thapsigargin and Leukemia--Lymphocytic--Chronic--B-Cell

Salinomycin, an antibiotic potassium ionophore, has been reported recently to act as a selective breast cancer stem cell inhibitor, but the biochemical basis for its anticancer effects is not clear. The Wnt/β-catenin signal transduction pathway plays a central role in stem cell development, and its aberrant activation can cause cancer. In this study, we identified salinomycin as a potent inhibitor of the Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomycin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Nigericin, another potassium ionophore with activity against cancer stem cells, exerted similar effects. In otherwise unmanipulated chronic lymphocytic leukemia cells with constitutive Wnt activation nanomolar concentrations of salinomycin down-regulated the expression of Wnt target genes such as LEF1, cyclin D1, and fibronectin, depressed LRP6 levels, and limited cell survival. Normal human peripheral blood lymphocytes resisted salinomycin toxicity. These results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal Wnt signaling by interfering with LPR6 phosphorylation, and thus impair the survival of cells that depend on Wnt signaling at the plasma membrane.

Topics: Apoptosis; beta Catenin; Calcium; Down-Regulation; Gene Expression Regulation, Leukemic; Genes, Neoplasm; HEK293 Cells; Humans; LDL-Receptor Related Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Low Density Lipoprotein Receptor-Related Protein-6; Nigericin; Phosphorylation; Pyrans; Signal Transduction; Thapsigargin; Wnt Proteins

A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8-mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.

Topics: Apoptosis; Caspases; Down-Regulation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Rearrangement, B-Lymphocyte; Heat-Shock Proteins; Humans; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Membrane Proteins; Models, Biological; RNA, Small Interfering; Signal Transduction; Stress, Physiological; Thapsigargin; Tunicamycin

To study defective signal transduction via the Ag receptor of B-chronic lymphocytic leukemia cells (B-CLL), we examine in this report the Ca2+ response triggered by anti-mu antibody in 23 patients previously classified in three phenotypic groups. Altered Ca2+ changes are essentially found in CLL group II whose leukemic cells are characterized by a marked expression of the CD11b Ag. B-CLL cells from patients with a defective Ca2+ response present an altered pattern of protein tyrosine phosphorylation after anti-mu stimulation in comparison with normal human B cells and B-CLL cells from patients having a normal Ca2+ response. Most of the proteins usually tyrosine phosphorylated after the triggering of cell-surface IgM are concerned. This includes the gamma 1 isoform of phospholipase C, although the protein is normally present in B-CLL cells. These findings suggest that the interruption of the phosphoinositide pathway in B-CLL cells is very proximal, at the level in the signaling cascade between activated surface Ig receptors and protein tyrosine kinases. Simultaneously, we demonstrate that some low responding patients exhibit a decreased Ca2+ response to thapsigargin, an agent known to release intracellular Ca2+ without inositol 1,4,5-trisphosphate production. This suggests that an altered functioning of the mechanism leading to the cell Ca2+ influx in B cells can be also involved in the decreased Ca2+ response observed in B-CLL cells.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antigens, CD; Calcium; CD11 Antigens; Humans; Immunoglobulin M; Leukemia, Lymphocytic, Chronic, B-Cell; Phenotype; Phosphorylation; Proteins; Rabbits; Receptors, Antigen, B-Cell; Terpenes; Thapsigargin; Tyrosine