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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

The interaction of Bak or AIF and endoG knockdown. (a) Cells were transfected with Ctrl or Bak siRNA for 48 h and treated with genistein (30 μM) for 48 h. Treated cells were used to detect Bak, AIF, and endoG expression. (b) Cells were transfected with Ctrl or double AIF or endoG siRNA for 48 h and treated with genistein (30 μM) for 48 h. Treated cells were used to detect Bak, AIF, and endoG expression. β-Actin was used as a protein loading control. (c) Cells were treated with single AIF, endoG, Bak, Ctrl siRNA, double AIF/endoG, or triple AIF/endoG/Bak 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)
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fig5: The interaction of Bak or AIF and endoG knockdown. (a) Cells were transfected with Ctrl or Bak siRNA for 48 h and treated with genistein (30 μM) for 48 h. Treated cells were used to detect Bak, AIF, and endoG expression. (b) Cells were transfected with Ctrl or double AIF or endoG siRNA for 48 h and treated with genistein (30 μM) for 48 h. Treated cells were used to detect Bak, AIF, and endoG expression. β-Actin was used as a protein loading control. (c) Cells were treated with single AIF, endoG, Bak, Ctrl siRNA, double AIF/endoG, or triple AIF/endoG/Bak 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)

Mentions: Our study revealed that Bak could induce the release of Cyt c from the mitochondria to the cytosol; thus, we detected whether Bak induced the release of AIF and endoG during apoptosis. We knocked down the expression of Bak with siRNAs in cancer cells. We found that Bak knockdown had little effect on the release of AIF and endoG (Figure 5a). Similarly, AIF and endoG knockdown had little effect on Bak activation (Figure 5b). These results indicate that Bak activation and the release of AIF and endoG are likely parallel apoptotic events. Indeed, we found that a Bak, AIF, and endoG triple knockdown almost completely inhibited apoptosis in cells treated with genistein (Figure 5c). We speculated that the other factors can cause Bak activation as well as the release of AIF and endoG during 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)

The interaction of Bak or AIF and endoG knockdown. (a) Cells were transfected with Ctrl or Bak siRNA for 48 h and treated with genistein (30 μM) for 48 h. Treated cells were used to detect Bak, AIF, and endoG expression. (b) Cells were transfected with Ctrl or double AIF or endoG siRNA for 48 h and treated with genistein (30 μM) for 48 h. Treated cells were used to detect Bak, AIF, and endoG expression. β-Actin was used as a protein loading control. (c) Cells were treated with single AIF, endoG, Bak, Ctrl siRNA, double AIF/endoG, or triple AIF/endoG/Bak 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)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4632302&req=5

fig5: The interaction of Bak or AIF and endoG knockdown. (a) Cells were transfected with Ctrl or Bak siRNA for 48 h and treated with genistein (30 μM) for 48 h. Treated cells were used to detect Bak, AIF, and endoG expression. (b) Cells were transfected with Ctrl or double AIF or endoG siRNA for 48 h and treated with genistein (30 μM) for 48 h. Treated cells were used to detect Bak, AIF, and endoG expression. β-Actin was used as a protein loading control. (c) Cells were treated with single AIF, endoG, Bak, Ctrl siRNA, double AIF/endoG, or triple AIF/endoG/Bak 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)
Mentions: Our study revealed that Bak could induce the release of Cyt c from the mitochondria to the cytosol; thus, we detected whether Bak induced the release of AIF and endoG during apoptosis. We knocked down the expression of Bak with siRNAs in cancer cells. We found that Bak knockdown had little effect on the release of AIF and endoG (Figure 5a). Similarly, AIF and endoG knockdown had little effect on Bak activation (Figure 5b). These results indicate that Bak activation and the release of AIF and endoG are likely parallel apoptotic events. Indeed, we found that a Bak, AIF, and endoG triple knockdown almost completely inhibited apoptosis in cells treated with genistein (Figure 5c). We speculated that the other factors can cause Bak activation as well as the release of AIF and endoG during 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