In present study, we found Icaritin, a compound purified from traditional herb medicine exhibited a potent anti-leukemia activities towards established CML cell line-K562 and primary bone marrow cells (including CD34+ cells) from CML patients. Icaritin effectively inhibited K562 growth in vitro. At concentration of 8 µM, Icaritin could lead to more than 50% of growth inhibition of K562. More importantly, we also observed that Icaritin exhibited strong efficacies on primary bone marrow cells from CML-CP and CML-BC patients while had no effect on growth and proliferation of normal bone marrow cells (), indicating Icaritin has low or no general cytotoxic effect on normal hematopoiesis. Consistent with this, Icaritin showed potent effects with low adverse reactions such as weight loss in vivo. We also checked the effects of Icaritin on Imatinib-resistant cells line and Imatinib-resistant primary cells from one CML patient, our results suggested that Icaritin evidently inhibited the growth of both Imatinib-resistant cells line and Imatinib-resistant primary cells. Furthermore, we also confirmed that Icaritin could induce Imatinib-resistant cells apoptosis. Although the results are preliminary; this will be a new clue for searching an alternative agent in overcoming Imatinib-resistance of Bcr/Abl+ cells.
Accumulating evidence indicated that many types of cancer, including leukemia, originate from and are maintained by a small of cancer stem/progenitor cells. These cancer stem/progenitor cells are often resistant to most therapeutic strategies. In this study, we enriched CML stem/progenitor cells from 3 patients with CML-BC using CD34 selection kit and successfully isolated CD34+ leukemia cells (the yield: 89.37%±6.79%). Here, we showed that Icaritin could effectively inhibit leukemic stem/progenitor cells proliferation and induce apoptosis, and thus suggesting that the effect of Icarintin on anti-leukemia activity may preferentially target to leukemic stem/progenitor cells.
It has been characterized that both extrinsic and intrinsic apoptotic pathways are involved in the activation of effectors caspases (casp-3, casp-2 and casp-7). The extrinsic pathways is initiated by binding death receptors, such as CD95/Fas, TNF or TRAIL receptor to activate caspase-8 and caspese-10, which in turn cleave and activate effectors caspases
. The involvement of intrinsic apoptotic pathway is more often events especially in cancer cells, which is characterized by the disruption of the mitochondrial membrane and proteins release 
. Our results demonstrated that Icaritin was able to induce apoptosis, both in K562 and primary CML cells. Icaritin inhibited Bcl-2 expression and up-regulated Bax expression, which resulted in a lower ratio of Bcl-2/Bax. We also found that cytochrome C levels were up-regulated, caspase-9 and caspase-3 were cleavaged and activated, Apaf-1 expression was down-regulated following Icaritin treatment, indicating the mitochondrial permeability is changed. Based on the observations, we suggested that mitochondrial-mediated caspase cascade pathway plays a major role in Icaritin-induced apoptosis.
K562 have been widely used as a model for leukemia differentiation. It is known to be induced to differentiate along either erythroid or megakaryocytic lineage 
. In our experiments, both morphologic and phenotypic analysis revealed that after Icaritin treatment for 6 to 8 days, a significant number of K562 exhibited erythroid-like features, including the change of cell volume, increased Hb concentration, RBC benziding staining and expression of erythroid specific markers, such as glycophorin-A (CD235a) and transferring receptor (CD71). It has been shown erythroid differentiation is a tightly regulated process that requires specific transcription factors 
. Accordingly, it has been reported that hydroxyurea induced erythroid differentiation of K562, which was associated with the activation of the p38 MAPK pathway 
. Davidson and Morange showed activation of the p38 pathway was necessary for cardiomyogenesis of the P19 embryonic carcinoma cell line during early stages 
. Recently, Ding et al showed that Icaritin-induced cardiomyocyte differentiation of murine embryonic stem cells was associated with enhanced phosphorylation of p38
. Consistent with these observations, we found that Icaritin significantly induced phosphorylation levels of p38 during K562 cell differentiation toward erythroid lineage, which was blocked by p38 inhibitor SB203580. We concluded Icaritin was able to induce CML cell differentiation presumably through the p38 pathway.
The potent anti-leukemia efficacy of Icaritin in murine model demonstrated that Icaritin was able to reduce the infiltration of leukemia cells and alleviate the load of leukemia cells in peripheral blood and spleen; effects were similar to those of Imatinib. Survival study also showed that Icaritin was able to significantly prolong the lifespan of mice loaded with leukemia. We found that Icaritin failed to influence Bcr/Abl expression in K562. However, whether Icaritin has influence on the downstream signal pathways of Bcr/Abl need to be further proven.
The anti-apoptotic activity of Bcr/Abl contributes greatly to the development of CML. Bcr/Abl may function either by enhancing the proliferation potential of hematopoietic progenitors or by protecting these progenitor cells from apoptosis 
. Accumulating evidence shows that the constitutive tyrosine kinase activity of Bcr-Abl is essential for its leukemogenic activity 
. Bcr-Abl-mediated signal transduction pathways could interfere with various cellular physiological processes, including cells proliferation, adhesion, and apoptosis 
. These processes are thought to involve in intracellular signaling pathways, such as MEK/ERK, JNK/SAPK and p38MAPK 
. It has been shown that the STAT family of transcription factors plays important role in transformation and antiapoptotic signaling stemming from constitutive activation of Bcr/Abl kinase 
. As a driving force for CML, the activated tyrosine kinase of Bcr/Abl is able to stimulate the Janus kinase (Jak) 2 pathway 
. Furthermore, PI3K/Akt pathway has emerged as one of the essential signaling mechanisms in Abl leukemogenesis as its downstream effectors are responsible for propagating the signals to promote myeloid and lymphoid transformation
. To assess whether the effect of Icaritin on anti-leukemia is involves in the inactivation of Jak-2/Stat-3/Akt axes, western blot was used for evaluating the expression of Jak-2, p-Stat-3 and p-Akt protein. It is noteworthy that the exposure of K562 cells to Icaritin resulted in diminished phospho-p38 or phospho-ERK expression, and induced activation of phospho-JNK or phospho-c-Jun, which contributes apparently to the apoptosis of Bcr/Abl+ cells. Significantly, Icaritin can obviously down-regulate the expression of Jak-2, phospho-Stat3 and phospho-Akt protein at a dose- and time-dependent manner, suggesting that the interruption of Jak-2/Stat-3/Akt signal network by Icaritin contributes to the growth-inhibition and diminished survival of K562 cells. Collectively, these observations suggest that transcriptional repression by Icaritin lowers the proliferation potential for Icaritin-mediated growth-inhibition of Bcr/Abl+ leukemia cells. On the other hand, the possibility that the action of Icaritin as an apoptosis-activator contribute to a diminished apoptotic threshold cannot be completely excluded. However, exact molecular mechanisms on anti-leukemia activity of Icaritin still need to be elucidated.
In conclusion, we have documented for the first time the anti-CML effects of Icaritin in vitro and in vivo. Our findings have shown that Icaritin was able to inhibit CML cell growth, and induce apoptosis and differentiation. The underlying mechanisms of Icaritin anti-CML activity are involved in the inhibition of MAPK/ERK/JNK signals and down-regulated kinase activity of Jak-2/Stat-3/Akt signal network. Further investigation of this novel anti-CML agent may offer insights into the pathogenetic mechanisms of CML and provide a new approach for CML treatment and a reversal to Imatinib-resistance.