Loss of mitochondrial transmembrane potential and caspase-9 activation during apoptosis induced by the novel styryl-lactone goniothalamin in HL-60 leukemia cells

Styryl-lactones such as goniothalamin represent a new class of compounds with potential anti-cancer properties. In this study, we investigated the mechanisms of goniothalamin (GTN), a plant styryl-lactone induced apoptosis in human promyelocytic leukemia HL-60 cells. This plant extract resulted in apoptosis in HL-60 cells as assessed by the externalisation of phosphatidylserine. Using the mitochondrial membrane dye (DIOC6) in conjunction with flow cytometry, we found that GTN treated HL-60 cells demonstrated a loss of mitochondrial transmembrane potential (Δψm). Further immunoblotting on these cells showed activation of initiator caspase-9 and the executioner caspases-3 and -7. Pretreatment with the pharmacological caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) abrogated apoptosis as assessed by all of the apoptotic features in this study. In summary, our results demonstrate that goniothalamin-induced apoptosis occurs via the mitochondrial pathway in a caspase dependent manner

Evaluation of the cytotoxic and genotoxic effects of goniothalamin in leukemic cell lines

The cytotoxic and genotoxic effects of goniothalamin, a plant styryllactone, were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the Alkaline Comet assay respectively in human leukemic cell lines. Following 72 h of treatment, the IC50 values of goniothalamin in human HL-60 promyelocytic leukemia cells and CEM-SS T-lymphoblastic cells were 4.5 μg/mL and 2.4 μg/mL respectively. The genotoxicity of goniothalamin in both HL-60 and CEM-SS cells was detected as early as 2 h following treatment at IC10 and IC25 concentrations. However, pretreatment with the antioxidant N-acetyl-cysteine (NAC) at 1 mM for 30 minutes did not abrogate genotoxicity of this compound. This result suggests that primary induction of DNA damage by goniothalamin may not involve oxidative damage. In conclusion, our results demonstrate genotoxic damage induced by goniothalamin in leukemic cells. Further studies are needed to ascertain the mode of action of goniothalamin in inducing DNA damage

Toxicity of tetramethylammonium hydroxide to aquatic organisms and its synergistic action with potassium iodide

The aquatic ecotoxicity of chemicals involved in the manufacturing process of thin film transistor liquid crystal displays was assessed with a battery of four selected acute toxicity bioassays. We focused on tetramethylammonium hydroxide (TMAH, CAS No. 75-59-2), a widely utilized etchant. The toxicity of TMAH was low when tested in the 72 h-algal growth inhibition test (Pseudokirchneriellia subcapitata, EC50 = 360 mg L−1) and the Microtox® test (Vibrio fischeri, IC50 = 6.4 g L−1). In contrast, the 24 h-microcrustacean immobilization and the 96 h-fish mortality tests showed relatively higher toxicity (Daphnia magna, EC50 = 32 mg L−1 and Oryzias latipes, LC50 = 154 mg L−1). Isobologram and mixture toxicity index analyses revealed apparent synergism of the mixture of TMAH and potassium iodide when examined with the D. magna immobilization test. The synergistic action was unique to iodide over other halide salts i.e. fluoride, chloride and bromide. Quaternary ammonium ions with longer alkyl chains such as tetraethylammonium and tetrabutylammonium were more toxic than TMAH in the D. magna immobilization test

A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2

A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd2+, Cu2+, Pb2+, Zn2+ and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 × 10−7 (arsenite), 1.0 × 10−4 (naphthalene), 1.0 × 10−4 (Cu2+), 3.8 × 10−4 (H2O2), 1.0 × 10−3 (Cd2+), 1.0 × 10−3 (Zn2+), 1.0 × 10−2 (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb2+), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation

Involvement of ICE-like protease(s) in TGF-beta 1 induced apoptosis in hepatocytes

Internucleosomal DNA cleavage is often regarded as the biochemical hallmark of apoptosis and can be reproduced in vitro in rat liver nuclei. This chromatin cleavage in rat liver nuclei was further characterised and studies described in this thesis showed that the DNA was initially cleaved into 700kbp, 200-250kbp and 30-50kbp fragments via a Mg2+ dependent process which was potentiated by Ca2+. Further investigations of the DNA cleavage processes were carried out in primary hepatocytes using TGF-1 to induce apoptosis. The studies showed for the first time that treatment of hepatocytes with TGF-1 resulted in multi-step DNA cleavage as seen in rat nuclei. Cycloheximide and an interleukin-1-converting enzyme (ICE)-like inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (Z-VAD.FMK) and Z-Asp-Glu-Val-Asp-(OMe) fluoromethyl ketone (Z-DEVD.FMK), a potent inhibitor of CPP32, blocked DNA cleavage and apoptosis. During apoptosis there was a time dependent increase in CPP32-like proteolytic activity in lysates isolated from TGF-1 treated hepatocytes, which was detected with the fluorogenic assay using Z-DEVD-amino-trifluoromethyl-coumarin (Z-DEVD.AFC). This activity was abolished when hepatocytes were pre-treated with either Z-VAD.FMK, Z-DEVD.FMK or cycloheximide. Unlike cycloheximide, Z-VAD.FMK and Z-DEVD.FMK were potent inhibitors of activated lysate from TGF-1 treated hepatocytes. Immunoblotting showed the processing of pro-CPP32 to its active form. In conclusion, this study shows for the first time that TGFF-1 mediated apoptosis involves the activation of ICE-like proteases and that cycloheximide inhibits apoptosis by blocking upstream of this ICE-like activity

Cytoprotective effects of (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) against 4-nitroquinoline 1-oxide-induced damage in CCD-18Co human colon fibroblast cells.

Stilbenes are a group of chemicals characterized with the presence of 1,2-diphenylethylene. Previously, our group has demonstrated that synthesized (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) possesses potential chemopreventive activity specifically inducing NAD(P)H:quinone oxidoreductase 1 (NQO1) protein expression and activity. In this study, the cytoprotective effects of BK3C231 on cellular DNA and mitochondria were investigated in normal human colon fibroblast, CCD-18Co cells. The cells were pretreated with BK3C231 prior to exposure to the carcinogen 4-nitroquinoline 1-oxide (4NQO). BK3C231 was able to inhibit 4NQO-induced cytotoxicity. Cells treated with 4NQO alone caused high level of DNA and mitochondrial damages. However, pretreatment with BK3C231 protected against these damages by reducing DNA strand breaks and micronucleus formation as well as decreasing losses of mitochondrial membrane potential (ΔΨm) and cardiolipin. Interestingly, our study has demonstrated that nitrosative stress instead of oxidative stress was involved in 4NQO-induced DNA and mitochondrial damages. Inhibition of 4NQO-induced nitrosative stress by BK3C231 was observed through a decrease in nitric oxide (NO) level and an increase in glutathione (GSH) level. These new findings elucidate the cytoprotective potential of BK3C231 in human colon fibroblast CCD-18Co cell model which warrants further investigation into its chemopreventive role