The Effect of Bioactive Phospholipids on Normal and Malignant Hematopoiesis—Modulation of Their Effects By Downregulating the Activities of Heme Oxygenase 1 (HO-1) and Inducible Nitric Oxygenase Synthase (iNOS)

Background. Bioactive phospholipids, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), lysophosphatidylcholine (LPC), and its derivative lysophosphatidic acid (LPA), haveemerged as important mediators regulating the trafficking of several types of normal and malignant cells. As we have demonstrated, these factors are upregulated in tissues, including in the bone marrow microenvironment after radio/chemotherapy [Leukemia 2010; 24: 976-985] . While the role of S1P in regulating the trafficking of normal and malignant hematopoietic cells is well established, in this work we focused on the biological effects of C1P, LPC, and LPA. Hypothesis. We hypothesized that not only S1P but also other bioactive phospholipids are involved in regulating hematopoietic cell trafficking and expansion within hematopoietic tissues. Moreover, based on our results showing negative effects of heme oxygenase 1 (HO-1) [ Stem Cell Rev . 2015, 11, 110-118 ] and inducible nitric oxygenase synthase (iNOS) [ Stem Cell Rev . 2017, 13, 92-103 ] on the trafficking of normal hematopoietic cells, we hypothesized that this process could be inhibited by upregulating HO-1 and iNOS in malignant hematopoietic cells.Materials and Methods. We employed eight human myeloid and lymphoid cell lines as well as cells from patients diagnosed with acute myeloid leukemia. We phenotyped these cells for expression of bioactive lipid receptors and tested the functionality of these receptors by assaying for activation of p42/44 MAPK and AKT after stimulation with S1P, C1P, LPC, and LPA. We also tested the effect of bioactive phospholipids on cell migration, adhesion, and proliferation. In parallel experiments, we evaluated the effect of bioactive phospholipids on the expression of HO-1 and iNOS in human leukemic cells and the involvement of p38 MAPK in these phenomena. Furthermore, in in vivo experiments the expression of HO-1 and iNOS was upregulated by administration of the p38 MAPK inhibitor SB203580. We also performed in vivo seeding efficiency studies in immunodeficient mice with cells stimulated by bioactive phospholipids in the presence or absence of SB203580. In parallel, we also tested the chemotactic responsiveness of normal human CD34⁺ cells and clonogenic blasts from AML patients to bioactive phospholipids in chemotaxis and adhesion experiments. Results. We demonstrate for the first time that, besides S1P receptors, human leukemic cells express functional receptors for other bioactive lipids and that C1P, LPC, and LPA are, as has been reported for S1P, important chemoattractants and pro-adhesive factors for human leukemic cells. In contrast only S1P and C1P, but not LPC or LPA stimulated migration and adhesion of normal human clonogeneic CD34⁺ cells. At the same time, we report that all of the tested phospholipids did not stimulate proliferation of human clonogeneic CD34⁺ or leukemic cells. We also demonstrate that these pro-migratory effects of bioactive phospholipids can be explained, at least partially, by downregulation HO-1 and iNOS activities in a p38 MAPK-dependent manner. Stimulation of leukemic cells before injection into immunodeficient mice with bioactive phospholipids increased their seeding efficiency to BM, liver, and lungs. This effect was significantly inhibited after exposure of these cells to SB203580. Conclusions. We demonstrate for the first time that, besides S1P receptors, human leukemic cells express other functional receptors that enable chemotaxis in response to, as well as adhesion to, several bioactive phospholipids. Since this effect is mediated, at least partially, by downregulating HO-1 and iNOS expression in a p38 MAPK-dependent manner, inhibitors of p38 MAPK may find application in inhibiting the spread of leukemic cells in situations in which S1P, C1P, LPC, and LPA become upregulated in the tissues. This may occur, for example, after radio- and chemotherapy, leading to the unwanted spread of leukemic cells that have escaped therapy.