sphingosine-1-phosphate and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Resveratrol, an important phytoalexin in many plants, has been reported to have cytotoxic effects on various types of cancer. Ceramide is a bioactive sphingolipid that regulates many signaling pathways, including cell growth and proliferation, senescence and quiescence, apoptosis, and cell cycle. Ceramides are generated by longevity assurance genes (LASS). Glucosylceramide synthase (GCS) and sphingosine kinase-1 (SK-1) enzymes can convert ceramides to antiapoptotic molecules, glucosylceramide, and sphingosine-1-phosphate, respectively. C8:ceramide, an important cell-permeable analogue of natural ceramides, increases intracellular ceramide levels significantly, while 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and SK-1 inhibitor increase accumulation of ceramides by inhibiting GCS and SK-1, respectively. Chronic myelogenous leukemia (CML) is a hematological disorder resulting from generation of BCR/ABL oncogene. In this study, we examined the roles of ceramide metabolizing genes in resveratrol-induced apoptosis in K562 CML cells. There were synergistic cytotoxic and apoptotic effects of resveratrol with coadministration of C8:ceramide, PDMP, and SK-1 inhibitor. Interestingly, there were also significant increases in expression levels of LASS genes and decreases in expression levels of GCS and SK-1 in K562 cells in response to resveratrol. Our data, in total, showed for the first time that resveratrol might kill CML cells through increasing intracellular generation and accumulation of apoptotic ceramides.

Topics: Apoptosis; Ceramides; Down-Regulation; Glucosylceramides; Glucosyltransferases; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lysophospholipids; Morpholines; Phosphotransferases (Alcohol Group Acceptor); Resveratrol; RNA, Neoplasm; Sphingosine; Stilbenes; Up-Regulation

The mechanisms by which sphingosine kinase-1 (SK-1)/sphingosine 1-phosphate (S1P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-1/S1P enhances Bcr-Abl1 protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl1 stability was enhanced by ectopic SK-1 expression. Conversely, siRNA-mediated SK-1 knockdown in K562/IMA-3 cells, or its genetic loss in SK-1(-/-) MEFs, significantly reduced Bcr-Abl1 stability. Regulation of Bcr-Abl1 by SK-1/S1P was dependent on S1P receptor 2 (S1P2) signaling, which prevented Bcr-Abl1 dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-1/S1P2 restored PP2A-dependent Bcr-Abl1 dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34(+) mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T315I) Bcr-Abl1 in situ. Accordingly, impaired SK-1/S1P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T315I-Bcr-Abl1-derived mouse allografts. Since SK-1/S1P/S1P2 signaling regulates Bcr-Abl1 stability via modulation of PP2A, inhibition of SK-1/S1P2 axis represents a novel approach to target wild-type- or mutant-Bcr-Abl1 thereby overcoming drug resistance.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Cell Line, Tumor; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lysophospholipids; Mice; Mice, SCID; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Piperazines; Protein Phosphatase 2; Pyrimidines; Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine; Ubiquitination

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. In order to investigate the role of sphingosine kinase-1 (SphK-1)/sphingosine 1-phosphate (S1P) signal pathway in the expression of CML cells, and to explore whether P210(bcr/abl) involved is activating SphK-1/S1P signal pathwey, the expressions of SphK-1 and S1P receptor mRNA in bcr/abl positive K562 cells and bcr/abl positive primary CML cells were detected by RT-PCR, the imatinib mesylate, the specific inhibitor of P210(bcr/abl) was employed to inhibit the P210(bcr/abl) tyrosine kinases of K562 cells and CML primary cells, and then the intracellular SphK-1 activity was assayed. The results indicated that after being cultured with 2.5 micromol/L imatinib mesylate for 0.5, 2, 6, 24 and 48 hours, the intensions of inhibiting SphK-1 activity were 0.007%, 38.9%, 34.6%, 28.1% and 76.1% resepectively. SphK-1 activity in CML cells also was reduced by 2.5 micromol/L imatinib mesylate (16.8% - 41.9% decrease). It is concluded that the CML cells express SphK-1 and different S1P receptor, and P210(bcr/abl) fusion protein in CML cells can activate SphK-1.

Topics: Benzamides; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Piperazines; Pyrimidines; RNA, Messenger; Signal Transduction; Sphingosine

In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -1 cells, which expressed approximately 2.3- and 19-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C18-ceramide, which is generated by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Inhibition of hLASS1 by small interfering RNA partially prevented imatinib-induced cell death in sensitive cells. In reciprocal experiments, overexpression of hLASS1, and not hLASS6, in drug-resistant cells caused a marked increase in imatinib-induced C18-ceramide generation, and enhanced apoptosis. Interestingly, there were no defects in the levels of mRNA and enzyme activity levels of hLASS1 for ceramide generation in K562/IMA-1 cells. However, expression levels of sphingosine kinase-1 (SK1) and generation of sphingosine 1-phosphate (S1P) were increased significantly in K562/IMA-1 cells, channeling sphingoid bases to the sphingosine kinase pathway. The partial inhibition of SK1 expression by small interference RNA modulated S1P levels and increased sensitivity to imatinib-induced apoptosis in resistant cells. On the other hand, forced expression of SK1 in K562 cells increased the ratio between total S1P/C18-ceramide levels approximately 6-fold and prevented apoptosis significantly in response to imatinib. Additional data indicated a role for SK1/S1P signaling in the up-regulation of the Bcr-Abl expression at the post-transcriptional level, which suggested a possible mechanism for resistance to imatinib-mediated apoptosis. In conclusion, these data suggest a role for endogenous C18-ceramide synthesis mainly via hLASS1 in imatinib-induced apoptosis in sensitive cells, whereas in resistant cells, alterations of the balance between the levels of ceramide and S1P by overexpression of SK1 result in resistance to imatinib-induced apoptosis.

Topics: Apoptosis; Benzamides; Ceramides; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lysophospholipids; Membrane Potential, Mitochondrial; Membrane Proteins; Piperazines; Pyrimidines; RNA, Small Interfering; Sphingosine; Sphingosine N-Acyltransferase; Time Factors; Up-Regulation