z-VAD-fmk-induced non-apoptotic cell death of macrophages: possibilities and limitations for atherosclerotic plaque stabilization

Macrophages play a pivotal role in atherosclerotic plaque destabilization in contrast to smooth muscle cells (SMCs). As a consequence, removal of macrophages from plaques via selective induction of cell death represents a promising approach to stabilize non-obstructive, rupture-prone atherosclerotic lesions. However, the mechanisms to initiate cell death in macrophages but not in other cell types of the plaque, in particular SMCs, are unknown. Recently, we have shown that the pan-caspase inhibitor z-VAD-fmk induces autophagy and necrotic cell death in J774A.1 and RAW264.7 macrophages as well as in IFN-gamma primed primary mouse peritoneal macrophages, but not in vascular SMCs or C2C12 myoblasts. The different sensitivity to z-VAD-fmk is largely based on differential expression of receptor-interacting protein 1 (RIP1). This finding suggests that caspase inhibition activates RIP1 which in turn initiates autophagy, although other explanations should be taken into account. z-VAD-fmk-treated J774A.1 macrophages overexpress and secrete several chemokines and cytokines, including TNFalpha. The combination of z-VAD-fmk and TNFalpha, but not TNFalpha alone, induces SMC necrosis. In this regard, z-VAD-fmk is detrimental and not beneficial for atherosclerotic plaque stability due to stimulation of inflammatory responses and indirect induction of SMC death. Future work is needed to determine the mechanism(s) that selectively trigger non-apoptotic cell death in plaque macrophages without evoking inflammation and SMC death.     

Everolimus-induced mTOR inhibition selectively depletes macrophages in atherosclerotic plaques by autophagy

Current pharmacological approaches to stabilize nonobstructive rupture-prone atherosclerotic plaques have only partially reduced the incidence of acute coronary syndromes and sudden death. Macrophages in these vulnerable plaques play a pivotal role in plaque destabilization, whereas smooth muscle cells promote plaque stability. In a recent study, we report that implantation of stents eluting everolimus, a mammalian target of rapamycin (mTOR) inhibitor, in atherosclerotic arteries of cholesterol-fed rabbits, led to a marked reduction in macrophage content without altering the amount of smooth muscle cells. Our in vitro studies showed that treatment of macrophages and smooth muscle cells with everolimus induced inhibition of translation of both cell types. However, cell death occurred only in macrophages and was characterized by bulk degradation of long-lived proteins, processing of microtubule associated protein light chain 3 (LC3), and cytoplasmic vacuolization, which are all markers of autophagy. Everolimus-induced autophagy was mediated by mTOR inhibition because cell viability was not affected using tacrolimus, an mTOR independent everolimus-analogue. These results provide proof-of-principle that macrophages in the vascular wall can be selectively cleared via induction of autophagy by mTOR inhibition. Therefore, stent-based delivery of an mTOR inhibitor may be a promising novel strategy for treatment of vulnerable atherosclerotic plaques.     

A Two-Carbon Switch to Sterol-Induced Autophagic Death

Although both cholesterol and plant sterols are abundant in our diets, our intestinal epithelial cells selectively and efficiently rid the body of plant sterols. However, a rare mutation in plant sterol excretion in humans results in the accumulation of plant sterols, particularly sitosterol, in the plasma and tissues. Sitosterol differs from cholesterol only in an extra ethyl group on the sterol side chain. Significantly, sitosterolemia is associated with accelerated atherothrombotic vascular disease, notably myocardial infarction. An important process that promotes atherothrombosis is advanced lesional macrophage death, leading to plaque necrosis. One of the causes of atherosclerotic macrophage death is sterol-induced cytotoxicity. We therefore compared the effects of excess intracellular sitosterol vs. cholesterol on macrophage death. Whereas excess cholesterol kills macrophages by caspase-dependent apoptosis, sitosterol kills macrophages by a caspase-independent pathway involving necroptosis and autophagy. The finding that an ethyl group on the sterol side chain fundamentally alters the way cells respond to excess sterols adds new insight into the mechanisms of sterol-induced cell death and may provide at least one explanation for the excess atherosclerotic heart disease in patients with sitosterolemia.

Addendum to:

Sitosterol-Containing Lipoproteins Trigger Free Sterol-Induced Caspase-Independent Death in ACAT-Competent Macrophages: Implications for Sterol Structure-Dependent Mechanisms of Cell Death and for Atherosclerotic Vascular Disease in Sitosterolemia

L. Bao, Y. Li, S.X. Deng, D. Landry and I. Tabas

J Biol Chem 2006; In press     

Autophagy plays a protective role in free cholesterol overload-induced death of smooth muscle cells

Smooth muscle cells (SMC) make up most of the vascular system. In advanced atherosclerotic plaques, dying SMCs undergo a complex death mode. In the present study, we examined the activation of autophagy in SMCs overloaded with excess free cholesterol (FC) and investigated the possible role which autophagy plays during the FC-induced cell death. After incubation with excess FC, a robust expression of autophagic vacuoles (AV) was detected using both fluorescence microscopy and transmission electron microscopy (TEM). The results revealed that FC induced a time-dependent upregulation of microtubule-associated protein-1 light chain 3-II (LC3-II). Inhibition of autophagy by 3-methyladenine (3-MA) enhanced both cell apoptosis and necrosis, while on the contrary, rapamycin inhibited cell death following cholesterol application. Furthermore, the impact of the colocalization of fragmented mitochondria with AVs was observed after cholesterol treatment. Our results also revealed that the modulation of autophagy directly influenced the cellular organellar stress. In conclusion, our findings demonstrated that excess FC induced the activation of autophagy in SMCs as a cellular defense mechanism, possibly through the degradation of dysfunctional organelles such as mitochondria and endoplasmic reticulum.     

Cell-cell interaction of macrophages and vascular smooth muscle cells in the synthesis of leukotriene b(4)

Biosynthesis of LTB(4) during cell-cell interaction between vascular smooth muscle cells (SMC) and alveolar macrophages (AM) has been investigated by use of both high-pressure Hquid chromatography (HPLC) and radtoimmunoassay (RIA). Both interleukin-beta (IL-beta) and tumour necrosis factor-alpha (TNFalpha) induced a time- and dose-dependent synthesis of 15-, and 5-hydroxyeicosatetraenoic acids (HETEs) from cultured SMC. However, neither TNFalpha nor IL-1beta induced a significant LTB(4) production in SMC alone or AM alone after 24 h of incubation. Addition of IL-1beta and TNFalpha simultaneously to SMC resulted in a dose-dependent synergistic increase of HETEs. Macrophages dose-dependently transformed extremely low concentrations of exogenous LTA(4) into LTB(4). Incubation of vascular SMC with various numbers of AM in the presence of IL-1beta (5 units/ml) and TNFalpha (10 units/ml) induced a great increase of LTB(4) synthesis in comparison with the detectable levels of LTB(4) produced by macrophages alone. Pretreatment of SMC with NDGA, cycloheximide, and actinomycin not only inhibited IL-1 and TNT induced HETEs synthesis but also abolished LTB(4) production when co-incubated with macrophages. These results suggest that LTB(4) in a mixture of SMC and macrophages could originate from a transcellular metabolism, i.e. macrophages transforming SMC-derived LTA(4) into LTB(4).     

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.     

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.     

Autophagy contributes to caspase-independent macrophage cell death

Macrophage cell death plays a role in many physiological and pathophysiological conditions. Previous work has shown that macrophages can undergo caspase-independent cell death, and this process is associated with Nur77 induction, which is involved in inducing chromatin condensation and DNA fragmentation. Here we show that autophagy is a cytosolic event that controls caspase-independent macrophage cell death. Autophagy was induced in macrophages treated with lipopolysaccharides (LPSs) and the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (Z-VAD), and the inhibition of autophagy by either chemical inhibitors or by the RNA interference knockdown of beclin (a protein required for autophagic body formation) inhibited caspase-independent macrophage cell death. We also found an increase in poly(ADP-ribose) (PAR) polymerase (PARP) activation and reactive oxygen species (ROS) production in LPS + Z-VAD-treated macrophages, and both are involved in caspase-independent macrophage cell death. We further determined that the formation of autophagic bodies in macrophages occurs downstream of PARP activation, and PARP activation occurs downstream of ROS production. Using macrophages in which receptor-interacting protein 1 (RIP1) was knocked down by small interfering RNA, and macrophages isolated from Toll/interleukin-1 receptor-domain-containing adaptor inducing IFN-beta (TRIF)-deficient mice, we found that TRIF and RIP1 function upstream of ROS production in LPS + Z-VAD-treated macrophages. We also found that Z-VAD inhibits LPS-induced RIP1 cleavage, which may contribute to ROS over-production in macrophages. This paper reveals that TRIF, RIP1, and ROS production, as well as PARP activation, are involved in inducing autophagy, which contributes to caspase-independent macrophage cell death.     

Regulator of G-Protein Signaling 19 (RGS19) and Its Partner Gα-Inhibiting Activity Polypeptide 3 (GNAI3) Are Required for zVAD-Induced Autophagy and Cell Death in L929 Cells

Autophagy has diverse biological functions and is involved in many biological processes. The L929 cell death induced by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD) was shown to be an autophagy-mediated death for which RIP1 and RIP3 were both required. It was also reported that zVAD can induce a small amount of TNF production, which was shown to be required for zVAD-induced L929 cell death, arguing for the contribution of autophagy in the zVAD-induced L929 cell death. In an effort to study RIP3 mediated cell death, we identified regulator of G-protein signaling 19 (RGS19) as a RIP3 interacting protein. We showed that RGS19 and its partner Gα-inhibiting activity polypeptide 3 (GNAI3) are involved in zVAD-, but not TNF-, induced cell death. The role of RGS19 and GNAI3 in zVAD-induced cell death is that they are involved in zVAD-induced autophagy. By the use of small hairpin RNAs and chemical inhibitors, we further demonstrated that zVAD-induced autophagy requires not only RIP1, RIP3, PI3KC3 and Beclin-1, but also RGS19 and GNAI3, and this autophagy is required for zVAD-induced TNF production. Collectively, our data suggest that zVAD-induced L929 cell death is a synergistic result of autophagy, caspase inhibition and autocrine effect of TNF.     

Leaky lysosomes in lung transplant macrophages: azithromycin prevents oxidative damage

Background

Lung allografts contain large amounts of iron (Fe), which inside lung macrophages may promote oxidative lysosomal membrane permeabilization (LMP), cell death and inflammation. The macrolide antibiotic azithromycin (AZM) accumulates 1000-fold inside the acidic lysosomes and may interfere with the lysosomal pool of Fe.

Objective

Oxidative lysosomal leakage was assessed in lung macrophages from lung transplant recipients without or with AZM treatment and from healthy subjects. The efficiency of AZM to protect lysosomes and cells against oxidants was further assessed employing murine J774 macrophages.

Methods

Macrophages harvested from 8 transplant recipients (5 without and 3 with ongoing AZM treatment) and 7 healthy subjects, and J774 cells pre-treated with AZM, a high-molecular-weight derivative of the Fe chelator desferrioxamine or ammonium chloride were oxidatively stressed. LMP, cell death, Fe, reduced glutathione (GSH) and H-ferritin were assessed.

Results

Oxidant challenged macrophages from transplants recipients without AZM exhibited significantly more LMP and cell death than macrophages from healthy subjects. Those macrophages contained significantly more Fe, while GSH and H-ferritin did not differ significantly. Although macrophages from transplant recipients treated with AZM contained both significantly more Fe and less GSH, which would sensitize cells to oxidants, these macrophages resisted oxidant challenge well. The preventive effect of AZM on oxidative LMP and J774 cell death was 60 to 300 times greater than the other drugs tested.

Conclusions

AZM makes lung transplant macrophages and their lysososomes more resistant to oxidant challenge. Possibly, prevention of obliterative bronchiolitis in lung transplants by AZM is partly due to this action.     

Osteopontin stimulates autophagy via integrin/CD44 and p38 MAPK signaling pathways in vascular smooth muscle cells

Osteopontin (OPN) exerts pro‐inflammatory effect and is associated with the development of abdominal aortic aneurysm (AAA). However, the molecular mechanism underlying this association remains obscure. In the present study, we compared gene expression profiles of AAA tissues using microarray assay, and found that OPN was the highest expressed gene (>125‐fold). Furthermore, the expression of LC3 protein and autophagy‐related genes including Atg4b, Beclin1/Atg6, Bnip3, and Vps34 was markedly upregulated in AAA tissues. To investigate the ability of OPN to stimulate autophagy as a potential mechanism involved in the pathogenesis of this disease, we treated vascular smooth muscle cells (SMCs) with OPN, and found that OPN significantly increased the formation of autophagosomes, expression of autophagy‐related genes and cell death, whereas blocking the signal by anti‐OPN antibody markedly inhibited OPN‐induced autophagy and SMC death. Furthermore, inhibition of integrin/CD44 and p38 MAPK signaling pathways markedly abrogated the biological effects of OPN on SMCs. These data for the first time demonstrate that OPN sitmulates autophagy directly through integrin/CD44 and p38 MAPK‐mediated pathways in SMCs. Thus, inhibition of OPN‐induced autophagy might be a potential therapeutic target in the treatment of AAA disease. J. Cell. Physiol. 227: 127–135, 2012. © 2011 Wiley Periodicals, Inc.     

A Role of RIP3-Mediated Macrophage Necrosis in Atherosclerosis Development

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Highlights? RIP3 deficiency in macrophages reduces advanced atherosclerotic lesions ? RIP3 deficiency reduces primary necrosis of macrophages in advanced lesions ? RIP3-dependent macrophage necrosis is not postapoptotic cell death ? Increased RIP3 converts apoptosis to necrosis in advanced lesional macrophages     

IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways.

Interleukin-10 (IL-10) limits inflammatory responses by inhibiting macrophage activation. In macrophages, IL-10 activates Stat1 and Stat3. We characterized IL-10 responses of the J774 mouse macrophage cell line, and of J774 cells expressing wild-type hIL-10R, mutant hIL-10R lacking two membrane-distal tyrosines involved in recruitment of Stat3 (hIL-10R-TyrFF), a truncated Stat3 (DeltaStat3) which acts as a dominant negative, or an inducibly active Stat3-gyraseB chimera (Stat3-GyrB). A neutralizing anti-mIL-10R monoclonal antibody was generated to block the function of endogenous mIL-10R. IL-10 inhibited proliferation of J774 cells and of normal bone marrow-derived macrophages, but not J774 cells expressing hIL-10RTyrFF. Dimerization of Stat3-GyrB by coumermycin mimicked the effect of IL-10, and expression of DeltaStat3 blocked the anti-proliferative activity of IL-10. For macrophage de-activation responses, hIL10R-TyrFF could not mediate inhibition of lipopolysaccharide-induced TNFalpha, IL-1beta or CD86 expression, while DeltaStat3 did not interfere detectably with these IL-10 responses. Thus signals mediating both anti-proliferative and macrophage de-activation responses to IL-10 require the two membrane-distal tyrosines of IL-10R, but Stat3 appears to function only in the anti-proliferative response.     

RNA interference in J774 macrophages reveals a role for coronin 1 in mycobacterial trafficking but not in actin-dependent processes

Macrophages are crucial for innate immunity, apoptosis, and tissue remodeling, processes that rely on the capacity of macrophages to internalize and process cargo through phagocytosis. Coronin 1, a member of the WD repeat protein family of coronins specifically expressed in leukocytes, was originally identified as a molecule that is recruited to mycobacterial phagosomes and prevents the delivery of mycobacteria to lysosomes, allowing these to survive within phagosomes. However, a role for coronin 1 in mycobacterial pathogenesis has been disputed in favor for its role in mediating phagocytosis and cell motility. In this study, a role for coronin 1 in actin-mediated cellular processes was addressed using RNA interference in the murine macrophage cell line J774. It is shown that the absence of coronin 1 in J774 macrophages expressing small interfering RNA constructs specific for coronin 1 does not affect phagocytosis, macropinocytosis, cell locomotion, or regulation of NADPH oxidase activity. However, in coronin 1-negative J774 cells, internalized mycobacteria were rapidly transferred to lysosomes and killed. Therefore, these results show that in J774 cells coronin 1 has a specific role in modulating phagosome-lysosome transport upon mycobacterial infection and that it is dispensable for most F-actin-mediated cytoskeletal rearrangements.     

Hsp70 protects macrophages infected with Salmonella choleraesuis against TNF-α-induced cell death

Hsp70 plays an important role in cytoprotection against tumor necrosis factor (TNF) α-mediated cytotoxicity. To investigate the role of Hsp70 in cytoprotein during Salmonella infection, we examined endogenous Hsp70 induction and TNF-α production in a monocyte/macrophage line, J774A.1, after infection with a virulent strain of Salm.choleraesuis RF-1 carrying a 50 kb virulent plasmid or the plasmid-cured avirulent strain 31N-1. Intracellular bacteria progressively increased in J774A.1 cells phagocytosing avirulent 31N-1 bacteria, whereas such progressive growth was not evident in J774A.1 cells phagocytosing avirulent 31N-1 bacteria. On the contrary, J774A.1 cells infected with virulent RF-1 bacteria expressed less Hsp70 than those infected with avirulent 31N-1 bacteria. The level of TNF-α production by J774A.1 infected with virulent RF-1 was much the same as that by J774A.1 infected with avirulent 31N-1. J774A.1 infected with virulent RF-1 died spontaneously; death was inhibited by the addition of anti-TNF-α mAb. Although the frequency of dead J774A.1 with hypodiploid DNA content increased only marginally after infection with avirulent 31N-1, treatment with Hsp70 anti-sense oligonucleotide resulted in a dramatic increase of dead cells in the infected macrophages. Taken together, these results suggest that Hsp70 induced macrophages plays an important role in host defense against Salmonella infection by protecting the macrophages against TNF α-induced cell death. Furthermore, cell death due to impaired endogenous Hsp synthesis in the phagocytes implies a novel pathogenic mechanism for virulence of Salm. choleraesuis RF-1.     

Identification of Small Molecules That Suppress Ricin-Induced Stress-Activated Signaling Pathways

Ricin is a member of the ribosome-inactivating protein (RIP) family of plant and bacterial toxins. In this study we used a high-throughput, cell-based assay to screen more than 118,000 compounds from diverse chemical libraries for molecules that reduced ricin-induced cell death. We describe three compounds, PW66, PW69, and PW72 that at micromolar concentrations significantly delayed ricin-induced cell death. None of the compounds had any demonstrable effect on ricin''s ability to arrest protein synthesis in cells or on ricin''s enzymatic activity as assessed in vitro. Instead, all three compounds appear to function by blocking downstream stress-induced signaling pathways associated with the toxin-mediated apoptosis. PW66 virtually eliminated ricin-induced TNF-α secretion by J774A.1 macrophages and concomitantly blocked activation of the p38 MAPK and JNK signaling pathways. PW72 suppressed ricin-induced TNF-α secretion, but not p38 MAPK and JNK signaling. PW69 suppressed activity of the executioner caspases 3/7 in ricin toxin- and Shiga toxin 2-treated cells. While the actual molecular targets of the three compounds have yet to be identified, these data nevertheless underscore the potential of small molecules to down-regulate inflammatory signaling pathways associated with exposure to the RIP family of toxins.     

Mercury inhibits nitric oxide production but activates proinflammatory cytokine expression in murine macrophage: differential modulation of NF-kappaB and p38 MAPK signaling pathways.

Mercury is well known to adversely affect the immune system; however, little is known regarding its molecular mechanisms. Macrophages are major producers of nitric oxide (NO) and this signaling molecule is important in the regulation of immune responses. The present study was designed to determine the impact of mercury on NO and cytokine production and to investigate the signaling pathways involved. The murine macrophage cell line J774A.1 was used to study the effects of low-dose inorganic mercury on the production of NO and proinflammatory cytokines. Cells were treated with mercury in the presence or absence of lipopolysaccharide (LPS). Mercury (5-20 microM) dose-dependently decreased the production of NO in LPS-stimulated cells. Concomitant decreases in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein were detected. Treatment of J774A.1 cells with mercury alone did not affect the production of NO nor the expression of iNOS mRNA or protein. Interestingly, mercury alone stimulated the expression of tumor necrosis factor alpha (TNFalpha), and increased LPS-induced TNFalpha and interleukin-6 mRNA expression. Mercury inhibited LPS-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) but had no effect alone. In contrast, mercury activated p38 mitogen-activated protein kinase (p38 MAPK) and additively increased LPS-induced p38 MAPK phosphorylation. These results indicate that mercury suppresses NO synthesis by inhibition of the NF-kappaB pathway and modulates cytokine expression by p38 MAPK activation in J774A.1 macrophage cells.     

Smac mimetic compounds potentiate interleukin-1beta-mediated cell death

Smac mimetic compounds (SMCs) potentiate TNFα-mediated cancer cell death by targeting the inhibitor of apoptosis (IAP) proteins. In addition to TNFα, the tumor microenvironment is exposed to a number of pro-inflammatory cytokines, including IL-1β. Here, we investigated the potential impact of IL-1β on SMC-mediated death of cancer cells. Synergy was seen in a subset of a diverse panel of 21 cancer cell lines to the combination of SMC and IL-1β treatment, which required IL-1β-induced activation of the NF-κB pathway. Elevated NF-κB activity resulted in the production of TNFα, which led to apoptosis dependent on caspase-8 and RIP1. In addition, concurrent silencing of cIAP1, cIAP2, and X-linked IAP by siRNA was most effective for triggering IL-1β-mediated cell death. Importantly, SMC-resistant cells that produced TNFα in response to IL-1β treatment were converted to an SMC-sensitive phenotype by c-FLIP knockdown. Reciprocally, ectopic expression of c-FLIP blocked cell death caused by combined SMC and IL-1β treatment in sensitive cancer cells. Together, our study indicates that a positive feed-forward loop by pro-inflammatory cytokines can be exploited by SMCs to induce apoptosis in cancer cells.