Cell Death ELISA Research Articles

Targeting Epidermal Growth Factor Receptor by MiRNA-145 Inhibits Cell Growth and Sensitizes NSCLC Cells to Erlotinib.

Background:Despite effective activity of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as erlotinib, all non-small cell lung cancer (NSCLC) patients eventually acquire resistance to these agents. Studies have demonstrated that down-regulation of miRNA-145 leads to enhancement of EGFR expression, cell proliferation and metastasis. The aim of this study was to investigate the effect of miRNA-145 on sensitivity of the A549 NSCLC cells to erlotinib. Methods:Quantitative real-time PCR was used to examine the effect of miRNA-145 on EGFR expression. The effect of miRNA-145 on cell growth and sensitivity the lung cancer cells to erlotinib was examined by trypan blue and MTT assays, respectively. The combination index was calculated using the non-constant method of Chou-Talalay. Apoptosis was determined by ELISA cell death assay. Results:We found that miRNA-145 was markedly suppressed the expression of EGFR and inhibited the cancer cell growth, relative to blank control and negative control miRNA (p<0.05). Pretreatment with miRNA-145 synergistically enhanced the sensitivity of the lung cancer cells to erlotinib. Results of apoptosis assay revealed that miRNA-145 can induce apoptosis and increase the erlotinib-mediated apoptosis. Conclusions:Our data demonstrate that miRNA-145 play a critical role in the lung cancer cell growth, survival and EGFR-TKIs resistance possibly by regulation of EGFR. Therefore, miRNA-145 replacement therapy can become a new therapeutic strategy in lung cancer.

Annona cherimola Seed Extract Activates Extrinsic and Intrinsic Apoptotic Pathways in Leukemic Cells.

Annona cherimola Mill is a large green fruit with black seeds widely known to possess toxic properties due to the presence of Annonaceous acetogenins. The present study investigates the anti-cancer properties of an Annona cherimola Mill ethanolic seed extract on Acute Myeloid Leukemia (AML) cell lines in vitro and elucidates the underlying cellular mechanism. The anti-proliferative effects of the extract on various AML cell lines and normal mesenchymal cells (MSCs) were assessed using WST-1 viability reagent. The pro-apoptotic effect of the extract was evaluated using Annexin V/PI staining and Cell Death ELISA. The underlying mechanism was deciphered by analyzing the expression of various proteins using western blots. Treatment with an A. cherimola seed ethanolic extract promotes a dose- and time-dependent inhibition of the proliferation of various AML cell lines, but not MSCs. Positive Annexin V staining, as well as DNA fragmentation, confirm an increase in apoptotic cell death by upregulating the expression of pro-apoptotic proteins which control both intrinsic and extrinsic pathways of apoptosis. GC/MS analysis revealed the presence of phytosterols, in addition to other bioactive compounds. In conclusion, Annona cherimola Mill seed extract, previously known to possess a potent toxic activity, induces apoptosis in AML cell lines by the activation of both the extrinsic and the intrinsic pathways.

Exosomes derived from cardiac parasympathetic ganglionic neurons inhibit apoptosis in hyperglycemic cardiomyoblasts.

Diabetic cardiomyopathy is known to involve two forms of cardiac cell death: apoptosis and necrosis. However, it remains unknown whether hyperglycemia-induced apoptosis in the H9c2 cell culture system is inhibited by parasympathetic ganglionic neurons (PGN) derived exosomes (exos). We isolated PGN and sympathetic ganglionic neurons (SGN) from the right stellate ganglion in rats, and derived exos from these sources. H9c2 cells were divided into 4 groups: (1) Control, (2) H9c2 + Glucose (100mmol/L), (3) H9c2 + Glucose + PGN-exos, and (4) H9c2 + Glucose + SGN-exos. We determined cell proliferation and viability with an MTT assay kit, and assessed apoptotic cell death with TUNEL staining and ELISA. Data were further confirmed by analyzing the presence of pro-apoptotic proteins Caspase-3 and Bax, and anti-apoptotic protein Bcl-2. Glucose exposed H9c2 cells significantly reduced cell viability, which was improved by PGN-exos, but not by SGN-exos. Furthermore, increased apoptosis in hyperglycemia in H9c2 cells was confirmed with TUNEL staining and cell death ELISA which demonstrated significantly (p < 0.05) reduction with PGN-exos treatment, but not with SGN-exos. Moreover, high expression of pro-apoptotic proteins Caspase-3 and Bax was reduced following treatment with PGN-exos; however, SGN-exos were unable to reduce the expression. Significantly reduced anti-apoptotic protein Bcl-2 following glucose treatment was improved with PGN-exos. Therefore, our data suggest that hyperglycemia induces apoptosis in H9c2 cells and decreases cell viability, and that PGN-exos are able to inhibit apoptosis, improve cell viability, and restore levels of anti-apoptotic protein Bcl-2.

Inhibitory Effects of Novel Oridonin Analog CYD0617‐2 on Hepatic Stellate Cell Activation through NF‐κB and Stat3 Pathways

IntroductionInhibition of activated hepatic stellate cell (HSC) proliferation is recognized as a promising therapeutic strategy for liver fibrosis. Previously, we reported that natural compound oridonin exhibits potent anti‐hepatic fibrosis activity, and that NF‐κB and Stat3 signaling contribute to HSC activation. In this study, we determined the anti‐fibrotic effects of a novel oridonin analog CYD0617‐2 in activated HSCs and the underlying mechanism.MethodsThe activated human and rat HSC lines LX‐2 and HSC‐T6 were treated with CYD0617‐2. Cell viability was measured by alamarBlue assay. Apoptosis was determined by Cell death ELISA. Cellular proteins were analyzed with Western blot. Cytokines were determined with ELISA.ResultsCYD0617‐2 treatment significantly inhibited proliferation and induced apoptosis in both cell lines in a dose‐dependent manner approximately 10 fold more potent than the parent compound oridonin. CYD0617‐2 stimulated apoptotic proteins p21, p27, and cleaved‐PARP, while suppressing extracellular matrix proteins fibronectin and laminin. CYD0617‐2 blocked LPS‐induced NF‐κB p65 nuclear translocation and DNA binding activity, prevented LPS‐induced NF‐κB inhibitory protein IκBα phosphorylation and degradation, and suppressed LPS‐stimulated IKKα/β phosphorylation. LPS‐induced NF‐κB cytokines IL‐6, MCP‐1, and IL‐1β were attenuated by CYD0617‐2. The endogenous and LPS‐induced NF‐κB p65 S536 phosphorylation was inhibited by CYD0617‐2. Importantly, NF‐κB chemical inhibitor Bay‐11‐0782 was found to inhibit proliferation and promote apoptosis in both cell lines. Moreover, CYD0617‐2 attenuated Stat3 activation by suppressing pStat3/T705 phosphorylation and target gene c‐myc.ConclusionThe potent anti‐hepatic fibrogenetic effect of CYD0617‐2 may be through suppression of IKK/IκBα/NF‐κB and Stat3 pathway.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA‑494 inhibits apoptosis of murine vascular smooth muscle cells invitro.

Apoptosis of vascular smooth muscle cells (VSMCs) is a process that regulates vessel remodeling in various cardiovascular diseases. The specific mechanisms that control VSMC apoptosis remain unclear. The present study aimed to investigate whether microRNA‑494 (miR‑494) is involved in regulating VSMC apoptosis and its underlying mechanisms. Cell death ELISA and terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling assays were used to detect apoptosis of murine VSMCs following stimulation with tumor necrosis factor‑α (TNF‑α). The results indicated that TNF‑α upregulated VSMC apoptosis in a dose‑dependent manner. Microarray analysis was used to evaluate the expression profile of microRNAs following TNF‑α stimulation in murine VSMCs. The expression of miR‑494 was downregulated, whereas B‑cell lymphoma-2‑like11(BCL2L11) protein expression levels were upregulated in VSMCs following treatment with TNF‑α. Luciferase reporter assays confirmed that BCL2L11 was a direct target of miR‑494. Transfection with miR‑494 mimics decreased VSMC apoptosis and downregulated BCL2L11 protein levels. Conversely, transfection with miR‑494 inhibitors increased cell apoptosis and upregulated BCL2L11 protein levels, suggesting that miR‑494 may function as an essential regulator of BCL2L11. The increase in apoptosis caused by miR‑494 inhibitors was abolished in cells co‑transfected with BCL2L11‑targeting small interfering RNA. The findings of the present study revealed that miR‑494 inhibited TNF‑α‑induced VSMC apoptosis by downregulating the expression of BCL2L11.

Induction of AMPK activation by N,N'-diarylurea FND-4b decreases growth and increases apoptosis in triple negative and estrogen-receptor positive breast cancers.

PurposeTriple negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer. AMP-activated protein kinase (AMPK) is a major energy regulator that suppresses tumor growth, and 1-(3-chloro-4-((trifluoromethyl)thio)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (FND-4b) is a novel AMPK activator that inhibits growth and induces apoptosis in colon cancer. The purpose of this project was to test the effects of FND-4b on AMPK activation, proliferation, and apoptosis in breast cancer with a particular emphasis on TNBC.Materials and methods(i) Estrogen-receptor positive breast cancer (ER+BC; MCF-7, and T-47D), TNBC (MDA-MB-231 and HCC-1806), and breast cancer stem cells were treated with FND-4b for 24h. Immunoblot analysis assessed AMPK, acetyl-CoA carboxylase (ACC), ribosomal protein S6, cyclin D1, and cleaved PARP. (ii) Sulforhodamine B growth assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h. Proliferation was also assessed by counting cells after 72h of FND-4b treatment. (iii) Cell death ELISA assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h.Results(i) FND-4b increased AMPK activation with concomitant decreases in ACC activity, phosphorylated S6, and cyclin D1 in all subtypes. (ii) FND-4b decreased proliferation in all cells, while dose-dependent growth decreases were found in ER+BC and TNBC. (iii) Increases in apoptosis were observed in ER+BC and the MDA-MB-231 cell line with FND-4b treatment.ConclusionsOur findings indicate that FND-4b decreases proliferation for a variety of breast cancers by activating AMPK and has notable effects on TNBC. The growth reductions were mediated through decreases in fatty acid synthesis (ACC), mTOR signaling (S6), and cell cycle flux (cyclin D1). ER+BC cells were more susceptible to FND-4b-induced apoptosis, but MDA-MB-231 cells still underwent apoptosis with higher dose treatment. Further development of FND compounds could result in a novel therapeutic for TNBC.

Clathrin-mediated Endocytosis of Alpha-1 Antitrypsin is Essential for its Protective Function in Islet Cell Survival.

Cytokine-induced pancreatic β cell death plays a pivotal role in both type 1 and type 2 diabetes. Our previous study showed that alpha-1 antitrypsin (AAT) inhibits β cell death through the suppression of cytokine-induced c-Jun N-terminal kinase (JNK) activation in an islet transplantation model. The aim of this study was to further understand how AAT impacts β cells by studying AAT endocytosis in human islets and a βTC3 murine insulinoma cell line.Methods: In vitro, human islets and βTC3 cells were stimulated with cytokines in the presence or absence of chlorpromazine (CPZ), a drug that disrupts clathrin-mediated endocytosis. Western blot, real-time PCR and cell death ELISA were performed to investigate β cell death. The oxygen consumption rate (OCR) was measured on human islets. In vivo, islets were harvested from C57BL/6 donor mice treated with saline or human AAT and transplanted into the livers of syngeneic mice that had been rendered diabetic by streptozotocin (STZ). Islet graft survival and function were analyzed.Results: AAT was internalized by β cells in a time- and dose-dependent manner. AAT internalization was mediated by clathrin as treatment with CPZ, profoundly decreased AAT internalization, cytokine-induced JNK activation and the downstream upregulation of c-Jun mRNA expression. Similarly, addition of CPZ attenuated cytokine-induced caspase 9 cleavage (c-casp 9) and DNA fragmentation, which was suppressed by AAT. Treatment of donor mice with AAT produced AAT internalization in islets, and resulted in a higher percentage of recipients reaching normoglycemia after syngeneic intraportal islet transplantation.Conclusion: Our results suggest that AAT is internalized by β cells through clathrin-mediated endocytosis that leads to the suppression of caspase 9 activation. This process is required for the protective function of AAT in islets when challenged with proinflammatory cytokines or after islet transplantation.

Palbociclib enhances radiosensitivity of hepatocellular carcinoma and cholangiocarcinoma via inhibiting ataxia telangiectasia–mutated kinase–mediated DNA damage response

AimPalbociclib is an oral cyclin-dependent kinase 4/6 inhibitor, which is efficacious in treating breast cancer. Currently, there are numerous active clinical trials testing palbociclib alone or in combination with other medications for treating various types of malignancies. Here, we evaluated the anti-cancer effect of palbociclib in combination with radiation therapy (RT) for treating human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) and addressed the molecular mechanism behind the combination therapy. MethodsImmunofluorescence staining of γH2AX or 53BP1 was used to determine the effect of palbociclib on double-strand break (DSB) repair. Clonogenic assays, sphere formation and cell death ELISA were performed to study the sensitising effect of palbociclib on radiation-induced cytotoxicity. Signal alteration in DSB repair pathways was examined by Western blot analysis. Finally, we evaluated the in vivo anti-cancer activity and the associated molecular events of the combination therapy in a preclinical HCC xenograft model. ResultsPalbociclib affected the kinetics of DNA repair and enhanced the radiation sensitivity of HCC and CCA cells. Importantly, we found that palbociclib inhibits ataxia telangiectasia–mutated (ATM) kinase, the key upstream kinase responding to RT-induced DSBs. Furthermore, we showed that the inhibitory effect of palbociclib on RT-induced ATM kinase activation is mediated by protein phosphatase 5 (PP5). Both in vitro and in vivo investigations revealed that the inhibition of the PP5-ATM axis by palbociclib after DNA damage is responsible for the synergism between palbociclib and RT. ConclusionOur findings provide a novel combination strategy against liver cancer cells. Clinical trials using palbociclib as an adjuvant in RT are warranted.

SPOT-008 Neurotensin receptor type 2 protects B-cell chronic lymphocytic leukaemia cells from apoptosis

Introduction B-cell chronic lymphocytic leukaemia (B-CLL) cells are resistant to apoptosis, and consequently accumulate to the detriment of normal B cells and patient immunity. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Material and methods B-cells were isolated from the peripheral blood of healthy donors and CLL patients (n=34) using the MACSxpress human B-CLL Cell Isolation Kit. Gene expression was assessed by RT-qPCR, protein expression and signalling pathway activation was assessed by Western Blot. Protein-protein interaction was confirmed by both Indirect Immuno-Fluorescence and co-immunoprecipitations. Cell death was assessed by Elisa Cell Death as well as Flow Cytometry. Studied receptors were inhibited either by RNA interference or pharmacological inhibitors. Results and discussions In this study, we identified neurotensin receptor 2 (NTSR2) as an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed in B-CLL cells, whereas expression of its natural ligand, neurotensin (NTS), was minimal in both B-CLL cells and patient plasma. Surprisingly, NTSR2 remained in a constitutively active phosphorylated state, caused not by a mutation-induced gain-of-function but rather by an interaction with the oncogenic tyrosine kinase receptor TrkB. Functional and biochemical characterisation revealed that the NTSR2–TrkB interaction acts as a conditional oncogenic driver requiring the TrkB ligand BDNF, which unlike NTS is highly expressed in B-CLL cells. Together, NTSR2, TrkB and BDNF induce autocrine and/or paracrine survival pathways that are independent of mutation status and indolent or progressive disease course. The NTSR2–TrkB interaction activates survival signalling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. When NTSR2 was downregulated, TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death. Conclusion Together, our findings demonstrate that the NTSR2–TrkB interaction and the sustained activation of the signalling pathways under the control of these two actors plays a crucial role in B-CLL cell survival, suggesting that inhibition of NTSR2 represents a promising targeted strategy for treating B-CLL malignancy.

Inhibition of Proliferation and Induction of Apoptosis by Thymoquinone via Modulation of TGF Family, p53, p21 and Bcl-2α in Leukemic Cells.

Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties. The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFβ1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL.

Benefits of Antibiotics During Viral Infections: Immuno‐Modulating Properties of Tulathromycin in Porcine Reproductive and Respiratory Syndrome

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive‐strand RNA virus that grows in primary alveolar macrophages and causes acute pneumonia in pigs. PRRSV is a major concern in the swine industry with a total cost of productivity losses estimated at $600 million annually in the U.S. alone. However, due to its high antigenic variability, and poorly understood immunopathogenesis, there is currently no treatment to control PRRSV infection. The common occurrence of PRRSV infection with bacterial infections, including Actinobacillus pleuropneumoniae, begs the question of the value of antibiotics for the treatment of the disease it causes. Tulathromycin, a macrolide used for the treatment and prevention of respiratory disease in pigs and cattle, has been shown to exhibit potent immuno‐modulating properties [1]. We hypothesize that tulathromycin attenuates the detrimental effects of PRRSV in porcine macrophages. This may help characterize novel mechanism through which an anti‐microbial agent may deliver clinical benefits in the context of a viral infection.Aims1) Determine if Tulathromycin has direct anti‐viral effects in PRRSV‐infected porcine macrophages. 2) Identify new immuno‐modulating effects of Tulathromycin in PRRSV‐infected macrophages.MethodsPorcine monocytes were isolated from peripheral blood of healthy piglets. Seven days‐old monocyte‐derived macrophages were treated with Tulathromycin (1 mg/ml) or untreated (control) and incubated for 1h at 37°C. Macrophages were then cultured in media alone, or with PRRSV (MOI of 1) for 2h to 24h. (1) Extracellular and intracellular Viral titers were measured at 2h, 24h, and 48h post‐infection. (2) Macrophage activation was monitored by morphological changes and confirmed with IL‐8 ELISA and ROS production assays at 2h, 6h, 12h or 24h. (3) Phagocytosis properties were assessed by measuring zymosan particles (1 μg/mL) or 3μM opsonized latex beads (ratio beads: cells, 10:1) engulfment assay (4) Cell death ELISA, Annexin V immunostaining and lactate dehydrogenase assay were performed to evaluate the effects of Tulathromycin on PRRSV‐induced necrosis and apoptosis.Results(1) Tulathromycin did not change PRRSV particle forming unit (pfu) in macrophages at any time of infection (2.1×104 PFU/mL with tulathromycin versus 1.9×104 PFU/mL for control) (2). Exposure to PRRSV increased macrophage‐induced production of IL‐8 and ROS by more than four times compared to control. Pre‐treatment with tulathromycin significantly attenuated PRRSV‐induced macrophage activation (i.e. 1.85 times compared to untreated macrophages). (3) Macrophage phagocytosis of both zymosan and latex beads was impaired by PRRSV. Tulathromycin was able to restore phagocytic functions in PRRSV‐infected macrophages to control values. (4) Finally, Tulathromycin reduced viral induced necrosis but synergized with the virus to induce apoptosis.ConclusionOur results demonstrate that Tulathromycin attenuates macrophage‐driven inflammatory response induced by PRRSV, restores PRRSV‐induced phagocytic impairment, and inhibits cell necrosis. These effects occur in the absence of a direct anti‐viral activity. Together, these data demonstrate the potential clinical benefits of Tulathromycin in the context of PRRSV‐induced pneumonia through the modulation of a viral‐induced macrophage‐driven inflammation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gallic Acid Induces Apoptosis in Human Gastric Adenocarcinoma Cells.

Gastric cancer is one of the most common malignant cancers with a poor prognosis and high mortality rate worldwide. Current treatment of gastric cancer includes surgery and chemotherapy as the main modalities, but the potentially severe side-effects of chemotherapy present a considerable challenge. Gallic acid is a trihydroxybenzoic acid found to exert an anticancer effect against a variety of cancer cells. The purpose of this study was to determine the anti-cancer activity of Galla chinensis and its main component gallic acid on human gastric adenocarcinoma cells. MTT assay and cell death ELISA were used to determine the apoptotic effect of Gallic Chinensis and gallic acid on human gastric adenocarcinoma cells. To determine the pathway and relevant components by which gallic acid-induced apoptosis is mediated through, cells were transfected with siRNA (Fas, FasL, DR5, p53) using Lipofectamine 2000. Reults: Gallic Chinensis and gallic acid induced apoptosis of human gastric adenocarcinoma cells. Gallic acid induced up-regulation of Fas, FasL, and DR5 expression in AGS cells. Transfection of cells with Fas, FasL, or DR5 siRNA reduced gallic acid-induced cell death. In addition, p53 was shown to be involved in gallic acid-mediated Fas, FasL, and DR5 expression as well as cell apoptosis in AGS cells. These results suggest that gallic acid has a potential role in the treatment of gastric cancer.

Thiamine deficiency activates hypoxia inducible factor-1α to facilitate pro-apoptotic responses in mouse primary astrocytes.

Thiamine is an essential enzyme cofactor required for proper metabolic function and maintenance of metabolism and energy production in the brain. In developed countries, thiamine deficiency (TD) is most often manifested following chronic alcohol consumption leading to impaired mitochondrial function, oxidative stress, inflammation and excitotoxicity. These biochemical lesions result in apoptotic cell death in both neurons and astrocytes. Comparable histological injuries in patients with hypoxia/ischemia and TD have been described in the thalamus and mammillary bodies, suggesting a congruency between the cellular responses to these stresses. Consistent with hypoxia/ischemia, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α) under physiological oxygen levels. However, the role of TD-induced HIF-1α in neurological injury is currently unknown. Using Western blot analysis and RT-PCR, we have demonstrated that TD induces HIF-1α expression and activity in primary mouse astrocytes. We observed a time-dependent increase in mRNA and protein expression of the pro-apoptotic and pro-inflammatory HIF-1α target genes MCP1, BNIP3, Nix and Noxa during TD. We also observed apoptotic cell death in TD as demonstrated by PI/Annexin V staining, TUNEL assay, and Cell Death ELISA. Pharmacological inhibition of HIF-1α activity using YC1 and thiamine repletion both reduced expression of pro-apoptotic HIF-1α target genes and apoptotic cell death in TD. These results demonstrate that induction of HIF-1α mediated transcriptional up-regulation of pro-apoptotic/inflammatory signaling contributes to astrocyte cell death during thiamine deficiency.

Therapeutic Effects of a Novel Agonist of Peroxisome Proliferator-Activated Receptor Alpha for the Treatment of Diabetic Retinopathy

PurposeClinical studies have shown that peroxisome proliferator-activated receptor alpha (PPARα) agonist fenofibrate has therapeutic effects on diabetic retinopathy (DR). The purpose of this study was to identify a novel PPARα agonist and to evaluate its beneficial effects on DR.MethodsThe transcriptional activity of PPARα was measured by a luciferase-based promoter assay. TUNEL was used to evaluate apoptosis in retinal precursor cells (R28). Diabetes was induced in rats by injection of streptozotocin. Retinal inflammation was examined using leukostasis assay, and retinal vascular leakage was measured using permeability assay. Retinal function was measured using electroretinogram (ERG) recording, and retinal apoptosis was quantified using the cell death ELISA. The anti-angiogenic effect was evaluated in the oxygen-induced retinopathy (OIR) model.ResultsA compound, 7-chloro-8-methyl-2-phenylquinoline-4-carboxylic acid (Y-0452), with a chemical structure distinct from existing PPARα agonists, activated PPARα transcriptional activity and upregulated PPARα expression. Y-0452 significantly inhibited human retinal capillary endothelial cell migration and tube formation. The compound also protected R28 cells against apoptosis and inhibited NF-κB signaling in R28 cells exposed to palmitate. In diabetic rats, Y-0452 ameliorated leukostasis and vascular leakage in the retina. In addition, Y-0452 preserved the retinal function and reduced retinal cell death in diabetic rats. Y-0452 also alleviated retinal neovascularization in the OIR model.ConclusionsY-0452 is a novel PPARα agonist and has therapeutic potential for DR.

Abstract 4300: Thymoquinone Inhibits Proliferation and induces apoptosis in leukemic cells

Abstract Introduction: Adult T-cell leukemia (ATL) is caused by human T-cell lymphotropic virus 1 (HTLV-1) and is an aggressive form of malignancy. So far, there is no effective treatment for ATL. Thymoquinone has been reported to exhibit many useful effects including anti-cancerous properties. Objective: The aim of this study is to investigated the effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. Materials and methods: Cells were treated with various concentrations of thymoquinone for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed using staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. Results: At non-cytotoxic concentrations of TQ, it resulted in inhibition of proliferation in a dose dependant manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increased DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFβ1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. Conclusion: Thymoquinone showed antiproliferative and proapoptotic potentials in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL. Citation Format: Steve Harakeh, Josiane Semaan, Marwan El Sabban, Soad K. Al Jaouni, Mona Diab-Assaf, Rania Azar. Thymoquinone Inhibits Proliferation and induces apoptosis in leukemic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4300. doi:10.1158/1538-7445.AM2017-4300

Tylvalosin Induces Apoptosis of Porcine Neutrophils and Macrophages, Efferocytosis by Porcine Macrophages and Modulation of Lipid Mediators of Inflammation in vitro: A New Class of Anti‐Microbial with Anti‐inflammatory and Pro‐resolution Properties?

Mucosal inflammatory diseases cause great economic losses in the livestock industry. Previous research has shown that some macrolides effectively modulate bacterial‐induced inflammation in animal models. Tylvalosin (TYL) is a new macrolide antibiotic, meant to treat porcine respiratory diseases, in which inflammation is a major component of pathophysiology. The immune modulating effects of tylvalosin are unknown. We hypothesized that TYL may promote the resolution of inflammation in pigs, hence making it an appealing anti‐infective therapeutic.AimsThe aims of the study were to characterize the effects of this macrolide on (1) apoptosis (2) necrosis and reactive oxygen species (ROS) production (3) cytokine, chemokine and lipid mediators of inflammation – leukotriene B4 (LTB−4), prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) (4) phagocytosis and efferocytosis of neutrophils (PMNs) by macrophages.MethodsPorcine blood was collected from the jugular vein for isolation of PMNs and monocytes. Isolated monocytes were grown and matured (verified by esterase staining) over the course of 7 days. Leukocytes were treated with vehicle, TYL, lincomyosin or positive controls: LPS, staurosporine or calcium ionophore. (1) Apoptosis was measured by western blot for cleaved‐caspase 3, TUNEL staining for nicked DNA, and cell death ELISA (2) lactate dehydrogenase (LDH) assay for necrosis detection was used to assess cell death. ROS levels were assessed using bioassays. (3) Multiplex‐bead based assays and qPCR were employed to assess macrophage cytokine and chemokine levels. Reverse phase‐HPLC and liquid chromatography tandem mass spectrometry were used to assess PMN lipid mediator secretions (4) Macrophage phagocytosis and efferocytosis were determined using zymosan (ZYM) phagocytosis assays, TUNEL staining, and myeloperoxidase (MPO) assay.Results(1) Porcine PMN and macrophage apoptosis were increased in cells exposed to TYL in a time and dose dependent manner (P&lt;0.05), but not in the other groups. (2) Tylvalosin had not effect on LDH or ROS levels in macrophages or PMNs. (3) Tylvalosin reduced CXCL8 mRNA transcription and protein secretion in macrophages. IL‐1α but not IL‐1β protein secretion was inhibited. TYL reduced levels of pro‐inflammatory LTB4 and PGE2, while inducing the production of pro‐resolving LXA4 in TYL‐treated porcine PMNs (4) Treated and untreated macrophages had similar levels of phagocytic uptake of ZYM particles. In contrast, MPO revealed a dose dependent increase in efferocytosis of PMN's in macrophages upon TYL treatment (P&lt;0.05).ConclusionsThese findings suggest that TYL promotes apoptosis and efferocytosis of porcine leukocytes, while having no significant effect on necrosis or ROS production. The effect appears to be, at least in part drug‐selective, and does not affect phagocytic function. TYL inhibits LTB4 and PGE‐2, while enhancing the production of LXA4. Together, these results suggest that tylvalosin may have anti‐inflammatory and pro‐resolving properties in porcine tissues.Support or Funding InformationECO Animal Health, Natural Sciences and Engineering Research Council &amp; University of Calgary

Targeted superparamagnetic nanoparticles coated with 2-deoxy-d-gloucose and doxorubicin more sensitize breast cancer cells to ionizing radiation

IntroductionNanoparticles are promising as a new approach to enhance chemo- radiotherapy efficiency in breast cancer mainly via targeted therapy. Materials & methodsSKBR3 and T47D breast cancer cells were treated with superparamagnetic mesoporous hydroxyapatite nanocomposites (SPmHANs)conjugated with 1 μM doxorubicin and 0.5 mM 2-Deoxy-d-Glucose and irradiated with 1 and 2 Gy gamma rays in vitro. The treatment toxicity and also the apoptosis/necrosis ratio were measured by MTT assay and also ELISA cell death detection PLUS, respectively. ResultsThe decreased cell viability with the combined treatment, with determined 42% loading efficiency for 200 ppm 2DG and 93% for5ppm doxorubicin on SPmHANs in PH about 7.4 and 5.5, were calculated to 60.9% and 68% compared to radiotherapy alone inT47D and SKBR3 cells (both with p < 0.05), respectively. ConclusionBreast cancer cure may boost from The combined targeted nanoparticle treatment with doxorubicin and 2-Deoxy-d-Glucose may boost breast cancer radiotherapy by improved chemodrug localization, increased cytotoxicity in tumor cells and decreased single modality treatment doses.

Microglia activation triggers oligodendrocyte precursor cells apoptosis via HSP60.

Reactive microglia are present in lesions of myelin-associated white matter disorders resulting in injuries to oligodendrocyte precursor cells (OPCs). Therefore, protection of OPCs from injury due to excessive activation of microglia is important in treating these diseases. Heat shock protein 60 (HSP60) has been demonstrated to be released extracellularly in the failing heart upon stress or injury. However, the role of HSP60 in the central nervous system and whether it participates in the toxic effects of microglia on OPCs remains unclear. The present study used the co-culture, cell death assays, binding assays, immunochemistry, western blot and ELISA. HSP60 was demonstrated to be released extracellularly by LPS-activated microglia and to bind to OPCs, triggering OPC apoptosis. When pretreated with toll-like receptor (TLR) 4 blocking antibody, the viability of OPCs increased, while the expression of nuclear factor κB (NFκB), caspase 3 and the release of proinflammatory cytokines triggered by HSP60 decreased. These results suggest that HSP60 released by microglia may mediate OPC apoptosis through binding to TLR4 on the surface of OPCs and subsequently activating the TLR4-NFκB signaling pathway. HSP60 may, therefore, serve as a potential target for treatment of myelin-associated neurodegenerative diseases that are accompanied by microglia activation.

Abstract 1010: Evaluation of small-molecule FASN inhibitors in preclinical models of colorectal cancer

Abstract Fatty Acid Synthase (FASN), a key enzyme of de novo lipid synthesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN activity is associated with decreased survival and increased disease recurrence. Recently, a first-in-class, oral FASN inhibitor (TVB-2640) entered a Phase I clinical trial (3V2640-CLIN-002) in solid tumor patients demonstrating a favorable tolerability profile with no significant adverse events; however, tumor characteristics that would indicate responsiveness to FASN inhibition are not fully understood. The purpose of our study was: (i) to determine the effect of novel, selective and reversible FASN inhibitors on proliferation of primary CRC cell cultures, established CRC cell lines, and CRC patient-derived xenografts (PDXs); and (ii) to identify potential biomarkers associated with CRC responsiveness to FASN inhibition. METHODS. The effect of TVB-3166, TVB-3664, and TVB-3693 (all developed by 3-V Biosciences) on the proliferation of primary cells (established from 1st generation PDX tumors) and CRC cell lines was assessed by cell count; apoptosis was assessed by Cell Death ELISA. In addition, tumor growth was assessed in PDX models established in NOD SCID gamma mice using freshly resected CRC specimens (either primary CRC or metastasis) from our patient population. Once the xenografts grew to ∼100 mm3, mice were randomized into two groups (n = 5) to receive either vehicle or TVB-3664 (3mg/kg) by gavage daily. Tumor volume and animal weights were measured weekly. Western blot analysis, immunohistochemistry and immunofluorescent staining were used to identify FASN-mediated changes in β-catenin, Akt and AMPK pathways. RESULTS. TVB compounds tested showed similar efficacy in primary and established CRC cells with a wide range of sensitivity to FASN inhibition. The 5 cell lines that were most responsive to FASN inhibition demonstrated a low basal level of pAMPK and pAkt as compared to the 5 least responsive cells. Moreover, we noted that increased FASN protein expression was also associated with increased sensitivity to FASN inhibition. Inhibition of proliferation by TVB compounds was associated with decreased expression of active β-catenin, c-MYC, pAkt, and survivin, while an increase in apoptosis was noted by induction of PARP cleavage. Consistent with our in vitro studies, TVB-3664 treatment significantly reduced tumor volume in vivo with no weight changes or toxicity observed. CONCLUSIONS. Our studies show that the novel FASN inhibitors, as a single agent, significantly inhibit CRC growth both in vitro and in vivo. Importantly, our results suggest that basal activation of AMPK and Akt may be predictive of responsiveness to FASN inhibition and may function as potential biomarkers to allow a more personalized treatment approach. Citation Format: Yekaterina Y. Zaytseva, Piotr G. Rychahou, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, Timothy S. Heuer, George Kemble, B. Mark Evers. Evaluation of small-molecule FASN inhibitors in preclinical models of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1010.

Abstract P5-05-05: Inhibition of enhanced glucose uptake and glycolysis by KU-55933 as a novel strategy against aggressive breast cancer

Abstract The ability of cancer cells to produce large amounts of lactate through aerobic glycosis (Warburg effect) is coupled to high rates of glucose uptake. Enhanced glucose uptake and glycolysis are closely correlated to increased breast tumor aggressiveness and poor prognosis. However, despite the importance of glucose uptake in supplying energy and preventing apoptosis of cancer cells, the majority of current efforts in searching for therapeutic agents targeting glucose metabolism have been aimed at modulating activities of different metabolic enzymes that are involved in glycolysis. Very limited studies have been done in developing novel therapeutic agents against glucose uptake in breast cancer cells. Ataxia-telangiectasia (A-T) is a monogenic, autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. The gene mutated in this disease, ATM (A-T, mutated), encodes a 370-kDa protein kinase. Although ATM is traditionally considered to be a nuclear protein that functions as a signal transducer in the cellular response to DNA damage, it is now known that ATM is also present in the cytoplasm and has important cytoplasmic functions. We previously discovered that ATM activates Akt, a main regulator of glucose uptake, by stimulating its phosphorylation at Ser473 following insulin treatment. We also found that ATM participates in insulin-mediated glucose uptake in muscle cells, and KU-55933, a specific inhibitor of ATM, strongly inhibits this process. Recently, we found that KU-55933 inhibits cell proliferation by inducing apoptosis in MDA-MB-231, a triple-negative breast cancer cell line. We have also found that KU-55933 inhibits migration of MDA-MB-231 by a cell invasion assay. Furthermore, we found that these cancer cells exhibit enhanced glucose uptake in response to insulin and the addition of KU-55933 leads to a dramatic reduction of insulin-mediated glucose uptake in these cells. To further test whether KU-55933's ability to induce apoptosis is linked to its inhibition of glucose uptake, we performed a cell death ELISA assay in MDA-MB-231 cells treated with KU-55933 and different concentrations of glucose. Our results show that KU-55933 induces apoptosis of MDA-MB-231 cells, resulting in a similar degree of cell death as glucose starvation, while cells treated with glucose in conjunction with KU-55933 have decreased apoptosis. Moreover, we performed a cell migration assay and found that KU-55933 strongly inhibits the migration of MDA-MB-231 cells (similar to that caused by glucose starvation), which is almost fully rescued by the extra glucose supplemented in the cell culture medium. We have also established a positional isotope labeling-based targeted metabolomics method that can directly measure the conversion from glucose to lactate through glycolysis in cancer cells. Our results show strong production of lactate from glucose in MDA-MB-231 cells even under normal aerobic growth conditions, and KU-55933 strongly inhibits this process. Our findings may lead to the development of KU-55933 and its analogs as a new generation of therapeutic agents against aggressive breast cancer. Citation Format: Yang D-Q, Harris B, Jiang S, Li Y, Freund D, Hegeman A, Cleary M. Inhibition of enhanced glucose uptake and glycolysis by KU-55933 as a novel strategy against aggressive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-05-05.