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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 Caspase-3 activity is increased significantly upon treatment with Syk inhibitors. a Caspase-3 activity was determined from whole cell lysates by the cleavage of the fluorogenic caspase substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC). The release of aminomethylcoumarin was determined by fluorometry using an excitation length of 360 nm and an emission wave-length of 460 nm. Syk-Inhibitors induce a significant concentration-dependent increase of caspase-3 activity. 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 Western Blot analyses of AMO-1 whole cell lysates treated with Piceatannol was utilized to analyze the expression of pro-caspase 3 and PARP-1 cleavage. After 24 h of incubation with Syk inhibitors, whole cell lysates were prepared and Western Blot analysis was performed. DMSO was used as a control. GAPDH was used to confirm equal amounts of protein. Results are representative of at least three independent experiments. Proteins detected are indicated by an arrow on the right.c Expression of xIAP, Mcl-1, cytochrome c and Survivin upon treatment with Syk inhibitors. Protein extracts were analyzed for the expression of proteins participating in apoptosis such as xIAP, Mcl-1, survivin and cytochrome c by immunoblotting. An increase of cytochrome c was observed, implicating that Piceatannol induces apoptosis via the internal, mitochondrial pathway. There was no effect of Piceatannol on the anti-apoptotic proteins survivin, Mcl-1 and xIAP. Results are representative of various independent experiments. Proteins detected are indicated by an arrow on the right.
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Fig5: a, b Caspase-3 activity is increased significantly upon treatment with Syk inhibitors. a Caspase-3 activity was determined from whole cell lysates by the cleavage of the fluorogenic caspase substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC). The release of aminomethylcoumarin was determined by fluorometry using an excitation length of 360 nm and an emission wave-length of 460 nm. Syk-Inhibitors induce a significant concentration-dependent increase of caspase-3 activity. 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 Western Blot analyses of AMO-1 whole cell lysates treated with Piceatannol was utilized to analyze the expression of pro-caspase 3 and PARP-1 cleavage. After 24 h of incubation with Syk inhibitors, whole cell lysates were prepared and Western Blot analysis was performed. DMSO was used as a control. GAPDH was used to confirm equal amounts of protein. Results are representative of at least three independent experiments. Proteins detected are indicated by an arrow on the right.c Expression of xIAP, Mcl-1, cytochrome c and Survivin upon treatment with Syk inhibitors. Protein extracts were analyzed for the expression of proteins participating in apoptosis such as xIAP, Mcl-1, survivin and cytochrome c by immunoblotting. An increase of cytochrome c was observed, implicating that Piceatannol induces apoptosis via the internal, mitochondrial pathway. There was no effect of Piceatannol on the anti-apoptotic proteins survivin, Mcl-1 and xIAP. Results are representative of various independent experiments. Proteins detected are indicated by an arrow on the right.

Mentions: Caspase-3 assays and Western Blot analysis revealed that the induction of apoptotic cell death was accompanied by caspase-3 activation and poly[ADP ribose]polymerase 1 (PARP-1) cleavage (Fig. 5a, b). In addition, an increased release of cytochrome c upon incubation with the compounds indicated that the apoptosis induction in tested MM cells was mediated via the mitochondrial signalling pathway (Fig. 5c). In contrast to previous findings in CLL cells, we could not detect any regulation of the expression of myeloid leukaemia cell differentiation protein MCL-1, the x-linked inhibitor of apoptosis protein xIAP or survivin (also known as BIRC5 or API4) by the used compounds (Fig. 5c).Fig. 5


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 Caspase-3 activity is increased significantly upon treatment with Syk inhibitors. a Caspase-3 activity was determined from whole cell lysates by the cleavage of the fluorogenic caspase substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC). The release of aminomethylcoumarin was determined by fluorometry using an excitation length of 360 nm and an emission wave-length of 460 nm. Syk-Inhibitors induce a significant concentration-dependent increase of caspase-3 activity. 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 Western Blot analyses of AMO-1 whole cell lysates treated with Piceatannol was utilized to analyze the expression of pro-caspase 3 and PARP-1 cleavage. After 24 h of incubation with Syk inhibitors, whole cell lysates were prepared and Western Blot analysis was performed. DMSO was used as a control. GAPDH was used to confirm equal amounts of protein. Results are representative of at least three independent experiments. Proteins detected are indicated by an arrow on the right.c Expression of xIAP, Mcl-1, cytochrome c and Survivin upon treatment with Syk inhibitors. Protein extracts were analyzed for the expression of proteins participating in apoptosis such as xIAP, Mcl-1, survivin and cytochrome c by immunoblotting. An increase of cytochrome c was observed, implicating that Piceatannol induces apoptosis via the internal, mitochondrial pathway. There was no effect of Piceatannol on the anti-apoptotic proteins survivin, Mcl-1 and xIAP. Results are representative of various independent experiments. Proteins detected are indicated by an arrow on the right.
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Related In: Results  -  Collection

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Fig5: a, b Caspase-3 activity is increased significantly upon treatment with Syk inhibitors. a Caspase-3 activity was determined from whole cell lysates by the cleavage of the fluorogenic caspase substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC). The release of aminomethylcoumarin was determined by fluorometry using an excitation length of 360 nm and an emission wave-length of 460 nm. Syk-Inhibitors induce a significant concentration-dependent increase of caspase-3 activity. 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 Western Blot analyses of AMO-1 whole cell lysates treated with Piceatannol was utilized to analyze the expression of pro-caspase 3 and PARP-1 cleavage. After 24 h of incubation with Syk inhibitors, whole cell lysates were prepared and Western Blot analysis was performed. DMSO was used as a control. GAPDH was used to confirm equal amounts of protein. Results are representative of at least three independent experiments. Proteins detected are indicated by an arrow on the right.c Expression of xIAP, Mcl-1, cytochrome c and Survivin upon treatment with Syk inhibitors. Protein extracts were analyzed for the expression of proteins participating in apoptosis such as xIAP, Mcl-1, survivin and cytochrome c by immunoblotting. An increase of cytochrome c was observed, implicating that Piceatannol induces apoptosis via the internal, mitochondrial pathway. There was no effect of Piceatannol on the anti-apoptotic proteins survivin, Mcl-1 and xIAP. Results are representative of various independent experiments. Proteins detected are indicated by an arrow on the right.
Mentions: Caspase-3 assays and Western Blot analysis revealed that the induction of apoptotic cell death was accompanied by caspase-3 activation and poly[ADP ribose]polymerase 1 (PARP-1) cleavage (Fig. 5a, b). In addition, an increased release of cytochrome c upon incubation with the compounds indicated that the apoptosis induction in tested MM cells was mediated via the mitochondrial signalling pathway (Fig. 5c). In contrast to previous findings in CLL cells, we could not detect any regulation of the expression of myeloid leukaemia cell differentiation protein MCL-1, the x-linked inhibitor of apoptosis protein xIAP or survivin (also known as BIRC5 or API4) by the used compounds (Fig. 5c).Fig. 5

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