cytochrome-c-t and 3-methylquercetin

It has been reported that apoptosis plays a very important role on anoxia/reoxygenation (A/R)-induced injury, and human silent information regulator type 1 (SIRT1) can inhibit the apoptosis of cardiomyocytes. It has been proved that isorhamnetin (IsoRN), 3'-O-methyl-quecetin, can protect the cardiomyocytes, but the mechanism is still not clear. The aim of the study was to explore whether the protective effects of IsoRN on the cardiomyocytes against the A/R-induced injury are mediated by SIRT1. The effects of IsoRN on cardioprotection against A/R injury in neonatal rat cardiomyocytes were monitored by cell viability, the levels of mitochondrial membrane potential (Δψm), apoptosis, and intracellular reactive oxygen species (ROS), the levels of lactate dehydrogenase (LDH), creatine phosphokinase (CPK) and mitochondrial permeability transition pores (mPTP). The effects on protein expression were measured by western blot assay. The results showed that IsoRN can reduce A/R-induced injury by decreasing the level of lactate dehydrogenase and creatine phosphokinase release from the cardiomyocytes, increasing cell viability and expression of SIRT1, reducing the generation of reactive oxygen species, inhibiting opening of mitochondrial permeability transition pores and loss of Δψm and activation of caspase-3, and decreasing the release of cytochrome c, and reducing apoptosis. In addition, sirtinol, a SIRT1 inhibitor, drastically reduced the protective effects of IsoRN on cardioprotective effects in cardiomocytes. In conclusion, we firstly demonstrated that SIRT1 may be involved in the protective effects of IsoRN on cardiomocytes against the A/R-induced injury.

Topics: Animals; Apoptosis; Caspase 3; Cell Hypoxia; Cell Survival; Cytochromes c; L-Lactate Dehydrogenase; Myocardial Reperfusion Injury; Myocytes, Cardiac; Quercetin; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sirtuin 1

Isorhamnetin (ISO) is a flavonoid from plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. To date, the anti‑tumor effects of ISO and the underlying mechanisms have not been elucidated in lung cancer cells. The present study investigated the inhibitory effects of ISO on the growth of human lung cancer A549 cells. Treatment of the lung cancer cells with ISO significantly suppressed cell proliferation and colony formation. ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner. Further investigation showed that the apoptosis proceeded via the mitochondria‑dependent pathway as indicated by alteration of the mitochondrial membrane potential, the release of cytochrome C and caspase activation. Of note, treatment with ISO also induced the formation of autophagosomes and light chain 3‑II protein in A549 cells. Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo. Thus, the results of the present study suggested that ISO is a potential anti‑lung cancer agent. In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

Topics: Adenine; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Drug Synergism; Epithelial Cells; Gene Expression; Humans; Hydroxychloroquine; Lung Neoplasms; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Microtubule-Associated Proteins; Mitochondria; Quercetin; Xenograft Model Antitumor Assays

Isorhamnetin is a flavanoid present in plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. Since the plasma level of isorhamnetin is maintained longer than quercetin, isorhamnetin may be a key metabolite to mediate the anti-tumor effect of quercetin. In the present study, we investigated the apoptotic mechanism of isorhamnetin in Lewis lung cancer (LLC) cells in vitro and established its in vivo anti-cancer efficacy. In cell culture, isorhamnetin significantly increased DNA fragmentation, and TUNEL positive apoptotic bodies and sub-G(1) apoptotic population in time- and dose-dependent manners. Western blot analyses revealed increased cleavage of caspase-3, and caspase-9 and PARP and increased cytosolic cytochrome C in isorhamnetin-treated cells. These events were accompanied by a reduced mitochondrial potential. Apoptosis was blocked by a general caspase inhibitor or the specific inhibitor of caspase-3 or -9. These in vitro results support mitochondria-dependent caspase activation to mediate isorhamnetin-induced apoptosis. Furthermore, an animal study revealed for the first time that isorhamnetin given by i.p. injection at a dose that is at least one order of magnitude lower than quercetin significantly suppressed the weights of tumors excised from LLC bearing mice. The in vivo anti-tumor efficacy was accompanied by increased TUNEL-positive and cleaved-caspase-3-positive tumor cells. Our data therefore support isorhamnetin as an active anti-cancer metabolite of quercetin in part through caspase-mediated apoptosis.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Lewis Lung; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Female; Flavonols; Injections, Intraperitoneal; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Quercetin; Time Factors