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Antitumoral effects of cyclin-dependent kinases inhibitors CR8 and MR4 on chronic myeloid leukemia cell lines.

Troadec S, Blairvacq M, Oumata N, Galons H, Meijer L, Berthou C - J. Biomed. Sci. (2015)

Bottom Line: Although Imatinib mesylate has revolutionized the treatment of chronic myeloid leukemia, some patients develop resistance with progression of leukemia.CR8 and MR4 induce dose-dependent apoptosis through mitochondrial pathway and further caspases 8/10 and 9 activation via down-regulation of short-lived survival and anti-apoptotic factors Mcl-1, XIAP and survivin which are strongly implicated in survival of Bcr-Abl transformed cells.These results suggest that CDK inhibitors may constitute a complementary approach to treat chronic myeloid leukemia.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Thérapie Cellulaire et Immunobiologie du Cancer, Université de Bretagne Occidentale, CHRU Morvan, 5 avenue Foch, 29609, Brest Cedex, France. samuel.troadec@univ-brest.fr.

ABSTRACT

Background: Although Imatinib mesylate has revolutionized the treatment of chronic myeloid leukemia, some patients develop resistance with progression of leukemia. Alternative or additional targeting of signalling pathways deregulated in Bcr-Abl-driven chronic myeloid leukemia may provide a feasible option for improving clinical response and overcoming resistance.

Results: In this study, we investigate ability of CR8 isomers (R-CR8 and S-CR8) and MR4, three derivatives of the cyclin-dependent kinases (CDKs) inhibitor Roscovitine, to exert anti-leukemic activities against chronic myeloid leukemia in vitro and then, we decipher their mechanisms of action. We show that these CDKs inhibitors are potent inducers of growth arrest and apoptosis of both Imatinib-sensitive and -resistant chronic myeloid leukemia cell lines. CR8 and MR4 induce dose-dependent apoptosis through mitochondrial pathway and further caspases 8/10 and 9 activation via down-regulation of short-lived survival and anti-apoptotic factors Mcl-1, XIAP and survivin which are strongly implicated in survival of Bcr-Abl transformed cells.

Conclusions: These results suggest that CDK inhibitors may constitute a complementary approach to treat chronic myeloid leukemia.

No MeSH data available.


Related in: MedlinePlus

Assessment of extrinsic- and mitochondrial-pathways in apoptosis induced by CDKs inhibitors in KCL22 cells. a KCL22 cells were treated with 10 μM of drugs for 24 h before western-blotting analysis of initiator and effector caspases and lamin A/C. b Kinetics of apoptotic process induced by Roscovitine-derived CDKs inhibitors. Time-dependent cleavage of caspases revealed by western-blotting analysis of KCL22 cells treated by 10 μM R-CR8 for indicated times. Actin was used as a sample loading control. c Analysis of KCL22 Annexin V-positive cells pretreated with 100 μM of caspase inhibitors then treated for 24 h with 10 μM R-CR8. Results are expressed as apoptotic cells fold increase versus negative untreated cells. Experiment was done twice
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Fig6: Assessment of extrinsic- and mitochondrial-pathways in apoptosis induced by CDKs inhibitors in KCL22 cells. a KCL22 cells were treated with 10 μM of drugs for 24 h before western-blotting analysis of initiator and effector caspases and lamin A/C. b Kinetics of apoptotic process induced by Roscovitine-derived CDKs inhibitors. Time-dependent cleavage of caspases revealed by western-blotting analysis of KCL22 cells treated by 10 μM R-CR8 for indicated times. Actin was used as a sample loading control. c Analysis of KCL22 Annexin V-positive cells pretreated with 100 μM of caspase inhibitors then treated for 24 h with 10 μM R-CR8. Results are expressed as apoptotic cells fold increase versus negative untreated cells. Experiment was done twice

Mentions: We then tried to determine whether an initiator caspase preceded caspase 3 cleavage. As demonstrated in Fig. 6a, R-CR8 or S-CR8 as well as MR4 treatment of CML cell lines induced activation of initiator procaspases 2, 8, 9 and 10, as well as other effector caspases 6 and 7. These results indicate that both extrinsic and mitochondrial pathways of apoptosis were triggered by the CDKs inhibitors. To investigate if one or some of the initiator procaspases was cleaved first and so, could be considered as the key event in drugs induced apoptosis, we analyzed the kinetics of caspases activation. As illustrated in Fig. 6b for the R-CR8 molecule on KCL22 cells, we observed by western blot experiments that all caspase events were visible from 4 h exposure of cells to the drugs. At this time, all studied caspases showed activation so we were unable to depict the scheme of apoptosis induction by these Roscovitine-derived molecules. Then, we performed Annexin V/PI staining experiments by flow cytometry, using specific caspases inhibitors. Figure 6c shows that pretreating cells with these caspases inhibitors only partially prevented R-CR8 induced apoptosis by less than 50 % for caspases 8 (z-IETD-fmk), 9 (z-LEHD-fmk), and 10 (z-AEVD-fmk) inhibitors and by approximately 70 % when these inhibitors were combined. These results seem to indicate that none of the extrinsic or mitochondrial pathways was predominant for apoptosis induction by the CDKs inhibitors. Moreover, the use of caspase 3 specific (z-DEVD-fmk), caspase 6 specific (z-VEID-fmk) as well as pan-caspase (z-VAD-fmk) inhibitors did not allow to prevent cell death, suggesting that CDKs inhibitors induced-apoptosis acts partially by a caspase-independent pathway.Fig. 6


Antitumoral effects of cyclin-dependent kinases inhibitors CR8 and MR4 on chronic myeloid leukemia cell lines.

Troadec S, Blairvacq M, Oumata N, Galons H, Meijer L, Berthou C - J. Biomed. Sci. (2015)

Assessment of extrinsic- and mitochondrial-pathways in apoptosis induced by CDKs inhibitors in KCL22 cells. a KCL22 cells were treated with 10 μM of drugs for 24 h before western-blotting analysis of initiator and effector caspases and lamin A/C. b Kinetics of apoptotic process induced by Roscovitine-derived CDKs inhibitors. Time-dependent cleavage of caspases revealed by western-blotting analysis of KCL22 cells treated by 10 μM R-CR8 for indicated times. Actin was used as a sample loading control. c Analysis of KCL22 Annexin V-positive cells pretreated with 100 μM of caspase inhibitors then treated for 24 h with 10 μM R-CR8. Results are expressed as apoptotic cells fold increase versus negative untreated cells. Experiment was done twice
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4504225&req=5

Fig6: Assessment of extrinsic- and mitochondrial-pathways in apoptosis induced by CDKs inhibitors in KCL22 cells. a KCL22 cells were treated with 10 μM of drugs for 24 h before western-blotting analysis of initiator and effector caspases and lamin A/C. b Kinetics of apoptotic process induced by Roscovitine-derived CDKs inhibitors. Time-dependent cleavage of caspases revealed by western-blotting analysis of KCL22 cells treated by 10 μM R-CR8 for indicated times. Actin was used as a sample loading control. c Analysis of KCL22 Annexin V-positive cells pretreated with 100 μM of caspase inhibitors then treated for 24 h with 10 μM R-CR8. Results are expressed as apoptotic cells fold increase versus negative untreated cells. Experiment was done twice
Mentions: We then tried to determine whether an initiator caspase preceded caspase 3 cleavage. As demonstrated in Fig. 6a, R-CR8 or S-CR8 as well as MR4 treatment of CML cell lines induced activation of initiator procaspases 2, 8, 9 and 10, as well as other effector caspases 6 and 7. These results indicate that both extrinsic and mitochondrial pathways of apoptosis were triggered by the CDKs inhibitors. To investigate if one or some of the initiator procaspases was cleaved first and so, could be considered as the key event in drugs induced apoptosis, we analyzed the kinetics of caspases activation. As illustrated in Fig. 6b for the R-CR8 molecule on KCL22 cells, we observed by western blot experiments that all caspase events were visible from 4 h exposure of cells to the drugs. At this time, all studied caspases showed activation so we were unable to depict the scheme of apoptosis induction by these Roscovitine-derived molecules. Then, we performed Annexin V/PI staining experiments by flow cytometry, using specific caspases inhibitors. Figure 6c shows that pretreating cells with these caspases inhibitors only partially prevented R-CR8 induced apoptosis by less than 50 % for caspases 8 (z-IETD-fmk), 9 (z-LEHD-fmk), and 10 (z-AEVD-fmk) inhibitors and by approximately 70 % when these inhibitors were combined. These results seem to indicate that none of the extrinsic or mitochondrial pathways was predominant for apoptosis induction by the CDKs inhibitors. Moreover, the use of caspase 3 specific (z-DEVD-fmk), caspase 6 specific (z-VEID-fmk) as well as pan-caspase (z-VAD-fmk) inhibitors did not allow to prevent cell death, suggesting that CDKs inhibitors induced-apoptosis acts partially by a caspase-independent pathway.Fig. 6

Bottom Line: Although Imatinib mesylate has revolutionized the treatment of chronic myeloid leukemia, some patients develop resistance with progression of leukemia.CR8 and MR4 induce dose-dependent apoptosis through mitochondrial pathway and further caspases 8/10 and 9 activation via down-regulation of short-lived survival and anti-apoptotic factors Mcl-1, XIAP and survivin which are strongly implicated in survival of Bcr-Abl transformed cells.These results suggest that CDK inhibitors may constitute a complementary approach to treat chronic myeloid leukemia.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Thérapie Cellulaire et Immunobiologie du Cancer, Université de Bretagne Occidentale, CHRU Morvan, 5 avenue Foch, 29609, Brest Cedex, France. samuel.troadec@univ-brest.fr.

ABSTRACT

Background: Although Imatinib mesylate has revolutionized the treatment of chronic myeloid leukemia, some patients develop resistance with progression of leukemia. Alternative or additional targeting of signalling pathways deregulated in Bcr-Abl-driven chronic myeloid leukemia may provide a feasible option for improving clinical response and overcoming resistance.

Results: In this study, we investigate ability of CR8 isomers (R-CR8 and S-CR8) and MR4, three derivatives of the cyclin-dependent kinases (CDKs) inhibitor Roscovitine, to exert anti-leukemic activities against chronic myeloid leukemia in vitro and then, we decipher their mechanisms of action. We show that these CDKs inhibitors are potent inducers of growth arrest and apoptosis of both Imatinib-sensitive and -resistant chronic myeloid leukemia cell lines. CR8 and MR4 induce dose-dependent apoptosis through mitochondrial pathway and further caspases 8/10 and 9 activation via down-regulation of short-lived survival and anti-apoptotic factors Mcl-1, XIAP and survivin which are strongly implicated in survival of Bcr-Abl transformed cells.

Conclusions: These results suggest that CDK inhibitors may constitute a complementary approach to treat chronic myeloid leukemia.

No MeSH data available.


Related in: MedlinePlus