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Analysis of the anti-proliferative and the pro-apoptotic efficacy of Syk inhibition in multiple myeloma.

Koerber RM, Held SA, Heine A, Kotthoff P, Daecke SN, Bringmann A, Brossart P - Exp Hematol Oncol (2015)

Bottom Line: Incubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways.Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c.In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs.

View Article: PubMed Central - PubMed

Affiliation: Medical Clinic III, Department of Hematology and Oncology, University Hospital Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.

ABSTRACT

Background: Multiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of malignant plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of immunomodulatory drugs like bortezomib or lenalidomide, that have been associated with improved survival, MM is still incurable and new treatment options are needed. In B cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma (DLBCL), Syk (spleen tyrosine kinase) inhibitors have shown promising in vitro and first clinical results. In our study, we analyzed the potential of Syk as a target in MM.

Methods: The MM cell lines AMO-1, U266 and RPMI8226 and primary MM cells were treated with the Syk inhibitors BAY61-3606, R406 or Piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Furthermore, we analyzed synergistic and additive effects of Syk inhibitors in combination with established anti-myeloma drugs and experimental inhibitors (e.g. PI-3-Kinase inhibitor NVP-BEZ235).

Results: Incubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways. Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c. In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs.

Conclusions: Our results show that Syk inhibition might represent a promising new treatment option in MM with an increased efficacy when combined with MAP kinase inhibitors. Furthermore, our study strongly underlines the potency of Syk inhibitors as a potential therapeutic treatment option for MM patients.

No MeSH data available.


Related in: MedlinePlus

a–c Inhibition of Syk induces apoptosis in MM cell lines. a–c Viability of MM cells was defined by the amount of nuclear fragmentation and compared to the respective DMSO control. MM cell lines [AMO-1 (a), U226 (b) and primary MM cells from patients with plasma cell leukemia (purity >90%) (c)] were treated with various concentrations of Syk inhibitors (Piceatannol, R406 and Bay61-3606) as indicated. After 24 h of incubation with Syk inhibitors, propidium iodide-staining was performed. Nuclear fragmentation was determined by flow cytometry. zVAD-FMK was added as confirmation for caspase activation (white bar graphs). c Primary MM cells were isolated from peripheral blood from patients with plasma cell leukemia (purity >90%) and analyzed as described above. Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of at least three. The significance is related to DMSO as control. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001.
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Fig4: a–c Inhibition of Syk induces apoptosis in MM cell lines. a–c Viability of MM cells was defined by the amount of nuclear fragmentation and compared to the respective DMSO control. MM cell lines [AMO-1 (a), U226 (b) and primary MM cells from patients with plasma cell leukemia (purity >90%) (c)] were treated with various concentrations of Syk inhibitors (Piceatannol, R406 and Bay61-3606) as indicated. After 24 h of incubation with Syk inhibitors, propidium iodide-staining was performed. Nuclear fragmentation was determined by flow cytometry. zVAD-FMK was added as confirmation for caspase activation (white bar graphs). c Primary MM cells were isolated from peripheral blood from patients with plasma cell leukemia (purity >90%) and analyzed as described above. Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of at least three. The significance is related to DMSO as control. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001.

Mentions: In order to analyze the effect of Syk inhibitors on the viability of established MM cell lines, Piceatannol, R406 or Bay61-3606 were added to the cell culture medium and flow cytometric propidium iodide (PI) analysis of DNA fragmentation was performed as described previously [33]. Incubation of cells with the compounds resulted in a concentration dependent cell death induction that was inhibited by the addition of the pan-caspase inhibitor zVAD-FMK. This indicates that caspase activity was indispensable for the observed apoptotic effects (Fig. 4a, b).Fig. 4


Analysis of the anti-proliferative and the pro-apoptotic efficacy of Syk inhibition in multiple myeloma.

Koerber RM, Held SA, Heine A, Kotthoff P, Daecke SN, Bringmann A, Brossart P - Exp Hematol Oncol (2015)

a–c Inhibition of Syk induces apoptosis in MM cell lines. a–c Viability of MM cells was defined by the amount of nuclear fragmentation and compared to the respective DMSO control. MM cell lines [AMO-1 (a), U226 (b) and primary MM cells from patients with plasma cell leukemia (purity >90%) (c)] were treated with various concentrations of Syk inhibitors (Piceatannol, R406 and Bay61-3606) as indicated. After 24 h of incubation with Syk inhibitors, propidium iodide-staining was performed. Nuclear fragmentation was determined by flow cytometry. zVAD-FMK was added as confirmation for caspase activation (white bar graphs). c Primary MM cells were isolated from peripheral blood from patients with plasma cell leukemia (purity >90%) and analyzed as described above. Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of at least three. The significance is related to DMSO as control. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
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Fig4: a–c Inhibition of Syk induces apoptosis in MM cell lines. a–c Viability of MM cells was defined by the amount of nuclear fragmentation and compared to the respective DMSO control. MM cell lines [AMO-1 (a), U226 (b) and primary MM cells from patients with plasma cell leukemia (purity >90%) (c)] were treated with various concentrations of Syk inhibitors (Piceatannol, R406 and Bay61-3606) as indicated. After 24 h of incubation with Syk inhibitors, propidium iodide-staining was performed. Nuclear fragmentation was determined by flow cytometry. zVAD-FMK was added as confirmation for caspase activation (white bar graphs). c Primary MM cells were isolated from peripheral blood from patients with plasma cell leukemia (purity >90%) and analyzed as described above. Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of at least three. The significance is related to DMSO as control. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001.
Mentions: In order to analyze the effect of Syk inhibitors on the viability of established MM cell lines, Piceatannol, R406 or Bay61-3606 were added to the cell culture medium and flow cytometric propidium iodide (PI) analysis of DNA fragmentation was performed as described previously [33]. Incubation of cells with the compounds resulted in a concentration dependent cell death induction that was inhibited by the addition of the pan-caspase inhibitor zVAD-FMK. This indicates that caspase activity was indispensable for the observed apoptotic effects (Fig. 4a, b).Fig. 4

Bottom Line: Incubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways.Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c.In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs.

View Article: PubMed Central - PubMed

Affiliation: Medical Clinic III, Department of Hematology and Oncology, University Hospital Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.

ABSTRACT

Background: Multiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of malignant plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of immunomodulatory drugs like bortezomib or lenalidomide, that have been associated with improved survival, MM is still incurable and new treatment options are needed. In B cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma (DLBCL), Syk (spleen tyrosine kinase) inhibitors have shown promising in vitro and first clinical results. In our study, we analyzed the potential of Syk as a target in MM.

Methods: The MM cell lines AMO-1, U266 and RPMI8226 and primary MM cells were treated with the Syk inhibitors BAY61-3606, R406 or Piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Furthermore, we analyzed synergistic and additive effects of Syk inhibitors in combination with established anti-myeloma drugs and experimental inhibitors (e.g. PI-3-Kinase inhibitor NVP-BEZ235).

Results: Incubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways. Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c. In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs.

Conclusions: Our results show that Syk inhibition might represent a promising new treatment option in MM with an increased efficacy when combined with MAP kinase inhibitors. Furthermore, our study strongly underlines the potency of Syk inhibitors as a potential therapeutic treatment option for MM patients.

No MeSH data available.


Related in: MedlinePlus