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Cytotoxic activity of the MK2 inhibitor CMPD1 in glioblastoma cells is independent of MK2

View Article: PubMed Central - PubMed

ABSTRACT

MAPK-activated protein kinase 2 (MK2) is a checkpoint kinase involved in the DNA damage response. MK2 inhibition enhances the efficacy of chemotherapeutic agents; however, whether MK2 inhibition alone, without concurrent chemotherapy, would attenuate survival of cancer cells has not been investigated. CMPD1 is a widely used non-ATP competitive inhibitor that prevents MK2 phosphorylation. We employed CMPD1 together with MK2 knock-down and ATP-competitive MK2 inhibitor III (MK2i) in a panel of glioblastoma cells to assess whether MK2 inhibition could induce cancer cell death. While CMPD1 was effective at selective killing of cancer cells, MK2i and MK2 knock-down had no effect on viability of glioblastoma cells. CMPD1 treatment induced a significant G2/M arrest but MK2i-treated cells were only minimally arrested at G1 phase. Intriguingly, at doses that were cytotoxic to glioblastoma cells, CMPD1 did not inhibit phosphorylation of MK2 and of its downstream substrate Hsp27. These results suggest that CMPD1 exhibits cytotoxic activity independently of MK2 inhibition. Indeed, we identified tubulin as a primary target of the CMPD1 cytotoxic activity. This study demonstrates how functional and mechanistic studies with appropriate selection of test compounds, combining genetic knock-down and pharmacological inhibition, coordinating timing and dose levels enabled us to uncover the primary target of an MK2 inhibitor commonly used in the research community. Tubulin is emerging as one of the most common non-kinase targets for kinase inhibitors and we propose that potential tubulin-targeting activity should be assessed in preclinical pharmacology studies of all novel kinase inhibitors.

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CMPD1 induces apoptosis and changes expression of Bcl-2 family proteins. (a) U87 cells were treated with CMPD1 for indicated time and the percentage of Annexin-V-positive cells was determined by flow cytometry. Data are expressed as mean±S.E.M. (n=3; *P<0.05, ***P<0.001, two-way ANOVA followed by Bonferroni post-test). (b) U87 and primary human glioblastoma cells were treated with CMPD1 (48 h) and analysed for expression of PARP (116 kDa) and cleaved PARP (85 kDa) using western blotting. Representative images of three independent experiments are shown. (c) U87 cells were treated with CMPD1 for indicated time and cell lysates analysed with western blotting using indicated antibodies. Representative images of three independent experiments are shown.
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fig3: CMPD1 induces apoptosis and changes expression of Bcl-2 family proteins. (a) U87 cells were treated with CMPD1 for indicated time and the percentage of Annexin-V-positive cells was determined by flow cytometry. Data are expressed as mean±S.E.M. (n=3; *P<0.05, ***P<0.001, two-way ANOVA followed by Bonferroni post-test). (b) U87 and primary human glioblastoma cells were treated with CMPD1 (48 h) and analysed for expression of PARP (116 kDa) and cleaved PARP (85 kDa) using western blotting. Representative images of three independent experiments are shown. (c) U87 cells were treated with CMPD1 for indicated time and cell lysates analysed with western blotting using indicated antibodies. Representative images of three independent experiments are shown.

Mentions: To investigate whether apoptosis underlies the decrease in viability of cells treated with CMPD1 (Figure 1a), we analysed apoptosis in U87 cells by Annexin-V staining and immunoblotting of the cleaved poly-(ADP-ribose)-polymerase (cPARP). CMPD1 (1 and 5 μM, 72 h) increased the amount of apoptotic cells to 38 and 56%, respectively (Figure 3a). In contrast, MK2i or SB203580 did not induce apoptosis in U87 cells (Supplementary Figures S3A and B). Consistent with an apoptotic mechanism for cell death, we observed a significant increase in the appearance of cleaved PARP after 48 h of cell treatment with CMPD1 (1 and 5 μM, Figure 3b).


Cytotoxic activity of the MK2 inhibitor CMPD1 in glioblastoma cells is independent of MK2
CMPD1 induces apoptosis and changes expression of Bcl-2 family proteins. (a) U87 cells were treated with CMPD1 for indicated time and the percentage of Annexin-V-positive cells was determined by flow cytometry. Data are expressed as mean±S.E.M. (n=3; *P<0.05, ***P<0.001, two-way ANOVA followed by Bonferroni post-test). (b) U87 and primary human glioblastoma cells were treated with CMPD1 (48 h) and analysed for expression of PARP (116 kDa) and cleaved PARP (85 kDa) using western blotting. Representative images of three independent experiments are shown. (c) U87 cells were treated with CMPD1 for indicated time and cell lysates analysed with western blotting using indicated antibodies. Representative images of three independent experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4979411&req=5

fig3: CMPD1 induces apoptosis and changes expression of Bcl-2 family proteins. (a) U87 cells were treated with CMPD1 for indicated time and the percentage of Annexin-V-positive cells was determined by flow cytometry. Data are expressed as mean±S.E.M. (n=3; *P<0.05, ***P<0.001, two-way ANOVA followed by Bonferroni post-test). (b) U87 and primary human glioblastoma cells were treated with CMPD1 (48 h) and analysed for expression of PARP (116 kDa) and cleaved PARP (85 kDa) using western blotting. Representative images of three independent experiments are shown. (c) U87 cells were treated with CMPD1 for indicated time and cell lysates analysed with western blotting using indicated antibodies. Representative images of three independent experiments are shown.
Mentions: To investigate whether apoptosis underlies the decrease in viability of cells treated with CMPD1 (Figure 1a), we analysed apoptosis in U87 cells by Annexin-V staining and immunoblotting of the cleaved poly-(ADP-ribose)-polymerase (cPARP). CMPD1 (1 and 5 μM, 72 h) increased the amount of apoptotic cells to 38 and 56%, respectively (Figure 3a). In contrast, MK2i or SB203580 did not induce apoptosis in U87 cells (Supplementary Figures S3A and B). Consistent with an apoptotic mechanism for cell death, we observed a significant increase in the appearance of cleaved PARP after 48 h of cell treatment with CMPD1 (1 and 5 μM, Figure 3b).

View Article: PubMed Central - PubMed

ABSTRACT

MAPK-activated protein kinase 2 (MK2) is a checkpoint kinase involved in the DNA damage response. MK2 inhibition enhances the efficacy of chemotherapeutic agents; however, whether MK2 inhibition alone, without concurrent chemotherapy, would attenuate survival of cancer cells has not been investigated. CMPD1 is a widely used non-ATP competitive inhibitor that prevents MK2 phosphorylation. We employed CMPD1 together with MK2 knock-down and ATP-competitive MK2 inhibitor III (MK2i) in a panel of glioblastoma cells to assess whether MK2 inhibition could induce cancer cell death. While CMPD1 was effective at selective killing of cancer cells, MK2i and MK2 knock-down had no effect on viability of glioblastoma cells. CMPD1 treatment induced a significant G2/M arrest but MK2i-treated cells were only minimally arrested at G1 phase. Intriguingly, at doses that were cytotoxic to glioblastoma cells, CMPD1 did not inhibit phosphorylation of MK2 and of its downstream substrate Hsp27. These results suggest that CMPD1 exhibits cytotoxic activity independently of MK2 inhibition. Indeed, we identified tubulin as a primary target of the CMPD1 cytotoxic activity. This study demonstrates how functional and mechanistic studies with appropriate selection of test compounds, combining genetic knock-down and pharmacological inhibition, coordinating timing and dose levels enabled us to uncover the primary target of an MK2 inhibitor commonly used in the research community. Tubulin is emerging as one of the most common non-kinase targets for kinase inhibitors and we propose that potential tubulin-targeting activity should be assessed in preclinical pharmacology studies of all novel kinase inhibitors.

No MeSH data available.


Related in: MedlinePlus