Notes: The significant loss of estrogen in women after menopause has been linked to increased incidence of Alzheimer’s disease. Here, the protective effect of β-ecdysterone (β-Ecd) on SH-SY5Y cell apoptosis in relation to Alzheimer’s disease is investigated. NF-κB and estrogen receptor activation was monitored in the presence of β-Ecd in Aβ-stress conditions using the Dual-Glo® Luciferase Assay System. Caspase activation was measured as a proxy for cell stress and apoptosis. Together, SH-SY5Y cell treatment with β-Ecd showed a protective effect, making β-Ecd a promising therapeutic candidate. (5169)

Notes: The effects of hydrogen sulfide on inflammasome activation were investigated. Lactate dehydrogenase levels and caspase activation were monitored using the CytoTox-ONE™ and Caspase-Glo® assays. Hydrogen sulfide was shown to decrease caspase activation and inflammasome activity both in vitro and in vivo. (5147)

Notes: These authors investigated the mechanism of action of the synthesized resveratrol analogue, (E)-N-(2-(4-methoxystyryl) phenyl) furan-2-carboxamide (CS) on colorectal cancer cells. They report that CS exerts a potent suppressive effect on HCT116 colorectal cancer cells, but not on normal colon cells. CS caused apoptosis via activation of an extrinsic pathway leading to caspase activation and cell cycle arrest from activated p53. The Caspase-Glo® 3/7, 8 and 9 Assays were used to detect caspase activation. (5045)

Notes: The role of the carcinogen Ochratoxin A in cellular inflammation was examined. HEK293 cells were treated with Ochratoxin A and cellular inflammatory markers were monitored. ATP levels and caspase activation were analyzed using the CellTiter Glo® and Caspase-Glo® assays. (5155)

Notes: A novel target of non-steroidal anti-inflammatory drugs (NSAIDs) was identified using the Caspase-Glo® -1, Caspase-Glo® 3/7, and Caspase-Glo® 9 assays. The authors observed that an inhibition of caspase activity lead to decreased cell death and inflammation. The CellTiter-Fluor™ and CellTiter-Glo®  assays were using to monitor cell viability. (5163)

Notes: Chagas disease is caused by the parasite Trypanosoma cruzi and can lead to Chronic Chagas disease cardiomyopathy (CCC). The ability of N,N-dimethylsphingosine (DMS) to decrease inflammation and function as a therapeutic for CCC was determined. As part of this study, Caspase-Glo® assays were used to detect inflammatory (caspase 1 and 8), or apoptotic (caspase 9 and 3/7) activities. (5166)

Notes: The authors utilized phosphoproteomics to identify host factors that are activated upon Influenza A infection. In total, 1116 proteins showed changes in regulation including cyclin-dependent kinases. The authors tested the effects of cyclin-dependent kinase inhibitors on cell viability and caspase activation using the CellTiter Glo® and Caspase-Glo® assays. (5154)

Notes: Caspase-Glo® 3/7 and Caspase-Glo® 8 Assays were used to assess activation of apoptosis pathways in MC3T3-E1 cells (clonal mouse), U-2 OS human osteoscarcoma cell line and CCL-51 cells (mouse mammary gland tumor cells). Although ibandronate reduced cell proliferation in all cell lines, its effect on activation of caspases was different in neoplastic versus non-neoplastic cells. Caspase-8 and caspase-3/7 activities were reduced in MC3T3-E1 cells after 72 hours treatment with ibandronate. In two tumor cell lines assayed, opposite results were seen: caspase-8 and caspase-3/7 activities increased in U-2 OS cells and in CCL-51 cells. Luminescence was detected using a GloMax® 96 Microplate Luminometer. (Figure 1 in the paper)

To analyze FAS promoter methylation levels, fragments of the targeted promoter regions were generated by digestion of genomic DNA using CpG methylation insensitive restriction enzymes MboII and PstI from cells cultured in the presence of ibandronate for varying lengths of time. The authors demonstrated that FAS promoter methylation is altered in tumor cells in the presence of ibandronate. (4253)

Notes: The authors of this study set out to describe the mechanism of cell death through which TNF-like weak inducer of apoptosis (TWEAK) exerts its apoptotic effect on certain cancer cells. The used the CellTiter-Glo® Cell Viability Assay and the Caspase-Glo® 3/7 Assay to investigate cell viability and mechanism of cell death in HSC3 cells treated with TWEAK. They looked at caspase-8 activity in cells treated with TWEAK in the presence or absence of a caspase-8 inhibitor using the Caspase-Glo® 8 Assay. They showed that TWEAK induces caspase-dependent apoptosis in these cells. (4170)

Notes: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) tends to cause apoptosis in tumor cells over normal cells, and so the TRAIL ligand and signaling pathway is an attractive pathway for developing cancer therapeutics. Bortezomib is a proteasome inhibitor that is used for therapy in many cancers. Studies indicate that it can sensitize tumor cells to the apoptotic effects of TRAIL. In this study, the authors investigated the ability of bortezomib on renal cell carcinoma (RCC). Growth inhibition of RCC in response to treatment with bortezomib followed by TRAIL treatment in the presence or absence of caspase inhibitors was assessed using the CellTiter® 96 AQ_ueous Non-Radioactive Cell Proliferation Assay. They assessed caspase activity in bortezomib/TRAI- treated RCC using the Caspase-Glo® 8 Assay. The effect of bortezomib on the proteasome of the RCCs was investigated using the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay. Their studies suggest that bortezomib can sensitize some RCC to TRAIL signaling. (4169)

Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

Notes: In this study, the Caspase-Glo^® 8 and Caspase-Glo^® 9 Assays were used to assess caspase activation in Zebrafish embryos after radiation exposure. After irradiation, caspase activation, morphological abnormalities, and DNA fragmentation were observed, all three of which could be partially reversed by treatment with the radiomodifier amifostine. The Caspase-Glo^® 9 Assay was shown to be sensitive enough to detect the effects of radiation on as few as 2 embryos. The authors concluded that the Caspase-Glo^® Assays provided an effective and convenient means for rapidly assessing the lethal effects of radiation on Zebrafish embryos, and for assaying the ability of the radiomodifier to counteract these effects. The assay could be performed within hours of radiation exposure directly on the embryos in a 96-well plate format. (3571)

Notes: Bacterial cytolethal distending toxin (CDT) is known to induce apoptosis of immune cells, but little is known of the toxin from Campylobacter jejuni. In this study, the authors examined the effects of C. jejuni CDT on cultured monocytes. The human monocyte line 28SC was inoculated with 2µg Campylobacter membrane proteins per milliliter of culture. After 8 hours, the cells were harvested and assessed for caspase-8 or caspase-9 activity using the Caspase-Glo^® 8 and Caspase-Glo^® 9 Assays, respectively. The 28SC cells were pulsed with 6.2 µM camptothecin for 2 hours as a positive control for apoptosis. (3288)

Notes: Researchers were interested in determining if knockdown of cellular FLICE inhibitory protein (c-FLIP) would affect caspase-8 activation. Instead of assaying cultured cells for the Caspase-Glo^® 8 Assay, the authors used siRNAs targeting c-FLIP_L, c-FLIP_S or nontarget control to transfect A549 cells. Equal number of cells were harvested and treated with FLAG-tagged Apo2L/TRAIL + anti-FLAG M2 antibody at 37°C for various times prior to lysis. The lysate was immunoprecipitated overnight using protein A/G beads. After washing with lysis buffer, the beads were resuspended in phosphate buffer and the immobilized death-inducing signaling complex (DISC) was assayed for caspase-8 activity in 96-well plates using an equal volume of Caspase-Glo^® 8 Reagent.
(3277)

Notes: In this paper, researchers examined the effect of activating mammalian homologues of the Drosophila Toll receptor gene (TDR) on apoptosis in the trophoblast cell lines HTR8 and 3A. Bacterial peptidoglycan (PDG) was used to indirectly activate caspase-3/7, -8 and -9 through TDRs. Caspase activity was measured using the Capase-Glo™ 3/7, 8 and 9 Assays. Caspase-3/7, -8 and -9 activities decreased in both the HTR8 and 3A cell lines transiently transfected with a Fas Associated Death Domain (FADD-DN). Data were expressed as relative light units and were normalized to protein levels in lysates. (3171)

Notes: The Caspase-Glo^® 3/7, -8 and -9 Assays were used to  measure Caspase-3, -8, and -9 activities in mouse liver homogenates. The authors describe a modified procedure for making liver homogenates in a hypotonic extraction buffer.  Homogenates were cleared with a 15 minute spin at 13,000 x g and normalized to a 1mg/ml concentration before use the assays. Data comparing control and cSN50 peptide-treated animals at various time points are presented.  (3213)