Abstract
BackgroundTreatment of acute leukemia is challenging and long-lasting remissions are difficult to induce. Innovative therapy approaches aim to complement standard chemotherapy to improve drug efficacy and decrease toxicity. Promising new therapeutic targets in cancer therapy include voltage-gated Kv1.3 potassium channels, but their role in acute leukemia is unclear. We reported that Kv1.3 channels of lymphocytes are blocked by memantine, which is known as an antagonist of neuronal N-methyl-D-aspartate type glutamate receptors and clinically applied in therapy of advanced Alzheimer disease. Here we evaluated whether pharmacological targeting of Kv1.3 channels by memantine promotes cell death of acute leukemia cells induced by chemotherapeutic cytarabine.MethodsWe analyzed acute lymphoid (Jurkat, CEM) and myeloid (HL-60, Molm-13, OCI-AML-3) leukemia cell lines and patients’ acute leukemic blasts after treatment with either drug alone or the combination of cytarabine and memantine. Patch-clamp analysis was performed to evaluate inhibition of Kv1.3 channels and membrane depolarization by memantine. Cell death was determined with propidium iodide, Annexin V and SYTOX staining and cytochrome C release assay. Molecular effects of memantine co-treatment on activation of Caspases, AKT, ERK1/2, and JNK signaling were analysed by Western blot. Kv1.3 channel expression in Jurkat cells was downregulated by shRNA.ResultsOur study demonstrates that memantine inhibits Kv1.3 channels of acute leukemia cells and in combination with cytarabine potentiates cell death of acute lymphoid and myeloid leukemia cell lines as well as primary leukemic blasts from acute leukemia patients. At molecular level, memantine co-application fosters concurrent inhibition of AKT, S6 and ERK1/2 and reinforces nuclear down-regulation of MYC, a common target of AKT and ERK1/2 signaling. In addition, it augments mitochondrial dysfunction resulting in enhanced cytochrome C release and activation of Caspase-9 and Caspase-3 leading to amplified apoptosis.ConclusionsOur study underlines inhibition of Kv1.3 channels as a therapeutic strategy in acute leukemia and proposes co-treatment with memantine, a licensed and safe drug, as a potential approach to promote cytarabine-based cell death of various subtypes of acute leukemia.
Highlights
Treatment of acute leukemia is challenging and long-lasting remissions are difficult to induce
Since MYC is a central oncogene inducing leukemic transformation in T cell acute lymphoid leukemia (T-ALL) and acute myeloid leukemia (AML) [11, 12], influencing AKT, ERK1/2 and MYC signaling may enhance the efficacy of chemotherapy in ALL and AML [13,14,15,16,17,18,19,20]
Analyzing acute lymphoid and myeloid leukemia cell lines and patients’ acute leukemic blasts, our data highlight the importance of Kv1.3 channels for the survival of acute leukemia cells and provide initial evidence that memantine and cytarabine co-treatment may be a potential therapeutic strategy to enhance the efficacy in acute leukemia treatment
Summary
Cell culture and determination of cell death Jurkat (JE6–1), F9, JMR [40], A3, C8 (I.9.2) [41], CEM, HL-60, Molm-13, OCI-AML-3 cells, primary cells from healthy donors, and primary leukemic cells were cultured in RPMI 1640 medium (Biochrom AG, Berlin, Germany) supplemented with 10% fetal calf serum. Cell lines or primary cells were cultured without drug, with memantine (Tocris Biosciences, Bristol, Great Britain), AraC (Department of Pharmacy, Medical Faculty Magdeburg), and a combination of memantine plus AraC. CEM and AML cell lines were incubated with human FcR block (Miltenyi) before staining with Kv1.3 antibodies. Memantine was kept in a constant concentration during recording in the fixed holding potential (− 80 mV) and the amplitude of the current was measured to determine membrane depolarization. Statistical analysis Statistics were performed with Cell Quest Pro software (BD Bioscience), HEKA FitMaster v2x53 and IgorPro for patch-clamp transient currents and GraphPad Prism for analysis of dose-response curves and Student’s t-test, with P* < 0.05, P** < 0.01, and P*** < 0.001.
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