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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, b Expression of Syk and phosphorylated Syk in different cell lines and patient samples. a Syk was immunoprecipitated from MM cell lines and primary MM cells. Immunoprecipitates were subjected to Western blotting using specific antibodies. Primary myeloma cells were obtained from peripheral blood of myeloma patients with plasma cell leukemia (PCL, purity >90%). Displayed is a representative immunoblot of Syk and pSyk in the absence of Syk inhibitors which shows Syk expression in the utilized MM cell lines and PCL-cells. b and c Addition of Syk inhibitors (Piceatannol, R406 and Bay61-3606) reduce the expression of pSyk in MM cell lines and PCL cells. Ponceau S staining was performed to confirm equal amounts of protein.
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Fig1: a, b Expression of Syk and phosphorylated Syk in different cell lines and patient samples. a Syk was immunoprecipitated from MM cell lines and primary MM cells. Immunoprecipitates were subjected to Western blotting using specific antibodies. Primary myeloma cells were obtained from peripheral blood of myeloma patients with plasma cell leukemia (PCL, purity >90%). Displayed is a representative immunoblot of Syk and pSyk in the absence of Syk inhibitors which shows Syk expression in the utilized MM cell lines and PCL-cells. b and c Addition of Syk inhibitors (Piceatannol, R406 and Bay61-3606) reduce the expression of pSyk in MM cell lines and PCL cells. Ponceau S staining was performed to confirm equal amounts of protein.

Mentions: Syk was shown to play an important role in the signal transduction via the BCR, cytokine receptors, integrins and Fc receptors on normal and malignant B cells. These receptors promote survival and are associated with the induction of proliferation, differentiation and migration. Using Immunoprecipitation, we found that phosphorylated Syk is expressed in the utilized multiple myeloma cell lines AMO-1, U266 and RPMI8226. Furthermore, Syk expression was also detected in primary MM cells of patients with plasma cell leukemia (PCL). In addition, we found that Syk can be downregulated when these myeloma cells were incubated with the inhibitors of Syk phosphorylation, i.e. Bay 61-3606 or Piceatannol and R406 (Fig. 1a, b).Fig. 1


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, b Expression of Syk and phosphorylated Syk in different cell lines and patient samples. a Syk was immunoprecipitated from MM cell lines and primary MM cells. Immunoprecipitates were subjected to Western blotting using specific antibodies. Primary myeloma cells were obtained from peripheral blood of myeloma patients with plasma cell leukemia (PCL, purity >90%). Displayed is a representative immunoblot of Syk and pSyk in the absence of Syk inhibitors which shows Syk expression in the utilized MM cell lines and PCL-cells. b and c Addition of Syk inhibitors (Piceatannol, R406 and Bay61-3606) reduce the expression of pSyk in MM cell lines and PCL cells. Ponceau S staining was performed to confirm equal amounts of protein.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4526421&req=5

Fig1: a, b Expression of Syk and phosphorylated Syk in different cell lines and patient samples. a Syk was immunoprecipitated from MM cell lines and primary MM cells. Immunoprecipitates were subjected to Western blotting using specific antibodies. Primary myeloma cells were obtained from peripheral blood of myeloma patients with plasma cell leukemia (PCL, purity >90%). Displayed is a representative immunoblot of Syk and pSyk in the absence of Syk inhibitors which shows Syk expression in the utilized MM cell lines and PCL-cells. b and c Addition of Syk inhibitors (Piceatannol, R406 and Bay61-3606) reduce the expression of pSyk in MM cell lines and PCL cells. Ponceau S staining was performed to confirm equal amounts of protein.
Mentions: Syk was shown to play an important role in the signal transduction via the BCR, cytokine receptors, integrins and Fc receptors on normal and malignant B cells. These receptors promote survival and are associated with the induction of proliferation, differentiation and migration. Using Immunoprecipitation, we found that phosphorylated Syk is expressed in the utilized multiple myeloma cell lines AMO-1, U266 and RPMI8226. Furthermore, Syk expression was also detected in primary MM cells of patients with plasma cell leukemia (PCL). In addition, we found that Syk can be downregulated when these myeloma cells were incubated with the inhibitors of Syk phosphorylation, i.e. Bay 61-3606 or Piceatannol and R406 (Fig. 1a, b).Fig. 1

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