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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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Mcl-1 degradation by Bay 61–3606 is independent of Syk.(A) MCF-7 cells were transfected with scrambled (siCTL) or Syk siRNA (siSyk) for 48 h, and expression of Syk and Mcl-1 was analyzed by Western blotting. (B) MCF-7 cells were exposed to Syk inhibitors for 6 h and Mcl-1 expression was assessed by Western blotting. (C) Three, breast-cancer cell lines were exposed to TRAIL (50 ng/ml) for 6 h, Syk mRNA expression was analyzed by RT-PCR (lower), and phosphorylation of Syk and expression of Mcl-1 were analyzed by Western blotting(upper). (D) T47D cells were exposed to increasing concentrations of Bay 61–3606 for 6 h, and expression of Mcl-1 was analyzed by Western blotting.
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pone.0146073.g003: Mcl-1 degradation by Bay 61–3606 is independent of Syk.(A) MCF-7 cells were transfected with scrambled (siCTL) or Syk siRNA (siSyk) for 48 h, and expression of Syk and Mcl-1 was analyzed by Western blotting. (B) MCF-7 cells were exposed to Syk inhibitors for 6 h and Mcl-1 expression was assessed by Western blotting. (C) Three, breast-cancer cell lines were exposed to TRAIL (50 ng/ml) for 6 h, Syk mRNA expression was analyzed by RT-PCR (lower), and phosphorylation of Syk and expression of Mcl-1 were analyzed by Western blotting(upper). (D) T47D cells were exposed to increasing concentrations of Bay 61–3606 for 6 h, and expression of Mcl-1 was analyzed by Western blotting.

Mentions: Bay 61–3606 is an inhibitor of spleen tyrosine kinase [21, 22], although it has a wide-ranging inhibitory profile against various kinases [23]. These multiple target spectra of Bay 61–3606 led us to investigate whether Syk could be involved in Bay 61-3606-mediated downregulation of Mcl-1. First, we examined whether downregulation of Syk expression can affect the Mcl-1 expression. Although we efficiently suppressed Syk expression via siRNA, the protein levels of Mcl-1 remained unchanged in MCF-7 cells (Fig 3A). In addition to genetic alteration using siRNA, we also tested three chemical inhibitors of Syk, i.e. S-II [2-(2-aminoethylamino)-4-(3-trifluoromethylanilino)-pyrimidine-5-carboxamide dihydrochloride dehydrate], Piceatannol, and curcumin [24, 25]. In our test, none of these Syk inhibitors reduced Mcl-1 expression in MCF-7 cells (Fig 3B).


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)

Mcl-1 degradation by Bay 61–3606 is independent of Syk.(A) MCF-7 cells were transfected with scrambled (siCTL) or Syk siRNA (siSyk) for 48 h, and expression of Syk and Mcl-1 was analyzed by Western blotting. (B) MCF-7 cells were exposed to Syk inhibitors for 6 h and Mcl-1 expression was assessed by Western blotting. (C) Three, breast-cancer cell lines were exposed to TRAIL (50 ng/ml) for 6 h, Syk mRNA expression was analyzed by RT-PCR (lower), and phosphorylation of Syk and expression of Mcl-1 were analyzed by Western blotting(upper). (D) T47D cells were exposed to increasing concentrations of Bay 61–3606 for 6 h, and expression of Mcl-1 was analyzed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0146073.g003: Mcl-1 degradation by Bay 61–3606 is independent of Syk.(A) MCF-7 cells were transfected with scrambled (siCTL) or Syk siRNA (siSyk) for 48 h, and expression of Syk and Mcl-1 was analyzed by Western blotting. (B) MCF-7 cells were exposed to Syk inhibitors for 6 h and Mcl-1 expression was assessed by Western blotting. (C) Three, breast-cancer cell lines were exposed to TRAIL (50 ng/ml) for 6 h, Syk mRNA expression was analyzed by RT-PCR (lower), and phosphorylation of Syk and expression of Mcl-1 were analyzed by Western blotting(upper). (D) T47D cells were exposed to increasing concentrations of Bay 61–3606 for 6 h, and expression of Mcl-1 was analyzed by Western blotting.
Mentions: Bay 61–3606 is an inhibitor of spleen tyrosine kinase [21, 22], although it has a wide-ranging inhibitory profile against various kinases [23]. These multiple target spectra of Bay 61–3606 led us to investigate whether Syk could be involved in Bay 61-3606-mediated downregulation of Mcl-1. First, we examined whether downregulation of Syk expression can affect the Mcl-1 expression. Although we efficiently suppressed Syk expression via siRNA, the protein levels of Mcl-1 remained unchanged in MCF-7 cells (Fig 3A). In addition to genetic alteration using siRNA, we also tested three chemical inhibitors of Syk, i.e. S-II [2-(2-aminoethylamino)-4-(3-trifluoromethylanilino)-pyrimidine-5-carboxamide dihydrochloride dehydrate], Piceatannol, and curcumin [24, 25]. In our test, none of these Syk inhibitors reduced Mcl-1 expression in MCF-7 cells (Fig 3B).

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