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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 Syk inhibition reduces phosphorylation of p38 and ERK1/2 in MM cell lines. a, b MM cell lines were treated with Piceatannol at concentrations of 10 and 25 µM. Phosphorylation status of down stream signaling mediators p38-Kinase and ERK1/2-Kinase (MAPK p42 and p44) was evaluated by Western blotting using antiphospho-p38 and antiphospho-ERK 1/2 antibodies. GAPDH was used to confirm equal amounts of protein. Results are representative of at least three independent experiments. c Nuclear translocation of NF-κB family members was determined by Western Blot analysis of nuclear extracts upon incubation with Syk inhibitors (c). Piceatannol was applied at 10 and 25 µM. DMSO was used as control. Proteins detected are indicated by an arrow on the right. Ponceau S staining was performed to confirm equal amounts of protein. Results are representative of at least three independent experiments.
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Fig3: a–c Syk inhibition reduces phosphorylation of p38 and ERK1/2 in MM cell lines. a, b MM cell lines were treated with Piceatannol at concentrations of 10 and 25 µM. Phosphorylation status of down stream signaling mediators p38-Kinase and ERK1/2-Kinase (MAPK p42 and p44) was evaluated by Western blotting using antiphospho-p38 and antiphospho-ERK 1/2 antibodies. GAPDH was used to confirm equal amounts of protein. Results are representative of at least three independent experiments. c Nuclear translocation of NF-κB family members was determined by Western Blot analysis of nuclear extracts upon incubation with Syk inhibitors (c). Piceatannol was applied at 10 and 25 µM. DMSO was used as control. Proteins detected are indicated by an arrow on the right. Ponceau S staining was performed to confirm equal amounts of protein. Results are representative of at least three independent experiments.

Mentions: In line with these results, we found that treatment of cells with the compounds resulted in a reduced activation of the downstream signalling events characterized by reduced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (Fig. 3a, b) and nuclear localized expression of transcription factors of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) family RelB and RelA (Fig. 3c). Representatively, the results are shown for AMO-1 cell line. However, all used MM cell lines (U266 and RPMI-8226) showed similar results in our experiments. The utilized concentrations of Syk inhibitors were adjusted to reported working concentrations in the literature.Fig. 3


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 Syk inhibition reduces phosphorylation of p38 and ERK1/2 in MM cell lines. a, b MM cell lines were treated with Piceatannol at concentrations of 10 and 25 µM. Phosphorylation status of down stream signaling mediators p38-Kinase and ERK1/2-Kinase (MAPK p42 and p44) was evaluated by Western blotting using antiphospho-p38 and antiphospho-ERK 1/2 antibodies. GAPDH was used to confirm equal amounts of protein. Results are representative of at least three independent experiments. c Nuclear translocation of NF-κB family members was determined by Western Blot analysis of nuclear extracts upon incubation with Syk inhibitors (c). Piceatannol was applied at 10 and 25 µM. DMSO was used as control. Proteins detected are indicated by an arrow on the right. Ponceau S staining was performed to confirm equal amounts of protein. Results are representative of at least three independent experiments.
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Related In: Results  -  Collection

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Fig3: a–c Syk inhibition reduces phosphorylation of p38 and ERK1/2 in MM cell lines. a, b MM cell lines were treated with Piceatannol at concentrations of 10 and 25 µM. Phosphorylation status of down stream signaling mediators p38-Kinase and ERK1/2-Kinase (MAPK p42 and p44) was evaluated by Western blotting using antiphospho-p38 and antiphospho-ERK 1/2 antibodies. GAPDH was used to confirm equal amounts of protein. Results are representative of at least three independent experiments. c Nuclear translocation of NF-κB family members was determined by Western Blot analysis of nuclear extracts upon incubation with Syk inhibitors (c). Piceatannol was applied at 10 and 25 µM. DMSO was used as control. Proteins detected are indicated by an arrow on the right. Ponceau S staining was performed to confirm equal amounts of protein. Results are representative of at least three independent experiments.
Mentions: In line with these results, we found that treatment of cells with the compounds resulted in a reduced activation of the downstream signalling events characterized by reduced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (Fig. 3a, b) and nuclear localized expression of transcription factors of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) family RelB and RelA (Fig. 3c). Representatively, the results are shown for AMO-1 cell line. However, all used MM cell lines (U266 and RPMI-8226) showed similar results in our experiments. The utilized concentrations of Syk inhibitors were adjusted to reported working concentrations in the literature.Fig. 3

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