Differential Proteomics Research Of Camptothecin Analog NSC606985 Inducing Apoptosis In U937 Cells

Acute myeloid leukemia(AML)is a heterogenous group of hematopoietic malignancies.Significant advances in understanding the biologic,molecular,and cytogenetic aspects of this malignancy have been achieved in the past 20 years.Meanwhile,substantial progress is also made in the treatment of patients with AML.However,most patients remain incurable.So development of new drugs for treatment of AML is very important.As a promising new class of anticancer drugs, camptothecins have advanced to the forefront of several areas of therapetitic and developmental chemotherapy.The last two years,our task group has studied the potential anti-leukemic effectiveness of NSC606985,a new class of camptothecin analogs,in vitro and in vivo.In order to explore further the molecular mechanisms of NSC606985 inducing AML cell apoptosis,the technology of differential proteomics has been used to analyze the differential proteins expression in U937 cells treated with(36 hours)or without 50nM NSC606985.As a result,we identified 28 proteins that were significantly deregulated by NSC606985. These proteins were mainly involved in cytoskeleton,RNA metabolism, DNA metabolism,protein metabolism,carbohydrate metabolism,energy metabolism,signal transduction,stress response,oxidation and reduction, differentiation and others.Cytoskeleton proteins occupied a great proportion of these differential proteins.We used western blot and two-dimensional gel electrophoresis plus western blot to validate the changes of cytoskeleton proteins in 2D-GE.The results revealed that the protein expression ofβ-tubulin,β-actin and transgelin-2 did not be changed.In tthe control group,these cytoskeleton proteins had several isoelectric points.After treated by NSC606985(the experimental group),the isoelectric points in the basic end transferred to the acid end and each of these cytoskeleton proteins had only one isoelectric point in the experimental group(it is equal to the acidest point in the control group).Because the phosphorylation is the commonest post-translational modification and it can induce isoelectric points to move to left,we presumed that these cytoskeleton proteins was been phosphorylated.Treated the total proteins of the experimental group by alkaline phosphotase,the great mass ofβ-tubulin,β-actin and transgelin-2 transferred to the basic end.It was according with the characteristic of phosphorylated proteins and it was proved that these three cytoskeleton proteins were been phosphorylated in the apoptotic process which was induced by NSC606985.Because the preliminary work proved that active cleaved PKCδplayed a key role in the apoptotic process and all of these three cytoskeleton proteins had recognizing motif (forcasting by scansite software),we observed that whether rottlerin(a specific inhibitor of PKC)pretreatment could inhibit the phosphorylationof these three cytoskeleton proteins.The results showed that rottlerin could totally inhibited the phosphorylation ofβ-tubulin andβ-actin,but it was unable to inhibited the phosphorylation of transgelin-2.It was suggested that the phosphorylation ofβ-tubulin andβ-actin were downstream to PKCδactivation,the phosphorylation of transgelin-2 was not one of the events which were,downstream to PKCδactivation.Peroxiredoxin 1 and thioredoxin,two antioxidant proteins,were increased significantly.It was suggested that ROS was produced.To confirm the change,mechanism and significance of ROS in NSC606985 inducing apoptotsis,we mensurated the content of ROS in the cells with DCF.The results showed that ROS of U937 cells was increased after NSC606985 treating 24 hours.This event was downstream to PKCδactivation because it was totally inhibited by rottlerin.Though NAC could eliminate ROS of U937 cells,it had no influence on NSC606985 inducing apoptotsis.In summary,this study showed that the proteomic technique was a powerful method to identify apoptotic related proteins,and it has unique superiority to explore post-translation modifications of proteins.The results of the present study are worthing further investigation to understand the mechanisms ofNSC606985 inducing apoptotsis.