Increase In Caspase-3 Activity Research Articles

An 8-aminoquinoline-naphthyl copper complex causes apoptotic cell death by modulating the expression of apoptotic regulatory proteins in breast cancer cells

Breast cancer is one of the most common cancers globally and a leading cause of cancer-related deaths among women. Despite the combination of chemotherapy with targeted therapy, including monoclonal antibodies and kinase inhibitors, drug resistance and treatment failure remain a common occurrence. Copper, complexed to various organic ligands, has gained attention as potential chemotherapeutic agents due to its perceived decreased toxicity to normal cells. The cytotoxic efficacy and the mechanism of cell death of an 8-aminoquinoline-naphthyl copper complex (Cu8AqN) in MCF-7 and MDA-MB-231 breast cancer cell lines was investigated. The complex inhibited the growth of MCF-7 and MDA-MB-231 cells with IC50 values of 2.54 ± 0.69 μM and 3.31 ± 0.06 μM, respectively. Nuclear fragmentation, annexin V binding, and increased caspase-3/7 activity indicated apoptotic cell death. The loss of mitochondrial membrane potential, an increase in caspase-9 activity, the absence of active caspase-8 and a decrease of tumour necrosis factor receptor 1(TNFR1) expression supported activation of the intrinsic apoptotic pathway. Increased ROS formation and increased expression of haem oxygenase-1 (HMOX-1) indicated activation of cellular stress pathways. Expression of p21 protein in the nuclei was increased indicating cell cycle arrest, whilst the expression of inhibitor of apoptosis proteins (IAPs); cIAP1, XIAP and survivin were decreased, creating a pro-apoptotic environment. Phosphorylated p53 species; phospho-p53(S15), phospho-p53(S46), and phospho-p53(S392) accumulated in MCF-7 cells indicating the potential of Cu8AqN to restore p53 function in the cells. In combination, the data indicates that Cu8AqN is a useful lead molecule worthy of further exploration as a potential anti-cancer drug.

Bortezomib suppresses acute myelogenous leukaemia stem-like KG-1a cells via NF-κB inhibition and the induction of oxidative stress.

Acute myelogenous leukaemia (AML) originates and is maintained by leukaemic stem cells (LSCs) that are inherently resistant to antiproliferative therapies, indicating that a critical strategy for overcoming chemoresistance in AML therapy is to eradicate LSCs. In this work, we investigated the anti-AML activity of bortezomib (BTZ), emphasizing its anti-LSC potential, using KG-1a cells, an AML cell line with stem-like properties. BTZ presented potent cytotoxicity to both solid and haematological malignancy cells and reduced the stem-like features of KG-1a cells, as observed by the reduction in CD34- and CD123-positive cells. A reduction in NF-κB p65 nuclear staining was observed in BTZ-treated KG-1a cells, in addition to upregulation of the NF-κB inhibitor gene NFΚBIB. BTZ-induced DNA fragmentation, nuclear condensation, cell shrinkage and loss of transmembrane mitochondrial potential along with an increase in active caspase-3 and cleaved PARP-(Asp 214) level in KG-1a cells. Furthermore, BTZ-induced cell death was partially prevented by pretreatment with the pancaspase inhibitor Z-VAD-(OMe)-FMK, indicating that BTZ induces caspase-mediated apoptosis. BTZ also increased mitochondrial superoxide levels in KG-1a cells, and BTZ-induced apoptosis was partially prevented by pretreatment with the antioxidant N-acetylcysteine, indicating that BTZ induces oxidative stress-mediated apoptosis in KG-1a cells. At a dosage of 0.1 mg/kg every other day for 2 weeks, BTZ significantly reduced the percentage of hCD45-positive cells in the bone marrow and peripheral blood of NSG mice engrafted with KG-1a cells with tolerable toxicity. Taken together, these data indicate that the anti-LSC potential of BTZ appears to be an important strategy for AML treatment.

Effects of thymoquinone on spinal cord injury in rats.

Spinal cord injury (SCI) is a condition that causes disturbances in normal sensory, motor, and autonomic functions. During SCI, damages occur such as, contusion, compression, distraction. The aim of this study was to investigate effects of the antioxidative thymoquinone on neuron and glia cells in SCI biochemically, immunohistochemically and ultrastructurally. Male Sprague-Dawley rats were divided into Control, SCI and SCI + Thymoquinone groups. After T10- T11 laminectomy was performed, a metal weight of 15 grams was left down the spinal tube for spinal damage. Immediately after the trauma, the muscles and skin incision were sutured. Thymoquinone was given to the rats by gavage as 30mg/kg/21days. Tissues fixed in 10% formaldehyde, embedded in paraffin wax and immunstained with Caspase-9 and phosphorylated signal transducer and activator of transcription 3 (pSTAT-3) antibodies. Remaining were stored at -80oC for biochemistry. Frozen spinal cord tissues were placed in a phosphate buffer solution and homogenized, centrifuged then used to measure malondialdehyde (MDA) levels, glutathione peroxidase (GSH) and Myeloperoxidase (MPO). In the SCI group, MDA, MPO, neuronal degeneration, vascular dilatation, inflammation, apoptotic appearance in the nucleus, loss of membrane and cristae in mitochondria, and dilatation in the endoplasmic reticulum were observed due to degeneration in the neuron structure. In the electron microscopic examination of the trauma + thymoquinone group, the membranes of the nuclei of the glial cells were thick and euchromatin, and mitochondria were shortened in length. In the SCI group, pyknosis and apoptotic changes were observed in neuronal structures and nuclei of glia cells in the substantia grisea and substantia alba region, along with positive Caspase-9 activity. An increase in Caspase-9 activity was observed in endothelial cells in blood vessels. In the SCI + thymoquinone group, Caspase-9 expression was positive in some of the cells in the ependymal canal while the cuboidal cells showed a negative Caspase-9 reaction in the majority. A few degenerated neurons in the substantia grisea region showed a positive reaction with Caspase-9. In SCI group, pSTAT-3 expression was positive in degenerated ependymal cells, neuronal structures, and glia cells. pSTAT-3 expression was positive in the endothelium and surrounding aggregated cells of the enlarged blood vessels. In the SCI+ thymoquinone group, pSTAT-3 expression was negative in most of the bipolar and multipolar neuron structures and glial cells in ependymal cells, enlarged blood vessel endothelial cells. It has been thought that thymoquinone application in spinal cord injuries may be an antioxidant that can be recommended as an alternative treatment in suppressing the apoptosis of neural cells by significantly reducing the inflammation process.

Effects of polyphenolic-rich extracts from Citrus hystrix on proliferation and oxidative stress in breast and colorectal cancer

Abstract Objectives The anti-proliferative effects of Citrus hystrix have been reported. However, information on breast and colorectal cancer is limited especially the mechanistic aspects. In this study, the antioxidant activities of hexane, ethyl acetate, methanol and water extracts of C. hystrix leaves and their growth inhibitory effects on colorectal (HCT 116) and breast cancer (MCF 7, MDA-MB 231 and HCC 1937) cells were analysed. Methods Antioxidant and oxidative stress status were measured using non-cellular and cellular assays. Caspase and gene expression were utilized to determine anti-proliferative effects. Polyphenolic content was analysed using LC-IT-TOF/MS. Results The water extract showed the highest polyphenolic content and antioxidant activities (FRAP, DPPH, ABTS, superoxide anion radical scavenging, ferrous ion chelation, cellular antioxidant assay). The ethyl acetate extracts of C. hystrix (CH-EA) demonstrated the highest anti-proliferative activity against all cancer cell lines (IC50<100 μg/mL). Increase in ROS was observed in CH-EA-treated HCT 116, MDA-MB 231 and HCC 1937 cells (p<0.05). Increase in caspase activities and upregulation of Bax, Bcl-2, Cdk-1, TP53 and TNF-α expression in HCT 116 cells indicated activation of apoptosis by CH-EA. LC-IT-TOF/MS analysis indicated presence of quercetin and rutin in CH-EA. Conclusions CH-EA showed anti-proliferative effects, possibly through modulation of oxidative stress and apoptosis.

Characterization of a Novel Oncolytic Virus for Cancer Therapy

BackgroundOncolytic viruses (OVs) that selectively replicate (oncoselectivity) and lyse (oncotoxicity) cancer cells are emerging as promising cancer therapeutics. A range of DNA and RNA OVs are in different stages of human clinical trials. The presence of preexisting immunity against OVs and their potential pathogenic effects are significant limitations. Using animal viruses that are non‐pathogenic to humans is considered an attractive strategy. We identified a naturally occurring animal virus with potent oncolytic ability, hereafter referred to as oncolytic virus 1 (OV1). We tested the oncoselectivity and oncotoxicity of OV1 using a range of human cancer cells, immortalized cells, and normal non‐cancerous cells. We further evaluated the oncolytic properties of OV1 in vivo using the murine triple‐negative breast cancer (TNBC) model, 4T1.2.MethodsHuman renal (769P), mammary (HCC 1500), colorectal (HRT‐18G), lung (A549), and pancreatic (BxPC‐3) cancer cells, and leukemia (THP‐1) cells, human immortalized embryonic kidney cells (293T), and human primary bronchial epithelial cells (PBEC) were infected with OV1 at a range of multiplicity of infection (MOI) from 0.01 to 5. After 72h of infection, MTS assay to determine cell viability and Caspase‐Glo 3/7 assay to quantify apoptosis were performed. OV1 replication in cultured 4T1.2 cells was assessed by infecting the cells at an MOI of 0.1 and quantifying viral nucleic acid in the supernatants at 72h. Female BALB/c mice were injected with 4T1.2 cells into the fourth mammary gland on the left side. At 19 days post‐tumor implantation, 105TCID50 units of OV1 in 50µl was injected half intratumorally and half intraperitoneally. Mice in the mock treatment group received 50µl of vehicle control. Tumor sizes were measured regularly and at day 35, mice were sacrificed and the number of live cells in the tumor were quantified by flow cytometry.ResultsThere was a significant decrease in the viability of OV1 infected 769P cells (MOI ≥ 0.1), A549 (MOI ≥ 1), THP‐1 (MOI ≥ 2.5), and HCC 1500 (MOI ≥ 2.5) at 72h compared with controls. Notably, there was no significant change in the viability of primary cells (PBEC) and immortalized 293T infected with OV1 at MOI of 0.1 to 2.5 compared to controls. There was a significant increase in caspase activity in 769P cells (at MOI ≥ 0.1), A549 (at MOI ≥ 1), THP‐1 (at MOI ≥ 1), BxPC3 (at MOI ≥ 2.5), HRT‐18G (at MOI ≥ 2.5) and HCC 1500 (at MOI ≥ 0.1). There was no increase in the caspase activity of PBEC at all the tested MOI. OV1 viral replication was observed in 4T1.2 cells in vitro, as evidenced by increasing viral nucleic acid in culture supernatants. BALB/c mice injected with OV1 showed a significant decrease in tumor size and a decrease in the live cell numbers in the tumor compared with mock treated mice.Conclusions and future directionsThe results established the oncoselectivity and oncotoxicity of OV1 and its oncolytic ability in vivo. Further studies are underway to investigate OV1 replication in cells, immune stimulatory properties of OV1 in vivo, and the generation of a recombinant OV1 with increased oncolytic ability.

Toxicological effects of tris(1,3-dichloro-2-propyl) phosphate in oyster Crassostrea gigas using proteomic and phosphoproteomic analyses

As a typical organophosphorus pollutant, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been widely detected in aquatic environment. Previous studies showed that protein phosphorylation might be a vital way of TDCIPP to exert multiple toxic effects. However, there is a lack of high-throughput investigations on how TDCIPP affected protein phosphorylation. In this study, the toxicological effects of TDCIPP were explored by proteomic and phosphoproteomic analyses together with traditional means in oysters Crassostrea gigas treated with 0.5, 5 and 50 μg/L TDCIPP for 28 days. Integration of omic analyses revealed that TDCIPP dysregulated transcription, energy metabolism, and apoptosis and cell proliferation by either directly phosphorylating pivotal proteins or phosphorylating their upstream signaling pathways. The U-shaped response of acetylcholinesterase activities suggested the neurotoxicity of TDCIPP in a hormesis manner. What’s more, the increase in caspase-9 activity as well as the expression or phosphorylation alterations in eukaryotic translation initiation factor 4E, cell division control protein 42 and transforming growth factor-β1-induced protein indicated the disruption of homeostasis between apoptosis and cell proliferation, which was consistent with the observation of shedding of digestive cells. Overall, combination of proteomic and phosphoproteomic analyses showed the capability of identifying molecular events, which provided new insights into the toxicological mechanisms of TDCIPP.

In vitro antioxidant and apoptotic activity of Lablab purpureus (L.) Sweet isolate and hydrolysates

Cancer is a disease that invades the lives of millions of people each year. Chemotherapy is currently the most effective treatment however resulting in many adverse effects to the human body. Alternative treatments are being explored to overcome this obstacle. Peptides that possess radical scavenging activity and bioactive properties can be advantageous in prevention and treatment of chronic diseases. In this study, Lablab purpureus isolate and its hydrolysates (trypsin, pepsin and alcalase) were analysed for radical scavenging potential (DPPH, ABTS, superoxide radical scavenging and FRAP) and antiproliferative activity. Antiproliferative activity was confirmed with the peptides ability to induce apoptosis (Caspase 3/7 activity and Annexin V-PI). The lowest inhibitory concentrations (IC50) for DPPH, ABTS and Superoxide radical scavenging ranged between 1.81-4.47, 1.73-2.42 and 1.36-4.41 mg/mL, respectively. FRAP ranged from 19.20 to 21.94 mg/mL. Generally, it is considered that a good antioxidant encompasses antiproliferative potential. Cell lines, A549, MCF-7 and HEK293, treated with pepsin hydrolysate showed (IC50 values of 119.6, 9.80 and 13.86 µg/mL). The isolate and pepsin were chosen for apoptotic studies. The pepsin hydrolysate showed the highest inhibition in the cancerous cell lines (A549 and MCF-7) without greatly effecting normal cells (HEK293), and the isolate was selected for comparative analysis. Annexin V-PI staining showed cells in different stages of apoptosis (cells during early apoptosis; A549, 42%; MCF-7, 17%; HEK, 34%). Caspase 3/7 assay demonstrated that the peptide causes an increase in caspase activity. Peptides have the potential to act as chemo-preventative agents due to their antioxidant and apoptotic abilities.

In vitro evaluation of the antiproliferative activity of Carpobrotus edulis on human neuroblastoma cells

Neuroblastoma is a solid neuroendocrine tumour located outside the cranial cavity and contributes about 15% of all cancer‑associated deaths in children. Treatment of neuroblastoma is quite challenging and involves the use of chemotherapy, surgery and radiotherapy. Despite treatment strategies, systemic toxicity are setbacks to patient well-being, hence the need for a new and affordable approach. Medicinal plants are of importance in the field of drug discovery for cancer as some notable anti-cancer agents have been isolated from them. In the present study, the anti-cancer activity of aqueous extract of Carpobrotus edulis (C. edulis), a ground-creeping edible medicinal plant was investigated in SK-N-BE(2) and SH-SY5Y neuroblastoma cells. The effect of C. edulis on cell viability and survival was determined using MTT (3-[4,5-dimethylthiazol-2-yl]−2,5 diphenyltetrazolium bromide) and clonogenic assays respectively. Apoptosis was determined using a Caspase-9 assay kit and flow cytometry was used to measure intracellular reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential. The results show that C. edulis inhibits cell viability (IC50 of 0.86 mg/ml and 1.45 mg/ml for SK-N-BE(2) and SHSY5Y cells respectively) and colony formation in the neuroblastoma cells as well as induce apoptosis, which is evidenced by an increase in caspase-9 activity in the cells. C. edulis also led to a loss of mitochondrial membrane potential and increased production of ROS. Collectively, these results suggest that C. edulis induces cell death via induction of mitochondrial‐mediated apoptosis and accumulation of intracellular ROS, thus providing a rationale for further investigations.

XIAP Knockdown in Alcohol-Associated Liver Disease Models Exhibits Divergent in vitro and in vivo Phenotypes Owing to a Potential Zonal Inhibitory Role of SMAC.

Alcohol-associated liver disease (ALD) has been recognized as the most common cause of advanced liver disease worldwide, though mechanisms of pathogenesis remain incompletely understood. The X-linked inhibitor of apoptosis (XIAP) protein was originally described as an anti-apoptotic protein that directly binds and inhibits caspases-3, 7, and 9. Here, we investigated the function of XIAP in hepatocytes in vitro using gain and loss-of-function approaches. We noted an XIAP-dependent increase in caspase activation as well as increased inflammatory markers and pro-inflammatory EV release from hepatocytes in vitro. Primary hepatocytes (PMH) from XiapAlb.Cre and XiaploxP mice exhibited higher cell death but surprisingly, lower expression of inflammation markers. Conditioned media from these isolated Xiap deleted PMH further decrease inflammation in bone marrow-derived macrophages. Also, interestingly, when administered an ethanol plus Fas-agonist-Jo2 model and an ethanol plus CCl4 model, these animals failed to develop an exacerbated disease phenotype in vivo. Of note, neither XiapAlb.Cre nor XiapAAV8.Cre mice presented with aggravated liver injury, hepatocyte apoptosis, liver steatosis, or fibrosis. Since therapeutics targeting XIAP are currently in clinical trials and caspase-induced death is very important for development of ALD, we sought to explore the potential basis of this unexpected lack of effect. We utilized scRNA-seq and spatially reconstructed hepatocyte transcriptome data from human liver tissue and observed that XIAP was significantly zonated, along with its endogenous inhibitor second mitochondria-derived activator of caspases (SMAC) in periportal region. This contrasted with pericentral zonation of other IAPs including cIAP1 and Apollon as well as caspases 3, 7, and 9. Thus providing a potential explanation for compensation of the effect of Xiap deletion by other IAPs. In conclusion, our findings implicate a potential zonallydependent role for SMAC that prevented development of a phenotype in XIAP knockout mice in ALD models. Targeting SMAC may also be important in addition to current efforts of targeting XIAP in treatment of ALD.

Long non-coding RNA TERRA influences DNA damage and survival of endothelial cells and cardiomyocytes

Abstract Ageing is the major risk factor for cardiovascular disease. Current therapies are mainly based on proteins, while targeting long non-coding RNAs (lncRNAs) is largely unexplored. Long non-coding RNAs are emerging as novel regulators of cellular functions and contributors to cardiovascular ageing. Although telomeres are heterochromatic regions, non-coding transcripts called Telomeric repeat-containing RNA (TERRA) are transcribed from the telomeres of most chromosomes. The transcription of TERRA starts at the subtelomere and ends in the telomere, leading to molecules of 0.2–10kb. This study aims to characterize the role of TERRA in the cardiovascular system. TERRA molecules from different chromosomes were upregulated in the hearts of old mice compared to young mice (p=0.002). An increased TERRA expression was also shown in heart tissue of patients with ischemic heart disease compared to donor heart tissue (p=0.001). In vitro an upregulation of the TERRA molecule transcribed from chromosome 20 (h20q-TERRA) was found in old passage human umbilical vein endothelial cells (HUVECs) (P15–17) compared to young HUVECs (P3) (p=0.014). IPSC-derived cardiomyocytes also increased the expression of h20q-TERRA with increasing passage (p=0.011). After knockdown of h20q-TERRA with LNA GapmeRs HUVECs show less sprout formation in a spheroid assay compared to negative control transfected HUVECs (p=0.002), without showing a change in migration (p=0.205) or proliferation (p=0.114). H20q-TERRA knockdown revealed that there was more apoptosis (p=0.015), more DNA damage as measured with the comet assay (p<0.001), increased γH2AX levels (p=0.029) and an increase in γH2AX colocalization with the telomere (p=0.011). The amount of phosphorylated P53 was also increased after knockdown (p=0.038), while it was decreased after TERRA overexpression (p=0.001). Inhibition of the ATM-yH2AX-P53 pathway did however not reduce the increased apoptosis after knockdown (KU60019 p=0.399; siP53 p=0.303). Silencing the m18-TERRA molecule in mouse endothelial H5V cells led to an increase in caspase activity (p=0.004) similar to what was shown in HUVECs. In addition, increased caspase activity (p=0.012), increased γH2AX levels (p<0.001) and increased γH2AX colocalization with the telomere (p=0.007) was also shown after silencing of h20q-TERRA in human cardiomyocytes. In summary, our data demonstrates that TERRA is upregulated with ageing and plays a role in endothelial and cardiomyocyte function and survival. These data show that TERRA transcripts are induced in cardiovascular ageing and are essential for endothelial cell function. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): Horizon 2020

Resveratrol for protection against statin toxicity in C2C12 and H9c2 cells.

Statins are among the most commonly prescribed drugs for the treatment of high blood cholesterol. Myotoxicity of statins in certain individuals is often a severe side effect leading to withdrawal. Using C2C12 and H9c2 cells, both exhibiting characteristics of skeletal muscle cells, we addressed whether resveratrol (RSV) can prevent statin toxicity. Statins decreased cell viability in a dose and time-dependent manner. Among the five statins tested, atorvastatin, simvastatin, lovastatin, pravastatin, and fluvastatin, simvastatin is the most toxic one. Simvastatin at 10 µM caused about 65% loss of metabolic activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in C2C12 cells or H9c2 cells. Inhibition of metabolic activity correlates with an increase in caspase activity. RSV was found to protect H9c2 cells from simvastatin-induced activation of caspase-3/7. However, such protection was not found in C2C12 cells. This cell type-dependent effect of RSV adds to the complexity in muscle cell toxicity of statins.

Inhibition mechanism of naphthylphenylamine derivatives acting on the CDC25B dual phosphatase and analysis of the molecular processes involved in the high cytotoxicity exerted by one selected derivative in melanoma cells

The dual phosphatases CDC25 are involved in cell cycle regulation and overexpressed in many tumours, including melanoma. CDC25 is a promising target for discovering anticancer drugs, and several studies focussed on characterisation of quinonoid CDC25 inhibitors, frequently causing undesired side toxic effects. Previous work described an optimisation of the inhibition properties by naphthylphenylamine (NPA) derivatives of NSC28620, a nonquinonoid CDC25 inhibitor. Now, the CDC25B•inhibitor interaction was investigated through fluorescence studies, shedding light on the different inhibition mechanism exerted by NPA derivatives. Among the molecular processes, mediating the specific and high cytotoxicity of one NPA derivative in melanoma cells, we observed decrease of phosphoAkt, increase of p53, reduction of CDC25 forms, cytochrome c cytosolic translocation and increase of caspase activity, that lead to the activation of an apoptotic programme. A basic knowledge on CDC25 inhibitors is relevant for discovering potent bioactive molecules, to be used as anticancer agents against the highly aggressive melanoma.

Epoxyeicosatrienoic acids protect pancreatic beta cells against pro-inflammatory cytokine toxicity

Pro-inflammatory cytokines contribute to pancreatic beta cell death in the pathogenesis of type 1 diabetes mellitus (DM). Cytochrome P450-derived epoxyeicosatrienoic acids (EETs), produced by selective epoxidation of arachidonic acid, display anti-inflammatory activity in numerous disease models, in part through inhibition of NFκB activity. No studies have directly assessed their roles in cellular models of pancreatic beta cell death and therefore we aimed to investigate the cytoprotective effects of the EET isomers 8(9)-, 11(12)- and 14(15)-EET and their corresponding vicinal diols (dihydroxyeicosatrienoic acids, DHETs) in a model of pro-inflammatory cytokine-toxicity using the rat pancreatic beta cell line BRIN-BD11. Co-treatment of cells with a cocktail of pro-inflammatory cytokines (IL-1β, IFNγ and TNFα) caused a marked increase in caspase activation and a reduction in cell viability, effects attenuated by inclusion of each EET; this was also associated with a reduction in cytokine-induced NFκB activation and nitrite accumulation. Surprisingly, of the DHET derivatives of EETs, 8(9)-DHET conferred similar protective effects against cytokine-induced caspase activation. This data therefore highlights a novel role of EETs and a surprising activity of 8(9)-DHET in attenuating cytokine-toxicity in pancreatic beta cells.

CED-4 CARD domain residues can modulate non-apoptotic neuronal regeneration functions independently from apoptosis

A major challenge in regenerative medicine is the repair of injured neurons. Regeneration of laser-cut C. elegans neurons requires early action of core apoptosis activator CED-4/Apaf1 and CED-3/caspase. While testing models for CED-4 as a candidate calcium-sensitive activator of repair, we unexpectedly discovered that amino acid substitutions affecting alpha-helix-6 within the CED-4 caspase recruitment domain (CARD) confer a CED-4 gain-of-function (gf) activity that increases axonal regrowth without disrupting CED-4 apoptosis activity. The in vivo caspase reporter CA-GFP reveals a rapid localized increase in caspase activity upon axotomy, which is absent in ced-4 and ced-3 loss-of-function mutants but present in the ced-4(gf) mutant. The ced-3 loss-of-function mutation can significantly suppress the axonal regrowth of the ced-4(gf) mutant, indicating that CED-4(gf) regeneration depends on CED-3 caspase. Thus, we identified a subdomain within the CED-4 CARD that regulates the dynamic and controlled caspase activity required for efficient regeneration.

Abstract 707: Emerging role of mitochondrial pathway in crosstalk between ferroptosis and apoptosis

Abstract Introduction: Several processes lead to cell death; ferroptosis, apoptosis, necrosis, and autophagy are the primary mechanisms. Each type of biological death has been believed to have a distinct biochemical and morphological fingerprint. However, emerging evidence suggests that these different types of cell death often share common pathways. In this study, we investigated whether mitochondrial signaling pathways play an important role in the intracellular communication between ferroptosis and apoptosis. Methods: The human colorectal carcinoma HCT116 cell line was used to evaluate the interplay between cell death pathways. Cells were treated with the apoptotic agent TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and ferroptotic agents such as artesunate and erastin. Cell death was determined by trypan blue exclusion assay. Apoptosis was measured by western blot. Ferroptosis was assessed using MDA assay. Bax oligomerization was performed by mitochondrial fractionation followed by SDS-PAGE under nondenaturing conditions. Results: We observed that the combinatorial treatment induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed an increase in pro-apoptotic Bax oligomerization in the mitochondrial fraction. The combined treatment-induced apoptosis was suppressed in Bax-deficient HCT116 cells. Ferroptotic agents enhanced the intracellular level of anti-apoptotic Mcl-1. However, the combined treatment decreased the level of Mcl-1. Interestingly, the combined treatment-induced apoptosis was abolished in HCT116 cells with knockin of Mcl-1 phosphorylation site mutant (S121A/E125A/S159A/T163A). Conclusions: These data suggest a novel mechanism for the combined treatment of the apoptotic agent TRAIL and ferroptotic agent erastin/artesunate-induced synergistic apoptosis through the mitochondrial pathway. Citation Format: Yong J. Lee. Emerging role of mitochondrial pathway in crosstalk between ferroptosis and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 707.

Abstract 2630: Inhibition of bone morphogenetic protein receptor 2 increases mitochondrial permeability and the downregulation of XIAP in lung cancer cells

Abstract Bone morphogenetic protein receptor (BMP) inhibitors have been shown to induce death of lung cancer cells, which involves the downregulation of x-linked inhibitor of apoptosis protein (XIAP). XIAP is a potent inhibitor of executioner caspases that promotes resistance to many chemotherapeutic agents. The mechanism by which inhibiting BMP signaling downregulates XIAP has not been elucidated. We find that the BMP inhibitors JL5 and DMH2 downregulate XIAP and enhances cell death of TRAIL, Taxol, and AEG a Smac mimetic. JL5, which inhibits both type I and type II BMP receptors, enhances cell death of cancer therapeutics by decreasing the expression of XIAP, which leads to an increase in caspase activity. The downregulation of XIAP is mediated by the inhibition of the BMP type II receptor and not the BMP type I receptors. Knockdown of BMPR2 but not the BMP type I receptors increases mitochondrial permeability resulting in the release of cytochrome c and Smac/Diablo into the cytosol. These studies suggest that the inhibition of BMPR2 increases cytosolic Smac/Diablo that inhibits XIAP enhancing apoptotic cell death of cancer therapeutics. Citation Format: Rachel E. NeMoyer, Elaine Langenfeld, John Langenfeld. Inhibition of bone morphogenetic protein receptor 2 increases mitochondrial permeability and the downregulation of XIAP in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2630.

Lycopene Inhibits Reactive Oxygen Species-Mediated NF-κB Signaling and Induces Apoptosis in Pancreatic Cancer Cells.

Generation of excess quantities of reactive oxygen species (ROS) caused by mitochondrial dysfunction facilitates rapid growth of pancreatic cancer cells. Elevated ROS levels in cancer cells cause an anti-apoptotic effect by activating survival signaling pathways, such as NF-κB and its target gene expression. Lycopene, a carotenoid found in tomatoes and a potent antioxidant, displays a protective effect against pancreatic cancer. The present study was designed to determine if lycopene induces apoptosis of pancreatic cancer PANC-1 cells by decreasing intracellular and mitochondrial ROS levels, and consequently suppressing NF-κB activation and expression of NF-κB target genes including cIAP1, cIAP2, and survivin. The results show that the lycopene decreased intracellular and mitochondrial ROS levels, mitochondrial function (determined by the mitochondrial membrane potential and oxygen consumption rate), NF-κB activity, and expression of NF-κB-dependent survival genes in PANC-1 cells. Lycopene reduced cell viability with increases in active caspase-3 and the Bax to Bcl-2 ratio in PANC-1 cells. These findings suggest that supplementation of lycopene could potentially reduce the incidence of pancreatic cancer.

E-Cigarette Vapor Decreases Cellular Proliferation through Nicotine-Dependent Mechanisms

Electronic cigarette (e-cigarette) use has risen as individuals look for healthier nicotine delivery systems than conventional cigarettes, but recent studies have demonstrated variable physiological responses to e-cigarette vapor. The safety of e-cigarette vapor remains an open question. We investigated the molecular cellular response of adenocarcinoma human alveolar basal epithelial (A549) cells to flavored e-cigarette vapor. E-cigarette and cigarette vapor were extracted via a vacuum-percolating system into PBS. The effects of a flavored e-liquid containing nicotine (eCSE) and nicotine free e-liquid (NFeCSE) were compared to cigarette smoke extract (CSE), pure nicotine, glycerol, and PBS. Cells exposed to eCSE and NFeCSE had decreased cell viability and proliferation. An increase in active caspase-3 was observed in cells exposed to NFeCSE, while cells exposed to eCSE and CSE displayed no change, suggesting nicotine-mediated protection from apoptosis. However, phosphorylated Akt, which regulates cellular proliferation, was elevated in response to eCSE, NFeCSE, CSE, and glycerol. As the glutathione-oxidative stress response was known to integrate cellular factors including carcinogenesis, inflammation, and cell proliferation, GSH/GSSG levels were measured in cells exposed to e-cigarette smoke. An increase in oxidative stress was observed in cells treated with CSE, but not eCSE, suggesting components in CSE independent of nicotine may be contributing to an imbalance in the stress response. These results indicate e-cigarette vapor affects A549 cell viability dependent on nicotine, and is distinct from CSE-induced mechanisms of oxidative-stress.

New Mutation of Coenzyme Q10 Monooxygenase 6 Causing Podocyte Injury in a Focal Segmental Glomerulosclerosis Patient.

Background:Focal segmental glomerulosclerosis (FSGS) is a kidney disease that is commonly associated with proteinuria and the progressive loss of renal function, which is characterized by podocyte injury and the depletion and collapse of glomerular capillary segments. The pathogenesis of FSGS has not been completely elucidated; however, recent advances in molecular genetics have provided increasing evidence that podocyte structural and functional disruption is central to FSGS pathogenesis. Here, we identified a patient with FSGS and aimed to characterize the pathogenic gene and verify its mechanism.Methods:Using next-generation sequencing and Sanger sequencing, we screened the causative gene that was linked to FSGS in this study. The patient's total blood RNA was extracted to validate the messenger RNA (mRNA) expression of coenzyme Q10 monooxygenase 6 (COQ6) and validated it by immunohistochemistry. COQ6 knockdown in podocytes was performed in vitro with small interfering RNA, and then, F-actin was determined using immunofluorescence staining. Cell apoptosis was evaluated by flow cytometry, the expression of active caspase-3 was determined by Western blot, and mitochondrial function was detected by MitoSOX.Results:Using whole-exome sequencing and Sanger sequencing, we screened a new causative gene, COQ6, NM_182480: exon1: c.G41A: p.W14X. The mRNA expression of COQ6 in the proband showed decreased. Moreover, the expression of COQ6, which was validated by immunohistochemistry, also had the same change in the proband. Finally, we focused on the COQ6 gene to clarify the mechanism of podocyte injury. Flow cytometry showed significantly increased in apoptotic podocytes, and Western blotting showed increases in active caspase-3 in si-COQ6 podocytes. Meanwhile, reactive oxygen species (ROS) levels were increased and F-actin immunofluorescence was irregularly distributed in the si-COQ6 group.Conclusions:This study reported a possible mechanism for FSGS and suggested that a new mutation in COQ6, which could cause respiratory chain defect, increase the generation of ROS, destroy the podocyte cytoskeleton, and induce apoptosis. It provides basic theoretical basis for the screening of FSGS in the future.

Inhibition of BET Bromodomain Proteins with GS-5829 and GS-626510 in Uterine Serous Carcinoma, a Biologically Aggressive Variant of Endometrial Cancer.

Purpose: Uterine serous carcinoma (USC) is a rare and aggressive variant of endometrial cancer. Whole-exome sequencing (WES) studies have recently reported c-Myc gene amplification in a large number of USCs, suggesting c-Myc as a potential therapeutic target. We investigated the activity of novel BET bromodomain inhibitors (GS-5829 and GS-626510, Gilead Sciences Inc.) and JQ1 against primary USC cultures and USC xenografts.Experimental Design: We evaluated c-Myc expression by qRT-PCR in a total of 45 USCs including fresh-frozen tumor tissues and primary USC cell lines. We also performed IHC and Western blot experiments in 8 USC tumors. USC cultures were evaluated for sensitivity to GS-5829, GS-626510, and JQ1 in vitro using proliferation, viability, and apoptosis assays. Finally, the in vivo activity of GS-5829, GS-626510, and JQ1 was studied in USC-ARK1 and USC-ARK2 mouse xenografts.Results: Fresh-frozen USC and primary USC cell lines overexpressed c-Myc when compared with normal tissues (P = 0.0009 and 0.0083, respectively). High c-Myc expression was found in 7 of 8 of primary USC cell lines tested by qRT-PCR and 5 of 8 tested by IHC. In vitro experiments demonstrated high sensitivity of USC cell lines to the exposure to GS-5829, GS-626510, and JQ1 with BET inhibitors causing a dose-dependent decrease in the phosphorylated levels of c-Myc and a dose-dependent increase in caspase activation (apoptosis). In comparative in vivo experiments, GS-5829 and/or GS-626510 were found more effective than JQ1 at the concentrations/doses used in decreasing tumor growth in both USC-ARK1 and USC-ARK2 mouse xenograft models.Conclusions: GS-5829 and GS-626510 may represent novel, highly effective therapeutics agents against recurrent/chemotherapy-resistant USC-overexpressing c-Myc. Clinical studies with GS-5829 in patients with USC harboring chemotherapy-resistant disease are warranted. Clin Cancer Res; 24(19); 4845-53. ©2018 AACR.