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The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

Ren H, Koo J, Guan B, Yue P, Deng X, Chen M, Khuri FR, Sun SY - Mol. Cancer (2013)

Bottom Line: API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1.Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1.However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiration, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi, P R China. chenmingwei@mail.xjtu.edu.cn.

ABSTRACT

Background: The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis.

Results: API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.

Conclusions: API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

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Related in: MedlinePlus

API-1 decreases Mcl-1 levels (B-D) in API-1-sensitive NSCLC cell lines (A). A, The given cell lines were treated with different concentrations of API-1 ranging from 10 to 0.5 μM for 3 days. Cell numbers were then estimated with the SRB assay. Data are means ± SDs of four replicate determinations. B-D, The given cell lines were treated with different concentrations of API-1 as indicated for 12 h (B and D) or 5 μM API-1 for the indicated times (C). The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect the indicated proteins.
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Figure 1: API-1 decreases Mcl-1 levels (B-D) in API-1-sensitive NSCLC cell lines (A). A, The given cell lines were treated with different concentrations of API-1 ranging from 10 to 0.5 μM for 3 days. Cell numbers were then estimated with the SRB assay. Data are means ± SDs of four replicate determinations. B-D, The given cell lines were treated with different concentrations of API-1 as indicated for 12 h (B and D) or 5 μM API-1 for the indicated times (C). The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect the indicated proteins.

Mentions: Human NSCLC cell lines exhibited varied sensitivities to API-1 as evaluated with the SRB assay after a 3-day incubation (Figure 1A). Among them, H1299, H522 and A549 were sensitive to API-1, whereas H226 and H1792 were quite resistant to API-1. Since our previous work investigated the effects of API-1 on the expression of several proteins (e.g., c-FLIP, DR4 and DR5) involved in the extrinsic apoptotic pathway [2], we focused our current study on determining the effects of API-1 on modulation of the levels of several proteins (e.g., Mcl-1 and survivin) involved in regulation of the intrinsic apoptotic pathway. In H1299 cells, API-1 decreased the levels of Mcl-1 and survivin at even 2.5 μM and the levels of Bcl-2 and Bcl-XL at 10 μM with no apparent increase in the levels of the pro-apoptotic proteins, Bax, Bad and Bim (Figure 1B). Moreover, we found that API-1 decreased Mcl-1 levels at 4 h, survivin levels at 8 h and Bcl-2 levels at 12 h post treatment (Figure 1C), indicating that Mcl-1 reduction is a rapid event ahead of survivin and Bcl-2 decrease in the process of API-1-induced apoptosis. By further comparing effects of API-1 on reducing Mcl-1 and survivin in 4 more NSCLC cell lines with different sensitivities to API-1, We found that API-1 reduced Mcl-1 levels effectively in H522 and A549 cells, which are sensitive to API-1, but only minimally in H226 and H1792 cells, which are insensitive to API-1. We detected survivin reduction in all four cell lines post API-1 treatment regardless of their sensitivities to API-1 (Figure 1D). These data emphasize the relevance and importance of Mcl-1 reduction in cell response to API-1.


The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

Ren H, Koo J, Guan B, Yue P, Deng X, Chen M, Khuri FR, Sun SY - Mol. Cancer (2013)

API-1 decreases Mcl-1 levels (B-D) in API-1-sensitive NSCLC cell lines (A). A, The given cell lines were treated with different concentrations of API-1 ranging from 10 to 0.5 μM for 3 days. Cell numbers were then estimated with the SRB assay. Data are means ± SDs of four replicate determinations. B-D, The given cell lines were treated with different concentrations of API-1 as indicated for 12 h (B and D) or 5 μM API-1 for the indicated times (C). The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect the indicated proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924345&req=5

Figure 1: API-1 decreases Mcl-1 levels (B-D) in API-1-sensitive NSCLC cell lines (A). A, The given cell lines were treated with different concentrations of API-1 ranging from 10 to 0.5 μM for 3 days. Cell numbers were then estimated with the SRB assay. Data are means ± SDs of four replicate determinations. B-D, The given cell lines were treated with different concentrations of API-1 as indicated for 12 h (B and D) or 5 μM API-1 for the indicated times (C). The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect the indicated proteins.
Mentions: Human NSCLC cell lines exhibited varied sensitivities to API-1 as evaluated with the SRB assay after a 3-day incubation (Figure 1A). Among them, H1299, H522 and A549 were sensitive to API-1, whereas H226 and H1792 were quite resistant to API-1. Since our previous work investigated the effects of API-1 on the expression of several proteins (e.g., c-FLIP, DR4 and DR5) involved in the extrinsic apoptotic pathway [2], we focused our current study on determining the effects of API-1 on modulation of the levels of several proteins (e.g., Mcl-1 and survivin) involved in regulation of the intrinsic apoptotic pathway. In H1299 cells, API-1 decreased the levels of Mcl-1 and survivin at even 2.5 μM and the levels of Bcl-2 and Bcl-XL at 10 μM with no apparent increase in the levels of the pro-apoptotic proteins, Bax, Bad and Bim (Figure 1B). Moreover, we found that API-1 decreased Mcl-1 levels at 4 h, survivin levels at 8 h and Bcl-2 levels at 12 h post treatment (Figure 1C), indicating that Mcl-1 reduction is a rapid event ahead of survivin and Bcl-2 decrease in the process of API-1-induced apoptosis. By further comparing effects of API-1 on reducing Mcl-1 and survivin in 4 more NSCLC cell lines with different sensitivities to API-1, We found that API-1 reduced Mcl-1 levels effectively in H522 and A549 cells, which are sensitive to API-1, but only minimally in H226 and H1792 cells, which are insensitive to API-1. We detected survivin reduction in all four cell lines post API-1 treatment regardless of their sensitivities to API-1 (Figure 1D). These data emphasize the relevance and importance of Mcl-1 reduction in cell response to API-1.

Bottom Line: API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1.Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1.However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiration, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi, P R China. chenmingwei@mail.xjtu.edu.cn.

ABSTRACT

Background: The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis.

Results: API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.

Conclusions: API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

Show MeSH
Related in: MedlinePlus