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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

AIF and endoG has an important role in apoptosis. (a) Cells were treated with genistein (30 μM) for 72 h, and subjected to subcellular fractionation. The cytosolic or mitochondrial fractions were immunoblotted for AIF and endoG detection. β-Actin and Cox IV were used as a protein loading control. (b) Cells were transfected with AIF or endoG siRNA for 48 h, and then transfected cells were immunoblotted for AIF and endoG detection. (c) HCT116 Bax KO or p53 KO cells were transfected with AIF and siRNA for 48 h, and then transfected cells were immunoblotted for AIF and endoG detection. β-Actin was used as a protein loading control. (d) HCT116 Bax KO cells were transfected with AIF, endoG siRNA, or double siRNA for 48 h, and then cells were treated with genistein for 72 h. Collected cells were stained with Annexin V for apoptosis analysis. (e) Cells were transfected with AIF, endoG siRNA, or double siRNA for 48 h, and then treated with genistein for 72 h. Cell apoptosis was quantitatively detected by a cell death ELISA kit as described in Materials and methods. Graphs showing results of quantitative analyses (n=3, mean±S.D., *P<0.05). Representative results of three experiments with consistent results are shown
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fig4: AIF and endoG has an important role in apoptosis. (a) Cells were treated with genistein (30 μM) for 72 h, and subjected to subcellular fractionation. The cytosolic or mitochondrial fractions were immunoblotted for AIF and endoG detection. β-Actin and Cox IV were used as a protein loading control. (b) Cells were transfected with AIF or endoG siRNA for 48 h, and then transfected cells were immunoblotted for AIF and endoG detection. (c) HCT116 Bax KO or p53 KO cells were transfected with AIF and siRNA for 48 h, and then transfected cells were immunoblotted for AIF and endoG detection. β-Actin was used as a protein loading control. (d) HCT116 Bax KO cells were transfected with AIF, endoG siRNA, or double siRNA for 48 h, and then cells were treated with genistein for 72 h. Collected cells were stained with Annexin V for apoptosis analysis. (e) Cells were transfected with AIF, endoG siRNA, or double siRNA for 48 h, and then treated with genistein for 72 h. Cell apoptosis was quantitatively detected by a cell death ELISA kit as described in Materials and methods. Graphs showing results of quantitative analyses (n=3, mean±S.D., *P<0.05). Representative results of three experiments with consistent results are shown

Mentions: Our experiments also found that genistein induced the release of AIF and endoG in Bax or p53 KO cancer cells (Figure 4a). AIF and endoG are important factors for mitochondrial apoptosis and can mediate caspase-dependent and -independent cell death.3, 37 We then used AIF siRNAs (si AIF), endoG siRNAs (si endoG), or both siRNAs (si AIF/endoG) to decrease AIF and endoG expression in cancer cells (Figures 4b and c). We found that knockdown of either AIF or endoG alone could not efficiently decrease apoptosis in cells treated with genistein. However, the AIF and endoG double knockdown substantially decreased apoptosis (Figures 4d and e). These results indicate that the release of mitochondrial AIF and endoG is necessary for genistein-induced Bax/p53-independent cell 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)

AIF and endoG has an important role in apoptosis. (a) Cells were treated with genistein (30 μM) for 72 h, and subjected to subcellular fractionation. The cytosolic or mitochondrial fractions were immunoblotted for AIF and endoG detection. β-Actin and Cox IV were used as a protein loading control. (b) Cells were transfected with AIF or endoG siRNA for 48 h, and then transfected cells were immunoblotted for AIF and endoG detection. (c) HCT116 Bax KO or p53 KO cells were transfected with AIF and siRNA for 48 h, and then transfected cells were immunoblotted for AIF and endoG detection. β-Actin was used as a protein loading control. (d) HCT116 Bax KO cells were transfected with AIF, endoG siRNA, or double siRNA for 48 h, and then cells were treated with genistein for 72 h. Collected cells were stained with Annexin V for apoptosis analysis. (e) Cells were transfected with AIF, endoG siRNA, or double siRNA for 48 h, and then treated with genistein for 72 h. Cell apoptosis was quantitatively detected by a cell death ELISA kit as described in Materials and methods. Graphs showing results of quantitative analyses (n=3, mean±S.D., *P<0.05). Representative results of three experiments with consistent results are shown
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: AIF and endoG has an important role in apoptosis. (a) Cells were treated with genistein (30 μM) for 72 h, and subjected to subcellular fractionation. The cytosolic or mitochondrial fractions were immunoblotted for AIF and endoG detection. β-Actin and Cox IV were used as a protein loading control. (b) Cells were transfected with AIF or endoG siRNA for 48 h, and then transfected cells were immunoblotted for AIF and endoG detection. (c) HCT116 Bax KO or p53 KO cells were transfected with AIF and siRNA for 48 h, and then transfected cells were immunoblotted for AIF and endoG detection. β-Actin was used as a protein loading control. (d) HCT116 Bax KO cells were transfected with AIF, endoG siRNA, or double siRNA for 48 h, and then cells were treated with genistein for 72 h. Collected cells were stained with Annexin V for apoptosis analysis. (e) Cells were transfected with AIF, endoG siRNA, or double siRNA for 48 h, and then treated with genistein for 72 h. Cell apoptosis was quantitatively detected by a cell death ELISA kit as described in Materials and methods. Graphs showing results of quantitative analyses (n=3, mean±S.D., *P<0.05). Representative results of three experiments with consistent results are shown
Mentions: Our experiments also found that genistein induced the release of AIF and endoG in Bax or p53 KO cancer cells (Figure 4a). AIF and endoG are important factors for mitochondrial apoptosis and can mediate caspase-dependent and -independent cell death.3, 37 We then used AIF siRNAs (si AIF), endoG siRNAs (si endoG), or both siRNAs (si AIF/endoG) to decrease AIF and endoG expression in cancer cells (Figures 4b and c). We found that knockdown of either AIF or endoG alone could not efficiently decrease apoptosis in cells treated with genistein. However, the AIF and endoG double knockdown substantially decreased apoptosis (Figures 4d and e). These results indicate that the release of mitochondrial AIF and endoG is necessary for genistein-induced Bax/p53-independent cell 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