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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 sensitizes MCF-7 cells to TRAIL by decreasing Mcl-1.(A, B) After MCF-7 cells were exposed to TRAIL (50 ng/ml) with or without Bay 61–3606 (2.5 μM) for 12 h, cells were extracted and analyzed by immunoblotting analysis using the indicated antibodies. (C) MCF-7 cells were exposed to several concentrations of Bay 61–3606 (upper) for 6 h or to 2.5 μM Bay 61–3606 for the indicated times (lower), and expression of Mcl-1 was analyzed by Western blotting. (D) MCF-7 cells were transfected with scrambled (siCTL) or Mcl-1 siRNA (siMcl-1). After 48 h, cells were exposed to TRAIL (50 ng/ml) for 24 h. The percentage of cell death was evaluated by trypan blue exclusion. Values are the mean ± SE from three separate experiments performed in triplicate. Asterisks indicate significant differences compared with the control (** P-values < 0.01).
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pone.0146073.g002: Bay 61–3606 sensitizes MCF-7 cells to TRAIL by decreasing Mcl-1.(A, B) After MCF-7 cells were exposed to TRAIL (50 ng/ml) with or without Bay 61–3606 (2.5 μM) for 12 h, cells were extracted and analyzed by immunoblotting analysis using the indicated antibodies. (C) MCF-7 cells were exposed to several concentrations of Bay 61–3606 (upper) for 6 h or to 2.5 μM Bay 61–3606 for the indicated times (lower), and expression of Mcl-1 was analyzed by Western blotting. (D) MCF-7 cells were transfected with scrambled (siCTL) or Mcl-1 siRNA (siMcl-1). After 48 h, cells were exposed to TRAIL (50 ng/ml) for 24 h. The percentage of cell death was evaluated by trypan blue exclusion. Values are the mean ± SE from three separate experiments performed in triplicate. Asterisks indicate significant differences compared with the control (** P-values < 0.01).

Mentions: We then analyzed the molecular mechanisms of the sensitization. The combination of Bay 61–3606 and TRAIL massively induced caspase-7 & -8 activation and cleavage of Bid, and PARP (Fig 2A). As shown in Fig 2A and 2B, there was no significant alteration in the expression levels of DR4, DR5, p53, and Bcl-2 family proteins, Bad, Bax, Bak, and Bcl-xL in MCF-7 cells. However, we could detect significantly Mcl-1 downregulation by Bay 61–3606 (Fig 2B) and Mcl-1 downregulation were concentration- and time-dependent (Fig 2C). Also, FLICE-inhibitory protein (FLIP) and Inhibitors of Apoptosis Proteins (IAP) family proteins, such as cIAP1, X-linked inhibitor of apoptosis protein (XIAP), and Survivin were downregulated by combination of Bay 61–3606 and TRAIL (Fig 2A and 2B). To examine the contribution of Mcl-1 to TRAIL resistance, the loss of function effect of Mcl-1 was tested. As shown in Fig 2D, Mcl-1 clearly knock downed the sensitized cells to TRAIL, implying that Mcl-1 contributes to the resistance to TRAIL. These results indicate that Bay 61–3606 sensitized MCF-7 cells to TRAIL by decreasing Mcl-1. Mcl-1 clearly knock downed the sensitized cells to TRAIL, implying that Mcl-1 contributes to the resistance to TRAIL. These results indicate that Bay 61–3606 sensitized MCF-7 cells to TRAIL by decreasing Mcl-1.


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 sensitizes MCF-7 cells to TRAIL by decreasing Mcl-1.(A, B) After MCF-7 cells were exposed to TRAIL (50 ng/ml) with or without Bay 61–3606 (2.5 μM) for 12 h, cells were extracted and analyzed by immunoblotting analysis using the indicated antibodies. (C) MCF-7 cells were exposed to several concentrations of Bay 61–3606 (upper) for 6 h or to 2.5 μM Bay 61–3606 for the indicated times (lower), and expression of Mcl-1 was analyzed by Western blotting. (D) MCF-7 cells were transfected with scrambled (siCTL) or Mcl-1 siRNA (siMcl-1). After 48 h, cells were exposed to TRAIL (50 ng/ml) for 24 h. The percentage of cell death was evaluated by trypan blue exclusion. Values are the mean ± SE from three separate experiments performed in triplicate. Asterisks indicate significant differences compared with the control (** P-values < 0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697837&req=5

pone.0146073.g002: Bay 61–3606 sensitizes MCF-7 cells to TRAIL by decreasing Mcl-1.(A, B) After MCF-7 cells were exposed to TRAIL (50 ng/ml) with or without Bay 61–3606 (2.5 μM) for 12 h, cells were extracted and analyzed by immunoblotting analysis using the indicated antibodies. (C) MCF-7 cells were exposed to several concentrations of Bay 61–3606 (upper) for 6 h or to 2.5 μM Bay 61–3606 for the indicated times (lower), and expression of Mcl-1 was analyzed by Western blotting. (D) MCF-7 cells were transfected with scrambled (siCTL) or Mcl-1 siRNA (siMcl-1). After 48 h, cells were exposed to TRAIL (50 ng/ml) for 24 h. The percentage of cell death was evaluated by trypan blue exclusion. Values are the mean ± SE from three separate experiments performed in triplicate. Asterisks indicate significant differences compared with the control (** P-values < 0.01).
Mentions: We then analyzed the molecular mechanisms of the sensitization. The combination of Bay 61–3606 and TRAIL massively induced caspase-7 & -8 activation and cleavage of Bid, and PARP (Fig 2A). As shown in Fig 2A and 2B, there was no significant alteration in the expression levels of DR4, DR5, p53, and Bcl-2 family proteins, Bad, Bax, Bak, and Bcl-xL in MCF-7 cells. However, we could detect significantly Mcl-1 downregulation by Bay 61–3606 (Fig 2B) and Mcl-1 downregulation were concentration- and time-dependent (Fig 2C). Also, FLICE-inhibitory protein (FLIP) and Inhibitors of Apoptosis Proteins (IAP) family proteins, such as cIAP1, X-linked inhibitor of apoptosis protein (XIAP), and Survivin were downregulated by combination of Bay 61–3606 and TRAIL (Fig 2A and 2B). To examine the contribution of Mcl-1 to TRAIL resistance, the loss of function effect of Mcl-1 was tested. As shown in Fig 2D, Mcl-1 clearly knock downed the sensitized cells to TRAIL, implying that Mcl-1 contributes to the resistance to TRAIL. These results indicate that Bay 61–3606 sensitized MCF-7 cells to TRAIL by decreasing Mcl-1. Mcl-1 clearly knock downed the sensitized cells to TRAIL, implying that Mcl-1 contributes to the resistance to TRAIL. These results indicate that Bay 61–3606 sensitized MCF-7 cells to TRAIL by decreasing Mcl-1.

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