cytochrome-c-t and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

Targeting mitochondrial redox homeostasis is an appealing methodology for cancer therapeutics because of the upregulated antioxidant capacity in drug resistance cases. By coupling triphenylamine (TPA) with an excellent fluorescent group BODIPY, a novel mitochondrial-targeted fluorescent probe, BODIPY-TPA (BTPA), was synthesized and characterized. Confocal microscopic colocalization imaging indicated that BTPA exhibited a subcellular mitochondrial distribution. Cytotoxicity experiments suggested that BTPA exhibited selective anticancer activity via the induction of mitochondrial dysfunction in BGC-823 cancer cells. BTPA induced alterations in mitochondrial redox homeostasis because of the electron-donating property of TPA and mitochondrial selectivity. In further studies, TrxR2 in the mitochondria was alternatively inhibited, which contributed to MtROS accumulation further attenuated PI3K/Akt signaling pathway. The resultant decline in mitochondrial antioxidant capacity aggravated mitochondrial oxidative stress, which is responsible for cytochrome C release and caspase-9 activation. NAC completely reversed BTPA-induced ROS-dependent mitochondrial-mediated intrinsic apoptosis. Therefore, BTPA was designed as a superior fluorescent cancer-imaging probe and a mitochondrial redox-targeting anticancer agent.

Topics: Aniline Compounds; Antineoplastic Agents; Antioxidants; Apoptosis; Boron Compounds; Cell Line, Tumor; Cytochromes c; Drug Resistance, Neoplasm; Humans; Microscopy, Confocal; Mitochondria; Neoplasms; Oxidation-Reduction; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Thioredoxin Reductase 2

Protein thiol modifications occur under both physiological and pathological conditions and have been shown to contribute to changes in protein structure, function, and redox signaling. The majority of protein thiol modifications occur on cysteine residues that have a low pK(a); these nucleophilic proteins comprise the "reactive thiol proteome." The most reactive members of this proteome are typically low-abundance proteins. Therefore, sensitive and quantitative methods are needed to detect and measure thiol modifications in biological samples. To accomplish this, we have standardized the usage of biotinylated and fluorophore-labeled alkylating agents, such as biotinylated iodoacetamide (IAM) and N-ethylmaleimide (NEM) and BODIPY-labeled IAM and NEM, for use in one- and two-dimensional proteomic strategies. Purified fractions of cytochrome c and glyceraldehyde-3-phosphate dehydrogenase were conjugated to a known amount of biotin or BODIPY fluorophore to create an external standard that can be run on standard SDS-PAGE gels, which allows for the quantification of protein thiols from biological samples by Western blotting or fluorescence imaging. A detailed protocol is provided for using thiol-reactive probes and making external standards for visualizing and measuring protein thiol modifications in biological samples.

Topics: Animals; Biotinylation; Blotting, Western; Boron Compounds; Cytochromes c; Electrophoresis, Polyacrylamide Gel; Ethylmaleimide; Fluorescent Dyes; Glyceraldehyde-3-Phosphate Dehydrogenases; Horses; Iodoacetamide; Microscopy, Fluorescence; Muscles; Myocardium; Protein Processing, Post-Translational; Proteomics; Sensitivity and Specificity; Sulfhydryl Compounds