The Identification of New c-FLIP Inhibitors for Restoring Apoptosis in TRAIL-Resistant Cancer Cells.

The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.

Bclaf1 regulates c-FLIP expression and protects cells from TNF-induced apoptosis and tissue injury.

TNF stimulation generates pro-survival signals through activation of NF-κB that restrict the build-in death signaling triggered by TNF. The competition between TNF-induced survival and death signals ultimately determines the fate of a cell. Here, we report the identification of Bclaf1 as a novel component of the anti-apoptotic program of TNF. Bclaf1 depletion in multiple cells sensitizes cells to TNF-induced apoptosis but not to necroptosis. Bclaf1 exerts its anti-apoptotic function by promoting the transcription of CFLAR, a caspase 8 antagonist, downstream of NF-κB activation. Bclaf1 binds to the p50 subunit of NF-κB, which is required for Bclaf1 to stimulate CFLAR transcription. Finally, in Bclaf1 siRNA administered mice, TNF-induced small intestine injury is much more severe than in control mice with aggravated signs of apoptosis and pyroptosis. These results suggest Bclaf1 is a key regulator in TNF-induced apoptosis, both in vitro and in vivo.

Chromomycins from soil-derived Streptomyces sp. inhibit the growth of human non-small cell lung cancer cells by targeting c-FLIP.

Three chromomycin derivatives, chromomycins A3 (1, CA3), A5 (2, CA5), and monodeacetylchromomycin A3 (3, MDA-CA3), were identified from the soil-derived Streptomyces sp. CGMCC 26516. A reinvestigation of the structure of CA5 is reported, of which the absolute configuration was unambiguously determined for the first time to be identical with that of CA3 based on nuclear magnetic resonance (NMR) data analysis as well as NMR and electronic circular dichroism calculations. Compounds 1-3 showed potent cytotoxicity against the non-small-cell lung cancer (NSCLC) cells (A549, H460, H157-c-FLIP, and H157-LacZ) and down-regulated the protein expression of c-FLIP in A549 cells. The IC50 values of chromomycins in H157-c-FLIP were higher than that in H157-LacZ. Furthermore, si-c-FLIP promoted anti-proliferation effect of chromomycins in NSCLC cells. In nude mice xenograft model, 1 and 2 both showed more potent inhibition on the growth of H157-lacZ xenografts than that of H157-c-FLIP xenografts. These results verify that c-FLIP mediates the anticancer effects of chromomycins in NSCLC.

Thiostrepton suppresses triple-negative breast cancer through downregulating c-FLIP/SMAD2/3 signaling pathway.

Triple-negative breast cancer (TNBC) is an aggressive subtype with poor prognosis of breast cancer. Thiostrepton exerts anti-tumor activities against several cancers including TNBC. Herein we discussed the new molecular mechanisms of thiostrepton in TNBC. Thiostrepton inhibited MDA-MB-231 cell viability, accompanied by a decrease of c-FLIP and p-SMAD2/3. c-FLIP overexpression reduced the sensitivity of MDA-MB-231 cells to thiostrepton, while SMAD2/3 knockdown increased the sensitivity of MDA-MB-231 cells to thiostrepton. Moreover, c-FLIP overexpression significantly increased the expression and phosphorylation of SMAD2/3 proteins and vice versa. In conclusion, our study reveals c-FLIP/SMAD2/3 signaling pathway as a novel mechanism of antitumor activity of thiostrepton.

(-)-Epigallocatechin-3-gallate induced apoptosis by dissociation of c-FLIP/Ku70 complex in gastric cancer cells.

Anti-cancer properties of (-)-epigallocatechin-3-gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE-inhibitory protein (c-FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti-cancer role of EGCG in gastric cancer (GC) cells through dissociation of c-FLIP/Ku70 complex. MKN-45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT-PCR. Protein expression of c-FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c-FLIP/Ku70 complex as well as Ku70 translocation were studied by sub-cellular fractionation and co-immunoprecipitation. EGCG induced apoptosis in MKN-45 cells with substantial up-regulation of P53 and P21, down-regulation of c-Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c-FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c-FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.

SCEL regulates switches between pro-survival and apoptosis of the TNF-α/TNFR1/NF-κB/c-FLIP axis to control lung colonization of triple negative breast cancer.

BACKGROUND: Patients with metastatic triple-negative breast cancer (mTNBC) have a higher probability of developing visceral metastasis within 5 years after the initial diagnosis. Therefore, a deeper understanding of the progression and spread of mTNBC is urgently needed. METHODS: The isobaric tag for relative and absolute quantitation (iTRAQ)-based LC-MS/MS proteomic approach was applied to identify novel membrane-associated proteins in the lung-tropic metastatic cells. Public domain datasets were used to assess the clinical relevance of the candidate proteins. Cell-based and mouse models were used for biochemical and functional characterization of the protein molecule Sciellin (SCEL) identified by iTRAQ to elucidate its role and underlying mechanism in promoting lung colonization of TNBC cells. RESULTS: The iTRAQ-based LC-MS/MS proteomic approach identified a membrane-associated protein SCEL that was overexpressed in the lung-tropic metastatic cells, and its high expression was significantly correlated with the late-stage TNBC and the shorter survival of the patients. Downregulation of SCEL expression significantly impaired the 3D colony-forming ability but not the migration and invasion ability of the lung colonization (LC) cells. Knockdown of SCEL reduced TNF-α-induced activation of the NF-κB/c-FLIP pro-survival and Akt/Erk1/2 growth signaling pathways in the LC cells. Specifically, knockdown of SCEL expression switched TNF-α-mediated cell survival to the caspase 3-dependent apoptosis. Conversely, ectopic expression of SCEL promoted TNF-α-induced activation of NF-κB/c-FLIP pro-survival and Akt/Erk1/2 pro-growth signaling pathway. The result of co-immunoprecipitation (Co-IP) and GST pull-down assay showed that SCEL could interact with TNFR1 to promote its protein stability. The xenograft mouse model experiments revealed that knockdown of SCEL resulted in increase of caspase-3 activity, and decrease of ki67 and TNFR1 expression as well as increase of tumor-associated macrophages in the metastatic lung lesions. Clinically, SCEL expression was found to be positively correlated with TNFR1 in TNBC tissues. Lastly, we showed that blocking TNF-α-mediated cell survival signaling by adalimumab effectively suppressed the lung colonization of the SCEL-positive, but not the SCEL-downregulated LC cells in the tail-vein injection model. CONCLUSIONS: Our findings indicate that SCEL plays an essential role in the metastatic lung colonization of TNBC by promoting the TNF-α/TNFR1/NF-κB/c-FLIP survival and Akt/Erk1/2 proliferation signaling. Thus, SCEL may serve as a biomarker for adalimumab treatment of TNBC patients.

TGR5 agonist inhibits intestinal epithelial cell apoptosis via cAMP/PKA/c-FLIP/JNK signaling pathway and ameliorates dextran sulfate sodium-induced ulcerative colitis.

Excessive apoptosis of intestinal epithelial cell (IEC) is a crucial cause of disrupted epithelium homeostasis, leading to the pathogenesis of ulcerative colitis (UC). The regulation of Takeda G protein-coupled receptor-5 (TGR5) in IEC apoptosis and the underlying molecular mechanisms remained unclear, and the direct evidence from selective TGR5 agonists for the treatment of UC is also lacking. Here, we synthesized a potent and selective TGR5 agonist OM8 with high distribution in intestinal tract and investigated its effect on IEC apoptosis and UC treatment. We showed that OM8 potently activated hTGR5 and mTGR5 with EC50 values of 202 ± 55 nM and 74 ± 17 nM, respectively. After oral administration, a large amount of OM8 was maintained in intestinal tract with very low absorption into the blood. In DSS-induced colitis mice, oral administration of OM8 alleviated colitis symptoms, pathological changes and impaired tight junction proteins expression. In addition to enhancing intestinal stem cell (ISC) proliferation and differentiation, OM8 administration significantly reduced the rate of apoptotic cells in colonic epithelium in colitis mice. The direct inhibition by OM8 on IEC apoptosis was further demonstrated in HT-29 and Caco-2 cells in vitro. In HT-29 cells, we demonstrated that silencing TGR5, inhibition of adenylate cyclase or protein kinase A (PKA) all blocked the suppression of JNK phosphorylation induced by OM8, thus abolished its antagonizing effect against TNF-α induced apoptosis, suggesting that the inhibition by OM8 on IEC apoptosis was mediated via activation of TGR5 and cAMP/PKA signaling pathway. Further studies showed that OM8 upregulated cellular FLICE-inhibitory protein (c-FLIP) expression in a TGR5-dependent manner in HT-29 cells. Knockdown of c-FLIP blocked the inhibition by OM8 on TNF-α induced JNK phosphorylation and apoptosis, suggesting that c-FLIP was indispensable for the suppression of OM8 on IEC apoptosis induced by OM8. In conclusion, our study demonstrated a new mechanism of TGR5 agonist on inhibiting IEC apoptosis via cAMP/PKA/c-FLIP/JNK signaling pathway in vitro, and highlighted the value of TGR5 agonist as a novel therapeutic strategy for the treatment of UC.

Tubeimoside-1 Enhances TRAIL-Induced Apoptotic Cell Death through STAMBPL1-Mediated c-FLIP Downregulation.

Tubeimoside-1 (TBMS-1), a traditional Chinese medicinal herb, is commonly used as an anti-cancer agent. In this study, we aimed to investigate its effect on the sensitization of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Our results revealed that even though monotherapy using TBMS-1 or TRAIL at sublethal concentrations did not affect cancer cell death, combination therapy using TBMS-1 and TRAIL increased apoptotic cell death. Mechanistically, TBMS-1 destabilized c-FLIP expression by downregulating STAMBPL1, a deubiquitinase (DUB). Specifically, when STAMBPL1 and c-FLIP bound together, STAMBPL1 deubiquitylated c-FLIP. Moreover, STAMBPL1 knockdown markedly increased sensitivity to TRAIL by destabilizing c-FLIP. These findings were further confirmed in vivo using a xenograft model based on the observation that combined treatment with TBMS-1 and TRAIL decreased tumor volume and downregulated STAMBPL1 and c-FLIP expression levels. Overall, our study revealed that STAMBPL1 is essential for c-FLIP stabilization, and that STAMBPL1 depletion enhances TRAIL-mediated apoptosis via c-FLIP downregulation.

TRAIL-Sensitizing Effects of Flavonoids in Cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents a promising anticancer agent, as it selectively induces apoptosis in transformed cells without altering the cellular machinery of healthy cells. Unfortunately, the presence of TRAIL resistance mechanisms in a variety of cancer types represents a major hurdle, thus limiting the use of TRAIL as a single agent. Accumulating studies have shown that TRAIL-mediated apoptosis can be facilitated in resistant tumors by combined treatment with antitumor agents, ranging from synthetic molecules to natural products. Among the latter, flavonoids, the most prevalent polyphenols in plants, have shown remarkable competence in improving TRAIL-driven apoptosis in resistant cell lines as well as tumor-bearing mice with minimal side effects. Here, we summarize the molecular mechanisms, such as the upregulation of death receptor (DR)4 and DR5 and downregulation of key anti-apoptotic proteins [e.g., cellular FLICE-inhibitory protein (c-FLIP), X-linked inhibitor of apoptosis protein (XIAP), survivin], underlying the TRAIL-sensitizing properties of different classes of flavonoids (e.g., flavones, flavonols, isoflavones, chalcones, prenylflavonoids). Finally, we discuss limitations, mainly related to bioavailability issues, and future perspectives regarding the clinical use of flavonoids as adjuvant agents in TRAIL-based therapies.

Induction of Breast Cancer Cell Apoptosis by TRAIL and Smac Mimetics: Involvement of RIP1 and cFLIP.

Smac mimetics are a group of compounds able to facilitate cell death in cancer cells. TNF-related apoptosis-inducing ligand (TRAIL) is a death receptor ligand currently explored in combination with Smac mimetics. The molecular mechanisms determining if the combination treatment results in apoptosis are however not fully understood. In this study, we aimed to shed light on these mechanisms in breast cancer cells. Three breast cancer cell lines, MDA-MB-468, CAMA-1 and MCF-7, were used to evaluate the effects of Smac mimetic LCL-161 and TRAIL using cell death assays and Western blot. The combination treatment induces apoptosis and caspase-8 cleavage in MDA-MB-468 and CAMA-1 but not in MCF-7 cells and downregulation of caspase-8 blocked apoptosis. Downregulation, but not kinase inhibition, of receptor-interacting protein 1 (RIP1) suppressed apoptosis in CAMA-1. Apoptosis is preceded by association of RIP1 with caspase-8. Downregulating cellular FLICE-like inhibitory protein (c-FLIP) resulted in increased caspase cleavage and some induction of apoptosis by TRAIL and LCL-161 in MCF-7. In CAMA-1, c-FLIP depletion potentiated TRAIL-induced caspase cleavage and LCL-161 did not increase it further. Our results lend further support to a model where LCL-161 enables the formation of a complex including RIP1 and caspase-8 and circumvents c-FLIP-mediated inhibition of caspase activation.

Solution structure of c-FLIP death effector domains.

The formation of death-inducing signaling complex (DISC) and death effector domain (DED) filament initiates extrinsic apoptosis. Recruitment and activation of procaspase-8 at the DISC are regulated by c-FLIP. The interaction between c-FLIP and procaspase-8 is mediated by their tandem DEDs (tDED). However, the structure of c-FLIPtDED and how c-FLIP interferes with procaspase-8 activation at the DISC remain elusive. Here, we solved the monomeric structure of c-FLIPtDED (F114G) at near physiological pH by solution nuclear magnetic resonance (NMR). Structural superimposition reveals c-FLIPtDED (F114G) adopts a structural topology similar to that of procaspase-8tDED. Our results provide a structural basis for understanding how c-FLIP interacts with procaspase-8 and the molecular mechanisms of c-FLIP in regulating cell death.

Expression, purification, and characterization of c-FLIP tandem death effector domains from Escherichia coli.

Cellular FLICE-like inhibitory protein (c-FLIP) regulates extrinsic apoptosis by controlling procaspase-8 activation through its tandem N-terminal death effector domains (DEDs). Here, we present the expression and purification of c-FLIP tandem DEDs (tDED) from Escherichia coli. We observed that the c-FLIPtDED maintains monomeric form under near-physiological pH condition in vitro. Our results also reveal a significant correlation between the pH conditions and the structure of c-FLIPtDED (F114A). The described methods and results would be helpful for follow-up study on the structural and functional of c-FLIP.

c-FLIP promotes drug resistance in non-small-cell lung cancer cells via upregulating FoxM1 expression.

The forkhead box M1 (FoxM1) protein, a transcription factor, plays critical roles in regulating tumor growth and drug resistance, while cellular FLICE-inhibitory protein (c-FLIP), an anti-apoptotic regulator, is involved in the ubiquitin-proteasome pathway. In this study, we investigated the effects of c-FLIP on the expression and ubiquitination levels of FoxM1 along with drug susceptibility in non-small-cell lung cancer (NSCLC) cells. We first showed that the expression levels of FoxM1 and c-FLIP were increased and positively correlated (R2 = 0.1106, P < 0.0001) in 90 NSCLC samples. The survival data from prognostic analysis demonstrated that high expression of c-FLIP and/or FoxM1 was related to poor prognosis in NSCLC patients and that the combination of FoxM1 and c-FLIP could be a more precise prognostic biomarker than either alone. Then, we explored the functions of c-FLIP/FoxM1 in drug resistance in NSCLC cell lines and a xenograft mouse model in vivo. We showed that c-FLIP stabilized FoxM1 by inhibiting its ubiquitination, thus upregulated the expression of FoxM1 at post-transcriptional level. In addition, a positive feedback loop composed of FoxM1, ß-catenin and p65 also participated in c-FLIP-FoxM1 axis. We revealed that c-FLIP promoted the resistance of NSCLC cells to thiostrepton and osimertinib by upregulating FoxM1. Taken together, these results reveal a new mechanism by which c-FLIP regulates FoxM1 and the function of this interaction in the development of thiostrepton and osimertinib resistance. This study provides experimental evidence for the potential therapeutic benefit of targeting the c-FLIP-FoxM1 axis for lung cancer treatment.

Antitumor effect of acanthoic acid against primary effusion lymphoma via inhibition of c-FLIP.

Acanthoic acid (AA) is an active substance that is extracted from Croton oblongifolius Roxb., a traditional plant in Thailand. The antiinflammatory effect of AA on NF-κB pathway has been exclusively reported, however, its anticancer effect is still lacking. PEL is a B cell lymphoma that is mostly found in HIV patients. The prognosis and progression of PEL patients are terribly poor with a median survival time less than 6 months, so the new effective treatment is urgently needed. In this study, we found that AA effectively inhibited PEL cell proliferation with IC50s at 120-130 µM in well-representative cells, while the IC50s of AA in PBMC were higher (>200 µM). AA increased percentages of Annexin V/PI positive cells, whereas adding of caspase inhibitor (Q-VD-OPh) prevented AA-induced cell death. The antiapoptotic protein, c-FLIP, was downregulated by AA which leading to the activation of caspase-8 and -3. Combination of AA and TRAIL dramatically enhanced apoptotic cell death. In PEL xenograft model, AA at the dose of 250 mg/kg effectively inhibited PEL tumor growth without detectable toxicities assessed by mice weight and appearance.

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells.

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd3+ ) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca2+ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.

The Fas/FasL Signaling Pathway: Its Role in the Metastatic Process and as a Target for Treating Osteosarcoma Lung Metastases.

Understanding how the tumor microenvironment participates in inhibiting or supporting tumor growth is critical for the development of novel therapies. Osteosarcoma (OS) metastasizes almost exclusively to the lung, an organ where Fas ligand (FasL) is constitutively expressed. This chapter focuses on our studies dedicated to the interaction of OS cells with the lung microenvironment. We will summarize our studies conducted over the past 20 years showing the importance of the Fas/FasL signaling pathway to the establishment and progression of OS metastases in the lung. We demonstrated that the FasL+ lung microenvironment eliminates Fas-positive (Fas+) OS cells that metastasize to the lungs, through apoptosis induced by Fas signaling following interaction of Fas on the tumor cell surface with FasL on the lung epithelial cells. Expression of the Fas receptor on OS cells inversely correlated with the ability of OS cells to form lung metastases. Blocking this pathway interferes with this process, allowing Fas+ cells to grow in the lung. By contrast, upregulation of Fas on Fas- OS cells inhibited their ability to metastasize to the lung. We demonstrated how the FasL+ lung microenvironment can be leveraged for therapeutic intent through the upregulation of Fas expression. To this end, we demonstrated that the histone deacetylase inhibitor entinostat upregulated Fas expression on OS cells, reduced their ability to form lung metastases, and induced regression of established micrometastases. Fas expression in OS cells is regulated epigenetically by the microRNA miR-20a. We showed that expressions of Fas and miR-20a are inversely correlated, and that delivery of anti-miR-20a in vivo to mice with established osteosarcoma lung metastases resulted in upregulation of Fas and tumor regression. Therefore, targeting the Fas signaling pathway may present therapeutic opportunities, which target the lung microenvironment for elimination of OS lung metastases. We have also shown that in addition to being critically involved in the metastatic potential, the Fas signaling pathway may also contribute to the efficacy of chemotherapy. We demonstrated that the chemotherapeutic agent gemcitabine (GCB) increased Fas expression in both human and mouse OS cells in vitro. In vivo, aerosol GCB therapy induced upregulation of Fas expression and the regression of established osteosarcoma lung metastases. The therapeutic efficacy of GCB was contingent upon a FasL+ lung microenvironment as aerosol GCB had no effect in FasL-deficient mice. Manipulation of Fas expression and the Fas pathway should be considered, as this concept may provide additional novel therapeutic approaches for treating patients with OS lung metastases.

Inhibition of Drp1 Sensitizes Cancer Cells to Cisplatin-Induced Apoptosis through Transcriptional Inhibition of c-FLIP Expression.

Mitochondrial fragmentation occurs during the apoptosis. Dynamin-related protein 1 (Drp1) acts as an important component in mitochondrial fission machinery and can regulate various biological processes including apoptosis, cell cycle, and proliferation. The present study demonstrates that dysfunction of mitochondrial dynamics plays a pivotal role in cisplatin-induced apoptosis. Inhibiting the mitochondrial fission with the specific inhibitor (Mdivi-1) did not affect apoptotic cell death in low concentrations (<10 µM). Interestingly, mdivi-1 enhanced cisplatin-induced apoptosis in cancer cells, but not in normal cells. Particularly in the presence of mdivi-1, several human cancer cell lines, including renal carcinoma cell line Caki-1, became vulnerable to cisplatin by demonstrating the traits of caspase 3-dependent apoptosis. Combined treatment induced downregulation of c-FLIP expression transcriptionally, and ectopic expression of c-FLIP attenuated combined treatment-induced apoptotic cell death with mdivi-1 plus cisplatin. Collectively, our data provide evidence that mdivi-1 might be a cisplatin sensitizer.

Magnolol Enhances the Therapeutic Effects of TRAIL through DR5 Upregulation and Downregulation of c-FLIP and Mcl-1 Proteins in Cancer Cells.

Magnolol is a biologically active compound, isolated from the Chinese herb Magnolia, that regulates antiproliferative, anticancer, antiangiogenic and antimetastatic activities. We found that magnolol sensitizes TRAIL-induced apoptotic cell death via upregulation of DR5 and downregulation of cellular FLICE-inhibitory protein (c-FLIP) and Mcl-1 in cancer cells, but not in normal cells. Mechanistically, magnolol increased ATF4-dependent DR5 expression at the transcription level, and knockdown of ATF4 markedly inhibited magnolol-induced DR5 upregulation. Silencing DR5 with siRNA prevented combined treatment with magnolol and TRAIL-induced apoptosis and PARP cleavage. Magnolol induced proteasome-mediated Mcl-1 downregulation, while magnolol-induced c-FLIP downregulation was regulated, at least in part, by lysosomal degradation. Our results revealed that magnolol enhanced TRAIL-induced apoptosis via ATF4-dependent DR5 upregulation and downregulation of c-FLIP and Mcl-1 proteins.

Delineating the role of c-FLIP/NEMO interaction in the CD95 network via rational design of molecular probes.

BACKGROUND: Structural homology modeling supported by bioinformatics analysis plays a key role in uncovering new molecular interactions within gene regulatory networks. Here, we have applied this powerful approach to analyze the molecular interactions orchestrating death receptor signaling networks. In particular, we focused on the molecular mechanisms of CD95-mediated NF-κB activation and the role of c-FLIP/NEMO interaction in the induction of this pathway. RESULTS: To this end, we have created the homology model of the c-FLIP/NEMO complex using the reported structure of the v-FLIP/NEMO complex, and rationally designed peptides targeting this complex. The designed peptides were based on the NEMO structure. Strikingly, the experimental in vitro validation demonstrated that the best inhibitory effects on CD95-mediated NF-κB activation are exhibited by the NEMO-derived peptides with the substitution D242Y of NEMO. Furthermore, we have assumed that the c-FLIP/NEMO complex is recruited to the DED filaments formed upon CD95 activation and validated this assumption in silico. Further insight into the function of c-FLIP/NEMO complex was provided by the analysis of evolutionary conservation of interacting regions which demonstrated that this interaction is common in distinct mammalian species. CONCLUSIONS: Taken together, using a combination of bioinformatics and experimental approaches we obtained new insights into CD95-mediated NF-κB activation, providing manifold possibilities for targeting the death receptor network.

Curcumin increased the expression of c-FLIP in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients.

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) disease is a chronic neuroinflammatory disease, which is associated with HTLV-1 infection. There is no effective and satisfactory treatment of HAM/TSP. It has been shown that curcumin exhibits modulatory effects on apoptosis and cytotoxicity-related molecules in HAM/TSP patients. In the present study, we examined the effect of curcumin on the gene expression of caspase-8, caspase-10, and anti-apoptotic protein c-FLIP, in HAM/TSP patients. Furthermore, we compared the expression of these molecules between HAM/TSP and asymptomatic carriers. Real-time PCR was performed to examine the mRNA expression of caspase-8, caspase-10, and c-FLIP in studied groups. The mRNA expression of caspase-8 and caspase-10 was similar before and after curcumin treatment in HAM/TSP patients (P > 0.05). The mRNA expression of c-FLIPL and c-FLIPs was higher after curcumin treatment compared with before treatment and significant differences were observed between the two groups (P = 0.004 and P = 0.044, respectively). The mRNA expression levels of caspase-8, caspase-10, c-FLIPL, and c-FLIPs were not statistically significant between HAM/TSP patients and asymptomatic carriers (P < 0.05). In conclusion, our results showed that curcumin increased the expression of c-FLIP in HAM/TSP patients which might suggest that, this molecule is involved in the apoptosis of HTLV-1-infected cells. Further studies with large sample size could be useful to clarify the role of this supplement in HAM/TSP patients.