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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

Combined treatment with NVP-BEZ235 or MAPK inhibitors increases induction of apoptosis. a Nuclear fragmentation of MM cells treated with either/and 500 nM NVP-BEZ235, 25 µM Piceatannol and 0.5 ng bortezomib. Nuclear fragmentation was determined by the method of Nicoletti and analysed by flow cytometry. In comparison to Piceatannol alone, a combination with the common anti-myeloma agent bortezomib exerts no further increase of nuclear fragmentation. The addition of NVP-BEZ235 (an orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) to Syk inhibitors increased nuclear fragmentation. Statistical analysis showed, that the combination of NVP-BEZ235 and Piceatannol is significantly higher than each drug alone. This works predominantly for AMO-1 cells. Calculational significant results in U266 cells can only be observed when correlated to Piceatannol, but not to NVP-BEZ235 (see Additional file 1). Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of three. DMSO was used as control. The significance is related to DMSO. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001. b–e Effects of Syk inhibition in combination with MAP-Kinase inhibitors are demonstrated. MM cell lines were incubated with 5 µM Bay61-3606 in addition to varying concentrations of U0126 (b), SP600125 (c), PD98059 (d) or SB202190 (e) alone as well as in combination. Combined treatment of Bay61-3606 with U0126 (a potent, selective inhibitor of MAP2K), SP600125 (a selective inhibitor of JNK), or PD98059 (an inhibitor of MAP2K/MEK) and SB203580 (a selective inhibitor of p38 MAPK) results in significant synergistic or additive effects. Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of three. DMSO was used for negative control. The significance is related to DMSO. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001.
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Fig7: Combined treatment with NVP-BEZ235 or MAPK inhibitors increases induction of apoptosis. a Nuclear fragmentation of MM cells treated with either/and 500 nM NVP-BEZ235, 25 µM Piceatannol and 0.5 ng bortezomib. Nuclear fragmentation was determined by the method of Nicoletti and analysed by flow cytometry. In comparison to Piceatannol alone, a combination with the common anti-myeloma agent bortezomib exerts no further increase of nuclear fragmentation. The addition of NVP-BEZ235 (an orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) to Syk inhibitors increased nuclear fragmentation. Statistical analysis showed, that the combination of NVP-BEZ235 and Piceatannol is significantly higher than each drug alone. This works predominantly for AMO-1 cells. Calculational significant results in U266 cells can only be observed when correlated to Piceatannol, but not to NVP-BEZ235 (see Additional file 1). Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of three. DMSO was used as control. The significance is related to DMSO. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001. b–e Effects of Syk inhibition in combination with MAP-Kinase inhibitors are demonstrated. MM cell lines were incubated with 5 µM Bay61-3606 in addition to varying concentrations of U0126 (b), SP600125 (c), PD98059 (d) or SB202190 (e) alone as well as in combination. Combined treatment of Bay61-3606 with U0126 (a potent, selective inhibitor of MAP2K), SP600125 (a selective inhibitor of JNK), or PD98059 (an inhibitor of MAP2K/MEK) and SB203580 (a selective inhibitor of p38 MAPK) results in significant synergistic or additive effects. Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of three. DMSO was used for negative control. The significance is related to DMSO. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001.

Mentions: In the next set of experiments we analyzed possible additive effects of drugs currently used in the treatment of MM with Syk inhibitors. We found no additive effects when Syk inhibitors were used with bortezomib (Fig. 7a). In addition, no additive effects were detected when Syk inhibitors were combined with thalidomide, lenalidomide, TRAIL (tumor necrosis factor related apoptosis inducing ligand) or dexamethasone (data not shown).Fig. 7


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)

Combined treatment with NVP-BEZ235 or MAPK inhibitors increases induction of apoptosis. a Nuclear fragmentation of MM cells treated with either/and 500 nM NVP-BEZ235, 25 µM Piceatannol and 0.5 ng bortezomib. Nuclear fragmentation was determined by the method of Nicoletti and analysed by flow cytometry. In comparison to Piceatannol alone, a combination with the common anti-myeloma agent bortezomib exerts no further increase of nuclear fragmentation. The addition of NVP-BEZ235 (an orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) to Syk inhibitors increased nuclear fragmentation. Statistical analysis showed, that the combination of NVP-BEZ235 and Piceatannol is significantly higher than each drug alone. This works predominantly for AMO-1 cells. Calculational significant results in U266 cells can only be observed when correlated to Piceatannol, but not to NVP-BEZ235 (see Additional file 1). Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of three. DMSO was used as control. The significance is related to DMSO. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001. b–e Effects of Syk inhibition in combination with MAP-Kinase inhibitors are demonstrated. MM cell lines were incubated with 5 µM Bay61-3606 in addition to varying concentrations of U0126 (b), SP600125 (c), PD98059 (d) or SB202190 (e) alone as well as in combination. Combined treatment of Bay61-3606 with U0126 (a potent, selective inhibitor of MAP2K), SP600125 (a selective inhibitor of JNK), or PD98059 (an inhibitor of MAP2K/MEK) and SB203580 (a selective inhibitor of p38 MAPK) results in significant synergistic or additive effects. Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of three. DMSO was used for negative control. The significance is related to DMSO. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001.
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Related In: Results  -  Collection

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Fig7: Combined treatment with NVP-BEZ235 or MAPK inhibitors increases induction of apoptosis. a Nuclear fragmentation of MM cells treated with either/and 500 nM NVP-BEZ235, 25 µM Piceatannol and 0.5 ng bortezomib. Nuclear fragmentation was determined by the method of Nicoletti and analysed by flow cytometry. In comparison to Piceatannol alone, a combination with the common anti-myeloma agent bortezomib exerts no further increase of nuclear fragmentation. The addition of NVP-BEZ235 (an orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) to Syk inhibitors increased nuclear fragmentation. Statistical analysis showed, that the combination of NVP-BEZ235 and Piceatannol is significantly higher than each drug alone. This works predominantly for AMO-1 cells. Calculational significant results in U266 cells can only be observed when correlated to Piceatannol, but not to NVP-BEZ235 (see Additional file 1). Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of three. DMSO was used as control. The significance is related to DMSO. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001. b–e Effects of Syk inhibition in combination with MAP-Kinase inhibitors are demonstrated. MM cell lines were incubated with 5 µM Bay61-3606 in addition to varying concentrations of U0126 (b), SP600125 (c), PD98059 (d) or SB202190 (e) alone as well as in combination. Combined treatment of Bay61-3606 with U0126 (a potent, selective inhibitor of MAP2K), SP600125 (a selective inhibitor of JNK), or PD98059 (an inhibitor of MAP2K/MEK) and SB203580 (a selective inhibitor of p38 MAPK) results in significant synergistic or additive effects. Results are expressed as the mean of three panels per pattern. Results are from one experiment representative of three. DMSO was used for negative control. The significance is related to DMSO. +P<1.000, *P < 0.1, **P < 0.01, ***P < 0.001.
Mentions: In the next set of experiments we analyzed possible additive effects of drugs currently used in the treatment of MM with Syk inhibitors. We found no additive effects when Syk inhibitors were used with bortezomib (Fig. 7a). In addition, no additive effects were detected when Syk inhibitors were combined with thalidomide, lenalidomide, TRAIL (tumor necrosis factor related apoptosis inducing ligand) or dexamethasone (data not shown).Fig. 7

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