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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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Enforced expression of ectopic Mcl-1 (A and B), but not survivin (C and D), protects cells from API-1-induced apoptosis including caspase activation. A and B, The indicated stable transfectants were exposed to 5 μM API-1 for 24 h and then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect caspase cleavage and Mcl-1 expression (A) and for detection of apoptosis (i.e., annexin V-positive cells) with flow cytometry (B). P, parental; V, vector. C, The indicated cell lines were exposed to the given concentrations of API-1 for 24 h and then harvested for preparation of whole cell protein lysates and subsequent Western blot analysis. D, The indicated H157 stable transfectants were treated with different concentrations of API-1 as indicated for 3 days. Cell numbers were estimated with SRB assay. The data are means ± SDs of four replicate determinations. CF, cleaved fragment.
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Figure 2: Enforced expression of ectopic Mcl-1 (A and B), but not survivin (C and D), protects cells from API-1-induced apoptosis including caspase activation. A and B, The indicated stable transfectants were exposed to 5 μM API-1 for 24 h and then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect caspase cleavage and Mcl-1 expression (A) and for detection of apoptosis (i.e., annexin V-positive cells) with flow cytometry (B). P, parental; V, vector. C, The indicated cell lines were exposed to the given concentrations of API-1 for 24 h and then harvested for preparation of whole cell protein lysates and subsequent Western blot analysis. D, The indicated H157 stable transfectants were treated with different concentrations of API-1 as indicated for 3 days. Cell numbers were estimated with SRB assay. The data are means ± SDs of four replicate determinations. CF, cleaved fragment.

Mentions: We next determined whether Mcl-1 and survivin reduction is indeed involved in mediating induction of apoptosis by API-1. To this end, we enforced the expression of ectopic Mcl-1 or survivin in NSCLC cells and then examined their protective effects on API-1-induced apoptosis. In both H1299 and A549 cells, API-1 treatment caused clear cleavage of caspase-8, caspase-9, caspase-3 and PARP in vector control cell lines; these effects were not observed or were drastically diminished in Mcl-1-transfected cell lines (Figure 2A). Moreover, API-1 did not or only minimally increased annexin V-positive (i.e., apoptotic) cell populations in both H1299/Mcl-1 and A549/Mcl-1 cell lines, but drastically in their matched control counterparts (Figure 2B). These results clearly indicate that enforced expression of ectopic Mcl-1 protects cells from undergoing API-1-induced apoptosis, suggesting that Mcl-1 reduction is indeed critical for mediating apoptosis induced by API-1. In contrast, enforced expression of ectopic survivin failed to protect cells from killing by API-1: survivin-transfected cells were even more sensitive than the control Lac Z-expressing cells to the effects of API-1 on PARP cleavage and cell survival (Figure 2C-D), suggesting that survivin reduction is less important than Mcl-1 in mediating induction of apoptosis by 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)

Enforced expression of ectopic Mcl-1 (A and B), but not survivin (C and D), protects cells from API-1-induced apoptosis including caspase activation. A and B, The indicated stable transfectants were exposed to 5 μM API-1 for 24 h and then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect caspase cleavage and Mcl-1 expression (A) and for detection of apoptosis (i.e., annexin V-positive cells) with flow cytometry (B). P, parental; V, vector. C, The indicated cell lines were exposed to the given concentrations of API-1 for 24 h and then harvested for preparation of whole cell protein lysates and subsequent Western blot analysis. D, The indicated H157 stable transfectants were treated with different concentrations of API-1 as indicated for 3 days. Cell numbers were estimated with SRB assay. The data are means ± SDs of four replicate determinations. CF, cleaved fragment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Enforced expression of ectopic Mcl-1 (A and B), but not survivin (C and D), protects cells from API-1-induced apoptosis including caspase activation. A and B, The indicated stable transfectants were exposed to 5 μM API-1 for 24 h and then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect caspase cleavage and Mcl-1 expression (A) and for detection of apoptosis (i.e., annexin V-positive cells) with flow cytometry (B). P, parental; V, vector. C, The indicated cell lines were exposed to the given concentrations of API-1 for 24 h and then harvested for preparation of whole cell protein lysates and subsequent Western blot analysis. D, The indicated H157 stable transfectants were treated with different concentrations of API-1 as indicated for 3 days. Cell numbers were estimated with SRB assay. The data are means ± SDs of four replicate determinations. CF, cleaved fragment.
Mentions: We next determined whether Mcl-1 and survivin reduction is indeed involved in mediating induction of apoptosis by API-1. To this end, we enforced the expression of ectopic Mcl-1 or survivin in NSCLC cells and then examined their protective effects on API-1-induced apoptosis. In both H1299 and A549 cells, API-1 treatment caused clear cleavage of caspase-8, caspase-9, caspase-3 and PARP in vector control cell lines; these effects were not observed or were drastically diminished in Mcl-1-transfected cell lines (Figure 2A). Moreover, API-1 did not or only minimally increased annexin V-positive (i.e., apoptotic) cell populations in both H1299/Mcl-1 and A549/Mcl-1 cell lines, but drastically in their matched control counterparts (Figure 2B). These results clearly indicate that enforced expression of ectopic Mcl-1 protects cells from undergoing API-1-induced apoptosis, suggesting that Mcl-1 reduction is indeed critical for mediating apoptosis induced by API-1. In contrast, enforced expression of ectopic survivin failed to protect cells from killing by API-1: survivin-transfected cells were even more sensitive than the control Lac Z-expressing cells to the effects of API-1 on PARP cleavage and cell survival (Figure 2C-D), suggesting that survivin reduction is less important than Mcl-1 in mediating induction of apoptosis by 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