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. Author manuscript; available in PMC: 2023 Nov 1.
Published in final edited form as: J Cell Physiol. 2022 Aug 22;237(11):4180–4196. doi: 10.1002/jcp.30863

Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis

Jin Hong Kim 1, Abdo J Najy 2,3, Jian Li 4, Xu Luo 4, Hyeong-Reh C Kim 2,3, M Haroon A Choudry 1, Yong J Lee 1
PMCID: PMC9691566  NIHMSID: NIHMS1829796  PMID: 35994698

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor-mediated extrinsic apoptosis, specifically in cancer cells, and Bid (BH3 interacting-domain death agonist) plays an important role in TRAIL-induced apoptosis. Ferroptosis is a newly defined form of regulated cell death known to be distinct from other forms of cell death. However, our previous studies have shown that ferroptosis shares common pathways with other types of programed cell death such as apoptosis. In this study, we investigated the role of Bid in the crosstalk between the ferroptotic agent-induced endoplasmic reticulum (ER) stress response and TRAIL-induced apoptosis. When human colorectal carcinoma HCT116 cells were treated with the ferroptosis-inducing agents artesunate and erastin in combination with TRAIL, TRAIL-induced activation of caspase-8 was enhanced, and subsequently, the truncation of Bid was increased. Similar results were observed when ovarian adenocarcinoma OVCAR-3 cells were treated with the ferroptotic agents in combination with TRAIL. Results from studies with Bid mutants reveal that the truncation of Bid and the presence of intact BH3 domains are critical for synergistic apoptosis. Non-functional Bid mutants were not able to activate the mitochondria-dependent apoptosis pathway, which is required for the conversion of p19 to p17, the active form of caspase-3. These results indicate that Bid plays a critical role in the crosstalk between the ferroptotic agent-induced ER stress response and TRAIL-induced apoptosis.

Keywords: TRAIL cytotoxicity, apoptosis, ferroptosis, endoplasmic reticulum stress, Bid

Introduction

Death receptor-mediated extrinsic apoptosis is initiated by extracellular tumor necrosis factor (TNF) super family ligands, including TNF-α, Fas ligand, and TNF-related apoptosis-inducing ligand (TRAIL) (Ashkenazi et al., 1998). TRAIL especially has been known as an attractive candidate for antitumor therapies because it selectively induces apoptosis in tumor cells, but not in normal cells (Ashkenazi et al., 1999; Walczak et al., 1999). Interaction between TRAIL and its receptors, death receptor 4 and death receptor 5 (DR5), induces receptor trimerization and recruitment of Fas-associated death domain (FADD) via interaction between death domains of the receptors and FADD. Then, the death effector domain of FADD engages procaspase-8 and forms the death-inducing signaling protein complex (DISC) (Ganten et al., 2004; Sprick et al., 2000). In DISC, procaspase-8 is activated by transcatalytic and autocatalytic cleavage and the activated caspase-8 triggers activation of downstream executioner caspases such as caspase-3, -6, and -7 (P. Li et al., 1997). The activation of caspase-8 is inhibited by cellular FLICE-like inhibitory protein (cFLIP), which is similar to procaspase-8 but lacks proteolytic activity (Irmler et al., 1997; Krueger et al., 2001). In addition, the activated caspase-8 is able to cleave Bid (BH3 interacting-domain death agonist) at Asp-60, a BH3-only pro-apoptotic protein of the Bcl-2 family (Gross et al., 1999; H. Li et al., 1998; Luo et al., 1998). The cleaved C-terminal fragment of Bid migrates to the mitochondria and induces Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP) (Wei et al., 2000; Wei et al., 2001). Although the translocation mechanism of truncated Bid (tBid) into the mitochondria is still unclear, previous studies have suggested that translocation of tBid is mediated by membrane or lipid (Lovell et al., 2008; Shamas-Din et al., 2013). After Bid is cleaved by activated caspase-8, it keeps its protein structure as a complex without dissociation of the cleaved parts by strong hydrophobic interaction (Chou et al., 1999; Kudla et al., 2000; McDonnell et al., 1999). Interaction of the cleaved Bid complex with membrane or liposome dissociates the complex and tBid is embedded on the mitochondrial outer membrane. Then, tBid experiences conformational change and exposes the BH3 domain, which can activate the mitochondria-dependent apoptosis pathway (H. Kim et al., 2009). Release of cytochrome c by MOMP facilitates apoptosome formation of Apaf1/procaspase-9 and then activates caspase-9 and caspase-3 sequentially (Baliga et al., 2003). Interestingly, TRAIL-induced apoptosis can be promoted by various agents such as matrix metalloprotease inhibitors (Nyormoi et al., 2003), other cytokines (Park et al., 2002), ionizing radiation (Chinnaiyan et al., 2000), and chemotherapeutic agents (Keane et al., 1999; Y. H. Kim et al., 2006; Nagane et al., 2000).

Ferroptosis is known as a unique, iron-dependent form of regulated cell death that is distinct from apoptosis, necrosis, and other forms of cell death in morphology, gene expression, and biochemistry (Dixon et al., 2012). Ferroptosis is driven by lipid peroxidation, which is caused by amino acid metabolism and accumulation of iron. The glutamate-cystine anti-transporter system, known as system Xc−, replaces intracellular glutamate with extracellular cystine. Cystine is converted to cysteine, which is used for the synthesis of glutathione (GSH). GSH plays a role in maintaining redox balance, and the concentration of GSH is controlled by glutathione peroxidase 4 (GPX4). Depletion of GSH and inactivation of GPX4 induce lipid peroxidation (Dixon et al., 2012; Lin et al., 2020; W. S. Yang et al., 2014). In addition, degradation of the iron storage protein ferritin by lysosomal autophagy, known as ferritinophagy, releases iron and breaks iron homeostasis. Accumulation of iron generates reactive oxygen species by Fenton reaction and then also causes lipid peroxidation (Braughler et al., 1986; Gao et al., 2016). Erastin (ERA) is one of the ferroptosis-inducing agents that inhibits system Xc− (Dixon et al., 2012). Unlike ERA, artesunate (ART) affects the lysozyme and induces the release of iron by lysosomal autophagy (N. D. Yang et al., 2014).

Although ferroptosis is distinct from other forms of regulated cell death, recent studies have suggested that ferroptosis shares common pathways with other cell death types (Hong et al., 2017; Lee et al., 2018; Lee et al., 2019; Linkermann et al., 2014; Nikoletopoulou et al., 2013). Previous studies report that ferroptosis-inducing agents cause the endoplasmic reticulum (ER) stress response and upregulate ER stress markers like phosphorylation of eIF2α and expression of ATF4 (activating transcription factor 4) and CHAC1 (cation transport regulator homolog 1) genes (Dixon et al., 2014; Rahmani et al., 2007). Data from microassays support the finding that ferroptosis-inducing agents promote expression of ATF4-dependent genes (Hong et al., 2017; Ohoka et al., 2005). The ATF4-CHOP (C/EBP-homologous protein) signaling pathway upregulates several pro-apoptotic proteins such as p53 upregulated modulator of apoptosis (PUMA), GADD34, Bim, and NOXA (Ghosh et al., 2012; Urra et al., 2013). In addition, CHOP binds to the promoter of DR5, also known as TRAIL-R2 (tumor necrosis factor-related apoptosis inducing ligand receptor 2) and induces upregulation of DR5 protein level (Lee et al., 2019; Lu et al., 2014; Martin-Perez et al., 2012; Sun et al., 2007; Yamaguchi et al., 2004). It is possible that these regulations by the ER stress response during ferroptosis play an important role in the crosstalk between ferroptosis and apoptosis.

In this study, we observed that Bid is a key molecule that regulates the crosstalk between the ferroptotic agent-induced ER stress response pathways and the TRAIL-induced mitochondria-dependent apoptotic pathways. Truncation of Bid and interaction between BH3 domains are especially critical for the synergistic apoptosis.

Materials and Methods

1. Cell lines and cell culture conditions

The human colorectal carcinoma HCT116 cell line and the ovarian adenocarcinoma OVCAR-3 cell line were obtained from American Type Culture Collection (ATCC, Manassas, VA). Bid-deficient HCT116 cells and Bid knockout (KO) HCT116 cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were provided by Dr. Luo (University of Nebraska Medical Center, Omaha, NE). CHOP-deficient (CHOP−/−) and corresponding wild-type (WT) mouse embryonic fibroblast (MEF) cell lines were provided by Dr. Randal J. Kaufman (Sanford Burnham Medical Research Institute, CA). HCT116 cells were maintained in McCoy’s 5A medium with 10% fetal bovine serum. OVCAR-3 cells were maintained in RPMI 1640 medium with 20% fetal bovine serum, 10 mM HEPES buffer, 1 mM sodium pyruvate, 25 mM glucose, 17.9 mM sodium bicarbonate, and 0.01 mg/ml human recombinant insulin. MEFs were maintained in DMEM medium with 10% fetal bovine serum. Three types of cells were incubated in a humidified atmosphere of 5% CO2 at 37°C.

2. Chemicals and reagents

Human recombinant TRAIL was produced as previously described (Hong et al., 2017). Mouse recombinant TRAIL was purchased from R&D systems (Minneapolis, MN). ART, ERA, and Z-IETD-FMK were purchased from Selleckchem (Houston, TX).

3. Cell death and viability assay

For quantification of the cell death rate, cells were trypsinized and stained with trypan blue followed by counting with a hemacytometer under a microscope. For fluorescence imaging, propidium iodide (PI) (Thermo Fisher Scientific, Waltham, TX) was used to stain dead cells. The nuclei of dead and live cells were counterstained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, TX).

4. Western blot analysis and antibodies

Immunoblotting was carried out as previously described (Kalimuthu et al., 2021). The following antibodies were used in this study: anti-PARP (#9532), anti-caspase-8 (#9746), anti-cleaved caspase-9 (#7237), anti-caspase-3 (#9664), anti-human Bid (#2002), anti-mouse Bid (#2003), anti-CHOP (#2895), and anti-DR5 (#69400) from Cell Signaling Technology (Danvers, MA); and anti-actin, goat anti-rabbit IgG-horseradish peroxidase (HRP), and goat anti-mouse IgG-HRP from Santa Cruz Biotechnology (Dallas, TX).

5. Densitometry analysis

To quantify the relative protein level, western blot analyses from three different experiments were performed and densitometry analysis from the interesting bands was performed using the ImageJ program.

6. Caspase 8 activity assay

Cell pellets were lysed with cell extract buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.5 mM EDTA, and 0.5% NP-40) for 30 minutes on ice. Then, samples were centrifuged at 15,000g for 10 minutes to collect soluble protein. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (ThermoFisher Waltham, MA). A total of 50 μg of protein was added to caspase buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 2.5 mM DTT, 25 μM Ac-IETD-AMC [Enzo Life Sciences, Farmingdale, NY]) and reaction incubated at 37°C for two hours. Caspase 8 activity was read using a Spectra Max Gemini fluorescence plate reader at 360 nm excitation and 460 nm emission (Molecular 25Devices, Menlo Park, CA). Samples were performed in triplicates and caspase 8 activity was plotted as average relative fluorescence units.

7. Mitochondria isolation

For mitochondrial fraction, cells were lysed by Dounce homogenizer in homogenization buffer (HB) containing 10 mM EGTA, 10 mM HEPES (pH 7.4), and 0.25 M sucrose and complete protease inhibitors. The cell lysates were centrifuged for 15 min at 1500×g at 4°C to remove nuclei. The supernatants were centrifuged for 15 min at 10,000g at 4°C and the pellet was saved. The mitochondrial pellets were resuspended in HB and then analyzed using immunoblotting.

8. Small interfering RNA

Bid small interfering RNA (siRNA) (ON-TARGETplus siRNA, Bid SMARTpool; L-004387-00-0005) and negative control siRNA (D-001810-10-05; ON-TARGETplus Non-targeting Control Pool) were purchased from Horizon Discovery. Cells were transfected with siRNA oligonucleotides using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. After 24 hours of transfection, cells were treated with ERA and TRAIL for further analysis.

9. JC-1 assay

To analyze mitochondrial membrane potential, cells were stained using the JC-1 Mitochondrial Membrane Potential Assay Kit (#10009172, Cayman Chemical, USA) according to the manufacturer’s instructions and examined using fluorescence microscopy.

10. Statistical analysis

All values are represented as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance or two-way analysis of variance followed by Tukey’s post hoc test as indicated using GraphPad Prism 8 software. p values of less than 0.05 were defined as statistically significant. Significance of p value is indicated as *P < 0.05; **P < 0.01; ***P < 0.001.

Results

Synergistic cytotoxicity induced by combinatorial treatment of ART and TRAIL is mediated through the Bid-dependent pathway

We previously reported that ferroptosis-inducing agents enhance TRAIL-induced apoptosis through crosstalk between the ER stress response signaling pathway and the Bax-mediated mitochondrial pathway (Lee et al., 2020). Since previous studies demonstrated that Bid is involved in TRAIL-induced mitochondria-dependent apoptosis, we investigated whether Bid plays an important role in the crosstalk between ferroptosis and TRAIL-induced apoptosis. For this study, human colon cancer HCT116 wild-type (WT) cells and their Bid-deficient (Bid KO) cells were treated with the ferroptotic agent ART in combination with TRAIL. In the first step, we observed cell morphological alterations under a light microscope during drug treatment (Figure 1A). HCT116 WT cells treated with both ART and TRAIL became round and detached compared to those treated with each single treatment. In contrast, there was no significant morphological alterations during single or combinatorial treatment in HCT116 Bid KO cells. To assess drug-induced cytotoxicity, we counted dead/live cells using trypan blue exclusion assay and observed PI/Hoechst stained cells under a fluorescence microscope. As shown in Figure 1B and 1C, a synergistic cell cytotoxic effect was observed with the combinatorial treatment compared to each single treatment in HCT116 WT cells. However, in HCT116 Bid KO cells, the combinatorial treatment did not induce the synergistic cytotoxicity. These results indicate that Bid is an essential molecule for the induction of synergistic cytotoxicity during combinatorial treatment.

Figure 1. Involvement of Bid in the enhancement of cytotoxicity during treatment of ART and TRAIL.

Figure 1.

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HCT116 wild-type (WT) and Bid knockout (KO) cells were pretreated with artesunate (ART, 50 μM) for 20 h and then exposed to human recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (2 ng/ml) for an additional 4 h. (A) Phase-contrast images were visualized under a bright-field microscope. Representative images are shown (scale bar: 100 μm). (B) Cell death was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two-way ANOVA followed by Tukey’s post hoc test was used. p-values: ***p < 0.001. (C) For fluorescence images, cells were stained with propidium iodide (PI) and Hoechst 33342. Representative images are shown (scale bar: 25 μm).

Combinatorial treatment with ferroptotic agent and TRAIL enhances activation of caspase-8 and truncation of Bid, which are important in the synergistic apoptosis

To confirm whether the combinatorial treatment-induced synergistic cytotoxicity is due to enhancement of apoptosis, we examined the activation of caspases and cleavage of poly (ADP-ribose) polymerase (PARP), hallmarks of apoptosis. When HCT116 WT cells were treated with ART and TRAIL, the synergistic apoptosis was observed in comparison to each single treatment, as assessed by increased cleavage of PARP. Activation of caspase-8/9/3 also increased during the combinatorial treatment. Moreover, the level of activated caspase-8-mediated tBid increased during the combinatorial treatment (Figure 2A,B). Most importantly, the combinatorial treatment increased the level of p17 fully mature caspase-3, which is important for apoptosis. Similar results were observed when HCT116 WT cells were treated with ERA and TRAIL (Figure 2C, D). In Bid KO cells, although activation of caspase-8 increased during the combinatorial treatment compared to only TRAIL treatment, only the p19 form and not the p17 form of caspase-3 was observed (Figure 2A,B). To examine the combinatorial treatment-induced mitochondrial damage, we used a JC-1 fluorescence probe to detect mitochondrial membrane potential (ΔΨm). In the JC-1 assay, the normal cells have high mitochondrial membrane potential and the JC-1 probe spontaneously forms complexes known as J-aggregates with intense red fluorescence. When cells undergo mitochondrial damage, the JC-1 probe remains in monomeric form with intense green fluorescence. ART-treated HCT116 WT and Bid KO cells showed both weak red fluorescence and weak green fluorescence. However, green fluorescence was stronger in the combined ART and TRAIL-treated HCT116 WT cells than in Bid KO cells. These results suggest that Bid plays an important role in the loss of mitochondrial membrane potential during the combinatorial treatment of ART and TRAIL (Figure 2E). To confirm the ferroptotic agents and TRAIL combinatorial treatment-induced synergistic apoptosis of other cancer cells, we employed ferroptotic agents, ART and ERA, and another cancer cell line, OVCAR-3. As shown in Figure 3, the combinatorial treatment of ERA and TRAIL induced synergistic cytotoxicity (Figure 3A) and increased apoptosis (increased cleavage of PARP, caspases, and Bid) (Figure 3B,C). Similar results were observed when OVCAR-3 WT cells were treated with ART and TRAIL (Figure 3D,E). The role of Bid in the combinatorial treatment of ERA and TRAIL-induced synergistic apoptosis was assessed using Bid knockdown OVCAR-3 cells. OVCAR-3 cells transfected with either negative control siRNA or Bid siRNA were treated with ERA and TRAIL. The amount of Bid protein was reduced by 40% in siRNA-transfected cells and synergistic apoptosis was also reduced in siRNA-transfected cells (Figure 3B). These results demonstrated that Bid plays an important role in the synergistic apoptosis of OVCAR-3 cells during treatment with ERA and TRAIL. Next, to examine whether truncation of Bid by activated caspase-8 is important in the synergistic apoptotic death, Z-IETD-FMK, a caspase-8 inhibitor, was employed. Despite treatment with ART and TRAIL, the synergistic cell cytotoxicity was inhibited by treatment with Z-IETD-FMK (Figure 4A). Moreover, cleavage of PARP, caspases, and Bid were completely inhibited by treatment with Z-IETD-FMK (Figure 4B,C). The effect of the caspase-8 inhibitor on the combinatorial treatment-induced caspase-8 activity was confirmed by quantitative analysis of caspase-8 activity. As shown in Figure 4D, Z-IETD-FMK inhibited activation of caspase-8 completely. These results demonstrated that truncation of Bid by caspase-8 activation is critical in synergistic apoptosis during treatment with ferroptosis-inducing agent and TRAIL.

Figure 2. Role of Bid in the synergistic interaction between ART/ERA and TRAIL in HCT116 cells.

Figure 2.

Figure 2.

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HCT116 WT and Bid KO cells were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. (A) Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies. (B) Densitometry analysis of the bands from western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two-way ANOVA followed by Tukey’s post hoc test was used. p-values: ***p < 0.001 vs. the Bid KO group. HCT116 WT cells were pretreated with ERA (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. (C) Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies. (D) Densitometry analysis of the bands from western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one-way ANOVA was used. p-values: ***p < 0.001 vs. the MOCK group. (E) Alteration of mitochondrial membrane potential was detected using JC-1 dye after treatment. Red and green fluorescence represents the aggregate and monomeric form of JC-1, respectively (scale bar: 50 μm).

Figure 3. Role of Bid in the synergistic interaction between ERA/ART and TRAIL in OVCAR-3 cells.

Figure 3.

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OVCAR-3 cells were transfected with negative control siRNA and Bid siRNA for 24 h. The transfected cells were pretreated with ERA (50 μM) for 20 h and then exposed to human recombinant TRAIL (50 ng/ml) for an additional 4 h. (A) Cell death was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two-way ANOVA followed by Tukey’s post hoc test was used. p-values: ***p < 0.001. (B) Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies. (C) Densitometry analysis of the bands from western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two-way ANOVA followed by Tukey’s post hoc test was used. p-values: ***p < 0.001 vs. the Bid siRNA group. OVCAR-3 WT cells were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (50 ng/ml) for an additional 4 h. (D) Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies. (E) Densitometry analysis of the bands from western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one-way ANOVA was used. p-values: ***p < 0.001 vs. the MOCK group.

Figure 4. Role of caspase-8 in the synergistic interaction between ART and TRAIL.

Figure 4.

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HCT116 WT cells were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. Caspase-8 inhibitor Z-IETD-FMK (20 μM) was added 2 h before TRAIL treatment. (A) Cell death was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one-way ANOVA was used. p-values: ***p < 0.001. (B) Whole-cell lysates were analyzed with immunoblotting assay using indicated antibodies. (C) Densitometry analysis of bands in western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one-way ANOVA was used. p-values: ***p < 0.001. (D) Caspase-8 activity was measured as described in Materials and Methods. All experiments were performed in triplicates and caspase 8 activity was plotted as average relative fluorescence units (RFUs).

Truncation of Bid and intact BH3 domain are important in synergistic apoptosis during the combinatorial treatment

Previous studies showed that Bid is able to be cleaved at Asp60 by activated caspase-8 and that the BH3 domain of Bid plays an important role in the mitochondria-dependent apoptosis pathway through interacting other Bcl-2 family proteins, including Bax and Bak (H. Kim et al., 2009; H. Li et al., 1998). To confirm the role of Bid truncation and interaction with other Bcl-2 family proteins in the synergistic apoptosis during treatment with ferroptosis-inducing agent and TRAIL, we used HCT116 Bid KO cells or their stable variants, which were created by infecting retroviral vectors containing control cDNA, Bid, Bid D60E (mutation at cleavage site), and Bid G94E (mutation in BH3 domain) (Huang et al., 2016). Figure 5 shows that synergistic apoptosis was observed in HCT116 WT and Bid-reconstituted HCT116 Bid KO cells during the combinatorial treatment with ART and TRAIL, as demonstrated by cleavage of PARP. Although activation of caspase-8 was enhanced during the combinatorial treatment in every cell line, no apoptosis was observed in Bid mutant-reconstituted HCT116 Bid KO cells. Bid D60E mutant cells failed to generate tBid. Unlike Bid D60E mutant cells, Bid G94E cells generated tBid by activated caspase-8. However, PARP cleavage was not observed in Bid G94E. These results suggest that truncation of Bid and its intact BH3 domain are required for the synergistic apoptosis by the combinatorial treatment.

Figure 5. Employment of Bid mutants for investigating the role of Bid in the synergistic apoptosis.

Figure 5.

Figure 5.

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HCT116 wild-type (WT) or Bid knockout (KO) cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. (A) Cell death was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two-way ANOVA followed by Tukey’s post hoc test was used. p-values: ***p < 0.001. (B, D) Cell lysates were analyzed with immunoblotting assay using indicated antibodies. (C, E) Densitometry analysis of bands in western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one-way ANOVA was used. p-values: **p < 0.01, ***p < 0.001 vs. the vector group in (C). p-values: ***p < 0.001 vs. the D60E group, ###p < 0.001 vs. the G94E group in (E).

Cleavage of Bid leads to translocation of tBid into the mitochondrial outer membrane, but not enough to induce mitochondria-dependent apoptosis

Previous studies showed that cleavage of Bid is a prerequisite to link death receptor-mediated intrinsic apoptosis (H. Li et al., 1998; Luo et al., 1998). Data from immunoblotting assay for mitochondria isolation show that tBid was produced in HCT116 WT, Bid/Bid KO, and Bid G94E/Bid KO cells and moved into mitochondria during the combinatorial treatment (Figure 6). As mentioned above, unlike WT and Bid/Bid KO cells, Bid G94E/Bid KO cells did not show apoptosis, as assessed by PARP cleavage. Cleaved caspase-9 signaling mediated by mitochondria-dependent apoptosis was also blocked in Bid G94E/Bid KO cells (Figure 5D,E). These results suggest that truncation of Bid leads to translocation of tBid into the mitochondrial outer membrane. However, it is not enough to form MOMP and induce the mitochondria-dependent apoptosis pathway without the functional BH3 domain of Bid in Bid G94E/Bid KO cells.

Figure 6. Translocation of truncated Bid to mitochondria during combined treatment of ART and TRAIL.

Figure 6.

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HCT116 WT or Bid KO cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. After treatments, mitochondrial and cytosol fractions were isolated as described in Materials and Methods. Each fraction was analyzed with immunoblotting assay using indicated antibodies.

Bid plays an important role in activating caspase-3 in the crosstalk between ferroptosis and TRAIL-induced apoptosis

As mentioned above, p17 mature caspase-3 was observed in HCT116 WT. However, unlike HCT116 WT cells, only the p19 intermediate form of caspase-3 was produced in Bid KO cells. Previous studies demonstrated that production of p17 mature caspase-3 is a sequential, two-step process and requires cytochrome c and dATP (Han et al., 1997; Liu et al., 1996). In particular, conversion of p19 to p17 contributes to its nuclear localization (Voss et al., 2007), and then the p17 mature caspase-3 cleaves PARP and promotes apoptosis (Lazebnik et al., 1994; Nicholson et al., 1995). Our results showed that the p17 form of caspase-3 was not observed in Vector/Bid KO, D60E/Bid KO, and G94E Bid KO cells and activation of caspase-9 mediated by the mitochondria-dependent apoptosis pathway was inhibited (Figure 5B-E). These results suggest that functional Bid is necessary for activation of caspase-3 in the crosstalk between ferroptosis and TRAIL-induced apoptosis.

Hierarchical profiles of ER stress response signaling pathways and cell death networks.

Our previous studies demonstrated that ferroptosis-inducing agents lead to activation of the ER stress response and the PERK-eIF2α-ATF4-CHOP signaling pathway, which results in upregulation of PUMA and DR5 (Hong et al., 2017; Lee et al., 2019). To examine the hierarchical profiles of ER stress response signaling pathways and cell death networks (Figure 10), we employed HCT116 WT and HCT116 Bid KO cells and then investigated the level of CHOP and DR5 expression during the combinatorial treatment. As shown in Figure 7, the level of CHOP and DR5 was upregulated during treatment with ART alone or combined ART and TRAIL, but not TRAIL alone. The upregulation of CHOP and DR5 occurred regardless of Bid status (deficient or mutant) or in the presence of caspase-8 inhibitor. These results suggest that since caspase-8 activation and Bid cleavage are downstream of DR5, an inhibitor of caspase-8 cannot block the ART-induced elevation of DR5 level. However, it can block ART-promoted TRAIL-induced apoptosis through inhibition of caspase-8 activity and Bid cleavage (Figure 4). To examine the role of CHOP in the cleavage of Bid during combined treatment of ART and TRAIL, we employed MEF WT and MEF CHOP KO cells. When these cells were treated with ART and TRAIL, Bid cleavage was observed in MEF WT cells, but not in MEF CHOP KO cells (Figure 8). These data suggest that CHOP plays an important role in the promotion of Bid truncation.

Figure 10. Diagram illustrating the role of Bid in the crosstalk between ferroptotic agent-induced ER stress response and TRAIL-induced apoptosis.

Figure 10.

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The ferroptotic agent ART-induced endoplasmic reticulum (ER) stress response enhances the activation of caspase-8 through promotion of DR5 expression and subsequently promotes cleavage of Bid. The cleaved Bid (cBid) translocates into the mitochondrial outer membrane. After dissociation of cBid by interaction with the mitochondrial outer membrane, truncated Bid (tBid) undergoes conformational change and exposes the BH3 domain, which interacts with Bax/Bak directly or indirectly and then induces mitochondrial outer membrane permeabilization (MOMP). Release of cytochrome c by MOMP forms the apoptosome. Conversion of p19 to p17, the active form of caspase-3, is mediated by mitochondria-dependent apoptosis. Fully activated caspase-3 induces apoptosis.

Figure 7. ART-induced endoplasmic reticulum stress response and DR5 expression.

Figure 7.

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HCT116 WT or Bid KO cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. Z-IETD-FMK (20 μM) was added 2 h before TRAIL treatment. (A-D) Cell lysates were analyzed with immunoblotting assay using indicated antibodies. Densitometry analysis of bands in western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one-way ANOVA was used. p-values: ***p < 0.001 vs. the MOCK group.

Figure 8. Truncation of Bid in CHOP-deficient cells.

Figure 8.

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MEF WT and CHOP KO cells were pretreated with ART (50 μM) for 20 h and then exposed to mouse recombinant TRAIL (100 ng/ml) for an additional 4 h. (A) Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies. (B) Densitometry analysis of the bands from western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two-way ANOVA followed by Tukey’s post hoc test was used. p-values: ***p < 0.001 vs. the CHOP KO group.

It is well known that activation of caspase-8 occurs in DISC, which consists of death receptor, FADD, procaspase-8, and cFLIP. A previous study showed that an ER stress-inducing agent downregulated cFLIP (Martin-Perez et al., 2012). To confirm the expression level of cFLIP during treatment with ART, HCT116 cells were treated with ART for various times. As shown in Figure 9, an ART-induced ER stress response occurred in a time-dependent manner. As treatment time increased, the level of CHOP expression increased. Interestingly, however, the level of cFLIPL and cFLIPS expression decreased (Figure 9). These results suggest that ART affects the expression of cFLIP, an anti-apoptotic molecule, and downregulation of cFLIP contribute to the synergistic interaction between ferroptotic agents and TRAIL.

Figure 9. Downregulation of cFLIP during ART treatment.

Figure 9.

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HCT116 cells were treated with ART (50 μM) for various times (4-24 h). Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies.

Discussion

In this study, we demonstrated that Bid plays a critical role in the crosstalk between ferroptosis and apoptosis. While the combinatorial treatment with ART/ERA and TRAIL induced synergistic apoptosis in HCT116 and OVCAR-3 cells, no apoptosis was observed in HCT116 Bid KO cells. The truncation of Bid and the presence of intact BH3 domain especially were necessary for the synergistic apoptosis. In addition, an increase in activated caspase-8 facilitated the cleavage of Bid, which is a key step to the connection between death receptor-mediated extrinsic apoptosis and mitochondria-dependent intrinsic apoptosis. This mitochondria-dependent apoptosis pathway promoted conversion of p19 caspase-3 to p17 mature caspase-3, which locates into the nucleus and cleaves PARP (Figure 10).

Our results reveal that ferroptosis-inducing agent enhances TRAIL-induced apoptosis via an increase in caspase-8 activation and subsequent Bid cleavage. Moreover, translocation of tBid to the mitochondria and interaction with the BH3 domain are obviously required for the synergistic apoptosis.

Our previous studies demonstrated that ferroptosis-inducing agents lead to activation of the ER stress response and the ATF4-CHOP signaling pathway, which results in upregulation of PUMA and DR5 (Hong et al., 2017; Lee et al., 2019). In this study, we confirmed upregulation of CHOP and DR5 during the treatment with ferroptosis-inducing agent ART (Figure 8). Moreover, we also showed downregulation of cFLIP during treatment with ART (Figure 9). Thus, it is possible that enhancement of caspase-8 activation during the combinatorial treatment is due not only to upregulation of DR5, but also to downregulation of cFLIP. The mechanism of alteration of gene expressions needs to be further examined in the near future.

The p17 form of activated caspase-3 is essential to cleave PARP and induce apoptosis because only the p17 form is able to translocate to the nucleus. A previous study showed that cytochrome c and dATP are required for activation of caspase-3 (Liu et al., 1996). Our results are consistent with those from previous reports showing that Bid is involved in activation of caspase-3. After truncation, tBid translocates into the mitochondrial outer membrane and induces the formation of MOMP by Bax/Bak oligomerization directly or indirectly. Through the MOMP, cytochrome c is released. Thus, the release of cytochrome c is dependent on the Bid-mediated pathway during combinatorial treatment with ferroptosis-inducing agent and TRAIL. These results clearly demonstrate that Bid plays a critical role in activating caspase-3 in the crosstalk between ferroptosis and TRAIL-induced apoptosis.

ART treatment induced cytotoxicity in HCT116 WT cells. Interestingly, ART-induced cytotoxicity was reduced in HCT116 Bid KO cells. Reduction of ART-induced cytotoxicity was observed in reconstituted Bid mutants, Bid D60E, and G94E/Bid KO cells. There observations are consistent with those from a previous study by Neitemeir at al. (2017). Their studies demonstrate that Bid links ferroptosis to mitochondrial damage in the oxidative neuronal cell death pathway. They treated HT-22 WT cells and Bid KO HT-22 cells with ERA and then observed that unlike WT cells, Bid KO cells preserved mitochondrial integrity and reduced cytotoxicity (Neitemeier et al., 2017). Nevertheless, whether Bid is involved in ferroptosis-induced mitochondrial damage in cancer cells is still ambiguous. This possibility needs to be further examined to understand the role of Bid in cancer cell ferroptosis.

In conclusion, we observed that the combinatorial treatment of ferroptosis-inducing agent and TRAIL induces synergistic apoptosis in human colon cancer cells and that the synergistic apoptosis is suppressed in Bid KO and Bid mutants cells. The ferroptosis-inducing agent leads to the ER stress response, upregulates DR5 expression, promotes activation of caspase-8, and subsequently enhances cleavage of Bid in the presence of TRAIL. In addition, we confirmed that non-functional Bid mutants could not convert the p19 form of activated caspase-3 to the p17 form and could not induce apoptosis. These results indicate that Bid plays an important role in the crosstalk between ferroptosis and TRAIL-induced apoptosis.

Acknowledgements

This work was supported by the following grants: National Cancer Institute (NCI) grants R21 CA259243 (Y.J.L.), R03 CA245171 (Y.J.L.), R03 CA212125 (Y.J.L.), R03 CA205496 (X.L.), R01GM118437 (X.L.), R01CA123362 (H-R.C.K.), and Department of Defense OC190038 (W81XWH-20-1-0190) (Y.J.L.). This project used the UPMC Hillman Cancer Center Core Facility and was supported in part by award P30CA047904.

The authors thank Christine Burr (Department of Surgery, University of Pittsburgh, Pittsburgh, PA) for her critical reading of the manuscript.

Abbreviations used in this paper:

ART

artesunate

ATCC

American Type Culture Collection

ATF4

activating transcription factor 4

Bid

BH3 interacting-domain death agonist

cBid

cleaved Bid

cFLIP

cellular FLICE-like inhibitory protein

CHAC1

cation transport regulator homolog 1

CHOP

CCAAT-enhancer-binding protein homologous protein

DISC

death-inducing signaling protein complex

DR5

death receptor 5

ER

endoplasmic reticulum

ERA

erastin

FADD

Fas-associated death domain

GPX4

glutathione peroxidase 4

GSH

glutathione

HB

homogenization buffer

HRP

horseradish peroxidase

KO

knockout

MEF

mouse embryonic fibroblast

MOMP

mitochondrial outer membrane permeabilization

PARP

poly (ADP-ribose) polymerase

PI

propidium iodide

PUMA

p53 upregulated modulator of apoptosis

RFU

relative fluorescence unit

SD

standard deviation

siRNA

small interfering RNA

tBid

truncated Bid

TNF

tumor necrosis factor

TRAIL

tumor necrosis factor-related apoptosis-inducing ligand

TRAIL-R2

tumor necrosis factor-related apoptosis inducing ligand receptor 2

WT

wild-type

Footnotes

Conflict of Interests

The authors declare no competing financial interests.

Data availability statement:

Data sources will be made freely available to the scientific research community from the corresponding author upon reasonable request as soon as they have been documented in a publication.

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Data Availability Statement

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