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Bay 61-3606 Sensitizes TRAIL-Induced Apoptosis by Downregulating Mcl-1 in Breast Cancer Cells.

Kim SY, Park SE, Shim SM, Park S, Kim KK, Jeong SY, Choi EK, Hwang JJ, Jin DH, Chung CD, Kim I - PLoS ONE (2015)

Bottom Line: First, Bay 61-3606 triggered ubiquitin-dependent degradation of Mcl-1 by regulating Mcl-1 phosphorylation.Second, Bay 61-3606 downregulates Mcl-1 expression at the transcription level.In this context, Bay 61-3606 acted as an inhibitor of Cyclin-Dependent Kinase (CDK) 9 rather than Syk.

View Article: PubMed Central - PubMed

Affiliation: ASAN Institute for Life Sciences, ASAN Medical Center, Seoul, Republic of Korea.

ABSTRACT
Breast cancer cells generally develop resistance to TNF-Related Apoptosis-Inducing Ligand (TRAIL) and, therefore, assistance from sensitizers is required. In our study, we have demonstrated that Spleen tyrosine kinase (Syk) inhibitor Bay 61-3606 was identified as a TRAIL sensitizer. Amplification of TRAIL-induced apoptosis by Bay 61-3606 was accompanied by the strong activation of Bak, caspases, and DNA fragmentation. In mechanism of action, Bay 61-3606 sensitized cells to TRAIL via two mechanisms regulating myeloid cell leukemia sequence-1 (Mcl-1). First, Bay 61-3606 triggered ubiquitin-dependent degradation of Mcl-1 by regulating Mcl-1 phosphorylation. Second, Bay 61-3606 downregulates Mcl-1 expression at the transcription level. In this context, Bay 61-3606 acted as an inhibitor of Cyclin-Dependent Kinase (CDK) 9 rather than Syk. In summary, Bay 61-3606 downregulates Mcl-1 expression in breast cancer cells and sensitizes cancer cells to TRAIL-mediated apoptosis.

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Bay 61–3606 downregulated Mcl-1 expression by inhibition of CDK9 and RNA polymerase II phosphorylation.(A) Cells were treated by Bay 61–3606 (2.5 μM) combined with TRAIL (50 ng/ml) for 6 h, and transcriptional downregulation of Mcl-1 was measured by RT-PCR (A, upper), and Mcl-1 luciferase reporter assay (A, lower). (B) MCF-7 cells were transfected with scrambled siRNA or three different CDK9 siRNAs (siCDK9 #1, #2, and #3). After 48 h of transfection, the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (C) Cell extracts were prepared from cells treated with in (A) and the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (D) MCF-7 cells were transfected with pcDNA3-WT-CDK9-HA or pcDNA3-DN-CDK9-HA. After 48 h of transfection, cells were treated with Bay 61–3606 (2.5 μM) for 6 h. Cell extracts were then analyzed by Western blotting using the indicated antibodies. Values are the mean ± SE from three separate experiments performed in triplicate. Asterisks indicate significant differences compared with the control (** P-values < 0.01, and *** P-values < 0.001).
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pone.0146073.g005: Bay 61–3606 downregulated Mcl-1 expression by inhibition of CDK9 and RNA polymerase II phosphorylation.(A) Cells were treated by Bay 61–3606 (2.5 μM) combined with TRAIL (50 ng/ml) for 6 h, and transcriptional downregulation of Mcl-1 was measured by RT-PCR (A, upper), and Mcl-1 luciferase reporter assay (A, lower). (B) MCF-7 cells were transfected with scrambled siRNA or three different CDK9 siRNAs (siCDK9 #1, #2, and #3). After 48 h of transfection, the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (C) Cell extracts were prepared from cells treated with in (A) and the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (D) MCF-7 cells were transfected with pcDNA3-WT-CDK9-HA or pcDNA3-DN-CDK9-HA. After 48 h of transfection, cells were treated with Bay 61–3606 (2.5 μM) for 6 h. Cell extracts were then analyzed by Western blotting using the indicated antibodies. Values are the mean ± SE from three separate experiments performed in triplicate. Asterisks indicate significant differences compared with the control (** P-values < 0.01, and *** P-values < 0.001).

Mentions: We investigated whether Bay 61–3606 modulates Mcl-1 expression at the transcription level. As shown in Fig 5A, downregulation of Mcl-1 mRNA expression by Bay 61–3606 was examined by RT-PCR and the Mcl-1 promoter assay. Moreover, Bay 61–3606 inhibited CDK9 kinase activity with an in vitro IC50 of 37 nM (S3 Fig). Based on this result, we investigated the molecular mechanisms in which Bay 61–3606 inhibits Mcl-1 transcription via CDK9. As shown in Fig 5B, knockdown of CDK9 by siRNAs inhibited RNA polymerase II (RNA pol II) phosphorylation at Ser2 and induces Mcl-1 downregulation. Incubation with Bay 61–3606 promoted the inhibition of CDK9 and RNA pol II phosphorylation, and resulted in Mcl-1 downregulation regardless of the TRAIL treatment (Fig 5C). This result is comparable with knockdown of CDK9 induces Mcl-1 downregulation by RNA pol II inhibition (Fig 5B), and thus implying that Bay 61–3606 has a negative role on CDK9 and RNA pol II activity.


Bay 61-3606 Sensitizes TRAIL-Induced Apoptosis by Downregulating Mcl-1 in Breast Cancer Cells.

Kim SY, Park SE, Shim SM, Park S, Kim KK, Jeong SY, Choi EK, Hwang JJ, Jin DH, Chung CD, Kim I - PLoS ONE (2015)

Bay 61–3606 downregulated Mcl-1 expression by inhibition of CDK9 and RNA polymerase II phosphorylation.(A) Cells were treated by Bay 61–3606 (2.5 μM) combined with TRAIL (50 ng/ml) for 6 h, and transcriptional downregulation of Mcl-1 was measured by RT-PCR (A, upper), and Mcl-1 luciferase reporter assay (A, lower). (B) MCF-7 cells were transfected with scrambled siRNA or three different CDK9 siRNAs (siCDK9 #1, #2, and #3). After 48 h of transfection, the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (C) Cell extracts were prepared from cells treated with in (A) and the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (D) MCF-7 cells were transfected with pcDNA3-WT-CDK9-HA or pcDNA3-DN-CDK9-HA. After 48 h of transfection, cells were treated with Bay 61–3606 (2.5 μM) for 6 h. Cell extracts were then analyzed by Western blotting using the indicated antibodies. Values are the mean ± SE from three separate experiments performed in triplicate. Asterisks indicate significant differences compared with the control (** P-values < 0.01, and *** P-values < 0.001).
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Related In: Results  -  Collection

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pone.0146073.g005: Bay 61–3606 downregulated Mcl-1 expression by inhibition of CDK9 and RNA polymerase II phosphorylation.(A) Cells were treated by Bay 61–3606 (2.5 μM) combined with TRAIL (50 ng/ml) for 6 h, and transcriptional downregulation of Mcl-1 was measured by RT-PCR (A, upper), and Mcl-1 luciferase reporter assay (A, lower). (B) MCF-7 cells were transfected with scrambled siRNA or three different CDK9 siRNAs (siCDK9 #1, #2, and #3). After 48 h of transfection, the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (C) Cell extracts were prepared from cells treated with in (A) and the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (D) MCF-7 cells were transfected with pcDNA3-WT-CDK9-HA or pcDNA3-DN-CDK9-HA. After 48 h of transfection, cells were treated with Bay 61–3606 (2.5 μM) for 6 h. Cell extracts were then analyzed by Western blotting using the indicated antibodies. Values are the mean ± SE from three separate experiments performed in triplicate. Asterisks indicate significant differences compared with the control (** P-values < 0.01, and *** P-values < 0.001).
Mentions: We investigated whether Bay 61–3606 modulates Mcl-1 expression at the transcription level. As shown in Fig 5A, downregulation of Mcl-1 mRNA expression by Bay 61–3606 was examined by RT-PCR and the Mcl-1 promoter assay. Moreover, Bay 61–3606 inhibited CDK9 kinase activity with an in vitro IC50 of 37 nM (S3 Fig). Based on this result, we investigated the molecular mechanisms in which Bay 61–3606 inhibits Mcl-1 transcription via CDK9. As shown in Fig 5B, knockdown of CDK9 by siRNAs inhibited RNA polymerase II (RNA pol II) phosphorylation at Ser2 and induces Mcl-1 downregulation. Incubation with Bay 61–3606 promoted the inhibition of CDK9 and RNA pol II phosphorylation, and resulted in Mcl-1 downregulation regardless of the TRAIL treatment (Fig 5C). This result is comparable with knockdown of CDK9 induces Mcl-1 downregulation by RNA pol II inhibition (Fig 5B), and thus implying that Bay 61–3606 has a negative role on CDK9 and RNA pol II activity.

Bottom Line: First, Bay 61-3606 triggered ubiquitin-dependent degradation of Mcl-1 by regulating Mcl-1 phosphorylation.Second, Bay 61-3606 downregulates Mcl-1 expression at the transcription level.In this context, Bay 61-3606 acted as an inhibitor of Cyclin-Dependent Kinase (CDK) 9 rather than Syk.

View Article: PubMed Central - PubMed

Affiliation: ASAN Institute for Life Sciences, ASAN Medical Center, Seoul, Republic of Korea.

ABSTRACT
Breast cancer cells generally develop resistance to TNF-Related Apoptosis-Inducing Ligand (TRAIL) and, therefore, assistance from sensitizers is required. In our study, we have demonstrated that Spleen tyrosine kinase (Syk) inhibitor Bay 61-3606 was identified as a TRAIL sensitizer. Amplification of TRAIL-induced apoptosis by Bay 61-3606 was accompanied by the strong activation of Bak, caspases, and DNA fragmentation. In mechanism of action, Bay 61-3606 sensitized cells to TRAIL via two mechanisms regulating myeloid cell leukemia sequence-1 (Mcl-1). First, Bay 61-3606 triggered ubiquitin-dependent degradation of Mcl-1 by regulating Mcl-1 phosphorylation. Second, Bay 61-3606 downregulates Mcl-1 expression at the transcription level. In this context, Bay 61-3606 acted as an inhibitor of Cyclin-Dependent Kinase (CDK) 9 rather than Syk. In summary, Bay 61-3606 downregulates Mcl-1 expression in breast cancer cells and sensitizes cancer cells to TRAIL-mediated apoptosis.

Show MeSH
Related in: MedlinePlus