The deubiquitinase Usp27x as a novel regulator of cFLIPL protein expression and sensitizer to death-receptor-induced apoptosis.

Death receptors are transmembrane proteins that can induce the activation of caspase-8 upon ligand binding, initiating apoptosis. Recent work has highlighted the great molecular complexity of death receptor signalling, in particular through ubiquitination/deubiquitination. We have earlier defined the deubiquitinase Ubiquitin-Specific Protease 27x (Usp27x) as an enzyme capable of stabilizing the pro-apoptotic Bcl-2 family member Bim. Here, we report that enhanced expression of Usp27x in human melanoma cells leads to the loss of cellular FLICE-like inhibitory protein (cFLIP) and sensitizes to Tumor necrosis factor receptor 1 (TNF-R1) or Toll-like receptor 3 (TLR3)-induced extrinsic apoptosis through enabling enhanced processing of caspase-8. The loss of cFLIPL upon overexpression of Usp27x was not due to reduced transcription, could be partially counteracted by blocking the ubiquitin proteasome system and was independent of the known cFLIPL destabilizing ubiquitin E3-ligases Itch and DTX1. Instead, Usp27x interacted with the E3-ligase TRIM28 and reduced ubiquitination of TRIM28. Reduction of cFLIPL protein levels by Usp27x-induction depended on TRIM28, which was also required for polyI:C-induced cell death. This work defines Usp27x as a novel regulator of cFLIPL protein expression and a deubiquitinase in fine tuning death receptor signalling pathways to execute apoptosis.

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.

Lactucin induces apoptosis through reactive oxygen species-mediated BCL-2 and CFLARL downregulation in Caki-1 cells.

BACKGROUND: Lactucin, a naturally occurring active sesquiterpene lactone, is abundantly found in chicory and romaine lettuce. A recent study reported that lactucin could induce apoptosis in leukemia cells. However, its cytotoxicity and potential molecular mechanisms underlying cancer cell death remain unclear. OBJECTIVE: Therefore, in this study, we aimed to investigate the direct effect and underlying mechanism of action of lactucin on renal cancer cells. METHODS: MTT assay and flow cytometry were performed to evaluate the rate of cell proliferation and apoptosis, respectively. Western blotting, reverse transcription polymerase chain reaction, and protein stability analyses were performed to analyze the effect of lactucin on the expression of apoptosis-related proteins such as B-cell lymphoma 2 (BCL-2) and CFLAR (CASP8 and FADD like apoptosis regulator) long isoform (CFLARL) in Caki-1 human renal cancer cells. In addition, reactive oxygen species (ROS) generation was evaluated using flow cytometry. RESULTS: Lactucin treatment induced apoptosis in Caki-1 cells in a dose-dependent manner via activation of the caspase pathway. It downregulated BCL-2 and CFLARL expression levels by suppressing BCL-2 transcription and CFLARL protein stability, respectively. Pretreatment with N-acetyl-1-cysteine, a ROS scavenger, attenuated the lactucin-induced apoptosis and restored the BCL-2 and CFLARL expression to basal levels. Lactucin-facilitated BCL-2 downregulation was regulated at the transcriptional level through the inactivation of the NF-κB pathway. CONCLUSIONS: Our study is the first to demonstrate that lactucin-induced apoptosis is mediated by ROS production, which in turn activates the caspase-dependent apoptotic pathway by inhibiting BCL-2 and CFLARL expression in Caki-1 cells.

Metformin induces caspase-dependent and caspase-independent apoptosis in human bladder cancer T24 cells.

Bladder cancer (BC) is the sixth most common cancer in men. Moreover, chemotherapy for BC leads to various side effects. Metformin is known to induce apoptosis in vitro in many types of cancer. Furthermore, it has feasibility as a drug repositioning used for the treatment of cancer. The molecular mechanism of metformin mediating apoptosis in BC is still unclear. In this study, we showed that metformin stimulated the caspase-dependent apoptotic signaling pathway in T24 cells, a human BC cell line. Moreover, the induced apoptosis was partially inhibited by a general caspase inhibitor, z-VAD-fmk, which suggested that metformin-induced apoptosis in T24 cells is partially caspase-independent. Notably, we observed the nuclear translocation of apoptosis-inducing factors (AIFs) in metformin-promoted apoptosis, which is a typical characteristic of the caspase-independent apoptotic pathway. In addition, we found that metformin-mediated apoptosis occurred via degradation of the cellular FADD-like interleukin-1ß-converting enzyme inhibitory protein (c-FLIP) by facilitating ubiquitin/proteasome-mediated c-FLIPL degradation. Furthermore, treatment with the reactive oxygen species scavenger N-acetylcysteine, failed to suppress metformin-induced apoptosis and c-FLIPL protein degradation in metformin-treated T24 cells. In conclusion, these results indicate that metformin-induced apoptosis was mediated through AIF-promoted caspase-independent pathways as well as caspase-dependent pathways in T24 cells. As such, metformin could be used as a possible apoptotic agent for the treatment of BC.

Magnetic nanoparticle-mediated gene therapy to induce Fas apoptosis pathway in breast cancer.

CD95 (Fas) is a complex integral protein that can be expressed in many cells. It induces apoptosis when interacted with its ligand CD95L (FasL). However, cancer cells are resistant to CD95-induced apoptosis because of the changes in death domain (DD) of CD95 (procaspase-8 and c-Flip). In this study, magnetic nanoparticles and lipid-based gene transfection methods were performed to provide active Fas expression in breast cancer cells. Plasmid DNA (pDNA), which can express both human Fas and GFP, was transfected to MCF-7 breast cancer cells. Expression of c-FLIP and caspase-8 and effect of monoclonal antibody FasL for apoptosis stimulation were investigated. Also transfection success of methods and effects on surface protein were compared. Western blot results indicated that MCF-7 cells do not express caspase-8 but express large amount of c-FLIPL. Both lipid-based and magnetic nanoparticle-mediated gene transfection methods successfully applied. Caspase-8 apoptosis pathway was activated on transfected cells. Magnetic nanoparticle-mediated gene transfer is a successful non-viral method for transfection, and it does not affect the expression of other cell proteins, such as beta actin and lamin-B1. The raised c-FLIPL concentration in cytosol inhibits apoptosis. However, transfection of CD95-GFP-tagged pDNA significantly increases apoptosis by activating caspase-8 pathway. FasL interaction indicated a slight increase of apoptosis in the transfected cells. The method and pDNA applied in this study have potentials to be used in gene therapy for breast cancer.

EOMES-positive CD4+ T cells are increased in PTPN22 (1858T) risk allele carriers.

The presence of the PTPN22 risk allele (1858T) is associated with several autoimmune diseases including rheumatoid arthritis (RA). Despite a number of studies exploring the function of PTPN22 in T cells, the exact impact of the PTPN22 risk allele on T-cell function in humans is still unclear. In this study, using RNA sequencing, we show that, upon TCR-activation, naïve human CD4+ T cells homozygous for the PTPN22 risk allele overexpress a set of genes including CFLAR and 4-1BB, which are important for cytotoxic T-cell differentiation. Moreover, the protein expression of the T-box transcription factor Eomesodermin (EOMES) was increased in T cells from healthy donors homozygous for the PTPN22 risk allele and correlated with a decreased number of naïve CD4+ T cells. There was no difference in the frequency of other CD4+ T-cell subsets (Th1, Th17, Tfh, Treg). Finally, an accumulation of EOMES+ CD4+ T cells was observed in synovial fluid of RA patients with a more pronounced production of Perforin-1 in PTPN22 risk allele carriers. Altogether, we propose a novel mechanism of action of PTPN22 risk allele through the generation of cytotoxic CD4+ T cells and identify EOMES+ CD4+ T cells as a relevant T-cell subset in RA pathogenesis.

Targeting of miR-20a against CFLAR to potentiate TRAIL-induced apoptotic sensitivity in HepG2 cells.

The article "Targeting of miR-20a against CFLAR to potentiate TRAIL-induced apoptotic sensitivity in HepG2 cells" by Y. Wang, Y.-R. Zhao, A.-Y. Zhang, J. Ma, Z.-Z. Wang, X. Zhang, published in Eur Rev Med Pharmacol Sci 2017; 21 (9): 2087-2097 has been withdrawn.

TNF Signaling in Peritoneal Mesothelial Cells: Pivotal Role of cFLIPL.

♦ BACKGROUND: Peritoneal dialysis (PD) coincides with high concentrations of proinflammatory cytokines, such as tumor necrosis factor (TNF), in the peritoneal cavity. During treatment, chronic inflammatory processes lead to damage of the peritoneal membrane and a subsequent ultrafiltration failure. Human peritoneal mesothelial cells (HPMCs) play a central role as mediators and targets of PD-related inflammatory changes. Although TNF Receptor 1 (TNFR1) is expressed in high numbers on the cells, TNF-induced apoptosis is inhibited. Here, the underlying molecular mechanisms of TNFR1 signaling in HPMCs are investigated. ♦ METHODS: Human peritoneal mesothelial cells were isolated from the omentum of healthy donors and the dialysis solution of PD patients. Flow cytometry was applied to determine cell surface expression of TNFR1 on HPMCS from healthy donors in absence or presence of TNF or PD fluid (PDF) and were compared to TNFR1 expression on cells from PD patients. To investigate TNFR1-mediated signaling, HPMCs were treated with PDF or TNF, and expression patterns of proteins involved in the TNFR1 signaling pathway were assessed by western blot. ♦ RESULTS: Incubation with PDF led to a significant up-regulation of TNFR1 on the cell surface correlating with elevated TNFR1 numbers on HPMCs from PD patients. Investigations of underlying molecular mechanisms of TNFR1 signaling showed that PDF affects TNFR1 signaling at the proapoptotic signaling pathway by upregulation of IκBα and downregulation of cFLIPL. In contrast, TNF exclusively induces the activation of NFκB by an increase of phosphorylated IκBα. ♦ CONCLUSIONS: Novel and relevant insights into the mechanisms of TNFR1-mediated signaling in HPMCs with an impact on our understanding of PD-associated damage of the peritoneal membrane are shown.

Expression of FADD and cFLIPL balances mitochondrial integrity and redox signaling to substantiate apoptotic cell death.

FADD and cFLIP both are pivotal components of death receptor signaling. The cellular signaling of apoptosis accomplished with death receptors and mitochondria follows independent pathways for cell death. FADD and cFLIP both have an important role in the regulation of apoptotic and non-apoptotic functions. Dysregulated expression of FADD and cFLIP is associated with resistance to apoptosis in cancer cells. Mitochondria are known to play critical role in maintaining cellular respiration and homeostasis in the cells as well as transduces various signals to determine the fate of cell death. However, involvement of FADD and cFLIP in regulation of mitochondrial integrity and programmed cell death signaling to define the fate of cells remains elusive. In the present study, we explored that, induced expression of FADD challenges the mitochondrial integrity and pulverizes the membrane potential by altering the expression of Bcl-2 and cytochrome c. In contrast, mutant of FADD was unable to affect the mitochondrial integrity. Interestingly, expression of FADD and cFLIP helps to balance redox potential by regulating the anti-oxidant levels. Further, we noticed that, knockdown of cFLIPL and induced expression of FADD rapidly accumulate intracellular ROS accompanied by JNK1 activation to substantiate apoptosis. Notably, the ectopic expression of cFLIPL resists the sensitivity of cancer cells against apoptosis inducers Etoposide and HA14-1. Altogether, our findings suggest that FADD and cFLIPL are important modulators of mitochondrial-associated apoptosis apart from the death receptor signaling.

Thymoquinone induces apoptosis through downregulation of c-FLIP and Bcl-2 in renal carcinoma Caki cells.

Renal carcinoma is a common and frequently fatal carcinoma occurring worldwide and death rates due to this carcinoma are increasing with time. In the present study, we investigated the potential of thymoquinone a natural compound to induce apoptosis in renal carcinoma Caki cells. Thymoquinone efficiently enhanced the apoptotic population of Caki cells in a dose-dependent manner. Moreover, thymoquinone-mediated apoptosis caused downregulation of c-FLIP and Bcl-2, the master regulators of the anti-apoptotic mechanism. However, we did not find any changes in mRNA expression level of c-FLIP, therefore; this regulation of c-FLIP was a result of post-translation modification by thymoquinone. In contrast, expression of the Bcl-2 protein was observed at both transcriptional and translational level. However, we also observed that thymoquinone enhanced the level of intracellular reactive oxygen species (ROS) in Caki cells, which resulted in reduction of mitochondrial membrane potential (MMP) and cytochrome c release into cytoplasm. Our results postulate that thymoquinone induces apoptosis through downregulating c-FLIP and Bcl-2 which can be utilized as a chemotherapeutic agent to treat renal carcinoma.

Overcoming resistance to TRAIL-induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs.

The discovery of the TRAIL protein and its death receptors DR4/5 changed the horizon of cancer research because TRAIL specifically kills cancer cells. However, the validity of TRAIL-based cancer therapies has yet to be established, as most cancer cells are TRAIL-resistant. In this report, we demonstrate that TRAIL-resistance of many cancer cell lines can be overcome after siRNA- or rocaglamide-mediated downregulation of c-FLIP expression and simultaneous inhibition of IAPs activity using AT406, a pan-antagonist of IAPs. Combined triple actions of the TRAIL, the IAPs inhibitor, AT406, and the c-FLIP expression inhibitor, rocaglamide (ART), markedly improve TRAIL-induced apoptotic effects in most solid cancer cell lines through the activation of an extrinsic apoptosis pathway. Furthermore, this ART combination does not harm normal cells. Among the 18 TRAIL-resistant cancer cell lines used, 15 cell lines become sensitive or highly sensitive to ART, and two out of three glioma cell lines exhibit high resistance to ART treatment due to very low levels of procaspase-8. This study provides a rationale for the development of TRAIL-induced apoptosis-based cancer therapies.

The pyrrolo-1,5-benzoxazepine, PBOX-15, enhances TRAIL­induced apoptosis by upregulation of DR5 and downregulation of core cell survival proteins in acute lymphoblastic leukaemia cells.

Apoptotic defects are frequently associated with poor outcome in pediatric acute lymphoblastic leukaemia (ALL) hence there is an ongoing demand for novel strategies that counteract apoptotic resistance. The death ligand TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) and its selective tumour receptor system has attracted exceptional clinical interest. However, many malignancies including ALL are resistant to TRAIL monotherapy. Tumour resistance can be overcome by drug combination therapy. TRAIL and its agonist antibodies are currently undergoing phase II clinical trials with established chemotherapeutics. Herein, we present promising therapeutic benefits in combining TRAIL with the selective anti-leukaemic agents, the pyrrolo-1,5-benzoxazepines (PBOXs) for the treatment of ALL. PBOX-15 synergistically enhanced apoptosis induced by TRAIL and a DR5-selective TRAIL variant in ALL-derived cells. PBOX-15 enhanced TRAIL-induced apoptosis by dual activation of extrinsic and intrinsic apoptotic pathways. The specific caspase-8 inhibitor, Z-IETD-FMK, identified the extrinsic pathway as the principal mode of apoptosis. We demonstrate that PBOX-15 can enhance TRAIL-induced apoptosis by upregulation of DR5, reduction of cellular mitochondrial potential, activation of the caspase cascade and downregulation of PI3K/Akt, c-FLIP, Mcl-1 and IAP survival pathways. Of note, the PI3K pathway inhibitor LY-294002 significantly enhanced the apoptotic potential of TRAIL and PBOX-15 validating the importance of Akt downregulation in the TRAIL/PBOX-15 synergistic combination. Considering the lack of cytotoxicity to normal cells and ability to downregulate several survival pathways, PBOX-15 may represent an effective agent for use in combination with TRAIL for the treatment of ALL.

Gambogic acid induces apoptosis and sensitizes TRAIL-mediated apoptosis through downregulation of cFLIPL in renal carcinoma Caki cells.

Gambogic acid (GA) is a natural compound derived from brownish gamboge resin that shows a range of bioactivity, such as antitumor and antimicrobial properties. Although, GA is already known to induce cell death in a variety of cancer cells, the molecular basis for GA-induced cell death in renal cancer cells is unclear. In this study, a treatment with GA induced cell death in human renal carcinoma Caki cells in a dose-dependent manner. Treatment of Caki cells with GA decreased the levels of antiapoptotic proteins, such as Bcl-2 and XIAP in a dose-dependent manner. In addition, GA decreased the expression of the cFLIPL protein, which was downregulated at the transcriptional level without any change in the levels of cFLIPs expression. z-VAD (pan-caspase inhibitor) partially blocked GA-mediated cell death. GA-induced apoptotic cell death in Caki cells is mediated partly by the AIF translocation from the mitochondria into the nucleus via a caspase-independent pathway. In contrast, N-acetylcysteine (NAC), a ROS scavenger, had no effect on GA-induced cell death. The restoration of cFLIPL attenuated GA-induced cell death in Caki cells. Furthermore, a sub-toxic dose of GA sensitized TRAIL-mediated apoptosis in Caki cells. Pretreatment with z-VAD completely blocked GA plus TRAIL-mediated apoptosis. On the contrary, pretreatment with NAC partially inhibited GA plus TRAIL-induced apoptosis. Our findings suggested that GA induces apoptosis via the downregulation of cFLIPL and sensitized TRAIL-mediated apoptosis in Caki cells.

Expression and localization of cFLIP anti-apoptotic protein in the porcine corpus luteum and corpora albicans during the estrous cycle and pregnancy.

We determined expression and localization of the anti-apoptotic cellular FLICE inhibitory protein (cFLIP) in the porcine corpora lutea (CL) and corpus albicans (CA) during estrous and pregnancy. The CL and CA were collected at different stages of estrous to determine cFLIP immunolocalization, and mRNA and protein expression. The mRNA expression of the short cFLIP isoform (cFLIPS) was higher at the early and mid CL stages, and lower by the late CL stage (P < 0.01); mRNA expression of the long cFLIP isoform (cFLIPL) was higher at the mid CL stage, and lower at the early and late CL stages (P < 0.01). Levels of cFLIPS and cFLIPL were steady and high during the early and mid CL stages, and had significantly decreased (P < 0.01) by the late stage. The cFLIP protein was highly expressed in the early and mid CL stages of estrous, but weakly ex-pressed in the late stage. Expression of cFLIPS showed no significant difference between preovulatory corpus albicans (CA1) and corpus albicans (CA2) coexistent with the CL from the previous estrus, but cFLIPL mRNA expression was higher during CA1 than CA2. The expression of cFLIPS showed no significant difference between CA1 and CA2, but cFLIPL was not detected. The cFLIP protein was weak-ly expressed in the CA. Expression of cFLIPS and cFLIPL mRNA and proteins was observed in the CL, and the cFLIP protein was highly expressed during pregnancy. We propose that cFLIPS/L acts as a survival factor, and performs an anti-apoptotic function in the porcine CL.

Temozolomide sensitizes stem-like cells of glioma spheres to TRAIL-induced apoptosis via upregulation of casitas B-lineage lymphoma (c-Cbl) protein.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has potent antitumor effects in glioma cell lines but has shown little clinical benefit for patients. We investigated whether the widely used chemotherapeutic agent temozolomide (TMZ) can sensitize glioma stem-like cells (GSCs) from human glioblastoma multiforme (GBM) to TRAIL-induced apoptosis. GSCs were isolated from GBM, and stem cell properties were confirmed by immunocytochemistry and in vivo tumorigenicity. Primary GSCs (PGCs) were produced by serum treatment of GBM-derived cells. Changes in expression levels of various TRAIL-related signaling factors before and after TRAIL or TRAIL + TMZ treatment were measured by Western blotting. Overexpression vectors and siRNAs were used to investigate mechanism of TRAIL sensitivity. GSCs showed greater resistance to TRAIL-induced apoptosis than PGCs and had lower basal caspase activity. Caspase knockdown in PGCs reduced TRAIL sensitivity. Expression levels of c-Fas-associated death domain-like interleukin 1-converting enzyme-like inhibitory protein long and short isoforms (c-FLIPL and c-FLIPS) were significantly higher in GSCs than PGCs, and siRNA-mediated c-FLIP knockdown in GSCs enhanced TRAIL-induced apoptosis. TMZ enhanced TRAIL-induced apoptosis in GSCs and downregulated c-FLIP expression. Add of TMZ also upregulated the expression of the E3 ubiquitin ligase casitas B-lineage lymphoma (c-Cbl). Moreover, overexpression of c-Cbl alone reduced c-FLIP expression, and c-Cbl knockdown both enhanced c-FLIP expression and reduced the potentiating effect of TMZ on TRAIL-induced apoptosis. The result indicated that TMZ may overcome TRAIL resistance in GSCs by suppressing c-FLIP expression through c-Cbl-mediated ubiquitination and degradation.

HDAC Inhibition Overcomes Acute Resistance to MEK Inhibition in BRAF-Mutant Colorectal Cancer by Downregulation of c-FLIPL.

PURPOSE: Activating mutations in the BRAF oncogene are found in 8% to 15% of colorectal cancer patients and have been associated with poor survival. In contrast with BRAF-mutant (MT) melanoma, inhibition of the MAPK pathway is ineffective in the majority of BRAFMT colorectal cancer patients. Therefore, identification of novel therapies for BRAFMT colorectal cancer is urgently needed. EXPERIMENTAL DESIGN: BRAFMT and wild-type (WT) colorectal cancer models were assessed in vitro and in vivo. Small-molecule inhibitors of MEK1/2, MET, and HDAC were used, overexpression and siRNA approaches were applied, and cell death was assessed by flow cytometry, Western blotting, cell viability, and caspase activity assays. RESULTS: Increased c-MET-STAT3 signaling was identified as a novel adaptive resistance mechanism to MEK inhibitors (MEKi) in BRAFMT colorectal cancer models in vitro and in vivo. Moreover, MEKi treatment resulted in acute increases in transcription of the endogenous caspase-8 inhibitor c-FLIPL in BRAFMT cells, but not in BRAFWT cells, and inhibition of STAT3 activity abrogated MEKi-induced c-FLIPL expression. In addition, treatment with c-FLIP-specific siRNA or HDAC inhibitors abrogated MEKi-induced upregulation of c-FLIPL expression and resulted in significant increases in MEKi-induced cell death in BRAFMT colorectal cancer cells. Notably, combined HDAC inhibitor/MEKi treatment resulted in dramatically attenuated tumor growth in BRAFMT xenografts. CONCLUSIONS: Our findings indicate that c-MET/STAT3-dependent upregulation of c-FLIPL expression is an important escape mechanism following MEKi treatment in BRAFMT colorectal cancer. Thus, combinations of MEKi with inhibitors of c-MET or c-FLIP (e.g., HDAC inhibitors) could be potential novel treatment strategies for BRAFMT colorectal cancer.

Combined treatment with ABT-737 and VX-680 induces apoptosis in Bcl-2- and c-FLIP-overexpressing breast carcinoma cells.

ABT-737, a BH3-mimetic small-molecule inhibitor, binds with very high affinity to Bcl-2, Bcl-xL and Bcl-w, and inhibits their activity. Aurora kinase is one of the serine/threonine kinase family members and is a vital and critical regulator of mitosis and meiosis. In the present study, we investigated the effects and mechanisms of a combined treatment of ABT-737 and VX-680 (Aurora kinase inhibitor) in human breast cancer MDA-MB­435S cells. ABT-737 plus VX-680 induced caspase-dependent apoptosis in the human breast cancer cells. Combined treatment with ABT-737 and VX-680 led to the downregulation of Bcl-2 expression at the transcriptional level and the downregulation of c-FLIP and Mcl-1 expression at the post-transcriptional level. Overexpression of Bcl-2 or c-FLIP could not block the induction of apoptosis caused by the combined treatment with ABT-737 and VX-680. However, overexpression of Mcl-1 partially inhibited the induction of apoptosis. In contrast, the combined treatment with ABT-737 and VX680 had no effect on the apoptosis in normal cells. Taken together, our study demonstrated that combined treatment with ABT-737 and VX-680 induced apoptosis in anti­apoptotic protein (Bcl-2 or c-FLIP)-overexpressing cells.

Carnosic acid sensitized TRAIL-mediated apoptosis through down-regulation of c-FLIP and Bcl-2 expression at the post translational levels and CHOP-dependent up-regulation of DR5, Bim, and PUMA expression in human carcinoma caki cells.

Carnosic acid is a phenolic diterpene from rosmarinus officinalis, and has multiple functions, such as anti-inflammatory, anti-viral, and anti-tumor activity. In this study, we examined whether carnosic acid could sensitize TRAIL-mediated apoptosis in human renal carcinoma Caki cells. We found that carnosic acid markedly induced TRAIL-mediated apoptosis in human renal carcinoma (Caki, ACHN, and A498), and human hepatocellular carcinoma (SK-HEP-1), and human breast carcinoma (MDA-MB-231) cells, but not normal cells (TMCK-1 and HSF). Carnosic acid induced down-regulation of c-FLIP and Bcl-2 expression at the post-translational levels, and the over-expression of c-FLIP and Bcl-2 markedly blocked carnosic acid-induced TRAIL sensitization. Furthermore, carnosic acid induced death receptor (DR)5, Bcl-2 interacting mediator of cell death (Bim), and p53 up-regulated modulator of apoptosis (PUMA) expression at the transcriptional levels via CCAAT/enhancer-binding protein-homologous protein (CHOP). Down-regulation of CHOP expression by siRNA inhibited DR5, Bim, and PUMA expression, and attenuated carnosic acid plus TRAIL-induced apoptosis. Taken together, our study demonstrates that carnosic acid enhances sensitization against TRAIL-mediated apoptosis through the down-regulation of c-FLIP and Bcl-2 expression, and up-regulation of ER stress-mediated DR5, Bim, and PUMA expression at the transcriptional levels.

Delaying mitotic exit downregulates FLIP expression and strongly sensitizes tumor cells to TRAIL.

Many of the current antitumor therapeutic strategies are based on the perturbation of the cell cycle, especially during mitosis. Antimitotic drugs trigger mitotic checkpoint activation, mitotic arrest and eventually cell death. However, mitotic slippage represents a major mechanism of resistance to these treatments. In an attempt to circumvent the process of slippage, targeting mitotic exit has been proposed as a better strategy to kill tumor cells. In this study, we show that treatments that induce mitotic checkpoint activation and mitotic arrest downregulate FLICE-like inhibitory protein (FLIP) levels and sensitize several tumor cell lines to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. Interestingly, we also demonstrate that in absence of mitotic checkpoint activation, mitotic arrest induced either by Cdc20 knockdown or overexpression of nondegradable cyclin B is sufficient to induce both FLIP downregulation and sensitivity to TRAIL. In summary, our data suggest that a combination of antimitotic drugs targeting cyclin B degradation and TRAIL might prevent mitotic slippage and allow tumor cells to reach the threshold for apoptosis induction, thereby facilitating tumor suppression.