Growth inhibitory effect of dihydroartemisinin on Bcr/Abl+ chronic myeloid leukemia K562 cells involve AKT, ERK and NF-κB modulation

Abstract

Purpose: In our previous publication, we have shown that dihydroartemisinin could significantly inhibit the growth of CML K562 cells by its anti-proliferative and inducing apoptotic effects. Given the pivotal effect of Bcr/Abl tyrosine kinase and its downstream signal factors on CML cell proliferation and survival, we extend our study to investigate the effect of DHA on Bcr/Abl and related signal factors to further illuminate the possible mechanisms of the effect of DHA on CML cells.

Methods: The expression of Bcr/Abl was analyzed with PCR and Western blotting methods at both mRNA and protein levels. Measurement of protein expression and tyrosine phosphorylation activity of Bcr/Abl, AKT, ERK1/2, NF-κB and cytochrome c were performed with Western blotting and immunoprecipitation methods. Using the activity kits analyzed the activity of caspase 9 and caspase 3.

Results: The treatment with DHA results in a significant suppression on Bcr/Abl expression and leads to a concentration-dependent reduction on the Bcr/Abl tyrosine activity. Moreover, it also results in a strong influence on the downstream signal factors of Bcr/Abl, which includes inhibition of tyrosine kinase activity of AKT and ERK1/2, suppression of NF-κB protein expression, promotion of the cytochrome c release and the consequential activation of caspase 3/9 in CML K562 cells.

Conclusions: Together with our previous report, our data show that the growth inhibitory effect of DHA on CML cells might be due to the influence on Bcr/Abl expression and its downstream signal factors. DHA might be a potential novel anti-CML drug candidate and worthy of further study.

Fig. 1

Dihydroartemisinin inhibits Bcr/Abl protein expression and tyrosine phosphorylation in CML K562 cells. a Western blotting analysis for P210^Bcr/Abl expression in K562 cells. Total protein of K562 cells pretreated with various concentrations of DHA for 48 h was electrophoresed on a SDS–PAGE gel and probed on a nitrocellulose membrane with antibody. Relative expression levels of P210^Bcr/Abl were expressed as the relative intensity compared with β-actin, which was analyzed as controls for protein loading. b The activation of Bcr/Abl tyrosine kinase of K562 cells was analyzed with immunoprecipitation (IP) method. P210^Bcr/Abl was precipitated from cell lysates of K562 cells treated with various concentrations of DHA for 48 h and was subjected to Western blotting analyses (IB). Upper panel shows the levels of Bcr/Abl tyrosine phosphorylation as detected by anti-phosphotyrosine (anti-pTyr) antibody. The lower panel shows analysis to control equal loading of proteins. Relative phosphorylation levels of P210^Bcr/Abl were expressed as the relative intensity compared with control group. ^# P < 0.05, ^## P < 0.01 versus the vehicle control group

Fig. 2

Effect of dihydroartemisinin on Bcr/Abl mRNA expression in K562 cells. Total RNA was isolated from K562 cells pretreated with vehicle or dihydroartemisinin. RT-PCR analysis was performed as described in materials and methods. Relative expression levels of Bcr/Abl mRNA were expressed as the relative intensity compared with β-actin. ^# P < 0.05, ^## P < 0.01 versus the vehicle control group

Fig. 3

Exposure of K562 cells to DHA results in down-regulation of NF-κB expression. The whole cell lysates of K562 cells after treated with DHA for 48 h in different concentrations were analyzed by Western blotting method, as shown in b. The protein expression levels were expressed as the relative intensity compared with β-actin which was analyzed as controls for protein loading (a). ^## P < 0.01 versus the vehicle control group

Fig. 4

Effect of DHA on tyrosine activity of ERKl/2 and AKT. Immunoprecipitation followed by Western blotting analyses was adopted to assay the tyrosine activity of ERKl/2 and AKT in K562 cells pretreated with different concentrations of DHA for 48 h. In b, c, upper panels show the levels of ERK1/2 and AKT tyrosine phosphorylation as detected by anti-phosphotyrosine (anti-pTyr) antibody. The lower panel was probed with anti-ERK1/2 and anti-AKT antibody to control equal loading of proteins. a Relative phosphorylation levels of ERKl/2 and AKT were expressed as the relative intensity compared with control group. ^## P < 0.01, ^### P < 0.001 versus the vehicle control group

Fig. 5

Effect of DHA on the release of mitochondria cytochrome c in K562 cells. After K562 cells was incubated with the different concentrations of DHA for 48 h, the release of mitochondria cytochrome c was analyzed with Western blotting method using anti-cytochrome c (cyto c) antibody. β-Actin was analyzed as control for protein loading. The protein expression levels were expressed as the relative intensity compared with β-actin. ^## P < 0.01, ^### P < 0.001 versus the vehicle control group

Fig. 6

Effect of DHA on caspase 3 and caspase 9 activities in K562 cells. K562 cell lysates were prepared after treated with 2, 5 and 10 μmol/L of DHA for 48 h. Assays were performed by using the caspase 3 and caspase 9 activity kits as described in materials and methods. The activity levels of caspase 3 and caspase 9 were expressed as the relative absorbance levels compared with control group. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 versus the control group