Promote Caspase Activation Research Articles

DIPG-15. CONCURRENT GB-13 (IL13.E13K-PE4E) IMPROVES THE EFFICACY OF RADIATION THERAPY IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse midline glioma with H3 K27 alterations (DMG) is the most aggressive primary malignant brain tumor in the pediatric population. Radiation therapy (RT) is the standard-of-care; however, it has not demonstrated a significant improvement in overall survival or stalled disease progression, and children usually succumb to disease within 12-15 months of diagnosis. Consequently, finding efficient treatments for DMG remains one of the most critical unmet needs in modern neuro-oncology. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface receptor upregulated in ~80% of DMG versus normal brain, posing a potentially promising therapeutic target. Immunotherapies harnessing IL-13Rα2 to selectively delivery cytotoxic payloads, such as pseudomonas exotoxin A (PE), to tumor cells have been previously demonstrated to be safe in DMG patients and efficacious in preclinical models of DMG with high IL-13Rα2 expression using convection-enhanced delivery (CED). Furthermore, there is evidence that PE-based therapies can sensitize cells to RT. In this study, we utilized in vitro and in vivo models of DMG to evaluate clinically-relevant combination treatments of RT and the IL-13/PE immunotoxin GB-13 (IL13.E13K-PE4E). GB-13 improved the effectiveness of low-dose RT in multiple DMG cell lines by promoting caspase activation, which boosted apoptotic cell death. Intratumoral administration of GB-13 via chronic CED decreased tumor burden and prolonged survival in both DMG patient-derived orthotopic xenograft and genetically engineered mouse models. When GB-13 and RT were administered concurrently, this impact became even more prominent, informing future preclinical and clinical investigation of a potentially efficacious therapeutic approach in a subset of DMG.

Cathepsin B mediates the lysosomal-mitochondrial apoptosis pathway in arsenic-induced microglial cell injury.

Arsenic is a prevalent environmental pollutant that targets the nervous system of living beings. Recent studies indicated that microglial injury could contribute to neuroinflammation and is associated with neuronal damage. Nevertheless, the neurotoxic mechanism underlying the arsenic-induced microglial injury requires additional research. This study explores whether cathepsin B promotes microglia cell damage caused by NaAsO2. Through CCK-8 assay and Annexin V-FITC and PI staining, we discovered that NaAsO2 induced apoptosis in BV2 cells (a microglia cell line). NaAsO2 was verified to increase mitochondrial membrane permeabilization (MMP) and promote the generation of reactive oxygen species (ROS) through JC-1 staining and DCFDA assay, respectively. Mechanically, NaAsO2 was indicated to increase the expression of cathepsin B, which could stimulate pro-apoptotic molecule Bid into the activated form, tBid, and increase lysosomal membrane permeabilization by Immunofluorescence and Western blot assessment. Subsequently, apoptotic signaling downstream of increased mitochondrial membrane permeabilization was activated, promoting caspase activation and microglial apoptosis. Cathepsin B inhibitor CA074-Me could mitigate the damage of microglial. In general, we found that NaAsO2 induced microglia apoptosis and depended on the role of the cathepsin B-mediated lysosomal-mitochondrial apoptosis pathway. Our findings provided new insight into NaAsO2-induced neurological damage.

BAX-dependent mitochondrial pathway mediates the crosstalk between ferroptosis and apoptosis.

Ferroptosis is considered a distinctive form of cell death compared to other types of death such as apoptosis. It is known to result from iron-dependent accumulation of lipid peroxides rather than caspase activation. However, we reported recently that ferroptosis interplays with apoptosis. In this study, we investigated a possible mechanism of this interplay between ferroptosis and apoptosis. Results from our studies reveal that combined treatment of the ferroptotic agent erastin and the apoptotic agent TRAIL effectively disrupted mitochondrial membrane potential (ΔΨm) and subsequently promoted caspase activation. The alterations of mitochondrial membrane potential are probably due to an increase in oligomerization of BAX and its accumulation at the mitochondria during treatment with erastin and TRAIL. Interestingly, the combined treatment-promoted apoptosis was effectively inhibited in BAX-deficient HCT116 cells, but not BAK-deficient cells. These results indicate that the BAX-associated mitochondria-dependent pathway plays a pivotal role in erastin-enhanced TRAIL-induced apoptosis.

ANTP-SmacN7 fusion peptide-induced radiosensitization in A549 cells and its potential mechanisms.

BackgroundRadioresistance in tumors limits the curative effect of the radiotherapy. Mimetic compounds of second mitochondria‐derived activator of caspase (Smac) are potential new tumor radiation‐sensitizing drugs because they can increase radiation‐induced tumor cell apoptosis. Here, we observed the radiosensitization effect of a new Smac mimetic Antennapedia protein (ANTP)‐SmacN7 fusion peptide in A549 cells and investigated the underlying mechanisms behind the effects of this protein on tumor cells.MethodsThe ANTP‐SmacN7 fusion peptide was synthesized and linked with fluorescein isothiocyanate to observe the protein's ability to penetrate cells. A549 cells were divided into the control, radiation‐only, ANTP‐SmacN7‐only and ANTP‐SmacN7 + radiation groups. The cells were exposed to 0, 2, 4 and 6 Gy, with 20 μmol/L of ANTP‐SmacN7. The radiation‐sensitizing effects of the ANTP‐SmacN7 fusion proteins were observed via clonogenic assay. Apoptosis was detected using flow cytometry. A comet assay was used to assess DNA damage. The levels and degrees of cytochrome‐c, PARP, H2AX, caspase‐8, caspase‐3, and caspase‐9 activation were detected via western blot assay. The radiation sensitization of the fusion peptide, expression of γ‐H2AX and C‐PARP were compared after adding the caspase inhibitor, Z‐VAD.ResultsANTP‐SmacN7 fusion proteins entered the cells and promoted A549 cell radiosensitization. Treatment with ANTP‐SmacN7 + radiation significantly reduced the A549 cell clone‐forming rate, increased the cytochrome‐c, cleaved caspase‐8, cleaved caspase‐3 and cleaved caspase‐9 expression levels, promoted caspase activation, and increased the rate of radiation‐induced apoptosis. The ANTP‐SmacN7 fusion peptide significantly increased radiation‐induced double‐stranded DNA rupture in the A549 cells and increased DNA damage. Adding Z‐VAD reduced the fusion peptide's proapoptotic effect but not the level of double‐stranded DNA breakage.ConclusionsThe ANTP‐SmacN7 fusion peptide exerted a remarkable radiosensitization effect on A549 cells. This protein may reduce tumor cell radioresistance by inducing caspase activation and may be a potential new Smac mimetic that can be applied in radiosensitization therapy.

The Zinc-Finger AN1-Type Domain 2a Gene Acts as a Regulator of Cell Survival in Human Melanoma: Role of E3-Ligase cIAP2.

The zinc-finger AN1-type domain-2a gene, also known as AIRAP (arsenite-inducible RNA-associated protein), was initially described as an arsenite-inducible gene in Caenorhabditis elegans and mammalian cells. Differently from the AIRAP worm homologue, aip-1, a gene known to play an important role in preserving animal lifespan and buffering arsenic-induced proteotoxicity, mammals have a second, constitutively expressed, AIRAP-like gene (AIRAPL), recently implicated in myeloid transformation. We have identified human AIRAP as a canonical heat-shock gene, whose expression, differently from AIRAPL, is strictly dependent on the proteotoxic-stress regulator heat-shock factor 1 (HSF1). AIRAP function is still not well defined and there is no information on AIRAP in cancer. Herein we show that bortezomib and next-generation proteasome inhibitors ixazomib and carfilzomib markedly induce AIRAP expression in human melanoma at concentrations comparable to plasma-levels in treated patients. AIRAP-downregulation leads to bortezomib sensitization, whereas AIRAP-overexpression protects melanoma cells from the drug, identifying AIRAP as a novel HSF1-regulated marker of chemotherapy resistance. More importantly, this study unexpectedly revealed that, also in the absence of drugs, AIRAP-silencing hinders melanoma clonogenic potential and spheroid growth, promoting caspase activation and apoptotic cell death, an effect independent of AIRAPL and linked to downregulation of the antiapoptotic protein cIAP2. Interestingly, AIRAP was found to interact with cIAP2, regulating its stability in melanoma. Taken together, the results identify AIRAP as a novel HSF1-dependent regulator of prosurvival networks in melanoma cells, opening new therapeutic perspectives in chemoresistant melanoma treatment. IMPLICATIONS: The findings identify ZFAND2A/AIRAP as a novel stress-regulated survival factor implicated in the stabilization of the antiapoptotic protein cIAP2 and as a new potential therapeutic target in melanoma.

Caspase-mediated cleavage of murine norovirus NS1/2 potentiates apoptosis and is required for persistent infection of intestinal epithelial cells.

Human norovirus (HNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after the resolution of symptoms. Elimination of persistent viral reservoirs has the potential to prevent outbreaks. Similar to HNoV, murine norovirus (MNV) is spread by persistent shedding in the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We now find that the function of CR6 NS1 is regulated by apoptotic caspase cleavage. Following induction of apoptosis in infected cells, caspases cleave the precursor NS1/2 protein, and this cleavage is prevented by mutation of caspase target motifs. These mutations profoundly compromise CR6 infection of IECs and persistence in the intestine. Conversely, NS1/2 cleavage is not strictly required for acute replication in extra-intestinal tissues or in cultured myeloid cells, suggesting an IEC-centric role. Intriguingly, we find that caspase cleavage of CR6 NS1/2 reciprocally promotes caspase activity, potentiates cell death, and amplifies spread among cultured IEC monolayers. Together, these data indicate that the function of CR6 NS1 is regulated by apoptotic caspases, and suggest that apoptotic cell death enables epithelial spread and persistent shedding.

RIPK1 can mediate apoptosis in addition to necroptosis during embryonic development

RIPK1 has emerged as a key effector in programmed necrosis or necroptosis. This function of RIPK1 is mediated by its protein serine/threonine kinase activity and through the downstream kinase RIPK3. Deletion of RIPK1 prevents embryonic lethality in mice lacking FADD, a signaling adaptor protein required for activation of Caspase 8 in extrinsic apoptotic pathways. This indicates that FADD-mediated apoptosis inhibits RIPK1-dependent necroptosis to ensure successful embryogenesis. However, the molecular mechanism for this critical regulation remains unclear. In the current study, a novel mouse model has been generated, by disrupting a potential caspase cleavage site at aspartic residue (D)324 in RIPK1. Interestingly, replacing D324 with alanine (A) in RIPK1 results in midgestation lethality, similar to the embryonic defect in FADD−/− mice but in stark contrast to the normal embryogenesis of RIPK1−/− null mutant mice. Surprisingly, disrupting the downstream RIPK3 alone is insufficient to rescue RIPK1D324A/D324A mice from embryonic lethality, unless FADD is deleted simultaneously. Further analyses reveal a paradoxical role for RIPK1 in promoting caspase activation and apoptosis in embryos, a novel mechanism previously unappreciated.

Structure-based design, synthesis and anticancer effect of cyclic Smac-polyarginine peptides.

The second mitochondria-derived activator of caspase (Smac/DIABLO) is a pro-apoptotic protein that released from mitochondria into the cytosol when cells undergo apoptosis. Smac promotes caspase activation by binding the inhibitors of apoptosis proteins (IAP), particularly XIAP and eliminating their inhibitory activity. Although the seven N-terminal amino acids AVPIAQK (SmacN7) of Smac protein is able to elicit an anticancer response by itself, it is neither cell-permeable nor stable in the cellular environment. Thus, the use of SmacN7 derivatives and mimetics is an alluring field for cancer therapy. In this study, heptamer Smac peptide was fused to a well-known octaarginine cell-penetrating peptide for promoting its intracellular access. Both therapeutic Smac part and cell-penetrating octaarginine parts of the peptide sequence constrained in a cyclic structure so as to enhance the apoptosis-inducing potential of the SmacN7 peptide. Biological assays interestingly showed that cyclic peptides P4, P5 and P7 gave rise to a significant level of cytotoxicity and apoptosis mediated cell death in multiple myeloma tumor cells (MM) comparing to linear peptide.

Varacin-1, a novel analog of varacin C, induces p53-independent apoptosis in cancer cells through ROS-mediated reduction of XIAP

Varacin C is a promising anticancer agent and possesses acid-promoted and photo-induced DNA-damaging activities. In this study, we synthesized an analog varacin-1 (VCA-1) and examined its anticancer potentials. The results demonstrated that VCA-1 caused dose-dependent apoptotic cell death in cancer cells. Note that this action is independent of p53 status, because VCA-1 induced similar levels of apoptosis in two different panels of cell lines (HCT116 p53- wild-type vs. HCT116 p53-knockout colon cancer cells, and p53-expressing U2OS vs. p53-deficient saos2 osteosarcoma cancer cells). VCA-1-induced apoptosis was found to be mainly via the extrinsic apoptosis pathway involving caspase-8 activation and XIAP reduction. Forced over-expression of XIAP markedly prevented apoptosis, indicating its essential role in VCA-1 induced apoptosis. On the other hand, VCA-1 treatment enhanced intracellular ROS (reactive oxygen species) generation also in a p53-independent manner, and consequently promoted caspase activation. Pretreatment of N-acetyl cysteine (an antioxidant), rather than z-VAD (specific caspase inhibitor), markedly prevented XIAP reduction, suggesting that XIAP reduction may be resulted from oxidative stress. In conclusion, data from this study reveal the essential roles of ROS generation and XIAP reduction in VCA-1-induced apoptosis in cancer cells. VCA-1 may be a novel cancer therapeutic agent, especially in p53-mutant human cancers.

Apigenin promotes TRAIL-mediated apoptosis regardless of ROS generation

Apigenin is a bioactive flavone in several herbs including parsley, thyme, and peppermint. Apigenin possesses anti-cancer and anti-inflammatory properties; however, whether apigenin enhances TRAIL-mediated apoptosis in cancer cells is unknown. In the current study, we found that apigenin enhanced TRAIL-induced apoptosis by promoting caspase activation and death receptor 5 (DR5) expression and a chimeric antibody against DR5 completely blocked the apoptosis. Apigenin also upregulated reactive oxygen species (ROS) generation; however, intriguingly, ROS inhibitors, glutathione (GSH) or N-acetyl-l-cysteine (NAC), moderately increased apigenin/TRAIL-induced apoptosis. Additional results showed that an autophagy inducer, rapamycin, enhanced apigenin/TRAIL-mediated apoptosis by a slight increase of ROS generation. Accordingly, NAC and GSH rather decreased apigenin-induced autophagy formation, suggesting that apigenin-induced ROS generation increased autophagy formation. However, autophagy inhibitors, bafilomycin (BAF) and 3-methyladenine (3-MA), showed different result in apigenin/TRAIL-mediated apoptosis without ROS generation. 3-MA upregulated the apoptosis but remained ROS levels; however, no changes on apoptosis and ROS generation were observed by BAF treatment. Taken together, these findings reveal that apigenin enhances TRAIL-induced apoptosis by activating apoptotic caspases by upregulating DR5 expression regardless of ROS generation, which may be a promising strategy for an adjuvant of TRAIL.

PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis.

Mitochondria drive apoptosis by releasing pro-apoptotic proteins that promote caspase activation in the cytosol. The rhomboid protease PARL, an intramembrane cleaving peptidase in the inner membrane, regulates mitophagy and plays an ill-defined role in apoptosis. Here, we employed PARL-based proteomics to define its substrate spectrum. Our data identified the mitochondrial pro-apoptotic protein Smac (also known as DIABLO) as a PARL substrate. In apoptotic cells, Smac is released into the cytosol and promotes caspase activity by inhibiting inhibitors of apoptosis (IAPs). Intramembrane cleavage of Smac by PARL generates an amino-terminal IAP-binding motif, which is required for its apoptotic activity. Loss of PARL impairs proteolytic maturation of Smac, which fails to bind XIAP. Smac peptidomimetics, downregulation of XIAP or cytosolic expression of cleaved Smac restores apoptosis in PARL-deficient cells. Our results reveal a pro-apoptotic function of PARL and identify PARL-mediated Smac processing and cytochrome c release facilitated by OPA1-dependent cristae remodelling as two independent pro-apoptotic pathways in mitochondria.

AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia.

The AKT (protein kinase B, PKB) family has been shown to participate in diverse cellular processes, including apoptosis. Previous studies demonstrated that protein kinase B2 (AKT2−/−) mice heart was sensitized to apoptosis in response to ischemic injury. However, little is known about the mechanism and apoptotic signaling pathway. Here, we show that AKT2 inhibition does not affect the development of cardiomyocytes but increases cell death during cardiomyocyte ischemia. Caspase-dependent apoptosis of both the extrinsic and intrinsic pathway was inactivated in cardiomyocytes with AKT2 inhibition during ischemia, while significant mitochondrial disruption was observed as well as intracytosolic translocation of cytochrome C (Cyto C) together with apoptosis-inducing factor (AIF) and endonuclease G (EndoG), both of which are proven to conduct DNA degradation in a range of cell death stimuli. Therefore, mitochondria-dependent cell death was investigated and the results suggested that AIF and EndoG nucleus translocation causes cardiomyocyte DNA degradation during ischemia when AKT2 is blocked. These data are the first to show a previous unrecognized function and mechanism of AKT2 in regulating cardiomyocyte survival during ischemia by inducing a unique mitochondrial-dependent DNA degradation pathway when it is inhibited.

Mitochondrial Omi/HtrA2 Promotes Caspase Activation Through Cleavage of HAX-1 in Aging Heart.

Mitochondrial homeostasis is a key process involved in cellular destiny and organic function. When mitochondrial status is abnormal, it will become a "death motor." Impaired mitochondria lead to the release of cytochrome c, and then trigger mitochondria-induced caspase activation. Omi/HtrA2, a serine protease, locates in mitochondria and involves in mitochondrial homeostasis. Increased Omi/HtrA2 is observed in aging cardiac tissues, and whether this has effects on mitochondrial status has not been reported. In this study, natural Sprague-Dawley rats (22 months) were used. We detected markedly increased proteolytic activity of Omi/HtrA2 and obvious activation of caspase-9 and caspase-3 in their myocardium. Then, we constructed stably transfected mitochondrial Omi/HtrA2 cells, and decreased mitochondrial membrane potential was detected by JC-1 (a probe for mitochondria) and tetramethylrhodamine methyl ester (TMRM) dyeing and significant release of cytochrome c was observed after separation of mitochondrial fraction and cytosolic fraction. Furthermore, ucf-101 (a special inhibitor of Omi/HtrA2) and HAX-1 siRNA could ameliorate those phenomena above. In conclusion, excessive Omi/HtrA2 in mitochondria induced decreased mitochondrial membrane potential by its proteolytic activity, followed by cytochrome c released from mitochondria into cytosol where cytochrome c promoted caspase activation. Also, Omi/HtrA2-HAX-1 chain played a significant role in mitochondrial homeostasis.

Deficient NLRP3 and AIM2 Inflammasome Function in Autoimmune NZB Mice.

Inflammasomes are protein complexes that promote caspase activation, resulting in processing of IL-1β and cell death, in response to infection and cellular stresses. Inflammasomes have been anticipated to contribute to autoimmunity. The New Zealand Black (NZB) mouse develops anti-erythrocyte Abs and is a model of autoimmune hemolytic anemia. These mice also develop anti-nuclear Abs typical of lupus. In this article, we show that NZB macrophages have deficient inflammasome responses to a DNA virus and fungal infection. Absent in melanoma 2 (AIM2) inflammasome responses are compromised in NZB by high expression of the AIM 2 antagonist protein p202, and consequently NZB cells had low IL-1β output in response to both transfected DNA and mouse CMV infection. Surprisingly, we also found that a second inflammasome system, mediated by the NLR family, pyrin domain containing 3 (NLRP3) initiating protein, was completely lacking in NZB cells. This was due to a point mutation in an intron of the Nlrp3 gene in NZB mice, which generates a novel splice acceptor site. This leads to incorporation of a pseudoexon with a premature stop codon. The lack of full-length NLRP3 protein results in NZB being effectively null for Nlrp3, with no production of bioactive IL-1β in response to NLRP3 stimuli, including infection with Candida albicans. Thus, this autoimmune strain harbors two inflammasome deficiencies, mediated through quite distinct mechanisms. We hypothesize that the inflammasome deficiencies in NZB alter the interaction of the host with both microflora and pathogens, promoting prolonged production of cytokines that contribute to development of autoantibodies.

The nucleic acid-sensing inflammasomes.

Inflammasomes are oligomeric signaling complexes that promote caspase activation and maturation of proinflammatory cytokines. Structural and biophysical studies have shed light on the mechanisms of nucleic acid recognition and signaling complex assembly involving the AIM2 (absent in myeloma 2) and IFI16 (γ-interferon-inducible protein 16) inflammasomes. However, our understanding of the mechanisms of the NLRP3 (nucleotide-binding oligomerization-like receptor family, pyrin domain-containing protein 3) activation, either by nucleic acids or by other reported stimuli, has remained elusive. Exciting recent progress on the filament formation by the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) pyrin domain and the IFI16-double stranded DNA complex has established that the formation of higher order polymers is one of the general mechanisms for signaling platform assembly in innate immune system. The paradigm-changing discovery of the extracellular function of the NLRP3-ASC inflammasome has opened the door for therapeutic targeting the inflammasome filament formation for various clinical conditions. Future characterization of the canonical and non-canonical inflammasome complexes will undoubtedly reveal more surprises on their structure and function and enrich our understanding of the molecular mechanisms of ligand recognition, activation, and regulation.

The Nuclear Splicing Factor RNA Binding Motif 5 Promotes Caspase Activation in Human Neuronal Cells, and Increases after Traumatic Brain Injury in Mice

Splicing factors (SFs) coordinate nuclear intron/exon splicing of RNA. Splicing factor disturbances can cause cell death. RNA binding motif 5 (RBM5) and 10 (RBM10) promote apoptosis in cancer cells by activating detrimental alternative splicing of key death/survival genes. The role(s) of RBM5/10 in neurons has not been established. Here, we report that RBM5 knockdown in human neuronal cells decreases caspase activation by staurosporine. In contrast, RBM10 knockdown augments caspase activation. To determine whether brain injury alters RBM signaling, we measured RBM5/10 protein in mouse cortical/hippocampus homogenates after controlled cortical impact (CCI) traumatic brain injury (TBI) plus hemorrhagic shock (CCI+HS). The RBM5/10 staining was higher 48 to 72 hours after injury and appeared to be increased in neuronal nuclei of the hippocampus. We also measured levels of other nuclear SFs known to be essential for cellular viability and report that splicing factor 1 (SF1) but not splicing factor 3A (SF3A) decreased 4 to 72 hours after injury. Finally, we confirm that RBM5/10 regulate protein expression of several target genes including caspase-2, cellular FLICE-like inhibitory protein (c-FLIP), LETM1 Domain-Containing Protein 1 (LETMD1), and amyloid precursor-like protein 2 (APLP2) in neuronal cells. Knockdown of RBM5 appeared to increase expression of c-FLIP(s), LETMD1, and APLP2 but decrease caspase-2.

Inactivated Sendai virus strain Tianjin induces apoptosis in breast cancer MCF-7 cells by promoting caspase activation and Fas/FasL expression.

Virotherapy represents a promising new approach for treating cancer. Here the authors have analyzed the effect of ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human breast cancer MCF-7 cells in vitro and in vivo. In vitro, UV-Tianjin inhibited the proliferation of MCF-7, MDA-MB-231, and T47D breast cancer cell lines, although MCF-7 cells were most susceptible to UV-Tianjin treatment. Hoechst staining and flow cytometric analysis of UV-Tianjin-treated MCF-7 cells revealed that UV-Tianjin induced apoptosis in a dose-dependent manner. Moreover, UV-Tianjin treatment resulted in reductions in the mitochondria membrane potential of MCF-7 cells and regulated the levels and activities of Bcl-2, Bax, cyt c, caspases, Fas, and Fas ligand (FasL). In vivo, UV-Tianjin inhibited the growth of MCF-7 tumors in nude mice and increased tumor cell apoptosis compared with saline-treated controls. In addition, the percentage of tumor cells positive for cleaved versions of caspase-7, caspase-8, and caspase-9 was higher in UV-Tianjin-treated tumors than in saline-treated controls. In summary, UV-Tianjin exhibited the antitumor activity in human breast cancer MCF-7 cells both in vitro and in vivo. The UV-Tianjin treatment seemed to induce apoptosis by activating both the mitochondrial and death receptor apoptotic pathways.

JNK1‑mediated phosphorylation of Smac/DIABLO at the serine 6 residue is functionally linked to its mitochondrial release during TNF‑α-‑induced apoptosis of HeLa cells.

The second mitochondria‑derived activator of caspases (Smac/DIABLO), is a mitochondrial protein that is released along with cytochrome c during apoptosis and promotes caspase activity. It has been reported that c‑Jun N‑terminal kinase (JNK) is involved in the regulation of Smac release during apoptosis; however, the specific role of JNK is not well understood. The aim of the present study was to investigate whether JNK1 is activated during apoptosis induced by tumor necrosis factor‑α (TNF‑α) and whether the activation of JNK1 is functionally associated with Smac release from the mitochondria in HeLa cells. It was determined that during apoptotic progression induced by TNF‑α, JNK1 is activated and translocated into the mitochondria. It was also shown that Smac release is markedly promoted by transient expression of JNK1, while it is suppressed by the expression of a dominant negative version of JNK1. Furthermore, expression of JNK1 also increases the levels of TNF‑α‑induced caspase‑3 activity. To determine whether JNK1 activity is directly involved in the release of Smac, an in vitro phosphorylation assay was performed with the N‑terminal 10‑mer peptide fragment of Smac (pep0110). The results indicated that pep0110 is phosphorylated in dose and time‑dependent manners by active JNK1, and that the phosphorylation of Smac at the N‑terminal serine 6 residue is functionally linked to Smac release during TNF‑α‑induced apoptosis. In conclusion, this suggests that Smac is a major physiological substrate of JNK1 in the regulation of apoptosis.

Perturbation of apoptosis upon binding of tRNA to the heme domain of cytochrome c

In response to apoptotic stimuli, cytochrome c, an inter-membrane space protein is released from mitochondria to activate the cascade of caspases that leads to apoptosis. Recent evidence suggests that cytochrome c interacts with tRNA in the cytoplasm and this interaction was shown to inhibit the caspase mediated apoptotic process. Interestingly, cytochrome c does not contain any putative RNA binding domain. In this report, we sought to define the structural component of cytochrome c that is involved in binding of tRNA. By using gel mobility shift assays, we show that holocytochrome c can interact with tRNA but not apocytochrome c that lacks the heme domain suggesting that heme is essential for the interaction of cytochrome c to tRNA. In addition, using in vitro cross linking and circular dichroism spectroscopic studies, we show that cytochrome c can undergo heme mediated oligomerization. Prevention of heme mediated oligomerization of cytochrome c by potassium ferricyanide treatment prevents the binding of tRNA and promotes caspase activation. Our studies provide a novel regulation of apoptosis by heme dependent tRNA interaction to cytochrome c.

Abstract 1599: Radiosensitization of GBM by a novel peptidomimetic of the Second Mitochondria-derived Activator of Caspases (SMAC).

Abstract TL32711 is a novel small molecule peptidomimetic of the SMAC (Second Mitochondria-derived Activator of Caspases) that promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway. Normally SMAC is located in the mitochondria; however it is released into the cytosol when cells undergo apoptosis. Release into the cytosol and cleavage of SMAC promotes caspase-9 activation by binding to inhibitors of apoptosis proteins, IAPs. Removing the inhibitory activity of IAPs increases cells’ sensitivity to apoptotic stimuli and abrogates NF-κB survival signaling. Thus, by targeting and eliminating IAPs, SMAC mimetics re-activate death signals in cancer cells, inhibit resistance mechanisms and induce tumor death by apoptosis. This study was undertaken to determine radiation sensitization potential of TL32711 in GBM using in vitro and in vivo approaches. TL32711 alone did not cause any killing effects in GBM cells. However, in vitro apoptosis was increased when TL32711 was combined with radiation in GBM cell lines. In vivo data demonstrated that TL32711 alone delayed the growth of implanted xenograft tumors that were further delayed when combined with radiation. The survival of mice was greatly increased in TL32711 plus radiation treated GBM intracranial orthotopic model when compared to TL32711 or radiation treatment alone. Mechanistically, TL32711 confers radiation sensitization by abrogating NFκB activity that is elevated by radiation-induced TNF-α. Taken together, this is the first report that provides strong evidence that TL32711 in combination with ionizing radiation has potential to increase the effectiveness of radiotherapy. Funded by NCI Contract No. HHSN261200800001E. Citation Format: David Cerna, Donna Carter, Naoko Takebe, C. Norman Coleman, Mansoor M. Ahmed, Stephen Yoo. Radiosensitization of GBM by a novel peptidomimetic of the Second Mitochondria-derived Activator of Caspases (SMAC). [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 1599. doi:10.1158/1538-7445.AM2013-1599