Effect of Turkish propolis extracts on proteome of prostate cancer cell line

Background

The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells.

Results

We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins.

Conclusion

The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.     

Identification of the proteins related to p53-mediated radioresponse in nasopharyngeal carcinoma by proteomic analysis

Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), and p53 is closely associated with the radiosensitivity of cancer, but the molecular mechanisms of p53-mediated radioresponse in NPC remains unclear. We previously established NPC CNE2sip53 cell line with p53 knockdown and paired control cell line CNE2/pSUPER, which provides a cell model system to investigate mechanisms of p53-mediated radioresponse in NPC. In this study, we first compared the radiosensitivity of CNE2sip53 and CNE2/pSUPER by a clonogenic survival assay, cell growth assay, and Hoechst 33258 staining and flow cytometry analysis of apoptotic cells. The results showed that the radiosensitivity of CNE2sip53 was significantly lower than that of CNE2/pSUPER, indicating that p53 plays a role in mediating NPC radiosensitivity. To search for the proteins associated with the p53-mediated radioresponse in NPC, a proteomic approach was performed to identify the radioresponsive proteins in CNE2sip53 and CNE2p/SUPER, respectively, and then the difference of radioresponsive proteins in CNE2sip53 and CNE2p/SUPER was compared. As a result, 14 differential radioresponsive proteins were identified in the two cell lines, 4 proteins of which were conformed by Western blot. Among them, 9 and 5 proteins were identified solely from CNE2p/SUPER and CNE2sip53, respectively. Furthermore, protein-protein interaction analysis showed that 7 differential radioresponsive proteins identified only in CNE2p/SUPER were related to p53 protein. Our results suggest that the differential radioresponsive proteins unique to CNE2p/SUPER may be involved in p53-mediated radioresponse in NPC, which will be helpful for elucidating the mechanisms of p53-mediated NPC cellular response to radiotherapy.     

5-Azacytidine Enhances the Radiosensitivity of CNE2 and SUNE1 Cells In Vitro and In Vivo Possibly by Altering DNA Methylation

The radioresistance of tumor cells remains a major cause of treatment failure in nasopharyngeal carcinoma (NPC). Recently, several reports have highlighted the importance of epigenetic changes in radiation-induced responses. Here, we investigated whether the demethylating agent 5-azacytidine (5-azaC) enhances the radiosensitivity of NPC cells. The NPC cell lines CNE2 and SUNE1 were treated with 1 μmol/L 5-azaC for 24 h before irradiation (IR); clonogenic survival was then assessed. Tumor growth was investigated in a mouse xenograft model in vivo. The apoptosis, cell cycle progression and DNA damage repair were examined using flow cytometry, immunofluorescent staining and western blotting. Promoter methylation and the expression of four genes epigenetically silenced during the development of NPC were evaluated by pyrosequencing and real-time PCR. We found that pretreatment with 5-azaC significantly decreased clonogenic survival after IR compared to IR alone; the sensitivity-enhancement ratio of 5-azaC was 1.4 and 1.2 for CNE2 and SUNE1 cells, respectively. The combined administration of 5-azaC and IR significantly inhibited tumor growth in the mouse xenograft model, and enhanced radiation-induced apoptosis in vitro compared to 5-azaC alone or IR alone. 5-AzaC also decreased promoter methylation and upregulated the expression of genes which are epigenetically silenced both in vitro and in vivo in NPC. Thus, 5-azaC enhance the radiosensitivity of both the CNE2 and SUNE1 cell lines, possibly by altering DNA methylation levels and increasing the ability of irradiated cells to undergo apoptosis. The use of 5-azaC combined with IR maybe represent an attractive strategy for the treatment of NPC.     

DNA-PK的活性与鼻咽癌细胞株CNE1/CNE2放射敏感性的关系

本文主要研究DNA依赖的蛋白激酶（DNA-dependent protein kinase,DNA-PK）与鼻咽癌细胞放射敏感性之间的关系。克隆形成实验分析鼻咽癌细胞CNEI／CNE2的剂量存活曲线,Signa TECT DNA-PK试剂盒检测DNA-PK活性,免疫荧光及激光显微共聚焦分析放疗前及放疗后15min、1h、6h、12h和24hCNE1／CNE2细胞中Kus及DNA-PKcs的亚细胞定位,Western blot分析两株细胞中Kus蛋白的表达。结果显示：CNE1细胞在每个剂量点的存活分数均高于CNE2细胞;同时发现放疗前后CNE1细胞中的DNA-PK活性也均高于CNE2细胞,但两株细胞中Ku70／Ku80蛋白表达无明显差异;放疗可使DNA-PK活性增加,且各个检测时间点CNE1细胞增加的幅度大于CNE2细胞;DNA-PK亚基可同时定位于胞浆和胞核,但主要位于胞核,细胞照射后Ku70、Ku80和DNA-PKcs从胞浆转运到胞核。结果表明：DNA-PK活性更高可能是CNE1细胞较CNE2细胞更能抵抗放射的原因之一;放疗所致DNA-PK活性增高可能与DNA-PK亚基从胞浆转运到胞核有关,而与Ku蛋白表达的总量无关。     

Downregulation of monocarboxylate transporter 1 inhibits the invasion and migration through suppression of the PI3K/Akt signaling pathway in human nasopharyngeal carcinoma cells

Monocarboxylate transporter 1 (MCT1) has been reported to be correlated wtih decreased survival and advanced stage of progression in a series of human tumor cells and primary cancers. Specifically, MCT1 has been documented to be involved in tumor progression, including invasion and migration. Here, we investigated the mechanism and effect of regulation of MCT1 on invasion and migration of nasopharyngeal carcinoma (NPC) cells. In the study, we firstly demonstrated that the expression of MCT1 in CNE2Z cells was obviously higher than that in HNE1 cells. Downregulation of MCT1 inhibited the invasion and migration in CNE2Z cells, upregulated the expression of E-cadherin, TIMP (tissue inhibitor of metalloproteinase)-2 and TIMP-1, and suppressed the expression of matrix metalloproteinases (MMP)-9 and MMP-2. Correspondingly, upregulation of MCT1 enhanced the invasive and migratory potential in HNE1 cells, increased the expression of MMP-9 and MMP-2, and downregulated the expression of E-cadherin, TIMP-2 and TIMP-1. The mechanistic study demonstrated that the effect of MCT1 might be correlated with PI3K/Akt signaling pathway. LY294002, a PI3K inhibitor, increased the inhibition of invasion and migration mediated by downregulation of MCT1 in CNE2Z cells. These findings collectively suggested that MCT1 might act as a new regulator to improve invasion and migration of NPC cells and be correlated with activating the PI3K/Akt pathway.     

A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin–p67phox–Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase.     

Interference effect of epigallocatechin-3-gallate on targets of nuclear factor kappaB signal transduction pathways activated by EB virus encoded latent membrane protein 1

AIM: To elucidate the interference effect of epigallocatechin-3-gallate (EGCG) on targets of nuclear factor kappaB (NF-kappaB) signal transduction pathway activated by EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma (NPC) cell lines. METHODS: The survival rates of CNE1 and CNE-LMP1 cell lines after the EGCG treatment were determined by MTT assay. NF-kappaB activation in CNE1 and CNE-LMP1 cells after EGCG treatment was analyzed by promoter luciferase reporter system. And then nuclear translocation of NF-kappaB (p65) after the EGCG treatment was analyzed by immunofluorescence and western blotting. Meanwhile, the changes of IkappaBalpha phosphorylation were observed after the EGCG treatment. EGFR promoter activity was analyzed by promoter luciferase reporter system and EGFR phosphorylation was observed by western blotting after the EGCG treatment. RESULTS: EGCG inhibited the survival rates of CNE1 and CNE-LMP1 cells and NF-kappaB activation caused by LMP1 in CNE-LMP1 cells. EGCG also suppressed the nuclear translocation of NF-kappaB (p65) and IkappaBalpha phosphorylation. Meanwhile, EGCG inhibited EGFR promoter activity and EGFR phosphorylation. CONCLUSIONS: EGCG inhibited not only the dose-dependent survival rate of NPC cells, but also the dose-dependent activation of NF-kappaB. This inhibition of LMP1-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha, and then EGCG inhibited EGFR activity which was a downstream gene from NF-kappaB. This study suggests that interference effect of EGCG on targets of signal transduction pathway plays an important role in the anticancer function.     

Rac1 GTPase is activated by hepatitis B virus replication--involvement of HBX

Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (betaPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.     

Oncogenic H-Ras enhances DNA repair through the Ras/phosphatidylinositol 3-kinase/Rac1 pathway in NIH3T3 cells. Evidence for association with reactive oxygen species

This study investigated the role of oncogenic H-Ras in DNA repair capacity in NIH3T3 cells. Expression of dominant-positive H-Ras (V12-H-Ras) enhanced the host cell reactivation of luciferase activity from UV-irradiated and cisplatin-treated plasmids and also increased the unscheduled DNA synthesis following cisplatin or UV treatment of cells. This observed enhancement of DNA repair capacity was inhibited by transient transfection with dominant-negative H-Ras (N17-H-Ras) or Rac1 (N17-Rac1) plasmids. Moreover, stable transfection of dominant-positive Rac1 (V12-Rac1) further enhanced DNA repair capacity. Because reactive oxygen species (ROS) are known to be a downstream effector of oncogenic Ras, we examined the role of ROS in DNA repair capacity. We found that ROS production by V12-H-Ras expression was mediated by the Ras/phosphatidylinositol 3-kinase (PI3K)/Rac1/NADPH oxidase-dependent pathway and that pretreatment of V12-H-Ras-transformed cells with an antioxidant (N-acetylcysteine) and an NADPH oxidase inhibitor (diphenyleneiodonium) decreased DNA repair capacity. Similarly, treatment with PI3K inhibitors (wortmannin and LY294002) inhibited the ability of oncogenic H-Ras to enhance DNA repair capacity. Furthermore, inhibition of the Ras/PI3K/Rac1/NADPH oxidase pathway resulted in increased sensitivity to cisplatin and UV in V12-H-Ras-expressing NIH3T3 cells. Taken together, these results provide evidence that oncogenic H-Ras activates DNA repair capacity through the Ras/PI3K/Rac1/NADPH oxidase-dependent pathway and that increased ROS production via this signaling pathway is required for enhancement of the DNA repair capacity induced by oncogenic H-Ras.     

A Highlights from MBoC Selection: Matrix compliance regulates Rac1b localization,NADPH oxidase assembly,and epithelial–mesenchymal transition

Epithelial–mesenchymal transition (EMT) is a form of epithelial plasticity implicated in fibrosis and tumor metastasis. Here we show that the mechanical rigidity of the microenvironment plays a pivotal role in the promotion of EMT by controlling the subcellular localization and downstream signaling of Rac GTPases. Soft substrata, with compliances comparable to that of normal mammary tissue, are protective against EMT, whereas stiffer substrata, with compliances characteristic of breast tumors, promote EMT. Rac1b, a highly activated splice variant of Rac1 found in tumors, localizes to the plasma membrane in cells cultured on stiff substrata or in collagen-rich regions of human breast tumors. At the membrane, Rac1b forms a complex with NADPH oxidase and promotes the production of reactive oxygen species, expression of Snail, and activation of the EMT program. In contrast, soft microenvironments inhibit the membrane localization of Rac1b and subsequent redox changes. These results reveal a novel mechanotransduction pathway in the regulation of epithelial plasticity via EMT.     

Antagonistic cross-talk between Rac and Cdc42 GTPases regulates generation of reactive oxygen species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.     

The HIV-1 Nef protein and phagocyte NADPH oxidase activation

Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia (cell line and primary culture) were transduced with lentiviral expression vectors. Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin). These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits. Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway. The following results suggest a key role for Rac in the priming effect of Nef. (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes. Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli.     

Up-regulation of heme oxygenase-1 expression through the Rac1/NADPH oxidase/ROS/p38 signaling cascade mediates the anti-inflammatory effect of 15-deoxy-delta 12,14-prostaglandin J2 in murine macrophages

We investigated the signaling pathway that leads to the expression of heme oxygenase-1 (HO-1) in murine macrophages in response to 15-deoxy-delta 12,14-prostaglandin J2 (15dPGJ2). 15dPGJ2 caused dose- and time-dependent activation of Rac1, followed by a transient increase in reactive oxygen species (ROS) via NADPH oxidase, which leads to downstream activation of p38 kinase. Inhibition of 15dPGJ2-dependent HO-1 expression significantly attenuated suppression by 15dPGJ2 of LPS-induced iNOS expression and subsequent production of nitric oxide (NO). Our findings strongly suggest that 15dPGJ2 exerts its anti-inflammatory activity through the Rac1-NADPH oxidase-ROS-p38 signaling to the up-regulation of HO-1 in an in vitro inflammation model.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Calcium/calmodulin-dependent regulation of Rac GTPases and Akt in histamine-induced chemotaxis of mast cells

Histamine induces chemotaxis of mast cells through the histamine H₄ receptor. This involves the activation of small GTPases, Rac1 and Rac2, downstream of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Activation of the H₄ receptor also results in phospholipase C (PLC)-mediated calcium mobilization; however, it is unclear whether the PLC‑calcium pathway interacts with the PI3K-Rac pathway. Here, we demonstrated that calcium mobilization regulates the PI3K-dependent activation of Rac GTPases through calmodulin. A PLC inhibitor (U73122) and an intracellular calcium chelator (BAPTA-AM) suppressed the histamine-induced activation of Rac, whereas the calcium ionophore ionomycin increased the active Rac GTPases, suggesting that intracellular calcium regulates the activation of Rac. The calmodulin antagonist (W-7) inhibited the histamine-induced activation of Rac and migration of mast cells, indicating that calmodulin mediates the effect of calcium. Inhibition of calcium/calmodulin signaling suppressed histamine-induced phosphorylation of Akt. The Akt inhibitor MK-2206 attenuated histamine-induced migration of mast cells. However, it did not suppress the activation of Rac GTPases. These results suggest that Rac GTPases and Akt play independent roles in the histamine-induced chemotaxis of mast cells. Our findings enable further elucidation of the molecular mechanism of histamine-induced chemotaxis of mast cells and help identify therapeutic targets for allergic and inflammatory conditions involving mast cell accumulation.     

Proteomics-based identification of proteins with altered expression induced by 12-O-tetradecanoylphorbol 13-acetate in nasopharyngeal carcinoma CNE2 cells

Nasopharyngeal carcinoma (NPC) is a malignancy with high incidence in Southern China and South-East Asia. Etiology studies indicate that chemical carcinogen promoters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), are important factors causing NPC development. However, the mechanism of the TPA effect on NPC remains unclear. In the present study, cells from a poorly differentiated squamous cell carcinoma NPC cell line, CNE2, were stimulated by TPA and proteomics technology was carried out to find protein discrepancies between control and TPA-treated cells. Results revealed that TPA treatment in CNE2 cells could upregulate the expression of ““““triosephosphate isomerase““““ and ““““14-3-3 protein sigma““““ and downregulate the expression of ““““reticulocalbin 1 precursor““““, ““““nucleophosmin““““, ““““mitochondrial matrix protein pl precursor““““, and ““““stathmin““““. The changes in the expression of these genes suggested that TPA induced CNE2 cells to antiproliferation and to apoptosis, which was confirmed by subsequent apoptosis detection. Therefore, the effects of TPA on nasopharyngeal carcinoma cells were distinct from the effects on primary epithelial cells and we suggest reasons for these differences.     

Death receptors 4 and 5 activate Nox1 NADPH oxidase through riboflavin kinase to induce reactive oxygen species-mediated apoptotic cell death

Stimulation of the proapoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, death receptors 4 (DR4) and 5 (DR5), conventionally induces caspase-dependent apoptosis in tumor cells. Here we report that stimulation of DR4 and/or DR5 by the agonistic protein KD548-Fc, an Fc-fused DR4/DR5 dual-specific Kringle domain variant, activates plasma membrane-associated Nox1 NADPH oxidase to generate superoxide anion and subsequently accumulates intracellular reactive oxygen species (ROS), leading to sustained c-Jun N-terminal kinase activation and eventual apoptotic cell death in human HeLa and Jurkat tumor cells. KD548-Fc treatment induces the formation of a DR4/DR5 signaling complex containing riboflavin kinase (RFK), Nox1, the Nox1 subunits (Rac1, Noxo1, and Noxa1), TNF receptor-associated death domain (TRADD), and TNF receptor-associated factor 2 (TRAF2). Depletion of RFK, but not the Nox1 subunits, TRADD and TRAF2, failed to recruit Nox1 and Rac1 to DR4 and DR5, demonstrating that RFK plays an essential role in linking DR4/DR5 with Nox1. Knockdown studies also reveal that RFK, TRADD, and TRAF2 play critical, intermediate, and negligible roles, respectively, in the KD548-Fc-mediated ROS accumulation and downstream signaling. Binding assays using recombinantly expressed proteins suggest that DR4/DR5 directly interact with cytosolic RFK through RFK-binding regions within the intracellular death domains, and TRADD stabilizes the DR4/DR5-RFK complex. Our results suggest that DR4 and DR5 have a capability to activate Nox1 by recruiting RFK, resulting in ROS-mediated apoptotic cell death in tumor cells.