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The effect of the dual Src/Abl kinase inhibitor AZD0530 on Philadelphia positive leukaemia cell lines.

Gwanmesia PM, Romanski A, Schwarz K, Bacic B, Ruthardt M, Ottmann OG - BMC Cancer (2009)

Bottom Line: An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl.AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15.Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl.

View Article: PubMed Central - HTML - PubMed

Affiliation: Med, Klinik II/Abt, Hämatologie, Johann Wolfgang Goethe-Universität, 60590 Frankfurt, Germany. Mambou@uni-heidelberg.de

ABSTRACT

Background: Imatinib mesylate, a selective inhibitor of Abl tyrosine kinase, is efficacious in treating chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL). However, most advanced-phase CML and Ph+ ALL patients relapse on Imatinib therapy. Several mechanisms of refractoriness have been reported, including the activation of the Src-family kinases (SFK). Here, we investigated the biological effect of the new specific dual Src/Abl kinase inhibitor AZD0530 on Ph+ leukaemic cells.

Methods: Cell lines used included BV173 (CML in myeloid blast crisis), SEM t(4;11), Ba/F3 (IL-3 dependent murine pro B), p185Bcr-Abl infected Ba/F3 cells, p185Bcr-Abl mutant infected Ba/F3 cells, SupB15 (Ph+ ALL) and Imatinib resistant SupB15 (RTSupB15) (Ph+ ALL) cells. Cells were exposed to AZD0530 and Imatinib. Cell proliferation, apoptosis, survival and signalling pathways were assessed by dye exclusion, flow cytometry and Western blotting respectively.

Results: AZD0530 specifically inhibited the growth of, and induced apoptosis in CML and Ph+ ALL cells in a dose dependent manner, but showed only marginal effects on Ph- ALL cells. Resistance to Imatinib due to the mutation Y253F in p185Bcr-Abl was overcome by AZD0530. Combination of AZD0530 and Imatinib showed an additive inhibitory effect on the proliferation of CML BV173 cells but not on Ph+ ALL SupB15 cells. An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl. AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15.

Conclusion: Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl.

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AZD0530 inhibits survival signalling pathways involved in Bcr-Abl leukaemogenesis. Whole cell lysates from AZD0530 and Imatinib treated BV173 cells (left) and SEM cells (right) were probed with antibodies (phosphorylated and non-phosphorylated forms) against Akt, Stat5 and Erk. Specificity of both inhibitors to Bcr-Abl was tested by comparing results of BV173 and SEM cells treated under similar conditions. In BV173 cells the activation of Akt, Stat5 and Erk was inhibited by AZD0530 and Imatinib, in contrast to SEM cells which showed no regulation of all three proteins. One representative experiment out of 3 is given.
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Figure 4: AZD0530 inhibits survival signalling pathways involved in Bcr-Abl leukaemogenesis. Whole cell lysates from AZD0530 and Imatinib treated BV173 cells (left) and SEM cells (right) were probed with antibodies (phosphorylated and non-phosphorylated forms) against Akt, Stat5 and Erk. Specificity of both inhibitors to Bcr-Abl was tested by comparing results of BV173 and SEM cells treated under similar conditions. In BV173 cells the activation of Akt, Stat5 and Erk was inhibited by AZD0530 and Imatinib, in contrast to SEM cells which showed no regulation of all three proteins. One representative experiment out of 3 is given.

Mentions: Activation of the Raf/Mek/Erk, PI3K/Akt and Stat pathways synergize to promote Ph+ leukaemic cell growth. To investigate if inactivation of Src kinases and Bcr-Abl by AZD0530 could interfere with the leukaemic survival signalling pathways, lysates from BV173 cells treated with AZD0530 or Imatinib were probed with antibodies (activated and whole protein) against Stat5, Erk, and Akt kinases. Results were compared to Ph- SEM cells (Figure 4). In the BV173 cells, both compounds inhibited phosphorylation of Stat5, Erk and Akt kinases. This correlated well with the stronger effect on the inhibition of SFKs. These results support the idea that the Src kinases couple Bcr-Abl to its substrate proteins, and AZD0530 may target more specifically the Src kinases. Neither AZD0530 nor Imatinib affected the activation of all three proteins in the SEM cells.


The effect of the dual Src/Abl kinase inhibitor AZD0530 on Philadelphia positive leukaemia cell lines.

Gwanmesia PM, Romanski A, Schwarz K, Bacic B, Ruthardt M, Ottmann OG - BMC Cancer (2009)

AZD0530 inhibits survival signalling pathways involved in Bcr-Abl leukaemogenesis. Whole cell lysates from AZD0530 and Imatinib treated BV173 cells (left) and SEM cells (right) were probed with antibodies (phosphorylated and non-phosphorylated forms) against Akt, Stat5 and Erk. Specificity of both inhibitors to Bcr-Abl was tested by comparing results of BV173 and SEM cells treated under similar conditions. In BV173 cells the activation of Akt, Stat5 and Erk was inhibited by AZD0530 and Imatinib, in contrast to SEM cells which showed no regulation of all three proteins. One representative experiment out of 3 is given.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654659&req=5

Figure 4: AZD0530 inhibits survival signalling pathways involved in Bcr-Abl leukaemogenesis. Whole cell lysates from AZD0530 and Imatinib treated BV173 cells (left) and SEM cells (right) were probed with antibodies (phosphorylated and non-phosphorylated forms) against Akt, Stat5 and Erk. Specificity of both inhibitors to Bcr-Abl was tested by comparing results of BV173 and SEM cells treated under similar conditions. In BV173 cells the activation of Akt, Stat5 and Erk was inhibited by AZD0530 and Imatinib, in contrast to SEM cells which showed no regulation of all three proteins. One representative experiment out of 3 is given.
Mentions: Activation of the Raf/Mek/Erk, PI3K/Akt and Stat pathways synergize to promote Ph+ leukaemic cell growth. To investigate if inactivation of Src kinases and Bcr-Abl by AZD0530 could interfere with the leukaemic survival signalling pathways, lysates from BV173 cells treated with AZD0530 or Imatinib were probed with antibodies (activated and whole protein) against Stat5, Erk, and Akt kinases. Results were compared to Ph- SEM cells (Figure 4). In the BV173 cells, both compounds inhibited phosphorylation of Stat5, Erk and Akt kinases. This correlated well with the stronger effect on the inhibition of SFKs. These results support the idea that the Src kinases couple Bcr-Abl to its substrate proteins, and AZD0530 may target more specifically the Src kinases. Neither AZD0530 nor Imatinib affected the activation of all three proteins in the SEM cells.

Bottom Line: An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl.AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15.Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl.

View Article: PubMed Central - HTML - PubMed

Affiliation: Med, Klinik II/Abt, Hämatologie, Johann Wolfgang Goethe-Universität, 60590 Frankfurt, Germany. Mambou@uni-heidelberg.de

ABSTRACT

Background: Imatinib mesylate, a selective inhibitor of Abl tyrosine kinase, is efficacious in treating chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL). However, most advanced-phase CML and Ph+ ALL patients relapse on Imatinib therapy. Several mechanisms of refractoriness have been reported, including the activation of the Src-family kinases (SFK). Here, we investigated the biological effect of the new specific dual Src/Abl kinase inhibitor AZD0530 on Ph+ leukaemic cells.

Methods: Cell lines used included BV173 (CML in myeloid blast crisis), SEM t(4;11), Ba/F3 (IL-3 dependent murine pro B), p185Bcr-Abl infected Ba/F3 cells, p185Bcr-Abl mutant infected Ba/F3 cells, SupB15 (Ph+ ALL) and Imatinib resistant SupB15 (RTSupB15) (Ph+ ALL) cells. Cells were exposed to AZD0530 and Imatinib. Cell proliferation, apoptosis, survival and signalling pathways were assessed by dye exclusion, flow cytometry and Western blotting respectively.

Results: AZD0530 specifically inhibited the growth of, and induced apoptosis in CML and Ph+ ALL cells in a dose dependent manner, but showed only marginal effects on Ph- ALL cells. Resistance to Imatinib due to the mutation Y253F in p185Bcr-Abl was overcome by AZD0530. Combination of AZD0530 and Imatinib showed an additive inhibitory effect on the proliferation of CML BV173 cells but not on Ph+ ALL SupB15 cells. An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl. AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15.

Conclusion: Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl.

Show MeSH
Related in: MedlinePlus