Death Receptor-mediated Apoptosis Pathway Research Articles

Optogenetic induction of caspase-8 mediated apoptosis by employing Arabidopsis cryptochrome 2

Apoptosis, a programmed cell death mechanism, is a regulatory process controlling cell proliferation as cells undergo demise. Caspase-8 serves as a pivotal apoptosis-inducing factor that initiates the death receptor-mediated apoptosis pathway. In this investigation, we have devised an optogenetic method to swiftly modulate caspase-8 activation in response to blue light. The cornerstone of our optogenetic tool relies on the PHR domain of Arabidopsis thaliana cryptochrome 2, which self-oligomerizes upon exposure to blue light. In this study, we have developed two optogenetic approaches for rapidly controlling caspase-8 activation in response to blue light in cellular systems. The first strategy, denoted as Opto-Casp8-V1, entails the fusion expression of the Arabidopsis blue light receptor CRY2 N-terminal PHR domain with caspase-8. The second strategy, referred to as Opto-Casp8-V2, involves the independent fusion expression of caspase-8 with the PHR domain and the CRY2 blue light-interacting protein CIB1 N-terminal CIB1N. Upon induction with blue light, PHR undergoes aggregation, leading to caspase-8 aggregation. Additionally, the blue light-dependent interaction between PHR and CIB1N also results in caspase-8 aggregation. We have validated these strategies in both HEK293T and HeLa cells. The findings reveal that both strategies are capable of inducing apoptosis, with Opto-Casp8-V2 demonstrating significantly superior efficiency compared to Opto-Casp8-V1.

Toxic effects of zinc oxide nanoparticles as a food additive in goat mammary epithelial cells

Zinc oxide nanoparticles (ZnO NPs) have recently been used as food preservatives and additives because of their good antibacterial and nutritional functions. This study performed RNA-seq analyses to evaluate the potential toxicity of ZnO NPs on goat mammary epithelial cells (GMECs) in vitro. Our results suggested that the ZnO NP treatment significantly reduced GMEC viability in a time- and dose-dependent manner. Transcriptomic analysis showed that ZnO NP exposure changed the expression levels of more than 500 genes in GMECs, including various biological pathways. We observed that decreased mitochondrial membrane potential caused mitochondrial dysfunction. Further study indicated that the treatment of cells with ZnO NPs resulted in the accumulation of reactive oxygen species (ROS), which led to oxidative stress. Meanwhile, the expression of genes (TNFα, TNFR1, FADD, Caspase 8 and Caspase 6) associated with the death receptor pathway was upregulated, which indicated the death receptor-mediated extrinsic apoptosis pathway was activated. Moreover, the expression levels of Bax, Cytc, Caspase 3 and Caspase 9 were upregulated, while the expression levels of Bcl2 were downregulated, which indicated mitochondria-mediated intrinsic apoptosis pathway was activated. More notably, ZnO NP exposure increased the expression levels of ER stress-related genes (PERK, ATF4, eIF2α and CHOP) and proteins (p-PERK, p-eIF2α, PERK and CHOP). Furthermore, gene ontology (GO) terms and genes related to autophagy were altered, suggesting that exposure to ZnO NPs might activate autophagy in GMECs. In summary, our findings showed that ZnO NPs could exert significant toxic effects on GMECs through multiple mechanisms. These pathways are related to each other and influence each other to participate in ZnO NPs-induced the damage of GMECs. Thus, their safe use in the feed and food industry should be considered. Meanwhile, RNA-seq might represent a new method of assessing the toxicity mechanisms of nanomaterials.

Calcium alleviates fluoride-induced kidney damage via FAS/FASL, TNFR/TNF, DR5/TRAIL pathways in rats

Long-term excessive intake of fluoride (F) can cause osseous and non-osseous damage. The kidney is the main fluoride excretion organ of the body. This study aimed to explore whether dietary calcium (Ca) supplementation can alleviate kidney damage caused by fluorosis and to further investigate the effects of Ca on the mitigation mechanism of renal cell apoptosis triggered by F. We evaluated the histopathological structure, renal function indicators, and gene and protein expression levels of death receptor-mediated apoptosis pathways in Sprague Dawley (SD) rats treated with sodium fluoride (NaF) and/or calcium carbonate (CaCO3) for 120 days. The results showed that 100 mg/L NaF induced kidney histopathological injury and apoptosis, increased the concentrations of Creatinine (CRE), uric acid (UA), blood urea nitrogen (BUN), potassium (K), phosphorus (P) and F (p < 0.05), and decrease the level of serum magnesium (Mg) (p < 0.05). Moreover, NaF increased the mRNA and protein expression levels of Fas cell surface death receptor (FAS), tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL), Caspase 8, Caspase 3 and poly ADP-ribose polymerase (PARP) (p < 0.01), which finally activated the death receptor pathway. Inversely, Ca supplementation reversed the decrease of CRE, BUN, UA, F and P levels induced by F, alleviated histopathological damage and apoptosis, and reduced the gene and protein expression levels of death receptor pathway-related markers. In conclusion, 1% Ca alleviates F-induced kidney apoptosis through FAS/FASL, TNFR/TNF, DR5/TRAIL signaling pathways.

The protective effect of harpagoside on angiotensin II (Ang II)-induced blood-brain barrier leakage in vitro.

Hypertension and its associated dysfunction of the blood-brain barrier (BBB) contribute to cerebral small vessel disease (cSVD). Angiotensin II (Ang II), a vasoactive peptide of the renin-angiotensin system (RAS), is not only a pivotal molecular signal in hypertension but also causes BBB leakage, cSVD, and cognitive impair. Harpagoside, the major bioactive constituent of Scrophulariae Radix, has been commonly used for the treatment of multiple diseases including hypertension in China. The effect of harpagoside on Ang II-induced BBB damage is unclear. We employed an immortalized endothelial cell line (bEnd.3) to mimic a BBB monolayer model in vitro and investigated the effect of harpagoside on BBB and found that harpagoside alleviated Ang II-induced BBB destruction, inhibited Ang II-associated cytotoxicity in a concentration-dependent manner and attenuated Ang II-induced reactive oxygen species (ROS) impair by downregulation of Nox2, Nox4, and COX-2. Harpagoside prevented Ang II-induced apoptosis via keeping Bax/Bcl-2 balance, decreasing cytochrome c release, and inactivation of caspase-8, caspase-9, and caspase-3 (the mitochondria-dependent and death receptor-mediated apoptosis pathways). Moreover, harpagoside can alleviate Ang II-induced BBB damage through upregulation of tight junction proteins and decrease of caveolae-mediated endocytosis. Thus, harpagoside might be a potential drug to treat Ang II-induced cSVD.

Apoptotic effects of norfloxacin on corneal endothelial cells.

Norfloxacin, a frequently used ocular antibiotic, might have cytotoxic effect on human corneal endothelial cells (HCECs), subsequently damage the cornea and finally impair human vision. However, the possible mechanisms of cytotoxicity of norfloxacin to HCEC line are unclear. Herein, we investigated the cytotoxicity of norfloxacin and its underlying cellular and molecular mechanisms using in vitro cultured non-transfected HCECs and verified the cytotoxicity with cat corneal endothelium in vivo. In the present study, the cytotoxicity of norfloxacin in the in vitro cultured HCECs was recognized by causing abnormal morphology such as cell shrinkage and detachment from plate bottom, and decline of viability of in vitro cultured HCECs. Then, its cytotoxicity was verified by inducing reduction of cell density and morphological abnormality of in vivo cat corneal endothelial cells. Furthermore, the cytotoxicity of norfloxacin in HCECs was corroborated as apoptosis by elevation of plasma membrane permeability, S phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in in vitro cultured HCECs and apoptosis-like swollen cells in the in vivo model. Moreover, norfloxacin induced extrinsic death receptor-mediated apoptosis pathway by activating caspase-2/-8/-3 and intrinsic mitochondrion-dependent apoptosis pathway by downregulating anti-apoptotic Bcl-2 and upregulating of pro-apoptotic Bad, which disrupted mitochondrial transmembrane potential, subsequently upregulated cytoplasmic cytochrome c and apoptosis-inducing factor and finally activated caspase-9/-3. Generally, norfloxacin induces HCE cell apoptosis via a death receptor-mediated and mitochondrion-dependent signaling pathway.

The first cloned sea cucumber FADD from Holothuria leucospilota: Molecular characterization, inducible expression and involvement of apoptosis

In this study, a sea cucumber Fas-associated death domain (FADD) named HLFADD was first cloned from Holothuria leucospilota. The full-length cDNA of HLFADD is 2137 bp in size, containing a 116-bp 5′-untranslated region (UTR), a 1334-bp 3′-UTR and a 687-bp open reading frame (ORF) encoding a protein of 228 amino acids with a deduced molecular weight of 26.42 kDa. HLFADD protein contains a conserved death effector domain at its N-terminal and a conserved death domain at its C-terminal, structurally similar to its counterparts in vertebrates. The over-expressed HLFADD protein could induce apoptosis in HEK293 cells, suggesting a possible death receptor-mediated apoptosis pathway in echinoderms adapted with FADD. Moreover, HLFADD mRNA is ubiquitously expressed in all examined tissues, with the highest transcript level in the coelomocytes, followed by intestine. In vitro experiments performed in the H. leucospilota coelomocytes, the expression of HLFADD mRNA was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLFADD might play important roles in the innate immune defense of sea cucumber against the invasion of bacteria and viruses.

C-Jun N-terminal kinase 3 deficiency protects axotomized retinal ganglion cells via affecting mitochondria involved apoptosis pathway.

To illustrate the isoform-specific role and mechanism of c-Jun N-terminal kinases (JNKs) in mouse optic nerve axotomy induced neurotrauma. We firstly investigated the expression of JNK1, JNK2, and JNK3 in the retinal ganglion cells (RGCs) by double-immunofluorescent staining. Then we created optic nerve axotomy model in wild type as well as JNK1, JNK2, JNK3, isoform specific gene deficiency mice. With that, we checked the protein expression profile of JNKs and its active form, and quantified the survival RGCs number by immunofluorescence staining. We further explored the molecules underlying isoform specific protective effect by real-time polymerase chain reaction (PCR) and Western blotting assay. We found that all the three isoforms of JNKs were expressed in the RGCs. Deficiency of JNK3, but not JNK1 or JNK2, significantly alleviated optic nerve axotomy induced RGCs apoptosis. We further established that expression of Noxa, a pro-apoptotic member of BH3 family, was significantly suppressed only in JNK3 gene deficiency mice. But tumor necrosis factor receptor 1 (TNFR1) and Fas, two key modulators of death receptor mediated apoptosis pathway, did not display obvious change in the expression. It is suggested that mitochondria mediated apoptosis, but not death receptor mediated apoptosis got involved in the JNK3 gene deficiency induced RGCs protection. Our study provides a novel insight into the isoform-specific role of JNKs in neurotrauma and indicates some cues for its therapeutics.

Mechanisms and Therapeutic Targets of Depression After Intracerebral Hemorrhage.

The relationship between depression and intracerebral hemorrhage (ICH) is complicated. One of the most common neuropsychiatric comorbidities of hemorrhagic stroke is Post-ICH depression. Depression, as a neuropsychiatric symptom, also negatively impacts the outcome of ICH by enhancing morbidity, disability, and mortality. However, the ICH outcome can be improved by antidepressants such as the frequently-used selective serotonin reuptake inhibitors. This review therefore presents the mechanisms of post-ICH depression, we grouped the mechanisms according to inflammation, oxidative stress (OS), apoptosis and autophagy, and explained them through their several associated signaling pathways. Inflammation is mainly related to Toll-like receptors (TLRs), the NF-kB mediated signal pathway, the PPAR-γ-dependent pathway, as well as other signaling pathways. OS is associated to nuclear factor erythroid-2 related factor 2 (Nrf2), the PI3K/Akt pathway and the MAPK/P38 pathway. Moreover, autophagy is associated with the mTOR signaling cascade and the NF-kB mediated signal pathway, while apoptosis is correlated with the death receptor-mediated apoptosis pathway, mitochondrial apoptosis pathway, caspase-independent pathways and others. Furthermore, we found that neuroinflammation, oxidative stress, autophagy, and apoptosis experience interactions with one another. Additionally, it may provide several potential therapeutic targets for patients that might suffer from depression after ICH.

A mini review of fluoride-induced apoptotic pathways

Fluorine or fluoride can have toxic effects on bone tissue and soft tissue at high concentrations. These negative effects include but not limited to cytotoxicity, immunotoxicity, blood toxicity, and oxidative damage. Apoptosis plays an important role in fluoride-induced toxicity of kidney, liver, spleen, thymus, bursa of Fabricius, cecal tonsil, and cultured cells. Here, apoptosis activated by high level of fluoride has been systematically reviewed, focusing on three pathways: mitochondrion-mediated, endoplasmic reticulum (ER) stress-mediated, and death receptor-mediated pathways. However, very limited reports are focused on the death receptor-mediated apoptosis pathways in the fluoride-induced apoptosis. Therefore, understanding and discovery of more pathways and molecular mechanisms of fluoride-induced apoptosis may contribute to designing measures for preventing fluoride toxicity.

An integrated approach to elucidate signaling pathways of dioscin-induced apoptosis, energy metabolism and differentiation in acute myeloid leukemia

Although the therapeutics have improved the rates of remission and cure of acute myelogenous leukemia (AML) in recent decades, there is still an unmet medical need for AML therapies because disease relapses are a major obstacle in patients who become refractory to salvage therapy. The development of therapeutic agents promoting both cytotoxicity and cell differentiation may provide opportunities to improve the clinical outcome. Dioscin-induced apoptosis in leukemic cells was identified through death receptor-mediated extrinsic apoptosis pathway. The formation of Bak and tBid, and loss of mitochondrial membrane potential were induced by dioscin suggesting the activation of intrinsic apoptotsis pathway. A functional analysis of transcription factors using transcription factor-DNA interaction array and IPA analysis demonstrated that dioscin induced a profound increase of protein expression of CCAAT/enhancer-binding protein α (C/EBPα), a critical factor for myeloid differentiation. Two-dimensional gel electrophoresis assay confirmed the increase of C/EBPα expression. Dioscin-induced differentiation was substantiated by an increase of CD11b protein expression and the induction of differentiation toward myelomonocytic/granulocytic lineages using hematoxylin and eosin staining. Moreover, both glycolysis and gluconeogenesis pathways after two-dimensional gel electrophoresis assay and IPA network enrichment analysis were proposed to dioscin action. In conclusion, the data suggest that dioscin exerts its antileukemic effect through the upregulation of both death ligands and death receptors and a crosstalk activation of mitochondrial apoptosis pathway with the collaboration of tBid and Bak formation. In addition, proteomics approach reveals an altered metabolic signature of dioscin-treated cells and the induction of differentiation of promyelocytes to granulocytes and monocytes in which the C/EBPα plays a key role.

XWL-1-48 exerts antitumor activity via targeting topoisomerase II and enhancing degradation of Mdm2 in human hepatocellular carcinoma

A novel podophyllotoxin derivative, XWL-1-48, was synthesized as an oral topoisomerase II inhibitor. kDNA decatenation assay indicated that XWL-1-48 significantly inhibited topoisomerase II activity in a concentration-dependent manner. Moreover, the cytotoxicity of XWL-1-48 is more potent than its congener GL331 and the IC50 values are from 0.34 ± 0.21 to 3.54 ± 0.54 µM in 10 cancer cell lines including KBV200 cells with P-gp overexpression. Noticeably, XWL-1-48 exerted potent antitumor activity in in vitro and in vivo human hepatocellular carcinoma (HCC) model. Further studies demonstrated that treatment of XWL-1-48 induced γ-H2AX and p-ATM expression, and further triggered DNA damage response through activation of ATM-p53-p21 and ATM-Chk2-Cdc25A pathways. Targeted inhibition of ATM by siRNA attenuated the ability of XWL-1-48 on inducing DNA damage. XWL-1-48 significantly suppressed Cyclin A and p-Cdk2 (Thr160) expression, increased p-Cdk2 (Thr14), led to inactivation of Cyclin A/Cdk2 complex, arrested cell cycle at S phase. Finally, XWL-1-48 elevated the ratio of Bax/Bcl2 and induced Fas and FasL, initiated mitochondria- and death receptor-mediated apoptosis pathway. Meanwhile, XWL-1-48 evidently enhanced degradation of Mdm2, blocked PI3K/Akt/Mdm2 pathway and suppressed HCC cell survival. Thus, XWL-1-48 may be a promising orally topoisomerase II inhibitor for treatment of HCC.

FLIP the Switch: Regulation of Apoptosis and Necroptosis by cFLIP.

cFLIP (cellular FLICE-like inhibitory protein) is structurally related to caspase-8 but lacks proteolytic activity due to multiple amino acid substitutions of catalytically important residues. cFLIP protein is evolutionarily conserved and expressed as three functionally different isoforms in humans (cFLIPL, cFLIPS, and cFLIPR). cFLIP controls not only the classical death receptor-mediated extrinsic apoptosis pathway, but also the non-conventional pattern recognition receptor-dependent apoptotic pathway. In addition, cFLIP regulates the formation of the death receptor-independent apoptotic platform named the ripoptosome. Moreover, recent studies have revealed that cFLIP is also involved in a non-apoptotic cell death pathway known as programmed necrosis or necroptosis. These functions of cFLIP are strictly controlled in an isoform-, concentration- and tissue-specific manner, and the ubiquitin-proteasome system plays an important role in regulating the stability of cFLIP. In this review, we summarize the current scientific findings from biochemical analyses, cell biological studies, mathematical modeling, and gene-manipulated mice models to illustrate the critical role of cFLIP as a switch to determine the destiny of cells among survival, apoptosis, and necroptosis.

Functional Consequences for Apoptosis by Transcription Elongation Regulator 1 (TCERG1)-Mediated Bcl-x and Fas/CD95 Alternative Splicing.

Here, we present evidence for a specific role of the splicing-related factor TCERG1 in regulating apoptosis in live cells by modulating the alternative splicing of the apoptotic genes Bcl-x and Fas. We show that TCERG1 modulates Bcl-x alternative splicing during apoptosis and its activity in Bcl-x alternative splicing correlates with the induction of apoptosis, as determined by assessing dead cells, sub-G1-phase cells, annexin-V binding, cell viability, and cleavage of caspase-3 and PARP-1. Furthermore, the effect of TCERG1 on apoptosis involved changes in mitochondrial membrane permeabilization. We also found that depletion of TCERG1 reduces the expression of the activated form of the pro-apoptotic mitochondrial membrane protein Bak, which remains inactive by heterodimerizing with Bcl-xL, preventing the initial step of cytochrome c release in Bak-mediated mitochondrial apoptosis. In addition, we provide evidence that TCERG1 also participates in the death receptor-mediated apoptosis pathway. Interestingly, TCERG1 also modulates Fas/CD95 alternative splicing. We propose that TCERG1 sensitizes a cell to apoptotic agents, thus promoting apoptosis by regulating the alternative splicing of both the Bcl-x and Fas/CD95 genes. Our findings may provide a new link between the control of alternative splicing and the molecular events leading to apoptosis.

Essential oil of Cephalotaxus griffithii needle inhibits proliferation and migration of human cervical cancer cells: involvement of mitochondria-initiated and death receptor-mediated apoptosis pathways

This study was conducted to determine the effect of Cephalotaxus griffithii needle essential oil (CGNO) on proliferation and migration of human cervical cancer (HCC) cells. CGNO treatment decreased the viability of all the tested HCC (HeLa, ME-180 and SiHa) cells. Morphological and DNA fragmentation analysis of CGNO-treated HeLa cells indicated the involvement of apoptosis in inducing HCC cell death. CGNO increased mitochondrial membrane depolarisation and upregulated the expression of caspase-9, caspase-8, caspase-3 and cleaved-PARP. The activity of caspase-8 and caspase-9 was also significantly increased. Wound healing and transwell migration assay demonstrated that CGNO significantly inhibited the migration of HeLa cells to close a scratched wound and also inhibited their migration through filter towards a chemotactic stimulus. Taken together, these results indicated that CGNO inhibited the proliferation and migration of HCC cells. Of note, CGNO induced HeLa cell death through mitochondria-initiated and death receptor-mediated apoptosis pathway.

Polysaccharide of Boschniakia rossica induces apoptosis on laryngeal carcinoma Hep2 cells

The aim of this study was to explore the anti-tumor potential of a polysaccharide isolated from Boschniakia rossica (BRP) in Hep2 human larynx squamous carcinoma cells. High performance size-exclusion chromatography analysis showed that BRP was a homogeneous polysaccharide and had a molecular weight of 22kDa. Total carbohydrate content in BRP was determined to be 96.9%, without the presence of protein and nucleic acid. BRP suppressed the proliferation of Hep2 cells in a time- and dose-dependent manner. Cell cycle analysis revealed that exposure to BRP (200μg/ml) caused a G0/G1 cell cycle arrest in Hep2 cells. Moreover, treatment with BRP at 100–400μg/ml for 24h induced a significant apoptosis Hep2 cells compared to untreated control cells, as determined by flow cytometry with annexin-V/propidium iodide double staining. Additionally, BRP treatment promoted the cleavage of pro-caspase-3, pro-caspase-8, and pro-caspase-9, coupled with increased expression of death receptor DR5 and Bax and reduced expression of Bcl-2. Taken together, our data demonstrate that BRP shows potent anti-tumor activity in human larynx squamous carcinoma, largely through induction of G0/G1 cell cycle arrest and apoptosis. Activation of both mitochondria-mediated and death receptor-mediated apoptosis pathways is involved in the cytotoxicity of BRP.

Naringenin에 의한 인체혈구암세포의 apoptosis 유발에 미치는 pro-apoptotic Bcl-2의 영향

감귤류에 많이 함유되어 있는 naringenin은 항암화학요법제로서 중요한 가능성을 가지고 있으나 항암활성에 대한 분자생물학적 기전에 대해서는 명확히 밝혀져 있지 않다. 본 연구에서는 인체 혈구암세포인 U937 세포에서 naringenin이 유발하는 항암효과 및 항암기전을 조사하였다. Naringenin 처리에 의한 U937 세포의 증식억제는 apoptosis 유발과 연관성이 있었으며, 이러한 현상은 caspases 활성화와 밀접한 관련이 있었다. 그러나 pan-caspase inhibitor인 z-VAD-fmk의 선처리에 의하여 U937 세포에서 naringenin이 유발하는 apoptosis가 억제되는 것으로 나타났으므로 caspases가 apoptosis 유발의 중요한 조절자라는 것을 알 수 있었다. 또한 U937 세포에 naringenin을 처리하였을 경우 pro-apoptotic Bcl-2 및 anti-apoptotic Bax의 발현에는 아무런 변화가 나타나지는 않았지만 Bcl-2가 과발현된 U937/Bcl-2 세포에서 naringenin에 의한 apoptosis가 억제되었다. 하지만 small-molecule Bcl-2 inhibitor인 HA14-1 및 naringenin을 같이 처리하였을 경우에는 XIAP 발현감소, Bid 단편화 및 caspase-3 활성화를 통하여 다시 apoptosis가 유발되었다. 따라서 HA14-1 및 naringenin에 의한 apoptosis 상승효과는 death receptor-mediated apoptosis pathway를 경유한다는 것을 제시하는 결과이다. Naringenin, a naturally occurring citrus flavonone, is a potentially valuable candidate for cancer chemotherapy. However, the cellular and molecular mechanisms responsible for its anticancer activity are largely unknown. In the present study, we attempted to elucidate the mechanisms responsible for naringenin-induced apoptosis in human leukemic U937 cells. We found that naringenin markedly inhibited the growth of U937 cells by decreasing cell proliferation and inducing apoptosis, which was associated with the activation of caspases. A pan-caspase inhibitor, z-VAD-fmk, significantly inhibited naringenin-induced U937 cell apoptosis, indicating that caspases are key regulators of apoptosis in response to naringenin in U937 cells. Although the levels of antiapoptotic Bcl-2 and proapoptotic Bax proteins remained unchanged in naringenin-treated U937 cells, Bcl-2 overexpression attenuated naringenin-induced apoptosis. Furthermore, combined treatment with naringenin and HA14-1, a small-molecule Bcl-2 inhibitor, effectively increased the apoptosis through enhancement of XIAP down-regulation, Bid cleavage, and caspase activation, suggesting that the synergistic effect was at least partially mediated through the death receptor-mediated apoptosis pathway.

Abstract 2953: Inhibition of calmodulin enhances TRA-8-induced apoptosis in pancreatic cancer cells .

Abstract Current chemotherapy and radiotherapy are not curative for pancreatic cancer. Tumor necrosis factor-related apoptosis-inducing ligand or agonist antibodies for death receptor 4/5 have been shown to be a promising cancer therapy. TRA-8, a humanized activating antibody for the death receptor 5 (DR5), is a potent apoptosis-inducing reagent for cancer cells. However, several pancreatic cell lines are resistant to TRA-8-induced apoptosis. We have previously demonstrated that calmodulin (CaM) antagonists enhance the Fas death receptor-mediated apoptosis pathway. Therefore, we investigated whether inhibition of CaM might affect DR5-induced apoptotic signaling in pancreatic cancer cells. We found that CaM antagonists, trifluoperazine (TFP) and tamoxifen (TMX), dose dependently enhanced TRA-8-induced apoptosis of the TRA-8-resistant PANC-1 pancreatic cells. Consistently, TFP and TMX increased TRA-8-induced activation of caspase 8, caspase 9 and caspase 3. Pretreatment of PANC-1 with Z-IETD-FMK, the caspase-8 inhibitor, decreased TFP-enhanced apoptosis by 2 fold, and markedly decreased caspase 3 activation. Immunoprecipitation analysis of the DR5-induced death inducing signaling complex (DISC) identified the interaction of CaM with DR5. Activation of DR-5 by TRA-8 increased the recruitment of CaM into the DISC, which was inhibited by TFP. In addition, TFP decreased the recruitment of the adaptor protein, FADD, and other DISC proteins that regulate cell survival, including Src and the caspase-8 inhibitory protein FLIP. Collectively, these results support an important role for CaM in DR5-induced DISC formation, thus regulating DR5-mediated survival and apoptotic signaling in pancreatic cancer cells. In summary, we have demonstrated that CaM antagonists sensitize pancreatic cancer cell to TRA 8-induced apoptosis, and that CaM binding to DR5 regulates DR5-induced DISC formation. Defining the function of CaM in regulating DR5-induced signaling pathways may lead to novel therapeutic strategies including the use of CaM antagonists with TRA-8 as combination therapy for pancreatic cancer. Citation Format: Kaiyu Yuan, Tong Zhou, Jay McDonald, Yabing Chen. Inhibition of calmodulin enhances TRA-8-induced apoptosis in pancreatic cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2953. doi:10.1158/1538-7445.AM2013-2953

Changes in Apoptotic Mechanisms Following Penetrating Ballistic-Like Brain Injury

We investigated apoptotic pathways in a model of severe traumatic brain injury, penetrating ballistic-like brain injury (PBBI). TUNEL staining identified increasing apoptosis within 24 h. From targeted arrays, 11 genes were identified for temporal mRNA evaluation. In addition, mRNA levels and enzyme activity for caspases 3, 8, and 9 were examined. In the death receptor-mediated apoptosis pathway, the relative quantities (RQs) of mRNA for tnfr1, fas, and tnf were upregulated while trail mRNA was downregulated. In the anti-apoptotic TNF-R2 pathway, tnfr2 and flip were upregulated while xiap was downregulated. These findings indicate that increases in tnf levels following injury are not only pro-apoptotic but may also signal competing anti-apoptotic mechanisms. For the mitochondria-mediated apoptosis pathway, RQs of anti-apoptotic factors bcl2a1d and birc3 were upregulated while both bcl2 and bax were downregulated. RQs for casp 3 and casp 8 increased while casp9 decreased. Enzymatic activity increased for caspases 3, 8, and 9. While multiple mechanisms promoting and inhibiting apoptosis are at play during the first week after a PBBI, the cumulative result remains increased apoptosis. The ability to understand and dissect these events will assist in the development and evaluation of treatments targeting apoptosis following severe brain injury.

Upregulation of SATB1 promotes tumor growth and metastasis in liver cancer

Special AT-rich binding protein-1 (SATB1) reprograms chromatin organization and transcription profiles to promote tumour growth and metastasis. This study aimed to confirm the effects of SATB1 on the growth and metastasis of liver cancer and its specific regulation mechanism. SATB1 expression was evaluated in human hepatoma tissue, adjacent noncancerous tissue and seven kinds of liver cancer cell lines. Cell cycle, cell proliferation, apoptosis and epithelial-mesenchymal transition (EMT) was investigated after enhanced or silenced expression of SATB1. The regulatory action of SATB1 on the expression of genes that are known to regulate cell cycle progression, apoptosis and EMT and the specific apoptotic pathway on which it acts were further analysed. Nude mice that received subcutaneous implantation were used to study the effects of SATB1 on tumour growth in vivo. Our data show that the high expression of SATB1 was observed in the human hepatocellular carcinoma tissue (26/45) and liver cancer cell lines with high metastatic potential. SATB1 upregulated CDK4 and downregulated p16 (INK) (4A) to promote cell cycle progression and cell proliferation and prevented apoptosis by inhibiting the FADD-caspase-8-caspase-3 death receptor-mediated apoptosis pathway. SATB1 also induced EMT concomitant with increased expression of Snail1, Slug, Twist and vimentin and decreased expression of E-cadherin, tight junction protein ZO-1 and desmoplakin. SATB1 promoted the growth of tumour in vivo. These data suggest that the SATB1 gene may play an important role in the development and progression of liver cancer by regulation of genes related to cell cycle progression, apoptosis and EMT.

Abstract 4885: mTOR complex 2 is involved in regulation of c-FLIP stability and sensitization of TRAIL-induced apoptosis

Abstract The mammalian target of rapamycin (mTOR), a serine-threonine kinase related tightly to the phosphoinositide 3-kinase family, acts as a central molecule in regulation of cell proliferation and survival through forming two complexes primarily with raptor (mTOR complex 1; mTORC1) or rictor (mTOR complex 2; mTORC2). Compared with the mTORC1, relatively little is known about the biological functions of mTORC2. However, it is clear that both of the mTOR signaling pathways are dysregulated in human cancers, including lung cancer. Accordingly, novel ATP-competitive mTOR kinase inhibitors such as PP242 that inhibit both mTORC1 and mTORC2 have been developed for cancer therapy. In this study, we focus on addressing whether mTORC2 regulates apoptosis, particularly induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potential cancer therapeutic protein tested in clinical trials. We found that PP242 synergized with TRAIL to augment apoptosis in human lung cancer cells. PP242 reduced the level of the short form of c-FLIP (FLIPS) and survivin with minimal effects on the expression of DR4 and DR5. Enforced expression of FLIPS, but not survivin, attenuated PP242/TRAIL-induced apoptosis; this suggests that FLIPS downregulation contributes to synergistic induction of apoptosis by the PP242 and TRAIL combination. Mechanistic studies revealed that PP242 decreased FLIPS stability, increased FLIPS ubiqutination and facilitated FLIPS degradation, suggesting that PP242 decreases FLIPS levels through promoting its degradation via an ubiqutination and proteasome-dependent mechanism. Interestingly, knockdown of rictor, but not raptor, mimicked PP242 in decreasing FLIPS levels and sensitizing cells to TRAIL. Moreover, rictor knockdown decreased the stability of FLIPS. These data together suggest that it is mTORC2 inhibition that mediates FLIPS downregulation and subsequent sensitization of TRAIL-induced apoptosis. Our findings hence provide the first evidence showing that mTORC2 positively regulates FLIPS expression, thereby, connecting mTORC2 signaling to regulation of the death receptor-mediated apoptosis pathway. (This study was supported by the Georgia Cancer Coalition Distinguished Cancer Scholar award and NIH/NCI R01 CA118450. FR Khuri and SY Sun are Georgia Cancer Coalition Distinguished Cancer Scholars) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4885. doi:1538-7445.AM2012-4885