Homharringtonine Affects The JAK2-STAT5 Associated Pathway In Human Leukemia Cells And Its Molecular Mechanism

Many cytokines and growth factors transduct signal through JAK/STAT(Janus kinase /signal transducers and activators of transcription)pathway,among which, JAK2 is most relative to hematopoiesis.STAT5 is the main downstream signal protein of JAK2,and been phosphorylated by JAK2,then dimerize and enter into the cell nucleus,thus regulating the transcription of relevant gene and supporting the growth, differention and proliferation of the cells.AML is frequently associated with chromosomal abnormalities and oncogenic proteins forming,which activated signal transduction pathways involved in the survival and proliferation of the malignant cells.Among which,constitutive activation of JAK2-STATs by overexpression or mutation was especially important to the malignant transformation of AML blasts.Pharmacologic inhibition of JAK2-STAT to abrogate AML cell proliferation may therefore represent an important part of targeted AML therapy.Homoharringtonine(HHT)is a plant alkaloid with antileukemia activity that has been used for treatment of acute leukemia,chronic leukemia,and myelodysplastic syndrome(MDS)since 1970's in China,and be significant effect.Although several cellular effects have been identified,including the inhibition of protein synthesis and telomerase activity,down-regulation of MCL-1,Survivin and VEGF which are close relevant to the malignancy of the cells,upregulation of pro-apoptotic bax and inducing caspase-3-mediated cleavage of poly(ADP-ribose)polymerase(PARP).But the influence on JAK2-STAT2 pathway of HHT has not been reported.In our study,the primary blasts of Acute mylogenous leukemia patients have been seperated and then incubated with HHT with different concentration(0,20,40, 80ng/ml)for 48 hr,the cell survival rates were tested by MTT.We found that:HHT obviously inhibited the proliferation of AML primary cells and shew a concentration-dependent manner(the inhibition rates were 16.49±2.70%,24.81±5.49%,47.58±4.31%respectively in each concerntration group),but not signifcant effect on healthy BM cells(the inhibition rates were 5.56±1.37%, 7.41±2.13%,12.04±3.21%in the 20,40,80ng/ml group respectively).We also observed the effect of HHT on AML cell lines HEL,K562 and HL-60,We found that 50%inhibiting concentration(IC50)of HHT on HEL,K562 and HL-60 are 64.74ng/ml,116.Sng/ml and 77.3ng/ml respectively for 48 hr.At the same time, apoptosis induction of HHT on HEL also been observed:after being incubated with HHT at 0,10,20,40,80,160ng/ml,typical DNA ladder appeared at 40 ng/ml,and with the increase of the concentration of HHT,DNA ladder increased too.While in the outcome of FC investigation,0,10,20,40,80ng/ml HHT were added into the HEL cells and cultured for 24 hr,and then be stained by Annexin-v and PI,the apoptosis shew a concentration-dependent manner,in 10,20,40,80ng/ml group,the apoptosis rate were 5.14±0.28%,10.58±0.22%,24.4±2.01%and 39.61±2.66% respectively,and all were higher than the control(1.51%).(p＜0.05)In the early study,researchers found that the infusion protein Bcr/Abl could activated JAK2-STAT5 pathway,even MAPK and PI3K pathway involved in the growth in the chronic myeloid leukemia cells,and JAK2V617F mutation in the patients with myeloproliferative disease could also constitutive activated multiple cell signal pathway involved in the growth(JAK-STAT,MAPK and PI3K and so on), among which,STAT5 is the main protein,and could activate and regulate the transcription of multiple genes after being activated by JAK2,including Bcl-xL,a antiapoptotic protein overexpressed in the progenitor cells of patients with polythemia vera and played an important role in the survial and proliferation of the cells.We supposed that HHT affect the JAK2-STAT5 pathway mainly at the protein level and used Western blot to investigateg the influence of HHT on protein expression of JAK2-STAT5.The results shew that:after AML primary cells,HEL and K562 cell lines being cultured with different concentration of HHT for 6 hours,the total protein level of JAK2 and STAT5 did not change,but the phosphorylation decreased obviously and in a concentration-dependent manner(in K562 cells,JAK2 also decreased).While in RT-PCR,We found that there were no changes in the expression of JAK2 and STAT5 mRNA.Besides,Bcl-xl also stable after being affectd by HHT in HEL,but when it has been prolonged to 24 hr,Bcl-xL decreased,so did in mRNA level.These results priliminarily suggested that HHT blocks the phosphorylation of JAK2-STAT5 through inhibiting the protein tyrosine kinase activity,thus inhibiting the transduction of the cell signal pathway associated with malignant proliferation of cells and inducing apoptosis.As MAPK and PI3K pathways also activated in most AML patients and are important transduction pathway involved in the survival and proliferation of malignant cells,we studied the effect of HHT on phosphorylated and total ERK1/2 and Akt activity in the myeloid leukemia cell lines.and we found that p-Akt also downregulated while the total Akt was constant.Some researchers found that jak2 plays a critical role in the signal transduction through bcr-abl/jak2/gab2/pi3k/akt network,and that when the activity of jak2 was inhibited,that of akt was restricted simultaneously.While no appreciable specific effects of HHT on ERK1/2 in HEL, K562 and primary cells,the clear mechanism remains unknown,may it be associated with the involvement of other adaptor proteins such as Gab2 and Grb2 in regulating the activity of ERK1/2.In order to confirm the apoptosis effect of HHT on AML cells were through inhibiting the phosphorylation of JAK2-STAT5,AG490 was used in HEL and K562 cells to see if JAK2 inhibition by using specific JAK2 kinase inhibitor could induce similar levels of apoptosis seen with the HHT.We found that:AG490 alone and be combined with HHT both could induce apoptosis.In western blot test,the phosphorylation of JAK2 and STAT5 decreased in HEL.While in K562,the phosphorylated-JAK2 downregulated but phosphorylated-STAT5 was stable,it could because in K562 cells JAK2 and STAT5 were activated by Bcr/Abl seperately.This result confirms that HHT acts as a wide spectrum PTK inhibitor no more than jak2 kinase inhibitor in our study.Based on the results above,conclusions could be brought forward.(1)HHT could notably inhibit the growth of AML primary cells and HEL,K562,HL-60 cell lines, and this growth inhibition was time and dose dependent.(2)10ng/mlHHY could induce the apoptosis of HEL cells,the apoptosis rate of HEL in 10,20,40,80ng/ml group,the apoptosis rates were 5.14±0.28%,10.58±0.22%,24.4±2.01%and 39.61±2.66%respectively,and all were higher than the control(1.51%) (p＜0.05).While investigating the apoptosis by DNA ladder observation,we found that typical DNA ladder appeared at 40 ng/ml,and with the increase of the concentration of HHT,DNA ladder increased too.(3)After being affected by different concentration of HHT,the total protein levels were stable in AML primary cells,HEL,K562,but the phosphorylation was downregulated and shew a concentration dependent manner.In RT-PCR,We found that the JAK2 and STAT5 proteins were both stable.Further research suggested that:Bcl-xL was stable after being incubated with HHT for 6 hours in HEL,while it been prolonged to 24 hours,Bcl-xL decreased,so did in mRNA level.Moreover,we also observed that the phosphorylation of AKT decreased in HEL and K562,but p-ERK1/2 shew no appreciable change.(4)Apoptosis was observed in HELand K562 after being affected by JAK2 specific inhibitor AG490 alone or in combination with HHT.In Western blot,the phosphorylated JAK2 and STAT5 were downregulated,while in K562,p-JAK2 decreased but there was no appreciable change in p-STAT5 proteins.