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

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.

W Guo 1,4 , Y Zhang 2,4 , Z Ling 3,4 , X Liu 1 , X Zhao 1 , Z Yuan* ,1 , C Nie* ,1 and Y Wei 1 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 genisteininduced 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. Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria. 1 Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG. [2][3][4] Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria. 2,[5][6][7] Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/ Bcl-xL/Mcl-1. [8][9][10][11][12] Upon apoptosis, the 'activator' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation. 8,11,12 Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation. 8,9 Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s, 13 liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization. 12,14,15 Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon 16,17 and prostate cancers, 18,19 contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis 20 and is necessary for tBid-induced cancer cell apoptosis. 21 Loss of Bax has been reported to promote tumor development in animal models. 22 Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers. [23][24][25] p53 has been reported to be a tumor suppressor, 26 and its mutant can cause chemoresistance in cancer cells. [27][28][29] Moreover, p53 is often inactivated in solid tumors via deletions or point mutations. 30,31 Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.
Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells. 32 Some studies have revealed that Bak is a determinant of cancer cell apoptosis. 33,34 Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction. 33,35 In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.

Results
Genistein induces Bax and p53-independent apoptosis in cancer cells. We first determined the apoptotic effects of genistein in HCT116 Bax KO and p53 KO cells. We treated the cells with genistein at the indicated time, and apoptosis was assessed by a DNA fragmentation ELISA. As depicted in Figure 1a, Figure 1A). Flow-cytometry analysis demonstrated that Bax deficiency also had little effect on cell death in p53 KO cells (Figure 2d). These results indicate that genistein caused apoptosis independent of Bax and p53.
Bak activation is important for genistein-induced apoptosis. Because Bak contributes to Bax-independent cell death, 33 Figure 1B). To further investigate the contribution of Bak to cell apoptosis, we transfected Bak siRNAs (si Bak) into HCT116 Bax KO or p53 KO cells to knock down Bak expression. Western blot analysis confirmed the lack of Bak expression in cancer cells ( Figure 3b). As illustrated in Figures 3b and c, Bak siRNAs efficiently decreased Cyt c release and caspase-3 activation. Meanwhile, Bak knockdown also decreased cell death in cancer cells treated with genistein ( Figure 3f). Further experiments revealed that Bcl-2 overexpression obviously inhibited Bak oligomerization and the conformational change as well as apoptosis by genistein (Figures 3d, e, and g). These results suggest that Bak has an important role in genisteininduced Bax/p53-independent cell apoptosis.
The release of AIF and endoG from the mitochondria to the cytosol mediates apoptosis. 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.
Bid causes Bak activation and the release of AIF and endoG during apoptosis. 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  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 genisteininduced 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.

Akt inactivation mediates Bid-induced cell apoptosis.
Previous studies have revealed that Akt activation could inhibit tBid appearance and Bid-induced apoptosis. 42,43 Moreover, a previous study showed that genistein induced apoptosis through the Akt pathway in anaplastic large-cell lymphoma. 44 Thus, we speculated that Akt is the upstream apoptotic regulator in our experiments. We first detected phosphorylated Akt (p-Akt) and total Akt (t-Akt) expression. We found that p-Akt was decreased during the time course of genistein treatment. Meanwhile, t-Akt had little change after treatment (Figure 7a). We then further detected the effects of Akt on apoptosis. We transfected a constitutively active Akt1 construct into cancer cells and found that Akt1 overexpression increased t-Akt in cells (Figure 7b). Moreover, Akt1 overexpression obviously inhibited the appearance of tBid, Bak oligomerization, caspase-3 cleavage, and the release of AIF and endoG (Figure 7c). Our experiments also revealed that LY294002, a PI3K/Akt pathway inhibitor that inactivates Akt, efficiently increased Bid activation, Bak oligomerization, caspase-3 cleavage, and the release of AIF and endoG (Figure 7c). These results indicate that Akt inactivation mediates Bid-induced downstream apoptotic events instigated by genistein.
Caspase-3 enhances Bid-induced Bak activation and the release of AIF and endoG. Previous reports have revealed that downstream apoptotic factors, such as Smac and caspase-9, could mediate upstream apoptotic events through positive feedback. 2   caspase-3 could enhance Bak activation. We first knocked down caspase-3 with siRNAs (si Cas-3). We found that caspase-3 expression was inhibited by siRNAs in cancer cells (Figure 8a). Our data indeed revealed that caspase-3 knockdown decreased Cyt c release and Bak oligomerization ( Figure 8b). However, interestingly, caspase-3 knockdown also obviously decreased the release of AIF and endoG (Figure 8b). Moreover, caspase-3 knockdown had little effect on Bid activation (Figure 8c). These results suggest that downstream caspase-3 could enhance Bid-induced subsequent apoptotic events, but did not affect Bid activation.
To further examine the effect of caspase-3 during apoptosis, we transfected an XIAP construct into cancer cells to antagonize caspase-3 activation. The cleavage of caspase-3 could be blocked by XIAP overexpression. XIAP overexpression could also decrease apoptosis. 46 Our data revealed that the XIAP construct increased XIAP overexpression and decreased apoptosis in cells treated with genistein ( Figure 8d). XIAP overexpression inhibited caspase-3 cleavage, Bak oligomerization, and the release of Cyt c. XIAP overexpression also decreased the release of AIF and endoG from the mitochondria (Figure 8e). These results further revealed that downstream caspase-3 could enhance Bidinduced apoptosis.

Discussion
Genistein, a naturally occurring isoflavone and prominent isoflavonoid found in soy products, is of interest because of its potent chemotherapeutic activities, including its ability to inhibit cell growth and induce apoptosis in a wide variety of cultured cancer cells. 44,[47][48][49][50][51] Moreover, genistein has chemopreventive effects in several human malignancies, including cancers of the breast, colon, and prostate. 49,52 Previous studies have revealed that genistein induces apoptosis in breast cancer cells by upregulating proapoptotic Bcl-2 proteins, such as Bax or Bak, and downregulating anti-apoptotic factors, such as Bcl-xL or Bcl-2. 48,53 Some studies have also revealed that genistein involves the Akt signaling pathway in apoptosis. 44,47 However, the exact function of genistein on cancer chemoresistance is still unknown.
In the present study, we first demonstrated that genistein can induce apoptosis independent of Bax and p53. Previous studies have demonstrated that genistein induced apoptosis independent of p53. 53 We focused on genistein and found out that genistein can induce cell death in the absence of Bax and p53. We found that Bak compensates for the loss of Bax in cancer cell apoptosis. However, Bak knockdown only partially    These results suggest that Bak may not be the sole decisive factor for genistein-induced apoptosis. Mitochondria respond to multiple death stimuli including those involving pro-apoptotic Bcl-2 family proteins, such as Bax and Bak, which induce mitochondrial membrane permeabilization and cause the release of apoptotic molecules. 2,6,33,38 Therefore, we speculated that Bak could mediate the release of some mitochondrial apoptotic factors, such as Cyt c, AIF, and endoG. Indeed, our data revealed that the release of AIF and endoG from that mitochondria was an important part of genistein-induced cell death. It has been reported that AIF and endoG translocate to the nucleus to trigger caspase-independent cell death. 37 Our data also revealed that Bak mediated the release of Cyt c. Bak knockdown efficiently inhibited the release of Cyt c. However, interestingly, Bak knockdown had little effect on the release of AIF and endoG. Conversely, an AIF and endoG double knockdown did not affect Bak activation. These results lead us to believe that another upstream factor mediates genisteininduced apoptosis.
Previous studies have also revealed that the Akt signaling pathway contributes to genistein-induced apoptosis 44,47 Our data further demonstrated that the Akt-Bid signaling pathway upregulates Bak activation and the release of AIF and endoG. Further experiments revealed that downstream caspase-3 could enhance Bak activation and the release of AIF and endoG. Arnoult et al. 3 revealed that mitocho-ndrial`release of AIF and endoG required caspase activation. Our study also confirmed this conclusion. Moreover, we also proved that Bak did not contribute to the release of AIF and endoG. Arnoult et al. also found that tBid could induce the release of Cyt c, but not AIF and endoG. However, our research revealed that tBid could induce the release of Cyt c, AIF, and endoG, as described previously. 39,54 Furthermore, we also found that caspase-3 inhibition decreased the release of mitochondrial apoptotic factors, whereas previous studies have shown that caspase inhibitors prevent the mitochondrial release of AIF and endoG, but not Cyt c. This result further suggests that we have found a different mechanism underlying the regulation of Bak activation and the release of AIF and endoG.
In conclusion, we explored, for the first time, the detailed molecular mechanisms of Bid-induced apoptosis after genistein treatment and demonstrate that caspase-3 could enhance genistein-induced apoptosis. Our findings explain how genistein induces apoptosis in Bax-and p53-deficient cancer cells. The Akt-Bid pathway initiates Bak activation and downstream caspase processing. Meanwhile, the Akt-Bid pathway also causes the release of AIF and endoG, which induces caspaseindependent cell death. Downstream caspase-3 reinforces Bak activation and the release of AIF and endoG through positive amplification loops (Figure 9). These positive regulatory feedback loops sensitize cancer cells to treatment with genistein. Genistein seems to have broader implications, even in a clinical perspective.  The constitutively active Akt1 construct HA-PKB-T308D/S473D was obtained as previously described. 5 1 μg of plasmid DNA or 100 nM siRNA and 4 μl of DMRIE-C reagent (Invitrogen, Carlsbad, CA, USA) were used per coverslip. The cells were incubated for 4 h in the transfection mixture, which was then replaced with fresh culture medium. For stable transfection, cells were transfected with the constructs as previously described. 2,6 Positive clones were selected with 600 μg/ml hygromycin (Invitrogen) for several weeks.
Apoptosis assays. Three methods were used to assess PL-induced apoptotic cell death: detection of DNA fragmentation with the Cell Death Detection ELISA kit (Roche Diagnostics, Castle Hill, NSW, Australia), Western blot analysis of caspase-3 cleavage, Cyt c, AIF, or endoG release and measurement of apoptotic cells by flow cytometry (Annexin/PI, Bak activation, or activated caspase-3). The Cell Death Detection ELISA quantified the apoptotic cells by detecting the histone-associated DNA fragments (mono-and oligo-nucleosomes) generated by the apoptotic cells. 5 Cell fractionation and western blot analysis. Mitochondria and cytoplasm from cells were fractionated by differential centrifugation as previously described. 5 Cytosol, mitochondria, total lysates, and immunoprecipitates were analyzed by western blot with antibody dilutions as follows: actin at 1:20 000; AIF, caspase-3, endoG, p53, Akt, p-Akt, Bid, XIAP, CoxIV at 1:2000; and Bax, Bak, Cyt c at 1:1000.
Bak oligomerization and bak conformational change. Cells were treated with agents and incubated with 1 mM BMH in 10% DMSO or DMSO alone for 30 min at 25°C. After centrifugation at 5000 × g for 25 min at 4°C, the reaction was split into supernatant and pellet fractions. The pelleted material (10 mg total protein) was separated by SDS-PAGE and immunoblotted with anti-Bak antibody to detect Bak oligomerization. 56 Bak conformational change was performed as described. 57 Briefly, cells were lysed in 1% CHAPS buffer, and 250 μg of protein was immunoprecipitated using anti-Bak (Ab-2), which only recognizes Bak that has undergone a conformation change. Immunoprecipitated protein was then subjected to immunoblot analysis by using anti-Bak as primary antibodies.
Statistical analysis. Statistical analysis of the differences between the groups was performed using the Student's t-test with Po0.05 considered as statistically significant.

Conflict of Interest
The authors declare no conflict of interest.  Figure 9 A diagram of signaling pathway for genistein-induced apoptosis in Bax and p53-independent manner. Genistein first induced Akt dephosphorylation. p-Akt decrease activated Bid to tBid. Bid cleavage induced the release of AIF and endoG as well as Bak activation. Activated Bak triggered caspase-dependent apoptosis, while AIF and endoG release initiated caspase-independent cell death. Activated caspase-3 mediated two positive feedback loops to enhance apoptosis. On one hand, caspase-3 feedback enhances Bak activation (the blue solid line); on the other hand, caspase-3 probably strengthened the release of AIF and endoG (the blue dotted line) Caspase-3 enhances Bid-dependent cell death W Guo et al