rottlerin and Multiple-Myeloma

The growth and proliferation of multiple myeloma (MM) cells are influenced by various cytokines produced by bone marrow stromal cells. As cytokine interaction between malignant plasma cells and neighbouring stromal cells is important in the pathogenesis of MM, the understanding of intracellular signalling events elicited by this interaction is of central importance. Recent reports have shown that protein kinase C (PKC) is directly involved in modulating apoptosis in different cells types, including those of haematopoietic neoplasms. In the present study, we analysed the expression patterns of PKC isoforms in the myeloma cell lines U266, RPMI-8226 and K620. This analysis demonstrated common expression of PKC-delta, PKC-iota, PKC- micro and PKC-zeta in all three myeloma cell lines. PKC-delta expression in plasma cells from 11 patients with MM was also shown by immunohistochemistry, utilizing a monoclonal mouse anti-human PKC-delta antibody. U266 cells treated with the broad PKC inhibitor safingol (l-threo-dihydrosphingosine) or the PKC-delta-specific inhibitor rottlerin (3'-[(8-Cinnamoyl-5,7-dihydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)methyl]-2',4',6'-trihydroxy-5'-methylacetophenone) showed decreased PKC-delta in the particulate fraction and resulted in significant apoptosis. Primary myeloma cells also showed apoptosis after treatment with the PKC inhibitors, as detected by both flow cytometric and morphological evaluation. Our results indicate that PKC-delta is commonly expressed in myeloma cells and plays an important role in plasma cell survival.

Topics: Acetophenones; Apoptosis; Benzopyrans; Blotting, Western; Down-Regulation; Enzyme Inhibitors; Humans; Multiple Myeloma; Protein Kinase C; Protein Kinase C-delta; Sphingosine; Tumor Cells, Cultured

The soluble interleukin 6 receptor alpha is an agonistic molecule of interleukin 6 (IL-6) and is important in the biology of multiple myeloma. More precisely, it potentiates the deleterious effects of IL-6 during tumour progression, facilitating angiogenesis and bone resorption. Because the mechanisms involved in the shedding of the interleukin 6 receptor alpha (IL-6Ralpha) in multiple myeloma are not known, we have investigated them in the XG-6 human myeloma cell line. Here we provide evidence that PMA-induced IL-6Ralpha shedding is controlled by a metalloproteinase and by protein kinase C (PKC) isoenzymes that do not require Ca(2+) for their activation. We show that XG-6 cells express PKC-delta, -eta and -zeta isoenzymes. However, after stimulation with PMA, only PKC-delta and PKC-eta are activated, as shown by their translocation to the membrane. Treatment with PMA induces an increase in PKC-delta phosphorylation in its active loop. In addition, by using rottlerin, a specific inhibitor of PKC-delta, we demonstrate that PKC-delta is involved in the PMA-induced shedding of IL-6Ralpha. With the use of UO126, a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway, we show that the PMA-induced IL-6Ralpha shedding is mediated in part by the MAPK pathway. Finally, whereas GF109203X, a general PKC inhibitor, inhibits the activation of ERK1/2 (extracellular signal-regulated protein kinase 1/2), rottlerin has no inhibitory effect, indicating that the Ras/MAPK activation is PKC-dependent but PKC-delta-independent. Taken together, these results suggest that the PMA-induced shedding of IL-6Ralpha is mediated by a PKC isoenzyme network.

Topics: Acetophenones; Antibodies, Monoclonal; Benzopyrans; Blotting, Western; Calcium; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Humans; Isoenzymes; MAP Kinase Signaling System; Microscopy, Fluorescence; Multiple Myeloma; Neovascularization, Pathologic; Phosphorylation; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Receptors, Interleukin-6; Subcellular Fractions; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured