Limits...
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.

Show MeSH

Related in: MedlinePlus

API-1 induces proteasome-mediated Mcl-1 degradation (B) and decreases Mcl-1 stability (C) without altering Mcl-1 mRNA levels (A). A, H1299 cells were exposed to the indicated concentrations of API-1 for 8 h. Total cellular RNA was then isolated from the cells for detection of Mcl-1 and GAPDH (internal control) mRNAs with RT-PCR. B, The indicated cell lines were pre-treated with 20 μM MG132 for 30 minutes prior to the addition of 5 μM API-1. After co-treatment for an additional 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. C, H1299 cells were treated with 5 μM API-1 for 8 h. The cells were then washed with PBS 3 times and refed with fresh medium containing 10 μg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J software (Bethesda, MA) and were normalized to actin. The results were plotted as the relative Mcl-1 levels compared to those at the time 0 of CHX treatment (right panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3924345&req=5

Figure 4: API-1 induces proteasome-mediated Mcl-1 degradation (B) and decreases Mcl-1 stability (C) without altering Mcl-1 mRNA levels (A). A, H1299 cells were exposed to the indicated concentrations of API-1 for 8 h. Total cellular RNA was then isolated from the cells for detection of Mcl-1 and GAPDH (internal control) mRNAs with RT-PCR. B, The indicated cell lines were pre-treated with 20 μM MG132 for 30 minutes prior to the addition of 5 μM API-1. After co-treatment for an additional 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. C, H1299 cells were treated with 5 μM API-1 for 8 h. The cells were then washed with PBS 3 times and refed with fresh medium containing 10 μg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J software (Bethesda, MA) and were normalized to actin. The results were plotted as the relative Mcl-1 levels compared to those at the time 0 of CHX treatment (right panel).

Mentions: To elucidate the mechanisms by which API-1 reduces Mcl-1 levels, we detected Mcl-1 mRNA expression in cells exposed to different concentrations of API-1 with RT-PCR and found that API-1 did not alter Mcl-1 mRNA levels (Figure 4A). Since Mcl-1 is known to be a protein subject to proteasomal degradation [23,24], we next determined whether API-1-induced Mcl-1 reduction is due to enhancement of its degradation. Hence, we compared the effects of API-1 on Mcl-1 reduction in the absence and presence of the proteasome inhibitor MG132 in a few of NSCLC cell lines. API-1 effectively decreased Mcl-1 levels in the absence of MG132, but failed to do so in the presence of MG132 (Figure 4B), indicating that API-1 indeed induces proteasomal degradation of Mcl-1. Moreover, we found that the half-life of Mcl-1 in API-1-treated cells (about 20 min) was shorter than that in DMSO-treated cells (about 90 min) (Figure 4C), indicating that API-1 decreases the stability of Mcl-1. Taken together, we conclude that API-1 facilitates proteasomal degradation of Mcl-1, resulting in Mcl-1 reduction.


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 induces proteasome-mediated Mcl-1 degradation (B) and decreases Mcl-1 stability (C) without altering Mcl-1 mRNA levels (A). A, H1299 cells were exposed to the indicated concentrations of API-1 for 8 h. Total cellular RNA was then isolated from the cells for detection of Mcl-1 and GAPDH (internal control) mRNAs with RT-PCR. B, The indicated cell lines were pre-treated with 20 μM MG132 for 30 minutes prior to the addition of 5 μM API-1. After co-treatment for an additional 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. C, H1299 cells were treated with 5 μM API-1 for 8 h. The cells were then washed with PBS 3 times and refed with fresh medium containing 10 μg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J software (Bethesda, MA) and were normalized to actin. The results were plotted as the relative Mcl-1 levels compared to those at the time 0 of CHX treatment (right panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: API-1 induces proteasome-mediated Mcl-1 degradation (B) and decreases Mcl-1 stability (C) without altering Mcl-1 mRNA levels (A). A, H1299 cells were exposed to the indicated concentrations of API-1 for 8 h. Total cellular RNA was then isolated from the cells for detection of Mcl-1 and GAPDH (internal control) mRNAs with RT-PCR. B, The indicated cell lines were pre-treated with 20 μM MG132 for 30 minutes prior to the addition of 5 μM API-1. After co-treatment for an additional 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. C, H1299 cells were treated with 5 μM API-1 for 8 h. The cells were then washed with PBS 3 times and refed with fresh medium containing 10 μg/ml CHX. At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J software (Bethesda, MA) and were normalized to actin. The results were plotted as the relative Mcl-1 levels compared to those at the time 0 of CHX treatment (right panel).
Mentions: To elucidate the mechanisms by which API-1 reduces Mcl-1 levels, we detected Mcl-1 mRNA expression in cells exposed to different concentrations of API-1 with RT-PCR and found that API-1 did not alter Mcl-1 mRNA levels (Figure 4A). Since Mcl-1 is known to be a protein subject to proteasomal degradation [23,24], we next determined whether API-1-induced Mcl-1 reduction is due to enhancement of its degradation. Hence, we compared the effects of API-1 on Mcl-1 reduction in the absence and presence of the proteasome inhibitor MG132 in a few of NSCLC cell lines. API-1 effectively decreased Mcl-1 levels in the absence of MG132, but failed to do so in the presence of MG132 (Figure 4B), indicating that API-1 indeed induces proteasomal degradation of Mcl-1. Moreover, we found that the half-life of Mcl-1 in API-1-treated cells (about 20 min) was shorter than that in DMSO-treated cells (about 90 min) (Figure 4C), indicating that API-1 decreases the stability of Mcl-1. Taken together, we conclude that API-1 facilitates proteasomal degradation of Mcl-1, resulting in Mcl-1 reduction.

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