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Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner.

Guo W, Zhang Y, Ling Z, Liu X, Zhao X, Yuan Z, Nie C, Wei Y - Cell Death Dis (2015)

Bottom Line: Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation.Conversely, AIF and endoG knockdown had little effect on Bak activation.Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Abdominal Oncology, State Key Laboratory of Biotherapy and Cancer Center, Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, 17# People's South Road, Chengdu, Chengdu 610041, PR China.

ABSTRACT
Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.

No MeSH data available.


Related in: MedlinePlus

Bid activation mediates AIF, endoG release, and Bak activation. (a) Cell were treated with genistein at indicated time and collected with western blot analysis. β-Actin was used as a protein loading control. (b) Cells were treated with genistein for 72 h and subjected to subcellular fractionation. The cytosolic (C) or mitochondrial (M) fractions were immunoblotted for Bid and tBid detection. (c) Cells were transfected with Bid and Ctrl siRNA for 48 h, and then treated with genistein for 72 h. One portion of cells was subjected to subcellular fraction to detect the release of AIF, endoG, and Cyt c. The other portion of cells was collected to detect Bak and caspase activation. β-Actin was used as a protein loading control. (d) Cells were transfected with si Ctrl or si Bid for 48 h and treated with genistein (30 μM) for 72 h. Cell death was detected with ELSIA. Graphs showing results of quantitative analyses (n=3, mean±S.D., **P<0.01). Representative results of three experiments with consistent results are shown
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fig6: Bid activation mediates AIF, endoG release, and Bak activation. (a) Cell were treated with genistein at indicated time and collected with western blot analysis. β-Actin was used as a protein loading control. (b) Cells were treated with genistein for 72 h and subjected to subcellular fractionation. The cytosolic (C) or mitochondrial (M) fractions were immunoblotted for Bid and tBid detection. (c) Cells were transfected with Bid and Ctrl siRNA for 48 h, and then treated with genistein for 72 h. One portion of cells was subjected to subcellular fraction to detect the release of AIF, endoG, and Cyt c. The other portion of cells was collected to detect Bak and caspase activation. β-Actin was used as a protein loading control. (d) Cells were transfected with si Ctrl or si Bid for 48 h and treated with genistein (30 μM) for 72 h. Cell death was detected with ELSIA. Graphs showing results of quantitative analyses (n=3, mean±S.D., **P<0.01). Representative results of three experiments with consistent results are shown

Mentions: Previous studies have demonstrated that activated Bid can mediate Bak activation and the release of AIF and endoG.38, 39, 40 We then detected whether Bid contributes to genistein-induced cell apoptosis. We first detected Bid expression and activation. Our data confirmed that Bid was cleaved into tBid (Figure 6a), which is the active truncated form of Bid,38 during the time course of genistein treatment. We also found that tBid translocated into the mitochondria from the cytosol (Figure 6b), which could induce the release of mitochondrial proteins.41 We then knocked down the expression of Bid with siRNAs. Our data revealed that Bid expression was inhibited by siRNA treatment (Figure 6c). Moreover, Bid knockdown affected Bak oligomerization; the release of Cyt c, AIF, and endoG; and caspase-3 cleavage in Bax or p53 KO cells (Figure 6c). Meanwhile, Bid knockdown also substantially decreased apoptosis in Bax or p53 KO cells treated with genistein (Figure 6d). We also transiently transfected Bid siRNAs into Bax KD/p53 KO or Bax KO/p53 KD cells. We found that Bid knockdown is sufficient to attenuate Bak oligomerization; the release of Cyt c, AIF, and endoG; and caspase-3 cleavage in cancer cells (Supplementary Figures 1C and D). These results demonstrated that Bid could upregulate Bak activation; the release of AIF and endoG; and subsequently apoptosis.


Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner.

Guo W, Zhang Y, Ling Z, Liu X, Zhao X, Yuan Z, Nie C, Wei Y - Cell Death Dis (2015)

Bid activation mediates AIF, endoG release, and Bak activation. (a) Cell were treated with genistein at indicated time and collected with western blot analysis. β-Actin was used as a protein loading control. (b) Cells were treated with genistein for 72 h and subjected to subcellular fractionation. The cytosolic (C) or mitochondrial (M) fractions were immunoblotted for Bid and tBid detection. (c) Cells were transfected with Bid and Ctrl siRNA for 48 h, and then treated with genistein for 72 h. One portion of cells was subjected to subcellular fraction to detect the release of AIF, endoG, and Cyt c. The other portion of cells was collected to detect Bak and caspase activation. β-Actin was used as a protein loading control. (d) Cells were transfected with si Ctrl or si Bid for 48 h and treated with genistein (30 μM) for 72 h. Cell death was detected with ELSIA. Graphs showing results of quantitative analyses (n=3, mean±S.D., **P<0.01). Representative results of three experiments with consistent results are shown
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fig6: Bid activation mediates AIF, endoG release, and Bak activation. (a) Cell were treated with genistein at indicated time and collected with western blot analysis. β-Actin was used as a protein loading control. (b) Cells were treated with genistein for 72 h and subjected to subcellular fractionation. The cytosolic (C) or mitochondrial (M) fractions were immunoblotted for Bid and tBid detection. (c) Cells were transfected with Bid and Ctrl siRNA for 48 h, and then treated with genistein for 72 h. One portion of cells was subjected to subcellular fraction to detect the release of AIF, endoG, and Cyt c. The other portion of cells was collected to detect Bak and caspase activation. β-Actin was used as a protein loading control. (d) Cells were transfected with si Ctrl or si Bid for 48 h and treated with genistein (30 μM) for 72 h. Cell death was detected with ELSIA. Graphs showing results of quantitative analyses (n=3, mean±S.D., **P<0.01). Representative results of three experiments with consistent results are shown
Mentions: Previous studies have demonstrated that activated Bid can mediate Bak activation and the release of AIF and endoG.38, 39, 40 We then detected whether Bid contributes to genistein-induced cell apoptosis. We first detected Bid expression and activation. Our data confirmed that Bid was cleaved into tBid (Figure 6a), which is the active truncated form of Bid,38 during the time course of genistein treatment. We also found that tBid translocated into the mitochondria from the cytosol (Figure 6b), which could induce the release of mitochondrial proteins.41 We then knocked down the expression of Bid with siRNAs. Our data revealed that Bid expression was inhibited by siRNA treatment (Figure 6c). Moreover, Bid knockdown affected Bak oligomerization; the release of Cyt c, AIF, and endoG; and caspase-3 cleavage in Bax or p53 KO cells (Figure 6c). Meanwhile, Bid knockdown also substantially decreased apoptosis in Bax or p53 KO cells treated with genistein (Figure 6d). We also transiently transfected Bid siRNAs into Bax KD/p53 KO or Bax KO/p53 KD cells. We found that Bid knockdown is sufficient to attenuate Bak oligomerization; the release of Cyt c, AIF, and endoG; and caspase-3 cleavage in cancer cells (Supplementary Figures 1C and D). These results demonstrated that Bid could upregulate Bak activation; the release of AIF and endoG; and subsequently apoptosis.

Bottom Line: Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation.Conversely, AIF and endoG knockdown had little effect on Bak activation.Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Abdominal Oncology, State Key Laboratory of Biotherapy and Cancer Center, Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, 17# People's South Road, Chengdu, Chengdu 610041, PR China.

ABSTRACT
Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.

No MeSH data available.


Related in: MedlinePlus