| Background:Chronic myeloid leukemia (CML), which accounts for approximately 20% of all adult leukemias, is characterized by the presence of the Philadelphia (Ph) chromosome derived from the juxtaposition of Abelson TK gene (Abl) on chromosome 9 to the break point cluster region (Bcr) gene on chromosome 22. The resultant oncogenic Bcr-Abl kinase is essential for the growth of CML cells and has become an attractive target for treatment of Ph+ CML cases. Inhibition of Bcr-Abl with Abl tyrosine kinase inhibitors (TKIs). such as imatinib(IM), is highly effective in controlling but not curing the disease. This is largely due to the inability of these kinase inhibitors to kill leukemia stem cell (LSC) (also known as leukemia-initiating cell) responsible for tumor initiation, drug resistance and relapse in myeloid leukemia. Therefore, eradication of LSCs is required for developing curative therapies for CML, which relies on identification of specific targets in leukemia stem cells. A number of targets, such as?-catenin, Hedgehog, NF-?B, have been shown to be involved in survival, self-renewal and apoptosis resistance of leukemia stem cells, but they also play similar roles in regulating normal hematopoietic stem cells(HSCs). Currently, it is very challenging to identify LSC-specific therapeutic targets involved in the key functional regulation of LSCs but not normal HSCs.Berbamine, a plant natural compound produced by Berberis amurensis, is a traditional Chinese medicine (TCM) that has been widely used to treat leucopenia and inflammation for decades in China. Our previous studies showed that berbamine and its derivatives potently inhibited the growth of imatinib-resistant CML cells but not normal hematopoietic cells. These data suggested that berbamine might have potential to target leukemia stem cells of chronic myeloid leukemia.Objects:1?To determine the in vivo anti-leukemia activity of the berbamine.2?To identify the specific protein target of Berbamine in leukemia, and understand the interaction between Berbamine and the specific target.3?To investigate the role of the identified target in survival, self-renewal and apoptosis resistance of LSCs, and eventually discover the molecular mechanism underlying the anti-leukemia activity of berbamine. Contents:1 The anti-chronic myeloid leukemia activity of the berbamine in vivo.To determine the in vivo anti-leukemia activity of the berbamine, we evaluated the effects of this compound on human CML cells K562 xenografts in nude mice by oral administration. We investigated the in vivo anti-leukemia activity of berbamine against IM sensitive-K562 (K562-S) leukemia cells in nude mice. The body weights in control and berbamine-treated groups were 16.69±2.92g and 17.61±1.70g, respectively. K562-R2 Berbamine could specifically interact with the CaMKII-y kinase in chronic myeloid leukemiaWe used berbamine as the bait to identify the putative targets that specifically regulate LSCs. The results shows that berbamine directly interacted with CaMKII y kinase in leukemia cells and inhibit its kinase activity. The phosphorylation level of CaMKII y decreased prominently in the CML cells with the berbamine treatment, while the total CaMKII y protein do not change. To gain an insight into the mechanism by which berbamine inhibited CaMKII?activity, we built a model of the CaMKII?in complex with calmodulin by using Modeller based on the X-ray crystal of CaMKII?/calmodulin complex. And the result shows that Berbamine molecule is located in the ATP binding pocket of CaMKII?kinase. 3 CaMKII y kinase regulates survival, self-renewal and apoptosis resistance of LSCs through GSK3/Wnt/p-catenin and mitochondrial/Caspase-2 pathways3.1 CaMKII y kinase is aberrantly activated in LSCs.To understand whether CaMKII y kinase expression is related with CML, we evaluated both total and phosphorylated CaMKII y protein levels in CD34+leukemia stem cells and normal hematopoietic stem cells. To detect CaMKII y levels in rare LSCs and HSCs, we sorted both CD34+leukemia stem cells and HSCs.We observed that CaMKII y kinase is aberrantly activated in LSCs.3.2 CaMKII y kinase is essential for survival of LSC and promotes self-renewal of LSCsWe evaluated the effect of CaMKII y expression inhibition on the growth of leukemia colonies using a colony forming assay, and observed that down-regulation of CaMKII y expression markedly inhibited the growth of CML cells in vitro, suggesting that down-regulation of CaMKII y expression diminishes the oncogenic potential of leukemia cells.To assess the effects of enhanced expression of CaMKII y on the self-renewal of tumor stem/progenitor cells of CML, we established a stable CML cell line with high CaMKII y expression using human CML K562 cells transfected with the CaMKII y-EGFP expression plasmid and G418 selection. We found that enhanced expression of CaMKII y led to an increase in the number of colonies. More importantly, the size of the colonies was larger in the cells stably transfected with CaMKII y-EGFP than that in the control. These results indicate that CaMKII y enhances the colony-forming ability and proliferative capacity of leukemia cells through promoting the survival and self-renewal of leukemia stem cells.3.3 CaMKII?kinase promotes survival and self-renewal of LSCs in vivoWe investigated the proliferation and differentiation of enhanced CaMKII?expression leukemia cells in vivo. Enhanced CaMKII?-EGFP expression K562 leukemia cells were transplanted into nude mice and EGFP expression leukemia cells were used as the control. As expected, the size of K562 leukemia xenograft tumors with enhanced CaMKII?-EGFP expression was larger than that of control K562 xenograft tumors at day 15. Unexpectedly, morphologically undifferentiated stem-like blast cells that highly express CaMKII?markedly increased in the tumor of enhanced CaMKII?-EGFP expression K562 xenografts as compared to control K562 xenografts.3.4 CaMKII y kinase regulates the expression level of phosphorylated GSK3-?,?-catenin and Caspase-2 protein.We examined?-catenin expression level, and found that enhanced expression of CaMKII?kinase greatly upregulated?-catenin protein level as compared to control with a concomitant increase of phosphorylated GSK3-?protein. These results suggest that CaMKII?upregulates?-catenin protein via inhibiting phosphorylation of GSK-3/?resulting in survival and self-renewal of LSCs. We also observed that enhanced expression of CaMKII?markedly reduced Caspase-2 protein level in leukemia cells. CaMKII?may also maintain apoptosis-resistance through inhibition of Caspase-2.3.5 CaMKII?kinase is an important molecular to GSK3/Wnt/?-catenin and mitochondrial/Caspase-2 signaling way.To obtain biochemical evidence for this signaling axis, co-immunoprecipitation experiments were carried out. Leukemia cell lysates were incubated with CaMKII?antibody or GSK3?antibody, and the immune complexes were then purified, separated by SDS-PAGE, and immunoblotted with GSK3?or CaMKII?or?-catenin antibody. We observed that GSK3p.?-catenin and Caspase-2 were present in the complex immunoprecipitated by the CaMKII y antibody. As expected, CaMKII y was also present in the complex in a reciprocal immunoprecipitate using GSK3?antibody. Neither CaMKII y nor GSK3P was detected in the immune complex associated with control IgG, validating the specificity of the observed co-association and the presence of CaMKII y/GSK3?/?-catenin signaling axis.3.6 CaMKII y inhibitor berbamine caused a decrease of?-catenin proteinP-catenin has been shown to be essential for survival and self-renewal of leukemia stem cells. We demonstrated that treatment of K562 leukemia cells with CaMKII y inhibitor berbamine caused a decrease of P-catenin protein.Conclusions:1?Berbamine eliminates both imatinib-sensitive and-resistant CML xenografts in nude mice. Berbamine showes significant retardation in tumor growth without obvious toxicity in all animal groups.2. Berbamine selectively interacts with CaMKII-y kinase in leukemia cells and directly targets the ATP binding site of CaMKII y kinase.3?CaMKII y kinase is an important molecular to GSK3/Wnt/p-catenin and mitochondrial/Caspase-2 signaling axis. CaMKII?upregulates?-catenin via inhibitory phosphorylation of GSK-3/?of leukemia stem cells. CaMKII?may also maintain apoptosis-resistance through inhibitin of Caspase-2.4?Berbamine directly targets the ATP binding site of CaMKII y kinase and inhibits the expression of phosphorylated CaMKIIy kinase. Thereby. Berbamine inhibits Wnt/?-Catenin signaling pathway, activates Caspase-2, and eventually causes the death of leukemia stem cell. |
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