Downregulation Of c-FLIP Expression Research Articles

Abstract 1980: Non-canonical regulation and function of anti-apoptotic protein c-FLIP

Abstract The metastasis of cancer cells to distant sites accounts for over 90% of cancer related deaths. In order to metastasize, cancer cells must be able to survive during extracellular matrix (ECM) detachment, namely by blocking anoikis (a caspase-dependent cell death) and restoring metabolic deficiencies. Such barriers during tumorigenesis are abrogated through activation of oncogenic signaling, and/or inhibition of tumor suppressors. Elucidating the signaling pathways involved in cancer cell metastasis is a crucial step in designing effective therapeutic interventions. Surprisingly, our investigation has revealed that cellular FLICE-like Inhibitory Protein (c-FLIP), a protein well known for its pro-survival function, instead antagonizes the viability of ECM-detached cancer cells. Additionally, we demonstrated that signaling emanating from oncogenic growth factor receptors results in the downregulation of c-FLIP expression providing additional evidence that cancer cells may benefit from diminished FLIP levels. Interestingly, this repression of c-FLIP expression by activated oncogenes is a consequence of PI3K signaling. Our study also suggests that low c-FLIP levels are beneficial in cancer cells due to capacity of c-FLIP to induce β-catenin degradation. Taken together, our study has revealed an unexpected role for c-FLIP in restricting the viability of tumor cells and raises the idea that the c-FLIP may have context-dependent pro- and anti-cell death roles during tumorigenesis. Citation Format: Matyas Abel Tsegaye, Kyle McGeehan, Mati Nemera, Zachary T. Schafer. Non-canonical regulation and function of anti-apoptotic protein c-FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1980.

Abstract 1444: c-FLIP antagonizes the survival of extracellular matrix detached cancer cells

Abstract The metastasis of cancer cells to distant sites accounts for over 90% of cancer related deaths. In order to metastasize, cancer cells must be able to survive during extracellular matrix (ECM) detachment, namely by blocking anoikis – caspase dependent cell death – and restoring metabolic deficiencies. Such barriers during tumorigenesis are abrogated through activation of oncogenes, and/or inhibition of tumor suppressors. Elucidating the signaling pathways involved in cancer cell metastasis is a crucial step in designing effective therapeutic interventions. Surprisingly, our investigation has revealed that cellular FLICE-like Inhibitory Protein (c-FLIP), a protein known for its anti-apoptotic function, antagonizes the survival of ECM-detached cancer cells. Additionally, we demonstrated that oncogenic signaling results in the downregulation of c-FLIP expression further suggesting that cancer cells can benefit from diminished FLIP levels. We observed that this repression of c-FLIP expression is a consequence of MEK-ERK signaling. Our study also suggests that the mechanism underlying this paradoxical c-FLIP pathway involves the induction of β-catenin degradation through inhibition of GSK-3β phosphorylation by p38 MAPK. This results in the ubiquitination and subsequent degradation of β-catenin which could contribute to reduced proliferation and migration. Citation Format: Matyas Abel Tsegaye, Kyle McGeehan, Mati Nemera, Zachary T. Schafer. c-FLIP antagonizes the survival of extracellular matrix detached cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1444.

Abstract 2057: Small molecule CB001 induces extrinsic apoptosis via downregulation of c-FLIP expression in mutant p53-expressing cancer cells

Abstract Cellular FADD-like IL-1β-converting enzyme-inhibitory protein (c-FLIP) is a master anti-apoptotic regulator in cancer cells. c-FLIP inhibits caspase 8 activation and suppresses cell apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) in malignant cells. Targeting c-FLIP is an attractive strategy for cancer therapy. We previously identified a small-molecule CB001 as a p53-restoring compound and found that CB001 induces cell death with little or no genotoxicity in human colorectal cancer cells. We now show that CB001 downregulates c-FLIP expression in cancer cells at the post-translational level via protein degradation. CB001 treatment induces an increase in caspase 8 activity which correlates with the decrease in c-FLIP at the protein level in mutant p53-expressing cancer cells. Knockdown of caspase 8 rescues cancer cells from CB001-induced apoptosis in mutant p53-expressing cancer cells, suggesting that downregulation of c-FLIP is one of the major mechanisms by which CB001 induces extrinsic cell apoptosis. Furthermore, our studies reveal a synergy between CB001 and TRAIL to induce apoptosis in TRAIL-resistant cancer cells, but not in normal cells at the tested doses. These results taken together suggest that CB001 is a promising lead for drug development with a strong capability to suppress tumor growth through targeting c-FLIP in cancer cells. Citation Format: Shengliang Zhang, Lanlan Zhou, Marie D. Ralff, Wafik S. El-Deiry. Small molecule CB001 induces extrinsic apoptosis via downregulation of c-FLIP expression in mutant p53-expressing cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2057.

C-FLIP and CD95 signaling are essential for survival of renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most-prominent tumor type of kidney cancers. Resistance of renal cell carcinoma (RCC) against tumor therapy is often owing to apoptosis resistance, e.g., by overexpression of anti-apoptotic proteins. However, little is known about the role of the apoptosis inhibitor c-FLIP and its potential impact on death receptor-induced apoptosis in ccRCC cells. In this study, we demonstrate that c-FLIP is crucial for resistance against CD95L-induced apoptosis in four ccRCC cell lines. Strikingly, downregulation of c-FLIP expression by short hairpin RNA (shRNA)interference led to spontaneous caspase activation and apoptotic cell death. Of note, knockdown of all c-FLIP splice variants was required to induce apoptosis. Stimulation of ccRCC cells with CD95L induced NF-κB and MAP kinase survival pathways as revealed by phosphorylation of RelA/p65 and Erk1/2. Interestingly, CD95L surface expression was high in all cell lines analyzed, and CD95 but not TNF-R1 clustered at cell contact sites. Downstream of CD95, inhibition of the NF-κB pathway led to spontaneous cell death. Surprisingly, knockdown experiments revealed that c-FLIP inhibits NF-κB activation in the context of CD95 signaling. Thus, c-FLIP inhibits apoptosis and dampens NF-κB downstream of CD95 but allows NF-κB activation to a level sufficient for ccRCC cell survival. In summary, we demonstrate a complex CD95-FLIP-NF-κB-signaling circuit, in which CD95-CD95L interactions mediate a paracrine survival signal in ccRCC cells with c-FLIP and NF-κB both being required for inhibiting cell death and ensuring survival. Our findings might lead to novel therapeutic approaches of RCC by circumventing apoptosis resistance.

Garcinol Enhances TRAIL-Induced Apoptotic Cell Death through Up-Regulation of DR5 and Down-Regulation of c-FLIP Expression.

Garcinol is a polyisoprenylated benzophenone derived from the Garcinia indica fruit that possess potential therapeutic effects such as inhibition of inflammation and tumor expansion. Here, we investigated whether garcinol induces TRAIL sensitization in renal carcinoma cells. Single treatment with garcinol or TRAIL did not effect on apoptosis. However, combined treatment with garcinol plus TRAIL significantly induced apoptosis in renal carcinoma (Caki, ACHN and A498), lung carcinoma (A549), and hepatoma (SK-Hep1) cells. In contrast, garcinol plus TRAIL did not alter cell viability in normal cells. Garcinol plus TRAIL induced up-regulation of DR5 and down-regulation of c-FLIP expression at post-translational levels. Furthermore, knock-down of DR5 by siRNA and ectopic expression of c-FLIP blocked apoptotic cell death induced by garcinol plus TRAIL. Overall, our study provides evidence that garcinol can be exploited as a potential TRAIL sensitizer.

Volasertib Enhances Sensitivity to TRAIL in Renal Carcinoma Caki Cells through Downregulation of c-FLIP Expression.

Polo-like kinase 1 (PLK1) plays major roles in cell cycle control and DNA damage response. Therefore, PLK1 has been investigated as a target for cancer therapy. Volasertib is the second-in class dihydropteridinone derivate that is a specific PLK1 inhibitor. In this study, we examined that combining PLK1 inhibitor with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) would have an additive and synergistic effect on induction of apoptosis in cancer cells. We found that volasertib alone and TRAIL alone had no effect on apoptosis, but the combined treatment of volasertib and TRAIL markedly induced apoptosis in Caki (renal carcinoma), A498 (renal carcinoma) and A549 (lung carcinoma) cells, but not in normal cells (human skin fibroblast cells and mesangial cells). Combined treatment induced accumulation of sub-G1 phase, DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and activation of caspase 3 activity in Caki cells. Interestingly, combined treatment induced downregulation of cellular-FLICE-inhibitory protein (c-FLIP) expression and ectopic expression of c-FLIP markedly blocked combined treatment-induced apoptosis. Therefore, this study demonstrates that volasertib may sensitize TRAIL-induced apoptosis in Caki cells via downregulation of c-FLIP.

Osthole enhances TRAIL-mediated apoptosis through downregulation of c-FLIP expression in renal carcinoma Caki cells.

Osthole, an active constituent isolated from the fruit of Cnidiummonnieri (L.) Cusson, has been shown to induce various beneficial biochemical effects such as anti-inflammatory and antitumor. In the present study, we examined whether osthole could sensitize TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human renal carcinoma Caki cells. We found that osthole and TRAIL alone, had no effect on apoptosis, but combined treatment with osthole and TRAIL markedly induced apoptosis in Caki (renal carcinoma), U251MG (glioma) and MDA-MB-231 (breast carcinoma) cells. In contrast, combined treatment with osthole and TRAIL did not induce apoptosis in normal human skin fibroblast cells. Osthole induced downregulation of cellular FLICE-like inhibitory protein (c-FLIP) expression, and overexpression of c-FLIP markedly blocked apoptosis induced by the combined treatment with osthole and TRAIL. In addition, osthole markedly reduced mitochondrial membrane potential levels, and increased cytosolic cytochromec release in combined treatment with osthole and TRAIL. Therefore, these data suggest that osthole may be an efficient TRAIL sensitizer.

YM155 sensitizes TRAIL-induced apoptosis through cathepsin S-dependent down-regulation of Mcl-1 and NF-κB-mediated down-regulation of c-FLIP expression in human renal carcinoma Caki cells.

YM155, a small-molecule survivin inhibitor, has been reported for its anti-cancer activity in various cancer cells. In this study, we investigated the effect of YM155 to enhance TRAIL-mediated apoptosis in human renal carcinoma cells. We found that YM155 alone had no effect on apoptosis, however, combined treatment with YM155 and TRAIL markedly induced apoptosis in human renal carcinoma cells (Caki, ACHN, and A498), breast cancer cells (MDA-MB231), and glioma cells (U251MG), but not normal cells [mesangial cell (MC) and human skin fibroblast (HSF)]. YM155 induced down-regulation of Mcl-1 expression at the post-translational levels, and the overexpression of Mcl-1 markedly inhibited YM155 plus TRAIL-induced apoptosis. Furthermore, YM155 induced down-regulation of c-FLIP mRNA expression through inhibition of NF-κB transcriptional activity. Ectopic expression of c-FLIP markedly blocked YM155-induced TRAIL sensitization. Taken together, our results suggested that YM155 sensitizes TRAIL-mediated apoptosis via down-regulation of Mcl-1 and c-FLIP expression in renal carcinoma Caki cells.

Abstract 4780: c-FLIP knockdown increases apoptosis in cervical cancer cell lines

Abstract Cervical Uterine cancer is a leading cause of cancer deaths in women worldwide. Its natural history begins gradually from low-grade and high-grades squamous intraepithelial lesions to an invasive disease. Decades of studies have confirmed a directly association between persistent high-risk Human Papillomavirus (HPV) infections and cervical cancer. However, high-risk HPV infections are not sufficient to induce cellular transformation and tumor occurrence. The cellular FLICE-like inhibitory protein (c-FLIP) has been founded overexpressed in several cancers and could be associated with cervical carcinogenesis. c-FLIP is a catalytically inactive caspase-8/-10 homologue, and when is overexpressed has the ability to inhibit the extrinsic pathway of apoptosis. Aim: To characterize c-FLIP participation in cervical carcinogenesis.Materials and Methods: Four cell lines were used in this study: ECT1 E6/E7 (squamous normal cervical epithelial), SiHa (cervical carcinoma containing an integrated HPV 16 genome), C-4I (cervical carcinoma containing an integrated HPV 18 genome) and C-33A (cervical carcinoma without HPV). c-FLIP expression in cell lines was determined by real time PCR and western blot. c-FLIP expression was transiently downregulated by siRNA directed against c-FLIP mRNA and the silencing effects on cell viability and apoptosis were analysed, by comparing with a control negative siRNA-transfected cells.Results: c-FLIP was found to be overexpressed in SiHa and C-4I cell lines and repressed in C-33A cell line. Only the long variant of c-FLIP was found in cervical cancer cell lines. Transient knockdown of c-FLIP resulted in significant reduction in cell viability (p< 0.001), compared with negative control siRNA-transfected cells. Knockdown cells lines exhibited an increased apoptotic rate in comparison with controls (p<0,001). Conclusions: The long variant of c-FLIP is overexpressed in cervical cancer cell lines with integrated genome of a high-risk HPV. Furthermore, the siRNA-mediated downregulation of c-FLIP expression induces apoptosis and decreases cell viability in cervical cancer cell lines. Therefore, this protein could be a potential therapeutic target in cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4780. doi:10.1158/1538-7445.AM2011-4780

Rocaglamide breaks TRAIL resistance in HTLV-1-associated adult T-cell leukemia/lymphoma by translational suppression of c-FLIP expression

The human T-cell leukemia virus type-1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATL) is incurable by currently known therapies. ATL samples and cell lines derived from ATL patients show restricted sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L). We have recently shown that HTLV-1-infected cells express elevated levels of cellular caspase-8 FLICE-inhibitory protein (c-FLIP) conferring resistance to receptor-mediated apoptosis. This finding underscores the demand to develop new strategies for treatment of ATL. In this study, we show that the naturally occurring herbal compound Rocaglamide (Roc) sensitizes CD95L- and TRAIL-induced apoptosis in HTLV-1-infected cells by downregulation of c-FLIP expression. Investigation of the molecular mechanism of Roc-mediated downregulation of c-FLIP revealed that it inhibits phosphorylation of the translation initiation factor 4E (eIF4E), a key factor that controls the rate-limiting step of translation, through inhibition of the MEK-ERK-MNK1 signaling pathway. This event prevents de novo synthesis of short-lived proteins such as c-FLIP in HTLV-1-infected cells. Our data suggest that Roc may serve as an adjuvant for TRAIL-based anticancer therapy.

Abstract 1280: Inhibition of cellular FLICE-inhibitory protein and induction of Death Receptor 5 expression is a major component of δ- tocotrienol-induced apoptosis in pancreatic cancer cells

Abstract Background: Previous pre-clinical studies have demonstrated that δ-tocotrienol, is the most bioactive natural vitamin E compound against pancreatic cancer. δ-Tocotrienol induces growth arrest and apoptosis in several human pancreatic cancer cell lines; however, the underlying mechanism remains unknown. In this study, we evaluated the role of the extrinsic and intrinsic apoptotic signaling pathways in δ-tocotrienol-induced apoptosis. Also, we investigated the modulation of cellular FLICE-inhibitory protein (c-FLIP) and death receptor 5 (DR5) by δ-tocotrienol, and the involvement of c-FLIP down-regulation and DR5 up-regulation in δ-tocotrienol-induced apoptosis. Methods: We examined δ-tocotrienol induced apoptosis in human pancreatic cancer cell lines in vitro and in vivo. We used western blot and immunohistochemistry to determine the expression of DR4, DR5, Caspase 8, Caspase 9, Caspase 3, c-FLIP, and PARP. The effect of combination treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and δ-tocotrienol was also determined. We examined the cellular effects of DR5 knockdown using small interfering RNA oligonucleotides, and c-FLIP overexpression using c-FLIP cDNA, on δ-tocotrienol-induced apoptosis using various assays. Results: Delta-tocotrienol activated caspase-8 and its downstream caspases but not caspase-9. Caspase-8 specific inhibitor abrogated δ-tocotrienol-induced apoptosis whereas caspase-9 specific inhibitor did not, indicating that δ-tocotrienol induces caspase-8 -dependent apoptosis. c-FLIP was rapidly down-regulated by δ-tocotrienol in all of the tested cell lines. Delta-tocotrienol up-regulated DR4 and DR5 significantly and enhanced TRAIL-induced apoptosis. Enforced expression of ectopic c-FLIP, and DR5 knockdown partially abolished δ-tocotrienol-induced apoptosis. δ-Tocotrienol inhibition of pancreatic tumor growth in SCID mice was associated with significant induction of apoptosis, activation of caspase 8, and down-regulation of c-FLIP expression. Conclusion: We conclude that DR5 up-regulation and c-FLIP down-regulation contributes to δ-tocotrienol-induced apoptosis in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1280.

Abstract 222: Inhibition of cellular FLICE-inhibitory protein and induction of death receptor 5 expression is a major component of δ-tocotrienol-induced apoptosis in pancreatic cancer cells

Abstract Background: Previous pre-clinical studies have demonstrated that δ-tocotrienol, is the most bioactive natural vitamin E compound against pancreatic cancer. δ-Tocotrienol induces growth arrest and apoptosis in several human pancreatic cancer cell lines; however, the underlying mechanism remains unknown. In this study, we evaluated the role of the extrinsic and intrinsic apoptotic signaling pathways in δ-tocotrienol induced apoptosis and investigated the modulation of cellular FLICE-inhibitory protein (c-FLIP) and death receptor 5 (DR5) by δ-tocotrienol and the involvement of c-FLIP down-regulation and DR5 up-regulation in δ-tocotrienol-induced apoptosis. Methods: We examined δ-tocotrienol induced apoptosis in human pancreatic cancer cell lines in vitro and in vivo. We used western blot and immunohistochemistry to determine the expression of DR4, DR5, Caspase 8, Caspase 9, Caspase 3, C-FLIPs and PARP. The effect of combination treatment with TRAIL and δ-tocotrienol was also determined. We examined the cellular effects of DR5 knockdown using small interfering RNA oligonucleotides, and c-FLIP overexpression using cDNA, on δ-tocotrienol-induced apoptosis using various assays. Results: Delta-tocotrienol activated caspase-8 and its downstream caspases but not caspase-9, whereas caspase-8 specific inhibitor abrogated δ-tocotrienol-induced apoptosis indicating that δ-tocotrienol induces caspase-8-dependent apoptosis. c-FLIP was rapidly down-regulated by δ-tocotrienol in all of the tested cell lines. δ-Tocotrienol up-regulated DR4 and DR5 significantly and enhanced tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Enforced expression of ectopic c-FLIP, and DR5 knockdown partially abolished δ-tocotrienol-induced apoptosis. δ-Tocotrienol inhibition of pancreatic tumor growth in SCID mice was associated with significant induction of apoptosis, activation of caspase 8, and down-regulation of c-FLIP expression. Conclusion: We conclude that DR5 up-regulation and c-FLIP down-regulation contributes to δ-tocotrienol-induced apoptosis in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 222.

The sesquiterpene lactone eupatolide sensitizes breast cancer cells to TRAIL through down-regulation of c-FLIP expression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapeutics due to its ability to induce apoptosis selectively in cancer cells. However, sensitivity of cancer cells for induction of apoptosis by TRAIL varies considerably. Therefore, it is important to develop agents that overcome this resistance. We show, for the first time, that eupatolide, the sesquiterpene lactone isolated from the medicinal plant Inula britannica, sensitizes human breast cancer cells to TRAIL-induced apoptosis. Treatment with TRAIL in combination with subtoxic concentrations of eupatolide enhanced the TRAIL-induced cytotoxicity in MCF-7, MDA-MB-231 and MDA-MB-453 breast cancer cells, whereas each reagent alone slightly induced cell death. The combination induced sub-G1 phase DNA content and annexin V-staining in MCF-7 cells, which are major features of apoptosis. Apoptotic characteristics induced by the combined treatment were significantly inhibited by a pan-caspase inhibitor. The sensitization to TRAIL-induced apoptosis was accompanied by the activation of caspase-8 and was concomitant with Bid and poly(ADP-ribose) polymerase (PARP) cleavage. Treatment of eupatolide alone significantly down-regulated the expression of cellular FLICE inhibitory protein (c-FLIP) in MCF-7 cells. Furthermore, enforced expression of c-FLIP significantly attenuated the apoptosis induced by this combination in MCF-7 cells, suggesting a key role for c-FLIP down-regulation in these events. We also observed that euaptolide inhibited AKT phosphorylation in a dose- and time-dependent manner. Moreover, inhibition of Akt by LY294002, a specific PI3K inhibitor, down-regulated c-FLIP expression in MCF-7 cells. Taken together, these results indicate that eupatolide could augment TRAIL-induced apoptosis in human breast cancer cells by down-regulating c-FLIP expression through the inhibition of AKT phosphorylation and be a valuable compound to overcome TRAIL resistance in breast cancer cells.

Herpes Simplex Virus Type 1 (HSV-1)-Induced Apoptosis in Human Dendritic Cells as a Result of Downregulation of Cellular FLICE-Inhibitory Protein and Reduced Expression of HSV-1 Antiapoptotic Latency-Associated Transcript Sequences

Herpes simplex virus type 1 (HSV-1) is one of the most frequent and successful human pathogens. It targets immature dendritic cells (iDCs) to interfere with the antiviral immune response. The mechanisms underlying apoptosis of HSV-1-infected iDCs are not fully understood. Previously, we have shown that HSV-1-induced apoptosis of iDCs is associated with downregulation of the cellular FLICE-inhibitory protein (c-FLIP), a potent inhibitor of caspase-8-mediated apoptosis. In this study, we prove that HSV-1 induces degradation of c-FLIP in a proteasome-independent manner. In addition, by using c-FLIP-specific small interfering RNA (siRNA) we show for the first time that downregulation of c-FLIP expression is sufficient to drive uninfected iDCs into apoptosis, underlining the importance of this molecule for iDC survival. Surprisingly, we also observed virus-induced c-FLIP downregulation in epithelial cells and many other cell types that do not undergo apoptosis after HSV-1 infection. Microarray analyses revealed that HSV-1-encoded latency-associated transcript (LAT) sequences, which can substitute for c-FLIP as an inhibitor of caspase-8-mediated apoptosis, are much less abundant in iDCs as compared to epithelial cells. Finally, iDCs infected with an HSV-1 LAT knockout mutant showed increased apoptosis when compared to iDCs infected with the corresponding wild-type HSV-1. Taken together, our results demonstrate that apoptosis of HSV-1-infected iDCs requires both c-FLIP downregulation and diminished expression of viral LAT.

Increased susceptibility to tumor necrosis factor‐α in butyric acid‐induced apoptosis is caused by downregulation of cFLIP expression in Jurkat T cells

Butyric acid is one of the major extracellular metabolites of periodontopathic Gram-negative bacteria. We previously demonstrated that butyric acid induced apoptosis in human T cells. In the present study, we examined the interaction between butyric acid and TNF-alpha in Jurkat T-cell apoptosis. Simultaneous treatment with TNF-alpha enhanced butyric acid-induced apoptosis by promoting caspase activity more than was achieved by either reagent alone. We examined which genes were associated with the increased susceptibility to TNF-alpha caused by butyric acid, and revealed that expression of cFLIP decreased with increased concentrations of butyric acid. Furthermore, exogenous expression of cFLIP protein suppressed the enhancing effect by TNF-alpha in the apoptosis. These results suggest that butyric acid downregulates cFLIP expression and increases the susceptibility to TNF-alpha by activating caspases via the death receptor signal.

The alkylphospholipid perifosine induces apoptosis of human lung cancer cells requiring inhibition of Akt and activation of the extrinsic apoptotic pathway

The Akt inhibitor, perifosine, is an alkylphospholipid exhibiting antitumor properties and is currently in phase II clinical trials for various types of cancer. The mechanisms by which perifosine exerts its antitumor effects, including the induction of apoptosis, are not well understood. The current study focused on the effects of perifosine on the induction of apoptosis and its underlying mechanisms in human non-small cell lung cancer (NSCLC) cells. Perifosine, at clinically achievable concentration ranges of 10 to 15 micromol/L, effectively inhibited the growth and induced apoptosis of NSCLC cells. Perifosine inhibited Akt phosphorylation and reduced the levels of total Akt. Importantly, enforced activation of Akt attenuated perifosine-induced apoptosis. These results indicate that Akt inhibition is necessary for perifosine-induced apoptosis. Despite the activation of both caspase-8 and caspase-9, perifosine strikingly induced the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 5, and down-regulated cellular FLICE-inhibitory protein (c-FLIP), an endogenous inhibitor of the extrinsic apoptotic pathway, with limited modulatory effects on the expression of other genes including Bcl-2, Bcl-X(L), PUMA, and survivin. Silencing of either caspase-8 or death receptor 5 attenuated perifosine-induced apoptosis. Consistently, further down-regulation of c-FLIP expression with c-FLIP small interfering RNA sensitized cells to perifosine-induced apoptosis, whereas enforced overexpression of ectopic c-FLIP conferred resistance to perifosine. Collectively, these data indicate that activation of the extrinsic apoptotic pathway plays a critical role in perifosine-induced apoptosis. Moreover, perifosine cooperates with TRAIL to enhance the induction of apoptosis in human NSCLC cells, thus warranting future in vivo and clinical evaluation of perifosine in combination with TRAIL in the treatment of NSCLC.

Sodium arsenite accelerates TRAIL-mediated apoptosis in melanoma cells through upregulation of TRAIL-R1/R2 surface levels and downregulation of cFLIP expression

AP-1/cJun, NF-κB and STAT3 transcription factors control expression of numerous genes, which regulate critical cell functions including proliferation, survival and apoptosis. Sodium arsenite is known to suppress both the IKK-NF-κB and JAK2-STAT3 signaling pathways and to activate the MAPK/JNK-cJun pathways, thereby committing some cancers to undergo apoptosis. Indeed, sodium arsenite is an effective drug for the treatment of acute promyelocytic leukemia with little nonspecific toxicity. Malignant melanoma is highly refractory to conventional radio- and chemotherapy. In the present study, we observed strong effects of sodium arsenite treatment on upregulation of TRAIL-mediated apoptosis in human and mouse melanomas. Arsenite treatment upregulated surface levels of death receptors, TRAIL-R1 and TRAIL-R2, through increased translocation of these proteins from cytoplasm to the cell surface. Furthermore, activation of cJun and suppression of NF-κB by sodium arsenite resulted in upregulation of the endogenous TRAIL and downregulation of the cFLIP gene expression (which encodes one of the main anti-apoptotic proteins in melanomas) followed by cFLIP protein degradation and, finally, by acceleration of TRAIL-induced apoptosis. Direct suppression of cFLIP expression by cFLIP RNAi also accelerated TRAIL-induced apoptosis in these melanomas, while COX-2 suppression substantially increased levels of both TRAIL-induced and arsenite-induced apoptosis. In contrast, overexpression of permanently active AKTmyr inhibited TRAIL-mediated apoptosis via downregulation of TRAIL-R1 levels. Finally, AKT overactivation increased melanoma survival in cell culture and dramatically accelerated growth of melanoma transplant in vivo, highlighting a role of AKT suppression for effective anticancer treatment.

A role for caspase-8 and c-FLIP L in proliferation and cell-cycle progression of primary hepatocytes

Growth factors are known to favor both proliferation and survival of hepatocytes. In the present study, we investigated if c-FLIP(L) (cellular FLICE-inhibitory protein, long isoform) could be involved in epidermal growth factor (EGF)-stimulated proliferation of rat hepatocytes since c-FLIP(L) regulates both cell proliferation and procaspase-8 maturation. Treatment with MEK inhibitors prevented induction of c-FLIP(L) by EGF along with total inhibition of DNA replication. However, EGF failed to inhibit processing of procaspase-8 in the presence of EGF suggesting that c-FLIP(L) does not play its canonical anti-apoptotic role in this model. Downregulation of c-FLIP expression using siRNA oligonucleotides strongly reduced DNA replication but did not result in enhanced apoptosis. Moreover, intermediate cleavage products of c-FLIP(L) and caspase-8 were found in EGF-treated hepatocytes in the absence of caspase-3 maturation and cell death. To determine whether the Fas/FADD/caspase-8/c-FLIP(L) complex was required for this activity, Fas, procaspase-8 and Fas-associated death domain protein (FADD) expression or function was inhibited using siRNA or constructs encoding dominant negative mutant proteins. Inhibition of any of these components of the Fas/FADD/caspase-8 pathway decreased DNA replication suggesting a function of these proteins in cell-cycle arrest. Similar results were obtained when the IETD-like caspase activity detectable in EGF-treated hepatocytes was inhibited by the pan-caspase inhibitor, z-ASP. Finally, we demonstrated co-immunoprecipitation between EGFR and Fas within 15 min following EGF stimulation. In conclusion, our results indicate that the Fas/FADD/c-FLIP(L)/caspase-8 pathway positively controls the G(1)/S transition in EGF-stimulated hepatocytes. Our data provide new insights into the mechanisms by which apoptotic proteins participate to mitogenic signals during the G(1) phase.