HERG K~+ Channels Functionally Linked With SDF-1 Induced Acute Leukemic Cells Migration And The Inhibitory Effect By Berberine

PartⅠHERG K~+ channels expression and analysis of itsregulation in leukemic cellsObjective: The human ether-a-go-go-related (herg) gene encoding K~+ channels(HERG) belongs to an evolutionarily conserved multigene family of voltage activated K~+channels and their contribution to the repolarization of the cardiac action potential are wellunderstood. Recent studies revealed that HERG K~+ channels are preferentially expressed indifferent histogenesis of tumor cells. Here we tried to explore the expression of herg inleukemic cells and CD34~+/CD38~- LSCs and tried to explore its regulation of leukemic cellsactivities such as proliferation, cell cycle and apoptosis.Method: Ficoll was used to isolate the bone marrow mononuclear cells, total RNAwas extracted and its purity and integrity was checked by running an aliquot on a 1.5%agarose gel and rt-PCR was used to evaluate the herg expression of different samples,CCK-8 kit was used to study cell proliferation. Flow cytometry was used to analyze HL-60cell cycle and apoptosis.Result: herg was aberrantly expressed in leukemic cell lines, CD34~+/CD38~- LSCs andprimary leukemic cells but not expressed in normal bone marrow mononuclear cells. Inaddition, the expression of herg mRNA was not associated with the clinical and cytogeneticfeature of leukemia. Our results showed that HL-60 cell proliferation was dramaticallyinhibited by blocking HERG K~+ channels with E-4031, a HERG K~+ channels specificinhibitor. Moreover, E-4031could cause a marked reduction of the cell populations in Sphase along with significant increase in cell numbers in G1 phase but did not cause cellsapoptosis. Conclusion: These data provided evidence for the oncogenic potential of HERG K~+channels and it may be a novel, potential marker of leukemia diagnosis in the future.PartⅡBerberine inhibits SDF-1-induced AML cells and leukemicstem cells migration via regulation of SDF-1 level in bone marrowstromal cellsObjective: Berberine is an isoquinoline derivative alkaloid isolated from manymedical herbs. Emerging evidences indicate the functional role of berberine on thedevelopment of cancer, berberine plays a prominent role in the control of tumor cellproliferation and invasion, but there is no report about berberine functional role onleukemia at present. SDF-1 is a homeostatic chemokine that signals through CXCR4 whichis expressed by hematopoietic tumor cells. The SDF-1/CXCR4 axis is involved in themigration process of leukemic cells. In this study, we investigated the effects of berberineon the SDF-1-induced HL-60 cells, primary AML cells and leukemic stem cells .(LSCs)migration.Method: Transwell migration chambers (8μm) were used to assess the role ofberberine on leukemic cell migration; Flow cytometry was used to analyze the role ofberberine on the CXCR4 expression and F-actin polymerlization; SDF-1 protein levelsecreted by bone marrow stromal cells (BMSCs) was evaluated by ELISA, western blottingwas used to detect the level of Akt and pAkt.Result: Our data demonstrated that berberine could partly inhibit SDF-1-inducedAML cells as well as LSCs migration. Berberine could reduce SDF-1 protein level secretedby BMSCs in the microenvironment but not affect CXCR4 expression on HL-60 cellmembrane. Berberine could inhibit F-actin polymerization and decrease the expression ofpAkt, SDF-lcould activate the Akt signal pathway and Akt inhibitor, LY2904002, couldinhibit the SDF-1 induced HL-60 cells migration as dose-dependent. Conclusion: Berberine could inhibit AML cells migration partly by reducing thesecreting of SDF-1 by BMSC and inhibiting HERG1 Kt channels of leukemic cells.Therefore, it is speculated that berberine might be a potentially effective agent forprevention of leukemia.PartⅢHERG K~+ channels role in cells migration anda functional link between HERG K~+ channels and SDF-1Objective: Stromal cell-derived factor-1 (SDF-1) and its unique receptor, CXCR4,regulate stem/progenitor cell migration and retention in the marrow and are required forhematopoiesis. Recent studies suggested that HERG K~+ channels were important regulatorsof non excitable cells migration and found in tumor cells. In this study, we investigatedwhether SDF-1-induced acute leukemic cell migration associated with HERG K~+ channels.Method: Transient transfected HEK293T was used to express HERG K~+ channels,transwell was used to acess cell migration, flow cytometry was used to F-actinpolymerization, HERG1 K~+ currents were measured by the standard two microelectrodevoltage clamp techniques.Result: Our data showed that berberine could specifically block HERG1 K~+ channelsand significantly blocked migration of HEK293T cells transfected with herg-pEGFP.Berberine could also decrease the F-actin polymerization in transient transfected HEK293T.SDF-1 increased HERG1 K~+ current expressed in HEK293T cells transfected withherg-pEGFP.Conclusion: The HERG K~+ current increased by SDF-1 might contribute to themechanism of SDF-1 induced cell migration and berberine which could be a specificHERG K~+ inhibitor decreased the cells migration induced by SDF-1. The data showed thatHERG K~+ channels were essential for cells migration induced by SDF-1.