Effects Of Neferine With Imatinib On The Pathway Of PI3K/AKT In CML Cells

Objective:To research the effects of Neferine with Imatinib on PI3K/AKT pathway in the chronic myelogenous leukemia (CML) cells,further explore the mechanism of neferine enhance Imatinib sensitivity when used in CML cells,underpin pharmacological basis of Nef as a sensitizer,and provide a new train of though for CML clinical treatment.Methods:1. Gene expression level were detected by reverse transcriptase quantitative real-time fluorescence polymerase chain reaction (RT-qPCR),such as the up-stream key genes (bcr/abl,PTEN and SHIP),the important signaling molecules in pathway(PI3K and Akt) and the down-stream target molecules(mTOR and Bcl-2family) in PI3K/AKT pathway.2. Protein relatively expreesion levels of Akt and p-akt was detected by Western blotting in PI3K/AKT pathway such as a key central factor.Results:1. Effects of Neferine with Imatinib on the gene expression of upstream bcr/abl, the negatively controlling genes of PTEN and SHIP(SHIP-1and SHIP-2) in PI3K/AKT pathway (1) Nef and Imatinib both can downregulate the mRNA expression level of bcr/abl in the up-stream of PI3K/AKT pathway,especially in combination(p<0.01).(2) Compared with control group, PTEN as a nagetive regulative gene those mRNA level are significantly higher in each drug group,except Nef(4μM) group.But to compare with Imatinib group,Nef(4μM) combined with Imatinib can up-regulate their expression level(P<0.05),and there is a significant different in Nef(8μM) combinated group(p<0.01).(3) To compare with control group,there is no different about the mRNA expression level of SHIP-1in each group(P>0.05).While SHIP-2gene expression are up-regulated in any group compared with control group and display more meaningfully. Nef(8μM) group,Nef(4μM) combinated group and Nef(4μM) combinated group,there are higher gene expression level when compared with Imatinib group(P<0.05).(4) The PTEN and SHIP-2shows a significant inverse correlation with bcr/abl mRNA expression level(Pearson R=-0.708,P<0.01and R=-0.586,P<0.01respectively).2. Effects of Neferine with Imatinib on the gene expression of PI3K/AKT/mTOR(1) PIK3CA and Akt in this pathway mRNA expression have no distinction in any group compared with control group(P>0.05).The akt total protein levels also do not chang in any group,while those phosphorylated protein are significantly decreased compared with control group,except Nef(4μM)(P<0.01).When Nef combines with Imatinib,to compare with Imatinib group, there are lower akt phosphorylated protein level(P<0.05).(2) Nef do not affect the mTOR gene expression in the pathway(P>0.05),but which can be decreased when it is combined with Imaitnib,especially Nef(8μM),to compare with Imatinib group(P<0.05).3. Effects of Neferine with Imatinib on the gene expression of Bcl-2family(1) In Nef group,Bcl-2and Bcl-xL as main effector molecules members of Bcl-2family in the downstream of PI3K/AKT pathway,those mRNA expression have no change compared with control group(P>0.05),while in combination with Imatinib they are down-regulated (P<0.01).(2) To compared with control group,other effector Bax have a higher mRNA expression level in any group(P<0.01),while this expression level in Nef(8μM) group and Imatinib combinated group have a significantly up-regulation expression level of mRNA as compared with Imatinib group(P<0.05).(3) In Nef group, those ratio of Bax/Bcl-2and Bax/Bcl-xL gene expression have up-regulated compared with control group(P<0.05),while in combination with Imatinib they are up-regulated significantly (P<0.01).Conclusions:1. Nef and Imatinib both can decrease the mRNA expression level of upstream bcr/abl of PI3K/AKT pathway.The negative regulation genes of PTEN and SHIP-2also have a significant up-regulation expression of mRNA,while the SHIP-1mRNA level have no change. The PTEN and SHIP-2show a significant inverse correlation with bcr/abl gene relative expression.2. There are no significantly in mRNA expression level of PIK3CA and Akt as well as the total protein expression level of Akt no matter the Nef and Imatinib utilization together or alone in this pathway,while two drugs both can step down the Akt phosphorylated protein level.while both can depress the mRNA expression level of mTOR in this pathway.3. For target gene Bcl-2family of the downstream in this pathway,Nef combine with Imatinib can depress the mRNA expression level of Bcl-2and Bcl-xL,while the two drugs also can up-regulated the mRNA expression level of Bax. Nef can up-regulatied the ratio of gene expression level of Bax/Bcl-2and Bax/Bcl-xL,while combine with Imatinib the two ratio also up-regulated significantly.