Downregulation Of c-FLIPL Research Articles

Dapagliflozin induces apoptosis by downregulating cFILPL and increasing cFILPS instability in Caki-1 cells.

Dapagliflozin is a sodium/glucose cotransporter 2 inhibitor used recently to treat patients with type 2 diabetes. A recent study has demonstrated that dapagliflozin induces apoptosis in human renal and breast tumor cells. However, to the best of our knowledge, the molecular mechanism underlying dapagliflozin-mediated apoptosis in Caki-1 human renal carcinoma cells has not been elucidated. The present study demonstrated that the dapagliflozin treatment dose-dependently increased cell death in Caki-1 cells. Dapagliflozin treatment also induced apoptosis as confirmed by FITC-conjugated Annexin V/PI staining. Additionally, treatment with dapagliflozin reduced the expression levels of anti-apoptotic proteins, cellular Fas-associated death domain-like interleukin-1-converting enzyme-inhibitory protein (cFLIP)L and cFLIPS in Caki-1 cells. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone inhibited dapagliflozin-induced apoptosis, implying that dapagliflozin–induced apoptosis is regulated by a caspase-dependent pathway. By contrast, N-acetylcysteine had no effect on dapagliflozin–induced apoptosis and downregulation of cFLIPL and cFLIPS expression. Furthermore, overexpression of cFLIPL, but not cFLIPS, partially inhibited apoptosis induced by dapagliflozin. cFLIPL and cFLIPS mRNA levels remained constant in Caki-1 cells after treatment with 0, 20, 40, 60, 80 and 100 µM dapagliflozin. Notably, it was confirmed that cFLIPS protein levels were reduced due to the increased cFLIPS instability in dapagliflozin-treated Caki-1 cells. The present study also demonstrated that dapagliflozin had no effect on HK-2 normal human kidney cells. Taken together, the present study revealed that dapagliflozin induced apoptosis via the downregulation of cFLIPL and an increase in cFLIPS instability, suggesting that dapagliflozin may be a feasible drug candidate for the treatment of human renal cancer.

Mathematical Modeling Reveals the Importance of the DED Filament Composition in the Effects of Small Molecules Targeting Caspase-8/c-FLIPL Heterodimer.

Procaspase-8 activation at the death-inducing signaling complex (DISC) triggers extrinsic apoptotic pathway. Procaspase-8 activation takes place in the death effector domain (DED) filaments and is regulated by c-FLIP proteins, in particular, by the long isoform c-FLIPL. Recently, the first-in-class chemical probe targeting the caspase-8/c-FLIPL heterodimer was reported. This rationally designed small molecule, FLIPin, enhances caspase-8 activity after initial heterodimer processing. Here, we used a kinetic mathematical model to gain an insight into the mechanisms of FLIPin action in a complex with DISC, in particular, to unravel the effects of FLIPin at different stoichiometry and composition of the DED filament. Analysis of this model has identified the optimal c-FLIPL to procaspase-8 ratios in different cellular landscapes favoring the activity of FLIPin. We predicted that the activity FLIPin is regulated via different mechanisms upon c-FLIPL downregulation or upregulation. Our study demonstrates that a combination of mathematical modeling with system pharmacology allows development of more efficient therapeutic approaches and prediction of optimal treatment strategies.

Down Regulation of c-FLIPL Enhance PD-1 Blockade Efficacy in B16 Melanoma.

Immune checkpoint blockade of programmed cell death protein 1 (PD-1) had an impressive long-lasting effect in a portion of advanced-stage melanoma patients, however, this therapy failed to induce responses in several patients; how to increase the objective response rate is very important. Cellular FLICE-inhibitory protein (c-FLIP) could inhibit apoptosis directly at the death-inducing signaling complex of death receptors and is also considered to be the main cause of immune escape. The overexpression of c-FLIPL occurs frequently in melanoma and its expression is associated with the prognosis. We found that the level of c-FLIPL expression was associated with the PD-1 blockade response rate in melanoma patients. Thus, we performed this research to investigate how c-FLIPL regulates immunotherapy in melanoma. We demonstrate that down regulation of c-FLIPL enhances the PD-1 blockade efficacy in B16 melanoma tumor model. Down regulation of c-FLIPL could increase the tumor apoptosis and enhance the antitumor response of T cells in the lymphocyte tumor cells co-culture system. Moreover, knockdown of c-FLIPL could decrease the expression of PD-L1 and recruit more effector T cells in the tumor microenvironment. Our results may provide a new combined therapeutic target for further improving the efficacy of PD-1 blockade in melanoma.

Novel 4-(3-phenylpropionamido), 4-(2-phenoxyacetamido) and 4-(cinnamamido) substituted benzamides bearing the pyrazole or indazole nucleus: synthesis, biological evaluation and mechanism of action

Based on some common structural features of known compounds interfering with p53 pathways and our previously synthesized benzamides, we synthesized new ethyl 5-(4-substituted benzamido)-1-phenyl-1H-pyrazole-4-carboxylates 26a-c, ethyl 5-(4-substituted benzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylates 27a-c and N-(1H-indazol-6-yl)-4-substituted benzamides 31a,b bearing in the 4 position of the benzamido moiety the 2-phenylpropanamido or 2-phenoxyacetamido or cinnamamido groups. A preliminary test to evaluate the antiproliferative activity against human lung carcinoma H292 cells highlighted how compound 26c showed the best activity. This last was therefore selected for further studies with the aim to find the mechanism of action. Compound 26c induces intrinsic apoptotic pathway by activating p53 and is also able to activate TRAIL-inducing death pathway by promoting increase of DR4 and DR5 death receptors, downregulation of c-FLIPL and caspase-8 activation.

Regulation of the antiapoptotic protein cFLIP by the glucocorticoid Dexamethasone in ALL cells.

We recently reported that the Smac mimetic BV6 and glucocorticoids, e.g. Dexamethasone (Dexa), synergize to induce cell death in acute lymphoblastic leukemia (ALL) in vitro and in vivo. Here, we discover that this synergism involves Dexa-stimulated downregulation of cellular FLICE-like inhibitory protein (cFLIP) in ALL cells. Dexa rapidly decreases cFLIPL protein levels, which is further enhanced by addition of BV6. While attenuating the activation of non-canonical nuclear factor-kappaB (NF-κB) signaling by BV6, Dexa suppresses cFLIPL protein but not mRNA levels pointing to a transcription-independent downregulation of cFLIPL by Dexa. Analysis of protein degradation pathways indicates that Dexa causes cFLIPL depletion independently of proteasomal, lysosomal or caspase pathways, as inhibitors of the proteasome, lysosomal enzymes or caspases all failed to protect from Dexa-mediated loss of cFLIPL protein. Also, Dexa alone or in combination with BV6 does not affect overall activity of the proteasome. Importantly, overexpression of cFLIPL to an extent that is no longer subject to Dexa-imposed downregulation rescues Dexa/BV6-mediated cell death. Vice versa, knockdown of cFLIP increases BV6-mediated cell death, thus mimicking the effect of Dexa. Altogether, these data demonstrate that Dexa-mediated downregulation of cFLIPL protein promotes Dexa/BV6-mediated cell death, thereby providing novel insights into the synergistic antitumor activity of this combination treatment.

Correction: The Multikinase Inhibitor Sorafenib Potentiates TRAIL Lethality in Human Leukemia Cells in Association with Mcl-1 and cFLIPL Down-regulation.

Correction: The Multikinase Inhibitor Sorafenib Potentiates TRAIL Lethality in Human Leukemia Cells in Association with Mcl-1 and cFLIPL Down-regulation.

Glycogen synthase kinase-3\u03b2 inhibition promotes lysosome-dependent degradation of c-FLIPL in hepatocellular carcinoma

Glycogen synthase kinase-3β (GSK-3β) is a ubiquitously expressed serine/threonine kinase involved in a variety of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK-3β inhibition triggered ASK1-JNK-dependent apoptosis in human hepatocellular carcinoma (HCC) cells. However, the comprehensive picture of downstream GSK-3β-regulated pathways/functions remains elusive. In this study, we showed that GSK-3β was aberrantly activated in HCC. Pharmacological inhibition and genetic depletion of GSK-3β suppressed the growth and induced caspase-dependent apoptosis in HCC cells. In addition, GSK-3β inhibition-induced apoptosis through downregulation of c-FLIPL in HCC, which was caused by biogenesis of functional lysosomes and subsequently c-FLIPL translocated to lysosome for degradation. This induction of the lysosome-dependent c-FLIPL degradation was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Moreover, GSK-3β inhibition-induced TFEB translocation acts through activation of AMPK and subsequently suppression of mTOR activity. Thus our findings reveal a novel mechanism by which inhibition of GSK-3β promotes lysosome-dependent degradation of c-FLIPL. Our study shows that GSK-3β may become a promising therapeutic target for HCC.

Abstract 2117: TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma

Abstract Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), the optimized form of the endogenous death ligand TRAIL, shows therapeutic potential for cancer due to its ability to induce apoptosis in cancer cells independent of p53, while exhibiting minimal toxicity to normal cells. Despite this, a majority of breast cancers display resistance to rhTRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors (DR) 4 and 5. To overcome rhTRAIL resistance, natural compounds have been investigated as sensitizing agents. This study considered the application of the naturally occurring flavonol Quercetin (Q). Q has been shown to have the ability to up-regulate DR5 and down-regulate anti-apoptotic proteins in cancer cells, and thereby, making Q a favorable choice to be employed as a sensitizing agent. The intention of this study was to ascertain the capacity of Q to sensitize rhTRAIL-resistant triple negative breast cancer BT-20 cells and hormone-dependent breast cancer MCF-7 cells to rhTRAIL-induced apoptosis and elucidate the underlying mechanism for Q’s sensitization. Q demonstrated the ability to intensify rhTRAIL’s pro-apoptotic effects in the breast cancer BT-20 and MCF-7 cell lines as detected through Annexin V/PI assays followed by FACS analysis. In comparison to single agent treatments, the cotreatment of Q and rhTRAIL enhanced the induction of the extrinsic pathway of apoptosis as marked by PARP cleavage (a hallmark of apoptosis), activation of caspase 8, and activation of the executioner caspases 3 and 7. The mechanism for Q’s augmentation in breast cancer was determined to be through the down-regulation of c-FLIPL (caspase 8 inhibitor) in a dose-dependent manner. Furthermore, Q promoted the ubiquitination of c-FLIPL facilitating the proteasome-mediated degradation of c-FLIPL in breast cancer. An additional mechanism for Q’s sensitization was displayed in breast cancer BT-20 cells. Q up-regulated DR5 membrane and protein expression in breast cancer BT-20 cells in a dose-dependent manner. RT-PCR analysis revealed that Q’s influence on DR5 expression occurred at the transcriptional level in those breast cancer cells. Thus, these data suggest that the cotreatment of Q and rhTRAIL possesses the therapeutic potential to be an effective anti-breast carcinoma regimen. Citation Format: Jasmine M. Manouchehri, Michael Kalafatis. TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2117. doi:10.1158/1538-7445.AM2017-2117

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.

Cordycepin induces autophagy-mediated c-FLIPL degradation and leads to apoptosis in human non-small cell lung cancer cells.

Cordycepin, a main active composition extracted from Cordyceps militaris, has been reported to exert anti-tumor activity in a broad spectrum of cancer types. However, the function of cordycepin on human non-small cell lung cancer cells is still obscure. Our present work showed that cordycepin inhibited cell growth by inducing apoptosis and autophagy in human NSCLC cells. Further study revealed that cordycepin triggered extrinsic apoptosis associated with down-regulation of c-FLIPL which suppresses the activity of caspase-8. And ectopic expression of c-FLIPL dramatically prevented cordycepin-caused apoptosis. Meanwhile, cordycepin stimulated autophagy through suppressing mTOR signaling pathway in lung cancer cells. When autophagy was blocked by Atg5 siRNA or PI3K inhibitor LY294002, the levels of apoptosis caused by cordycepin were obviously attenuated. In addition, suppression of autophagy could also elevate the level of c-FLIPL which indicated cordycepin-triggered autophagy promoted the degradation of c-FLIPL. Therefore, we conclude that cordycepin induces apoptosis through autophagy-mediated downregulation of c-FLIPL in human NSCLC cells. Taken together, our findings provide a novel prospect on the anti-tumor property of cordycepin, which may further prompt cordycepin to serve as a promising therapeutic approach in NSCLC treatment.

The Long Form of Cellular FLIP Renders NK-Cell Lymphoma Cells Resistant to Fas-Mediated Apoptosis

Activation-induced cell death mediated by Fas/Fas ligand (FasL) system plays a key role in regulating immune response. Although normal NK cells employ this system for their homeostasis, malignant NK cells derived from extranodal NK/T-cell lymphoma, nasal type (ENKL) seem to disrupt the process. ENKL cells usually express both Fas andFasL, whereas they seldom undergo apoptosis. Previous studies showed that loss-of-function mutations of Fas gene are considered a major mechanism of resistance to Fas-mediated apoptosis in ENKL (Shen et al., Am J Pathol 2002; Takakuwa et al., Oncogene 2002). In addition, recent findings indicated that the long form of cellular FLIP (c-FLIPL) is a crucial modulator of procaspase-8 and has the potency to interfere with the Fas-mediated apoptotic pathway in several cancer cells (reviewed by Lavrik and Krammer, Cell Death Differ 2012). We focused on c-FLIPL and examined whether down-regulation of c-FLIPL leads to efficient cell death in ENKL-derived cells.We used two ENKL-derived cell lines, NK-YS and Hank1, and Fas-sensitive Jurkat as a control. To determine the expression of Fas and FasL, flow cytometry (FCM) was performed and showed the co-expression Fas and FasL in both NK-cell lines. Western blot analysis detected the components of the death-inducing signaling complex (DISC), including FADD, procaspase-8, and c-FLIPL in the three lines. We next confirmed that NK-YS and Hank1 were resistant to the apoptotic stimuli induced by direct Fas cross-linking. Annexin V staining assay by FCM detected that above 40% of Jurkat cells were positive for annexin V, whereas most NK-YS and Hank1 cells failed to show apoptotic changes after Fas ligation.To determine whether Fas-mediated signaling is functional, we investigated the DISC formation in the NK-cell lines. After Fas ligation, FADD and cleaved caspase-8 were coimmunoprecipitated with Fas in NK-YS and Hank1 as well as in Jurkat. Confocal fluorescence imaging also detected fusion signals showing colocalization between Fas and caspase-8 in the three lines. In contrast, cleaved c-FLIPL was coimmunoprecipitated only in NK-YS and Hank1. Confocal microscopy detected that the colocalization of Fas and c-FLIPL was significantly increased in the NK-cell lines, compared with Jurkat. These results showed that c-FLIPL plays a critical role in inhibition of the proximal Fas-mediated signaling pathway in NK-YS and Hank1. Therefore, we next performed RNA interference for c-FLIPL in the NK-cell lines. Although the cells transfected control siRNA hardly showed apoptotic change even after Fas ligation, knockdown of c-FLIPL slightly increased apoptosis in NK-YS and Hank1 (Figure 1). Furthermore, the suppression of c-FLIPL remarkably sensitized the NK cell lines to the Fas stimulus. FCM detected that above 50% of cells came to be positive for annexin V in both NK-cell lines (Figure 1). Here, we demonstrated another mechanism of resistance to Fas-mediated apoptosis in ENKL cells.Our findings indicate that two ENKL-derived cell lines are similar to activated normal NK cells in being primed to undergo Fas-mediated apoptosis. However, they constitutively express and exploit c-FLIPL, which prevents their Fas-mediated apoptosis. The specific down-regulation of c-FLIPL can induce the autonomous death of ENKL cells. c-FLIPL may be a therapeutic target against ENKL. [Display omitted] DisclosuresKomatsu:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Speakers Bureau.

Potential Role of CD133 Expression in the Susceptibility of Human Liver Cancer Stem-Like Cells to TRAIL.

Hepatocellular carcinoma (HCC) is one of the most common malignancies, with a poor prognosis and high recurrence rate. In the present study, we identified CD133, one of the markers of cancer stem cells, as a novel molecular target of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In four human HCC cell lines established from primary HCC tumors, we found that CD133-high human liver cancer stem-like cells (CD133hi) derived from the SNU-475 cell line were highly susceptible to TRAIL compared to other HCC cell lines with a small population of CD133. CD133hi SNU-475 cells showed upregulation of TRAIL receptor DR5 and stemness-related genes such as c-Myc and ABC transporters compared to their CD133-low (CD133lo) cells. Hypersensitivity of CD133hi cells to TRAIL was associated with c-Myc-mediated upregulation of DR5 and downregulation of c-FLIPL in the cells. Knockdown of CD133 expression in CD133hi cells resulted in the downregulation of c-Myc, and depletion of c-Myc caused a decrease in the cell surface expression of DR5 and an increase in the expression of c-FLIPL and, consequently, attenuated TRAIL-induced cytotoxicity and apoptosis of CD133hi cells. These results suggest that TRAIL may provide a new strategy for CD133hi CSCs of HCC-targeted therapies and, potentially, for therapies of other CD133-expressing types of cancer.

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.

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

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. 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. 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. 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.

Ca2+ influx-mediated dilation of the endoplasmic reticulum and c-FLIPL downregulation trigger CDDO-Me-induced apoptosis in breast cancer cells.

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-C28-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we show that treatment with CDDO-Me induces progressive endoplasmic reticulum (ER)-derived vacuolation in various breast cancer cells and ultimately kills these cells by inducing apoptosis. We found that CDDO-Me-induced increases in intracellular Ca2+ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca2+ levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca2+ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. In addition, CDDO-Me rapidly reduced the protein levels of c-FLIPL (cellular FLICE-inhibitory protein) and overexpression of c-FLIPL blocked CDDO-Me-induced cell death, but not vacuolation. These results suggest that c-FLIPL downregulation is a key contributor to CDDO-Me-induced apoptotic cell death, independent of ER-derived vacuolation. Taken together, our results show that ER-derived vacuolation via Ca2+ influx and ROS generation as well as caspase activation via c-FLIPL downregulation are responsible for the potent anticancer effects of CDDO-Me on breast cancer cells.

The Histone Deacetylase Inhibitor Trichostatin A Sensitizes Human Renal Carcinoma Cells to TRAIL-Induced Apoptosis through Down-Regulation of c-FLIPL.

Histone acetylation plays a critical role in the regulation of transcription by altering the structure of chromatin, and it may influence the resistance of some tumor cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by regulating the gene expression of components of the TRAIL signaling pathway. In this study, we investigated the effects and molecular mechanisms of trichostatin A (TSA), a histone deacetylase inhibitor, in sensitizing TRAIL-induced apoptosis in Caki human renal carcinoma cells. Our results indicate that nontoxic concentrations of TSA substantially enhance TRAIL-induced apoptosis compared with treatment with either agent alone. Cotreatment with TSA and TRAIL effectively induced cleavage of Bid and loss of mitochondrial membrane potential (MMP), which was associated with the activation of caspases (-3, -8, and -9) and degradation of poly (ADP-ribose) polymerase (PARP), contributing toward the sensitization to TRAIL. Combined treatment with TSA and TRAIL significantly reduced the levels of the cellular Fas-associated death domain (FADD)-like interleukin-1β-converting enzyme (FLICE) inhibitory protein (c-FLIP), whereas those of death receptor (DR) 4, DR5, and FADD remained unchanged. The synergistic effect of TAS and TRAIL was perfectly attenuated in c-FLIPL-overexpressing Caki cells. Taken together, the present study demonstrates that down-regulation of c-FLIP contributes to TSA-facilitated TRAIL-induced apoptosis, amplifying the death receptor, as well as mitochondria-mediated apoptotic signaling pathways.

DR-02 * TEMOZOLOMIDE SENSITIZES GLIOMA STEM CELLS TO TRAIL-INDUCED APOPTOSIS THROUGH UPREGULATION OF c-CBL

TRAIL-induced apoptosis is inhibited in GSCs. In this study, we investgated whether or not the widely used chemotherapeutic agent-TMZ can sensitize TRAIL treatment in GSCs, related mechanism was investigated in cultured glioma stem cells (GSCs) and primary glioma cells (PGCs) from patients. We found that the expression of c-Fas-associated death domain-like interleukin 1-converting enzyme-like inhibitory protein long and short isoforms (c-FLIPL and c-FLIPS, respectively) was significantly higher in GSCs than in PGCs, and was associated with greater tolerance to TRAIL treatment in the former. Application of the chemotherapeutic drug temozolomide (TMZ) induced the upregulation of casitas B-lineage lymphoma(c-Cbl) and downregulation of c-FLIPL and c-FLIPS expression in GSCs, and when combined with TRAIL promoted GSC apoptosis. These results suggest that combination therapy with TMZ and TRAIL may have clinical applicability for glioma treatment.

Inhibition of the mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent in selectively killing tumor cells. However, TRAIL monotherapy has not been successful as many cancer cells are resistant to TRAIL. Chemotherapeutic agents, such as doxorubicin have been shown to act synergistically with TRAIL, but the exact mechanisms of actions are poorly understood. In this study, we performed high-throughput small interfering RNA screening and genome-wide gene expression profiling on doxorubicin-treated U1690 cells to explore novel mechanisms underlying doxorubicin-TRAIL synergy. The screening and expression profiling results were integrated and dihydroorotate dehydrogenase (DHODH) was identified as a potential candidate. DHODH is the rate-limiting enzyme in the pyrimidine synthesis pathway, and its expression was downregulated by doxorubicin. We demonstrated that silencing of DHODH or inhibition of DHODH activity by brequinar dramatically increased the sensitivity of U1690 cells to TRAIL-induced apoptosis both in 2D and 3D cultures, and was accompanied by downregulation of c-FLIPL as well as by mitochondrial depolarization. In addition, uridine, an end product of the pyrimidine synthesis pathway was able to rescue the sensitization effects initiated by both brequinar and doxorubicin. Furthermore, several other cancer cell lines, LNCaP, MCF-7 and HT-29 were also shown to be sensitized to TRAIL by brequinar. Taken together, our findings have identified a novel protein target and its inhibitor, brequinar, as a potential agent in TRAIL-based combinatorial cancer therapy and highlighted for the first time the importance of mitochondrial DHODH enzyme and pyrimidine pathway in mediating TRAIL sensitization in cancer cells.

The chalcone 2′‐hydroxy‐4′,5′‐dimethoxychalcone activates death receptor 5 pathway and leads to apoptosis in human nonsmall cell lung cancer cells

Natural chalcones have been proved to inhibit cancer cells with therapeutic potential, but the underlying molecular mechanism is still largely unexplored. Here, we identified a novel chalcone, 2'-hydroxy-4',5'-dimethoxychalcone (HDMC) and demonstrated that HDMC induced apoptosis in various nonsmall cell lung cancer cells. Further study showed that HDMC elevated cellular reactive oxygen species (ROS) levels, thus inducing expressions of ATF4 and C/EBP homologous protein (CHOP). Then, death receptor 5 (DR5) was upregulated through ATF4-CHOP axis and eventually resulted in apoptosis. We also found that downregulation of c-FLIPL contributed to HDMC-induced apoptosis. In conclusion, HDMC induces apoptosis in human nonsmall cell lung cancer cells via activation of DR5 signaling pathway, and ROS-mediated ATF4-CHOP axis is involved in the process. Our results further supported the potential for HDMC to be developed as a new antitumor agent for cancer therapy or chemoprevention.

Wogonin enhances antitumor activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo through ROS-mediated downregulation of cFLIPL and IAP proteins

Combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with other agents is a promising strategy to overcome TRAIL resistance in malignant cells. Wogonin, a flavonoid originated from Scutellaria baicalensis Georgi, has been shown to enhance TRAIL-induced apoptosis in malignant cells in in vitro studies. However, whether wogonin enhances TRAIL's antitumor activity in vivo has never been studied. In this study, the effect of combination of TRAIL and wogonin was tested in a non-small-cell lung cancer xenografted tumor model in nude mice. Consistent with the in vitro study showing that wogonin sensitized A549 cells to TRAIL-induced apoptosis, wogonin greatly enhanced TRAIL-induced suppression of tumor growth, accompanied with increased apoptosis in tumor tissues as determined by TUNEL assay. The expression levels of antiapoptotic proteins including long form of cellular FLICE-like inhibitory protein (cFLIPL), X-linked inhibitor of apoptosis protein (XIAP), and cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) were markedly reduced in both cultured cells and xenografted tumor tissues after co-treatment with wogonin and TRAIL. The down-regulation of these antiapoptotic proteins was likely mediated by proteasomal degradation that involved intracellular reactive oxygen species (ROS), because wogonin robustly induced ROS accumulation and ROS scavengers butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) and the proteasome inhibitor MG132 restored the expression of these antiapoptotic proteins in cells co-treated with wogonin and TRAIL. These results show for the first time that wogonin enhances TRAIL's antitumor activity in vivo, suggesting this strategy has an application potential for clinical anticancer therapy.