Targeting Lipid Raft And AMPK In Elimitaing Of Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is a lethal hematological malignancy resulting from transformation of a primitive hematopoietic cell caused by constitutely activity of BCR-ABL tyrosine kinase. Imatinib (IM, Gleevec), a selective BCR-ABL inhibitor, is a successful front-line treatment for CML. Although IM treatment results in achievement of complete cytogenetic response, there are still about 40% of CML patients receive drug resistance after imatinib targeting treatment in clinic. A growing body of evidence suggests that the reason imatinib does not cure CML is that it is unable to eradicate the leukemic stem cells which are in a quiescent state and not insensitive to imatinib despite inhibition of BCR-ABL activity. Thus there is a need for developing approaches to overcome and prevent resistance to imatinib. As muptile pathway cascades are invovled in regulation of selfrenwal and survival of CML progenitors and stem cells, targeting multiple pathways are essential for eliminating these cells.Lipid microdomains rich in sphingolipids and cholesterol, also known as lipid rafts, have been proposed as regions within plasma membranes that are important for cellular signaling and play important roles in regulating cell fuctions. In view of the importance of lipid rafts in tumor cell biology, MβCD, a selective inhibitor of lipid rafts was to explore the effects of MβCD on Imatinib -sensitive and -insensitive CML cells, as well as the combined effects with Imatinib. First, we examined the anti-proliferative effects of MβCD in K562 and K562R cells which expressing bcr-abl gene. The data showed that MβCD treatment significantly inhibited cell viability, colony formation and induced G2/M phase arrest at a concentration of 1 mM. Using Annexin V-PI double staining and Western Blot, we verified that MβCD can induce apoptosis in K562 and K562R cells. We also confirmed that MβCD induced autophagy in CML cells by Western Blot and Immunocytochemistry. Then, we detected the expression and activity of ERK and SPK1 through Western Blot and Kinase activity kit, respectively. To further clarify the mediation of SPK1 in MβCD induced apoptosis, we firstly tranduced K562 cells with adenovirus carrying spk1 gene. The K562 cells overexpressing SPK1 were treated with MβCD and detected for apoptosis. MβCD-induced apoptosis in the group of overexpressing SPK1 K562 cells is lower significantly than that in the control group. These results indicate that ERK/SPK1 signaling is not only involved in MβCD induced apoptosis, but partly mediates this progress. Because BCR-ABL protein with tyrosine kinase activity is regulated by signals of lipid rafts, we hypothesized that there may have synergistic effects of MβCD and Imatinib on the BCR-ABL positive CML cells, even of bcr-abl point mutation cells. To verify this hypothesis, we not only investigated the synergistic effects of MβCD and Imatinib on K562 and K562R cells proliferation, apoptosis and autophagy, but tested the synergism of MβCD and Imatinib on expressing BCR-ABLT315I BaF3 cells. Finally, we detected that the expression of BCR-ABL, its targeted protein p-CrkL, C-MYC, and activity of ERK and SPK1. These data imply that ERK/SPK1 signaling is synergistically regulated by MβCD and Imatinib. Summary, we revealed that MβCD could not only kill BCR-ABL positive CML cells, but combined with Imatinib to in part overcome Imatinib resistance.AMPK is a key regulator of cellular energy metabolism, and plays an important role in maintaining homeostasis in tumor. The effect of anticancer has been proved by AMPK activators, such as Metformin, AICAR and Resveratrol. However, the role of AMPK-mediated protection remains a key factor in drug resistance. Thus, we speculated that inhibition of AMPK may be beneficial to the anti-cancer effect of AMPK activators. In view of this, we introduced the selective inhibitor of AMPK, Compound C to do some research. Above all, we investigated the effects of Compound C alone on CML cell function. Using CCK-8 and colony formation assay, we found that Compound C could inhibit significantly CML cells proliferation. It is verified that Compound C induced cell apoptosis associated with caspase3 activation and mitochondrial pathway by using the methods of Annexin V-PI double staining, Cleaved caspase3 expression, JC-1 and electron microscopy. In addition, cell autophagy was induced by Compound C, which detected by MDC staining, immunocytochemistry and electron microscopy. Autophagic inhibitors including 3-MA and CQ, and RNA interference were used to suppressed Compound C induced autophagy, respectively, we found that levels of apoptosis were significantly increased in Compound C treated CML cells. It indicates that autophagy induced by Compound C is protective. In order to clarify the medicated role of AMPK in Compound C treated CML cells, we assayed cell apoptosis by adding AICAR and transfecting siAMPK. The data demonstrate that AICAR could not reverse Compound C induced apoptosis and autophagy. Besides, knockdown of AMPK even enhanced the effects of Compound C, suggesting that AMPK does not involve in the Compound C induced apoptosis and autophagy. Then, we investigated the impact on CML cells treated with Compound C and another AMPK activator, Resveratrol. The results showed that the combination of Compound C and Resveratrol could inhibit K562 and K562R cell proliferation significantly. And the similar effect was also investigated on apotosis of BaF3 cells expressing BCR-ABLT315I by the cotreatment of these two drugs. Furthermore, the expression of signal proteins including P-STAT5, P-AKT and P-CrkL was downregulated synergistically by treatment with Compound C in combination with Resveratrol. All these results indicate that the effect of combination therapy of Compound C and Resveratrol is more effective than that of the single drug. To clear the role of AMPK in combination of Compound C and Resveratrol, AICAR and siAMPK were applied to inhibit the AMPK the apoptosis was detected. The data indicate that AMPK pathway is not necessary for Compound C and/or Resveratrol to eliminating BCR-ABL positive CML cells.Taken together, by using the subject of BCR-ABL positive CML cells, the potential anti-cancer effects of two inhibitors, MβCD and Compound C, on BCR-ABL transforming activity and imatinib response were investigated.Targeting lipid raft and AMP leads to decrease of BCR-ABL activity and induces apoptosis of CML cells. Our results define a novel regulatory mechanism of BCR-ABL transforming activity suggesting that lipid raft and AMPK might be molecular targets for CML therapy.