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Sorafenib Sensitizes Glioma Cells to the BH3 Mimetic ABT-737 by Targeting MCL1 in a STAT3-Dependent Manner.

Kiprianova I, Remy J, Milosch N, Mohrenz IV, Seifert V, Aigner A, Kƶgel D - Neoplasia (2015)

Bottom Line: In analogous fashion, these synergistic effects were observed when we combined ABT-737 with the STAT3 inhibitor WP-1066.In contrast, the constitutively active STAT3 mutant STAT3-C was able to significantly enhance MCL1 levels under sorafenib treatment to retain cell survival.They also suggest that targeting STAT3 in combination with inducers of the intrinsic pathway of apoptosis may be a promising novel strategy for the treatment of malignant glioma.

View Article: PubMed Central - PubMed

Affiliation: Experimental Neurosurgery, Goethe University Hospital, Frankfurt am Main, Germany.

No MeSH data available.


Related in: MedlinePlus

MCL1 knockdown sensitizes glioma cell lines to ABT-737ā€“induced apoptotic cell death. Establishment of a stable lentiviral MCL1 knockdown in LN229 and U87 glioma cell lines, as determined by Western blotting (A). LN229 control cells (Ƙ) and MCL1-KD cells were treated with 5Ā Ī¼M ABT-737 in a time course experiment (4, 8, 16, and 24Ā hours), and cell death was quantified by FACS analysis of Annexin/PI (B). Effector caspase activation as determined by DEVD cleavage in LN229 empty vector control cells (Ƙ) and MCL1-KD cells after treatment for 1, 2, 4, 8, and 12Ā hours with 5Ā Ī¼M ABT-737 (C, left panel). Analysis of MTT activity in LN229 empty vector control cells (Ƙ) and MCL1-KD cells after treatment with 5Ā Ī¼M ABT-737 for 24Ā hours (C, right panel). Where indicated, cells were additionally treated with the pan-caspase inhibitor zVAD. U87 control cells (Ƙ) and MCL1-KD cells were treated with 5Ā Ī¼M ABT-737 in a time course experiment (4, 8, 16, and 24Ā hours), and cell death was quantified by FACS analysis of Annexin/PI (D). In all parts of the figure, graphs represent means of nĀ =Ā 4-8 culturesĀ +Ā SEM. *PĀ <Ā .05, significant difference to untreated controls; #PĀ <Ā .05, significant difference to respective control cell line.
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f0005: MCL1 knockdown sensitizes glioma cell lines to ABT-737ā€“induced apoptotic cell death. Establishment of a stable lentiviral MCL1 knockdown in LN229 and U87 glioma cell lines, as determined by Western blotting (A). LN229 control cells (Ƙ) and MCL1-KD cells were treated with 5Ā Ī¼M ABT-737 in a time course experiment (4, 8, 16, and 24Ā hours), and cell death was quantified by FACS analysis of Annexin/PI (B). Effector caspase activation as determined by DEVD cleavage in LN229 empty vector control cells (Ƙ) and MCL1-KD cells after treatment for 1, 2, 4, 8, and 12Ā hours with 5Ā Ī¼M ABT-737 (C, left panel). Analysis of MTT activity in LN229 empty vector control cells (Ƙ) and MCL1-KD cells after treatment with 5Ā Ī¼M ABT-737 for 24Ā hours (C, right panel). Where indicated, cells were additionally treated with the pan-caspase inhibitor zVAD. U87 control cells (Ƙ) and MCL1-KD cells were treated with 5Ā Ī¼M ABT-737 in a time course experiment (4, 8, 16, and 24Ā hours), and cell death was quantified by FACS analysis of Annexin/PI (D). In all parts of the figure, graphs represent means of nĀ =Ā 4-8 culturesĀ +Ā SEM. *PĀ <Ā .05, significant difference to untreated controls; #PĀ <Ā .05, significant difference to respective control cell line.

Mentions: To analyze the putative role of MCL1 for cell death resistance of glioma cells, we established a stable lentiviral knockdown of MCL1 in the two glioblastoma cells lines LN229 and U87 (FigureĀ 1A). The effect of ABT-737 single-agent treatment (5Ā Ī¼M) was subsequently assayed by flow cytometric quantification of total cell death in a time course experiment (4, 8, 16, and 24Ā hours). Knockdown of MCL1 evoked a significant increase in ABT-737ā€“induced cell death in LN229 cells (FigureĀ 1B). We also observed a moderate induction of cell death in MCL1-proficient LN229 wt cells. In LN229 MCL1 KD cells, ABT-737 induced a transient activation of effector caspases, peaking at 4 to 8Ā hours (FigureĀ 1C, left panel). In line with this observation, an MTT viability assay revealed a significantly lower MTT activity after treatment with ABT-737 in LN229 MCL1-KD cells which could be rescued with the pan-caspase-inhibitor zVAD (FigureĀ 1C, right panel). The potentiating effect of the MCL1 knockdown on ABT-737ā€“induced apoptosis was also confirmed in U87 cells (FigureĀ 1D). To rule out possible off-target effects of the applied shRNA, we also transiently knocked down MCL1 with two different siRNA oligos. In analogy to the data obtained with the stable knockdowns of MCL1, both siRNas significantly enhanced effector caspase activity and cell death in response to ABT-737 (Suppl. FigureĀ 1, Aā€“C). Collectively, these data provide evidence that, in glioma cells, MCL1 confers resistance to apoptotic cell death induced by ABT-737.


Sorafenib Sensitizes Glioma Cells to the BH3 Mimetic ABT-737 by Targeting MCL1 in a STAT3-Dependent Manner.

Kiprianova I, Remy J, Milosch N, Mohrenz IV, Seifert V, Aigner A, Kƶgel D - Neoplasia (2015)

MCL1 knockdown sensitizes glioma cell lines to ABT-737ā€“induced apoptotic cell death. Establishment of a stable lentiviral MCL1 knockdown in LN229 and U87 glioma cell lines, as determined by Western blotting (A). LN229 control cells (Ƙ) and MCL1-KD cells were treated with 5Ā Ī¼M ABT-737 in a time course experiment (4, 8, 16, and 24Ā hours), and cell death was quantified by FACS analysis of Annexin/PI (B). Effector caspase activation as determined by DEVD cleavage in LN229 empty vector control cells (Ƙ) and MCL1-KD cells after treatment for 1, 2, 4, 8, and 12Ā hours with 5Ā Ī¼M ABT-737 (C, left panel). Analysis of MTT activity in LN229 empty vector control cells (Ƙ) and MCL1-KD cells after treatment with 5Ā Ī¼M ABT-737 for 24Ā hours (C, right panel). Where indicated, cells were additionally treated with the pan-caspase inhibitor zVAD. U87 control cells (Ƙ) and MCL1-KD cells were treated with 5Ā Ī¼M ABT-737 in a time course experiment (4, 8, 16, and 24Ā hours), and cell death was quantified by FACS analysis of Annexin/PI (D). In all parts of the figure, graphs represent means of nĀ =Ā 4-8 culturesĀ +Ā SEM. *PĀ <Ā .05, significant difference to untreated controls; #PĀ <Ā .05, significant difference to respective control cell line.
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f0005: MCL1 knockdown sensitizes glioma cell lines to ABT-737ā€“induced apoptotic cell death. Establishment of a stable lentiviral MCL1 knockdown in LN229 and U87 glioma cell lines, as determined by Western blotting (A). LN229 control cells (Ƙ) and MCL1-KD cells were treated with 5Ā Ī¼M ABT-737 in a time course experiment (4, 8, 16, and 24Ā hours), and cell death was quantified by FACS analysis of Annexin/PI (B). Effector caspase activation as determined by DEVD cleavage in LN229 empty vector control cells (Ƙ) and MCL1-KD cells after treatment for 1, 2, 4, 8, and 12Ā hours with 5Ā Ī¼M ABT-737 (C, left panel). Analysis of MTT activity in LN229 empty vector control cells (Ƙ) and MCL1-KD cells after treatment with 5Ā Ī¼M ABT-737 for 24Ā hours (C, right panel). Where indicated, cells were additionally treated with the pan-caspase inhibitor zVAD. U87 control cells (Ƙ) and MCL1-KD cells were treated with 5Ā Ī¼M ABT-737 in a time course experiment (4, 8, 16, and 24Ā hours), and cell death was quantified by FACS analysis of Annexin/PI (D). In all parts of the figure, graphs represent means of nĀ =Ā 4-8 culturesĀ +Ā SEM. *PĀ <Ā .05, significant difference to untreated controls; #PĀ <Ā .05, significant difference to respective control cell line.
Mentions: To analyze the putative role of MCL1 for cell death resistance of glioma cells, we established a stable lentiviral knockdown of MCL1 in the two glioblastoma cells lines LN229 and U87 (FigureĀ 1A). The effect of ABT-737 single-agent treatment (5Ā Ī¼M) was subsequently assayed by flow cytometric quantification of total cell death in a time course experiment (4, 8, 16, and 24Ā hours). Knockdown of MCL1 evoked a significant increase in ABT-737ā€“induced cell death in LN229 cells (FigureĀ 1B). We also observed a moderate induction of cell death in MCL1-proficient LN229 wt cells. In LN229 MCL1 KD cells, ABT-737 induced a transient activation of effector caspases, peaking at 4 to 8Ā hours (FigureĀ 1C, left panel). In line with this observation, an MTT viability assay revealed a significantly lower MTT activity after treatment with ABT-737 in LN229 MCL1-KD cells which could be rescued with the pan-caspase-inhibitor zVAD (FigureĀ 1C, right panel). The potentiating effect of the MCL1 knockdown on ABT-737ā€“induced apoptosis was also confirmed in U87 cells (FigureĀ 1D). To rule out possible off-target effects of the applied shRNA, we also transiently knocked down MCL1 with two different siRNA oligos. In analogy to the data obtained with the stable knockdowns of MCL1, both siRNas significantly enhanced effector caspase activity and cell death in response to ABT-737 (Suppl. FigureĀ 1, Aā€“C). Collectively, these data provide evidence that, in glioma cells, MCL1 confers resistance to apoptotic cell death induced by ABT-737.

Bottom Line: In analogous fashion, these synergistic effects were observed when we combined ABT-737 with the STAT3 inhibitor WP-1066.In contrast, the constitutively active STAT3 mutant STAT3-C was able to significantly enhance MCL1 levels under sorafenib treatment to retain cell survival.They also suggest that targeting STAT3 in combination with inducers of the intrinsic pathway of apoptosis may be a promising novel strategy for the treatment of malignant glioma.

View Article: PubMed Central - PubMed

Affiliation: Experimental Neurosurgery, Goethe University Hospital, Frankfurt am Main, Germany.

No MeSH data available.


Related in: MedlinePlus