5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and thiazolyl-blue

Previous studies in malignant cells have shown that irradiation-induced upregulation of telomerase activity, not only protected damaged telomeres, but also contributed to DNA damage repair by chromosomal healing and increased resistance to irradiation. The purpose of the present study was to investigate the radiosensitizing effect of telomerase inhibitor MST-312 and the corresponding mechanism in the human hepatoma cell line HepG2.. Cell proliferation, telomerase activity, cell cycle distribution, DNA damage and repair, expression of p53, mitochondrial membrane potential, and cell apoptosis were measured with the MTT assay, real-time fluorescent quantitative PCR, flow cytometry, immunofluorescence, western blots, JC-1 staining, and Hoechst 33258 staining, respectively.. MST-312 effectively inhibited telomerase activity and showed relative weak toxicity to HepG2 cells at 4 μM. Compared with irradiation alone, 4 μM MST-312 pretreatment, followed by X-ray treatment, significantly reduced clonogenic potential. Aggravated DNA damage and increased sub-G1 cell fractions were observed. Further investigation found that homologous recombination (HR) repair protein Rad51 foci nuclear formation was blocked, and expression of p53 was elevated. These led to the collapse of mitochondrial membrane potential, and enhanced the apoptotic rate.. These data demonstrated that disturbances of telomerase function could enhance the radiosensitivity of HepG2 cells to X-ray irradiation by impairing HR repair processes. In addition, telomerase inhibitor MST-312 may be useful as an adjuvant treatment in combination with irradiation.

Topics: Analysis of Variance; Apoptosis; Benzamides; Benzimidazoles; Bisbenzimidazole; Blotting, Western; Carbocyanines; Cell Proliferation; DNA Damage; DNA Primers; Flow Cytometry; Fluorescent Antibody Technique; Hep G2 Cells; Humans; Membrane Potential, Mitochondrial; Radiation-Sensitizing Agents; Real-Time Polymerase Chain Reaction; Telomerase; Tetrazolium Salts; Thiazoles; X-Rays

Doxorubicin (DOX) is a highly effective antineoplastic drug. However, DOX-induced apoptosis in cardiomyocytes leads to irreversible degenerative cardiomyopathy and heart failure, which limits DOX clinical application. Leonurine is a special alkaloid for Herba leonuri, a traditional herb with cardioprotective effects. In current study, we investigated possible protective effects of Leonurine against DOX-induced cardiomyopathy in H9c2 cells. DOX-injured H9c2 cell model was made by application of 2 microM DOX. Leonurine was added to cells 2 h before DOX treatment. Pre-treated with Leonurine could attenuate DOX-induced apoptotic death of H9c2 cell, reduce MDA formation and intracellular Ca2+ overload. Leonurine also attenuated DOX-induced high expression of Bax, increased Bcl-2 expression in both protein and mRNA level. Myocardial mitochondrion is the target organelle of DOX-induced toxicity in cardiomyocytes. Leonurine moderated the dissipation of mitochondrial membrane potential (DeltaPsim) caused by DOX treatment. Our results indicated that Leonurine attenuated DOX-induced apoptosis in H9c2 cell by increasing anti-oxidant, anti-apoptotic ability and protecting mitochondrial function.

Topics: Animals; Apoptosis; Benzimidazoles; Bisbenzimidazole; Calcium; Carbocyanines; Cardiotonic Agents; Cell Nucleus; Cell Survival; Cells, Cultured; Coloring Agents; Doxorubicin; Fluorescent Dyes; Gallic Acid; Heart Ventricles; Malondialdehyde; Membrane Potential, Mitochondrial; Myocytes, Cardiac; Rats; Tetrazolium Salts; Thiazoles

Hypoglycemia sometimes occurs in patients with diabetes mellitus who receive excessive doses of insulin. Severe hypoglycemia has been known to induce mitochondrial swelling followed by neuronal death in the brain. Since L-carnitine effectively preserves mitochondrial function in various cells both in vitro and in vivo, we investigated its effects on the neuronal damage induced by hypoglycemic insult in male Wistar rats. Animals were given L-carnitine-containing water (0.1%) for 1 week and then received insulin (20 U/kg, i.p.) to induce hypoglycemia. Although L-carnitine did not affect the mortality of animals that developed hypoglycemic shock, it improved the cognitive function of the survived animals as assessed by the Morris water-maze test. L-carnitine effectively inhibited the increase in oxidized glutathione and mitochondrial dysfunction in the hippocampus and prevented neuronal injury. L-carnitine also inhibited the decrease in mitochondrial membrane potential and the generation of reactive oxygen species in hippocampal neuronal cells cultured in glucose-deprived medium. These results suggest that L-carnitine prevents hypoglycemia-induced neuronal damage in the hippocampus, presumably by preserving mitochondrial functions. Thus, L-carnitine may have therapeutic potential in patients with hypoglycemia induced by insulin overdose.

Topics: Aldehydes; Analysis of Variance; Animals; Apoptosis; Benzimidazoles; Brain Injuries; Carbocyanines; Carnitine; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Embryo, Mammalian; Glucose; Glutathione; Hippocampus; Hypoglycemia; Immunohistochemistry; In Situ Nick-End Labeling; Insulin; Male; Maze Learning; Membrane Potentials; Mitochondria; Neurons; Rats; Rats, Wistar; Reaction Time; Reactive Oxygen Species; Respiration; Tetrazolium Salts; Thiazoles; Time Factors

Bioreduction of water-soluble tetrazolium salts (e.g., MTS, XTT, and MTT) to their respective formazans is generally regarded as an indicator of cell "redox activity." The reaction is attributed mainly to mitochondrial enzymes and electron carriers. However, MTT reduction may also be catalyzed by a number of other nonmitochondrial enzymes. The goal of this work was to establish the sites of MTT reduction in intact HepG2 human hepatoma cells in culture.. In order to establish the subcellular localization of the sites of reduction of MTT, we imaged the formation of MTT-formazan deposits using backscattered light confocal microscopy. Mitochondria were visualized in viable cells using fluorescent dyes that bind in a manner dependent (JC-1 and TMRE) or independent (NAO) of mitochondrial electric potential.. Only 25-45% of MTT-formazan was associated with mitochondria after 25 min of incubation. No more than 25% of the mitochondrial area on images was occupied by MTT-formazan. Mitochondrial fluorescence of TMRE, NAO, and the monomeric form of JC-1 decreased rapidly in cells incubated with MTT. However, the intensity of fluorescence of JC-1 aggregates dropped by less than 30% at the onset of incubation and remained constant as reduction of MTT proceeded further.. (1) Most of MTT-formazan deposits are not coincident with mitochondria. (2) Monomeric JC-1, as well as TMRE and NAO, accumulating in mitochondria may be displaced by MTT. Thus, the presence of positively charged organic compounds (like MTT) may distort measurements of mitochondrial transmembrane electric potential, which are based on accumulation of fluorescent dyes.

Topics: Aminoacridines; Benzimidazoles; Carbocyanines; Cell Compartmentation; Energy Metabolism; Eukaryotic Cells; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Intracellular Membranes; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Organometallic Compounds; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured