of artesunate in leukemia treatment.

Ying:Nature science foundation of Hebei Province: Research Funding. Nan:Nature science foundation of Hebei Province: Research Funding. Jun:Nature science foundation of Hebei Province: Research Funding. Hai:Nature science foundation of Hebei Province: Research Funding. Kai:Nature science foundation of Hebei Province: Research Funding. Xu:Nature science foundation of Hebei Province: Research Funding. Pan:Nature science foundation of Hebei Province: Research Funding.