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. Author manuscript; available in PMC: 2016 Jun 6.
Published in final edited form as: J Cell Biochem. 2015 Nov 2;117(5):1136–1144. doi: 10.1002/jcb.25397

Autophagy-mediated degradation of IAPs and c-FLIPL potentiates apoptosis induced by combination of TRAIL and Chal-24

Jennings Xu 1,#, Xiuling Xu 1,#, Shaoqing Shi 1,2,#, Qiong Wang 1,2,#, Bryanna Saxton 1, Weiyang He 3, Xin Gou 3, Jun-Ho Jang 2,4, Toru Nyunoya 2,4, Xia Wang 2, Chengguo Xing 5, Lin Zhang 2,*, Yong Lin 1,*
PMCID: PMC4894717  NIHMSID: NIHMS790657  PMID: 26448608

Abstract

Combination chemotherapy is an effective strategy for increasing anticancer efficacy, reducing side effects and alleviating drug resistance. Here we report that combination of the recently identified novel chalcone derivative, chalcone-24 (Chal-24), and TNF-related apoptosis-inducing ligand (TRAIL) significantly increases cytotoxicity in lung cancer cells. Chal-24 treatment significantly enhanced TRAIL-induced activation of caspase-8 and caspase-3, and the cytotoxicity induced by combination of these agents was effectively suppressed by the pan-caspase inhibitor z-VAD-fmk. Chal-24 and TRAIL combination suppressed expression of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (c-FLIPL) and cellular inhibitor of apoptosis proteins (c-IAPs), and ectopic expression of c-FLIPL and c-IAPs inhibited the potentiated cytotoxicity. In addition, TRAIL and Chal-24 cooperatively activated autophagy. Suppression of autophagy effectively attenuated cytotoxicity induced by the Chal-24 and TRAIL combination, which was associated with attenuation of c-FLIPL and c-IAPs degradation. Altogether, these results suggest that Chal-24 potentiates the anticancer activity of TRAIL through autophagy-mediated degradation of c-FLIPL and c-IAPs, and that combination of Chal-24 and TRAIL could be an effective approach in improving chemotherapy efficacy.

Keywords: TRAIL, Chal-24, autophagy, apoptosis, c-IAP, c-FLIP

1. Introduction

While chemotherapy is one of the main approaches for treating cancer patients, it is still a challenge to increase its anticancer efficacy, minimize side effects and alleviate drug resistance. Most anticancer therapeutics suppress cancer by directly killing cancer cells via activation of apoptosis [Fulda, 2009; Ghavami et al., 2009; Ocker and Hopfner, 2012; Seve and Dumontet, 2005]. However, therapy failure is common, which is often due to apoptosis evasion that blunts the anticancer activity of chemotherapeutics [Hanahan and Weinberg, 2011; Lonning, 2010]. Therefore, strategies enhancing apoptosis in cancer cells will increase the efficacy of anticancer chemotherapeutics and alleviate chemoresistance [Ocker and Hopfner, 2012]. Combining drugs that target different pathways would significantly improve anticancer efficacy, potentially offering an effective strategy for improving chemotherapy.

Apoptosis can be induced by different stimuli, most of which activate either the intrinsic or extrinsic apoptosis pathways. Many DNA-damaging anticancer drugs activate the intrinsic apoptosis pathway, which is mediated by damage of the mitochondrial membrane, loss of mitochondrial potential and release of mitochondrial proteins cytochrome C and SMAC [Chang, 2011; Wang et al., 2004]. In contrast, the extrinsic pathway is mediated by death receptors that belong to the tumor necrosis factor (TNF) family of cytokine receptors. The binding of a ligand to a death receptor causes formation of the death-inducing signaling complex (DISC), which mediates activation of the caspase-8 and -3 cascade [Aggarwal, 2003; Wang and Lin, 2008]. The activation of caspase-8 in DISC is negatively regulated by cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP), and thus, suppressing c-FLIP would promote DISC-mediated apoptosis [Stegehuis et al., 2010; Wang et al., 2008b]. As a member of the TNF cytokine superfamily that has selective toxicity in transformed or tumor cells, TNF-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent that is currently vigorously tested in preclinical studies and clinical trials. However, primary and acquired TRAIL resistance substantially hampers its clinical application [Chen et al., 2010; Li et al., 2011; Wang et al., 2008b; Zhang and Fang, 2005]. Thus, combination of TRAIL with other therapeutics could be exploited for improving the anticancer activity of TRAIL.

Autophagy is the cellular process for removing unwanted cellular components, including organelles and proteins, which can lead to either cell survival or cell death [Mizushima and Komatsu, 2011; Todde et al., 2009]. In accordance with its contradictory roles in cell death control, the role of autophagy in the response of cancer cells to chemotherapy is also complex: it can act either for or against therapeutic-induced cytotoxicity [Wu et al., 2011]. Although autophagy was initially categorized as type II programmed cell death, the term “autophagic cell death” is uncommon [Shen and Codogno, 2011]. Instead, autophagy mediates apoptosis and necroptosis [He et al., 2014; Shen and Codogno, 2012].

Chal-24 is a novel chalcone derivative that has potential anticancer activity [He et al., 2014; Srinivasan et al., 2009]. At high concentrations, (>4 μM) Chal-24 activates autophagy-mediated necroptosis in cancer cells [He et al., 2014], but at lower concentrations (≤1 μM), it potentiates cisplatin-induced apoptosis [Shi et al., 2015]. It is apparent that Chal-24 kills cancer cells through a different mechanism than that of TRAIL. Thus, it is likely that a significant potentiation of anticancer activity will be achieved when these two agents are combined. This hypothesis was tested in this study, and the results show that the combination of Chal-24 and TRAIL induced strong apoptotic cytotoxicity, which was associated with autophagy-mediated degradation of IAPs and c-FLIP. Data from this study suggest that Chal-24 potentiates anticancer activity of TRAIL, and combination of Chal-24 and TRAIL could be an effective approach in improving chemotherapy efficacy.

2. Materials and methods

2.1. Reagents

Glutathione S-transferase (GST) TRAIL was prepared as described previously [Lin et al., 2000]. Antibodies against c-IAP-1 (AF8181), c-IAP2 (552782), FADD (556402), caspase-8 (551242), caspase-3 (559565) and p62 (610832) were from BD Biosciences (San Diego, CA, USA). Antibodies against GAPDH (sc-32233) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against XIAP (2042) was from Cell Signaling (Danvers, MA, USA). Anti-poly (ADP-ribose) polymerase (PARP, ALX-210-222) and c-FLIP (ALX-804-961-0100) were from Enzo Life Sciences (Farmingdale, NY, USA). Antibody against LC3B (L7543) was purchased from Sigma-Aldrich (St Louis, MO, USA). Wortmannin (12-338) were Calbiochem (La Jolla, CA, USA). Chloroquine (C6628) and 3MA (M9281) were from Sigma-Aldrich. The pan-caspase inhibitor z-VAD-fmk (ALX-260-039) was from Enzo Life Sciences. Caspase-Glo® 3/7 Assay Systems (G8090) and Caspase-Glo® 8 Assay Systems (G8200) were from Promega (Madison, WI 53711 USA). The FLAG-cIAP1, FLAG-cIAP2 and pEBB-XIAP (which expresses FLAG-XIAP) plasmids were from Addgene (Cambridge MA) [Hu et al., 2006; Hu and Yang, 2003; Yang et al., 2000]. The pEGFP-C1 plasmid was from Clontech (Mountain View, CA). The V5-c-FLIP plasmid (HsCD00445121) was purchased from DNASU Plasmid Repository.

2.2. Cell culture

A549 and H460 cells were obtained from America Type Culture Collection (Manassas, VA, USA) and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U penicillin and 100 μg/ml streptomycin. All cells were cultured in standard incubator conditions at 37°C with 5% CO2.

2.3. Cytotoxicity assay

Cytotoxicity assay was conducted with a cytotoxicity detection kit (Promega) based on the release of lactate dehydrogenase (LDH). Cells were seeded in a 48-well plate at 40–50% confluence. After overnight culture, cells were treated as indicated in each figure legend. LDH release was measured as described previously [Wang et al., 2006]. Ectopic expression was used to examine the effect of IAPs and c-FLIP on cytotoxicity induced by the Chal-24 and TRAIL combination. A549 cells were transfected with expression plasmids expressing EGFP and IAPs or FLIP. The pcDNA plasmid was transfected with EGFP as a negative control. EGFP was used as a transfection marker. Twenty-four hours post-transfection, the cells were treated with TRAIL (100 ng/ml) and Chal-24 (0.5 μM) for 40 h and examined under a fluorescence microscope. The percentage of live cells in the treated samples relative to their respective untreated cells was calculated as described previously [Wang et al., 2014].

2.4. Western blot

Cell lysates were prepared by suspending cells in M2 buffer (20 mM Tris-HCl pH 7.6, 0.5% NP40, 250 mM NaCl, 3 mM EDTA, 2 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, 20 mM β-glycerophosphate, 1 mM sodium vanadate, and 1 µg/ml leupeptin). Equal amounts of protein from each cell lysate were resolved by 8% or 12% SDS-PAGE and analyzed by Western blot. The proteins were visualized with enhanced chemiluminescence (Millipore) following the instructions of the manufacturer.

2.5. Caspase activity assay

Cells were lysed in M2 buffer, the activities of caspase-8 and -3/7 were measured with the Caspase-Glo® 3/7 Assay Systems and Caspase-Glo® 8 Assay Systems, respectively following the instructions of the manufacturer. The caspase activity in each sample was normalized with total protein concentration. The relative caspase activity was calculated by taking that of untreated cells as 1.

2.6. Statistical analysis

All data were expressed as mean ± SD and statistical significance was examined with one-way analysis of variance (ANOVA) pairwise comparison. p<0.05 was considered statistically significant.

3. Results

3.1. Potentiated cytotoxicity induced by the combination of TRAIL and Chal-24

The effect of combination of Chal-24 and TRAIL in killing cancer cells was examined and quantified by LDH assay. Treating the cells with increasing concentrations of either TRAIL (50–200 ng/ml) or Chal-24 (0.5–2.0 μM) and a fixed concentration of the other (100 ng/ml for TRAIL and 0.5 μM for Chal-24), a clear potentiation in cytotoxicity in A549 cells was seen (Fig. 1A, 1B). A similar observation was made in H460 cells, even with lower concentrations of TRAIL (25–100 ng/ml) (Fig. 1C, 1D). These results suggest that combination of Chal-24 and TRAIL results in potentiated cytotoxicity in human lung cancer cells.

Figure 1. Cytotoxicity induced by the combination of Chal-24 and TRAIL in lung cancer cells.

Figure 1

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A, A549 cells were treated with indicated concentrations of TRAIL and Chal-24 (0.5 μM) for 24 h. B, A549 cells were treated with indicated concentrations of Chal-24 and TRAIL (100 ng/ml) for 24 h. C, H460 cells were treated with indicated concentrations of TRAIL and Chal-24 (0.5 μM) for 24 h. D, H460 cells were treated with indicated concentrations of Chal-24 and TRAIL (50 ng/ml) for 24 h. Cell death was measured by LDH release assay. Data shown are mean ± SD. Data shown are mean ± SD, representative of three independent experiments.

3.2. TRAIL and Chal-24 combination induces apoptotic cell death

Because TRAIL mainly induces apoptosis in cancer cells, we then investigated if Chal-24 potentiates this pathway for the cytotoxicity in cells treated with the combination of Chal-24 and TRAIL. The results strongly supported this hypothesis: Chal-24 potently enhanced TRAIL-induced activity of caspase-8 and -3/7 (Fig. 2A, 2B, 2C) and cleavage of the caspase-3 substrate PARP (Fig. 2C); and the pan-caspase inhibitor z-VAD-fmk effectively suppressed cytotoxicity induced by combination of TRAIL and Chal-24 (Fig 2D). These results suggest that Chal-24 significantly enhanced TRAIL-induced apoptotic cell death.

Figure 2. Chal-24 and TRAIL co-treatment induces apoptotic cell death.

Figure 2

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A and B, A549 cells were treated with TRAIL (100 ng/ml) and Chal-24 (1 μM) for 24 h, the activities of caspase-8 and caspase-3/7 were measured. The relative activity was shown, in which the activity of the untreated sample was taken as 1. *p<0.05. C, the cells were treated with TRAIL (100 ng/ml for A549 and 50 ng/ml for H460 cells) and Chal-24 (1 μM) for 24 h. PARP, caspase-8 and active caspase-3 were detected by Western blot. GAPDH was detected as a loading control. D, A549 cells were pretreated with z-VAD-fmk (10 μM) for 30 min, followed by 24 h treatment with TRAIL (100 ng/ml), Chal-24 (1 μM) or in combination, cell death was detected by LDH assay. *p < 0.05.

3.3. Blocking the NF-κB pathway does not affect the cytotoxicity induced by TRAIL and Chal-24 combination

Because NF-κB suppresses TRAIL-induced apoptosis in certain cells and Chal-24 potentially suppresses NF-κB [Lin et al., 2000; Srinivasan et al., 2009; Zhang and Fang, 2005], we investigated whether NF-κB was involved in TRAIL/Chal-24-induced cytotoxicity. For this purpose, A549 and H460 cells stably transfected with the NF-κB super-suppressor IκBαAA were utilized, in which basal and TNF-induced NF-κB activity was suppressed [Wang et al., 2006]. Overexpression of the NF-κB super-suppressor IκBαAA did not impact the cell death induced by TRAIL and Chal-24 combination (Fig. 3A, 3B). Similarly, the specific NF-κB inhibitor SC514 did not affect the cytotoxicity induced by this drug combination (Fig. 3C). Consistently, the expression of the potential NF-κB targets IAP1 and cFLIP for cell survival was not altered in these cells (Data not shown). Thus, the NF-κB pathway is unlikely involved in the potentiated cytotoxicity induced by TRAIL and Chal-24 combination.

Figure 3. NF-κB is not involved in the cell death induced by the Chal-24 and TRAIL combination.

Figure 3

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A and B, A549 and H460 cells stably transfected with IκBαAA or an empty vector were treated with TRAIL (100 ng/ml for A549 and 50 ng/ml for H460 cells) and Chal-24 (1 μM) for 24 h, cell death was measured by LDH assay. Insert, expression of transfected protein was confirmed by Western blot. C, A549 cells were pretreated with SC514 (10 μM) for 30 min, then treated with TRAIL (100 ng/ml), Chal-24 (1 μM) or both for an additional 24 h, cell death was measured by LDH assay.

3.4. The cytotoxicity induced by combination of TRAIL and Chal-24 is dependent on autophagy

Our previous studies have shown that both TRAIL and Chal-24 induce autophagy, and autophagy has distinct roles in cytotoxicity induced by TRAIL or Chal-24 individually [He et al., 2014; He et al., 2012]. Therefore, we investigated whether autophagy is involved, and what the role of autophagy is, in the potentiated cytotoxicity induced by TRAIL and Chal-24 combination. While either TRAIL or Chal-24 moderately activated autophagy, the co-treatment of these agents cooperatively enhanced autophagy, which was shown as increased LC3 II expression and p62 degradation (Fig. 4A). Consistently, the TRAIL and Chal-24 combination strongly triggered GFP-LC3 punctuation (Figs. 4B, S1). The activation of autophagy was further confirmed by autophagic flux assay (Fig 4C). The autophagy inhibitors 3MA, CQ and WTM, which suppress autophagy by targeting different steps along the autophagy activation pathway, significantly suppressed cell death induced by the TRAIL and Chal-24 combination (Fig. 4D). Because the chemical inhibitor 3MA may have complex roles in autophagy regulation [Wu et al., 2010], a distinct approach using RNAi was employed in autophagy suppression. Indeed, knockdown of the key autophagy factor ATG7 effectively suppressed cell death induced by the TRAIL and Chal-24 combination (Fig. 4E). Altogether, these results suggest that the cytotoxicity induced by combination of TRAIL and Chal-24 depends on autophagy.

Figure 4. Autophagy is required for the cell death induced by Chal-24 and TRAIL combination.

Figure 4

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A, A549 cells were treated with TRAIL (100 ng/ml), Chal-24 (1 μM) alone or in combination for 24 h. The indicated proteins were detected by Western blot. GAPDH was used as a loading control. B, the cells were transfected with GFP-LC3 plasmid for 24 h, and treated with TRAIL (100 ng/ml), Chal-24 (2 μM) or both for an additional 1 h. Quantification of cell numbers with GFP-LC3 puncta was performed under a fluorescence microscope, **p < 0.01. C, the cells were pretreated with chloroquine (CQ, 20 μM) for 30 min, and then treated with TRAIL and Chal-24 (4 h or 24 h for upper panel for LC3 detection and lower panel for p62 detection, respectively). The indicated proteins were detected by Western blot. The intensity of the individual bands was quantified by densitometry and normalized to the corresponding loading control (GAPDH) bands. Relative protein expression was calculated with the untreated samples taken as 1. D, the cells were transfected with Beclin 1 or Atg 7 siRNA (20 nM) for 30 h, followed by TRAIL (100 ng/ml) and Chal-24 (1 μM) co-treatment for an additional 24 h, cell death was measured by LDH assay. **p < 0.01. Insert, the confirmation of the knockdown of Beclin 1 and Atg 7. E, A549 cells were pretreated with autophagy inhibitors (CQ, 20 μM; WTM, 1 μM; 3MA, 10 μM) for 30 min, followed by TRAIL (100 ng/ml) and Chal-24 (1 μM) co-treatment for an additional 24 h, cell death was measured by LDH assay. *p < 0.05.

3.5. Autophagy mediates degradation of IAPs and c-FLIPL induced by combination of TRAIL and Chal-24

The IAP proteins are the main cellular inhibitors of apoptosis [LaCasse et al., 2008; Varfolomeev et al., 2008; Vince et al., 2007; Wang et al., 2008a]. Our previous studies demonstrated that Chal-24 at high concentrations causes autophagy-dependent degradation of IAPs [He et al., 2014]. Thus, we examined whether TRAIL and Chal-24 combination affects the expression of IAPs. While TRAIL or Chal-24 alone had little effect, co-treatment with these two agents significantly suppressed expression of IAPs (Fig 5A). Suppressing autophagy with CQ, but not suppressing proteasome with MG132, effectively alleviated degradation of c-IAP1, c-IAP2 and XIAP (Fig. 5B), which is consistent with our previous reports that autophagy is involved in IAP degradation induced by either TRAIL or Chal-24 [He et al., 2014; He et al., 2012]. Similarly, the TRAIL and Chal-24 co-treatment resulted in decrease of c-FLIPL expression (Fig. 5C). CQ, but not MG132, effectively restored c-FLIPL expression in the co-treated cells (Fig. 5D). These results suggest that autophagy mediates degradation of IAPs and c-FLIPL induced by combination of TRAIL and Chal-24.

Figure 5. TRAIL and Chal-24 combination promotes autophagy-mediated degradation of cFLIPL and IAPs.

Figure 5

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A and C, A549 cells were treated with TRAIL (100 ng/ml), or Chal-24 (1 μM), or in combination for 24 h, the indicated proteins were detected by Western blot, β-tubulin was as a loading control. B and D, the cells were pretreated with CQ (20 μM) or MG132 (5 μM) for 30 min, followed by TRAIL (100 ng/ml) and Chal-24 (1 μM) co-treatment for an additional 24 h, the indicated proteins were detected by Western blot. β-tubulin was detected as a loading control. The intensity of the individual bands was quantified by densitometry and normalized to the corresponding loading control (β-tubulin) bands. Relative protein expression was calculated with the untreated samples taken as 1.

3.6. Ectopic expression of IAPs and c-FLIPL decreases cytotoxicity induced by combination of TRAIL and Chal-24

To confirm the role of suppressed IAPs and cFLIP in cytotoxicity induced by TRAIL and Chal-24 combination, ectopic expression of IAPs or cFLIP was performed before the combination treatment. The rationale is that, if IAPs or cFLIP play an inhibitory role against the cytotoxicity induced by TRAIL and Chal-24 combination, increasing their expression will attenuate the extent of cell death. Indeed, both ectopic IAPs and cFLIP clearly suppressed cell death (Fig. 6A, 6B). Together with the result that suppressing autophagy retained levels of IAPs and cFLIP and expressed cytotoxicity, these results suggest that autophagy-mediated suppression of IAPs and cFLIP contributes to cytotoxicity induced by TRAIL and Chal-24 combination.

Figure 6. Ectopic expression of IAPs and c-FLIPL decreases cytotoxicity induced by combination of TRAIL and Chal-24.

Figure 6

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A, A549 cells were transfected with plasmids expressing the indicated proteins. The expression of V5-cFLIP was detected by Western bot with anti-V5 antibody. The IAPs were detected with anti-FLAG antibody. β-Actin was detected as a loading control. B, 549 cells were transfected as in A, and treated with TRAIL (200 ng/ml) and Chal-24 (0.5 μM) for 24 h. Survival of EGFP-positive cells was quantified by counting live cells with green fluorescence. Data shown are mean ± SD. **p < 0.01.

4. Discussion

In this study, we explored the possibility of combining Chal-24 and TRAIL for improving anticancer therapy. The results validate our hypothesis by demonstrating that this combination significantly potentiates apoptotic cytotoxicity in cancer cells and by establishing a mechanism that involves autophagy and degradation of IAPs and c-FLIPL to release the apoptosis brake. This novel drug combination could be a potentially effective approach for improving anticancer therapeutic efficacy.

While TRAIL is a potential anticancer agent, innate and acquired TRIAL-resistances are common. Importantly, in TRAIL-resistant cancer cells, TRAIL does not only lose its anticancer activity, but also promotes proliferation and metastasis, converting TRAIL from an anticancer agent to a tumor promoter [Malhi and Gores, 2006]. Thus, preventing and overcoming TRAIL resistance is extremely important. The results of this study imply that the Chal-24 and TRAIL combination may have a potential in overcoming TRAIL chemoresistance.

TRAIL kills cancer cells through the extrinsic apoptosis pathway that is initiated from caspase-8 activation. Chal-24 enhanced TRAIL-induced caspase-8 activation, which was associated with suppression of the caspase-8 inhibitor c-FLIP. In addition, the Chal-24 and TRAIL combination suppressed the expression of the apoptosis suppressors c-IAP1, c-IAP2 and XIAP. Ectopic expression of IAPs and c-FLIPL decreases cytotoxicity induced by combination of TRAIL and Chal-24. The balance of pro- and anti-apoptosis signaling is key in determining the cellular fate in cells responding to apoptosis-inducing stimuli [Fulda, 2009; Ocker and Hopfner, 2012; Smolewski and Robak, 2011]. Our results suggest that Chal-24 sensitizes TRAIL-induced apoptosis mainly through releasing the apoptosis brake operated by IAPs and c-FLIP.

Our previous studies suggested that autophagy has a pro-survival role in TRAIL-treated cells, but a pro-death role in Chal-24-treated cells [He et al., 2014; He et al., 2012]. Interestingly, the combination of TRAIL and Chal-24 abolished the pro-survival role while retaining the pro-death role of autophagy. Consistent with this function of autophagy, our recent study showed a pro-death role of autophagy when Chal-24 was combined with cisplatin [Shi et al., 2015]. These results strongly suggest that the roles of autophagy in chemoresponse are dependent on stimulation types, and the autophagy-mediated cell response of a drug could be altered when it is combined with different anticancer agents. Additionally, other autophagy promotion approaches such as the mTOR inhibitor rapamycin or its analogs may be examined for sensitization of TRAIL and Chal-24 combination in therapy against different cancers.

The IAP proteins are well-characterized mediators for the NF-κB cell survival pathway, which suppresses cell death during death receptor signaling [Bertrand et al., 2008; LaCasse et al., 2008; Vanlangenakker et al., 2012]. However, it is unlikely the NF-κB pathway is involved in the potentiated effect of the TRAIL and Chal-24 combination. It is likely other apoptosis-suppressing mechanisms of IAPs are involved. Remarkably, the expression of IAPs are often increased in cancer cells, which is involved in chemoresistance, disease progression and poor prognosis [Darding and Meier, 2012]. Thus, suppressing IAPs could be an effective approach for improving cancer chemotherapy [Straub, 2011]. Similarly, overexpression of c-FLIP has been identified in different tumor types, and thus, c-FLIP is also proposed as a therapeutic target to restore apoptotic response in cancer cells [Shirley and Micheau, 2013]. Therefore, the concurrent suppression of IAPs and c-FLIP by the combination of TRAIL and Chal-24 could be an ideal approach to achieve potentiated anticancer efficacy.

5. Conclusion

Altogether, our results establish the combination of TRAIL and Chal-24 as a potential effective anticancer strategy, which may be exploited for improving chemotherapy efficacy. It is warranted to determine the anticancer effectiveness and chemoresistance attenuation potential of this drug combination in in vivo studies.

Supplementary Material

Figure S1. Combination of TRAIL and Chal-24 strongly triggered GFP-LC3 punctuation. A549 cells were transfected with a GFP-LC3-expressing plasmid for 24 h, and treated with TRAIL (100 ng/ml), Chal-24 (2 μM) or both for an additional 1 h. The representative photographs were taken under a fluorescence microscope.

Acknowledgments

This study was partly supported by grants from NIEHS/NIH (R01ES017328), NCI/NIH (R01CA142649), and the Office of Science (BER), U.S. Department of Energy (DE-FG02-09ER64783). S. Shi was a recipient of the joint student training award sponsored by China Scholarship Council.

Abbreviations

c-FLIP

cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein

CQ

Chloroquine

DISC

death-inducing signaling complex

GST

Glutathione S-transferase

IAP

inhibitor of apoptosis proteins

LDH

lactate dehydrogenase

TNF

tumor necrosis factor

TRAIL

TNF-related apoptosis-inducing ligand

WTM

Wortmannin

Footnotes

Conflict of interest

All authors declare no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Combination of TRAIL and Chal-24 strongly triggered GFP-LC3 punctuation. A549 cells were transfected with a GFP-LC3-expressing plasmid for 24 h, and treated with TRAIL (100 ng/ml), Chal-24 (2 μM) or both for an additional 1 h. The representative photographs were taken under a fluorescence microscope.

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