== Cleavage of pro-caspase 8, pro-caspase 3, and PARP (H) was inhibited by GSH and NAC in both HL-60 and HL-60/MX2 cells. and elevated the expression of phosphorylated-H2AX in a dose-dependent manner. In Topo II-deficient HL60/MX2 cells, however, G226-induced DNA damage, apoptosis and cytotoxicity were only partially reduced, suggesting that Topo II was not essential for the anti-tumor effects of G226. Furthermore, G226 (0.1252 mol/L) dose-dependently elevated the intracellular levels of H2O2andin the cancer cells, and pretreatment with GSH, NAC or DTT not only blocked G226-induced intracellular accumulation of ROS, but also abrogated G226-mediated phosphorylation of H2AX, apoptosis and cytotoxicity. == Conclusion: == G226-mediated ROS production contributes to the anti-cancer activity of this compound. Keywords:G226, epipolythiodioxopiperazine, 11-deoxyverticillin A, anti-cancer agent, Topo II, DNA damage, apoptosis, ROS, antioxidant == Introduction == DNA topoisomerase II (Topo II) is a nuclear enzyme that corrects topological DNA errors in replication, transcription, recombination, and chromosome condensation and decondensation via a DNA breakage/reunion mechanism1. Although the biological functions of Topo II are important for ensuring genomic integrity, the ability to interfere with Topo II and generate enzyme-mediated DNA damage is an effective strategy for cancer chemotherapy2,3. In fact, most clinically active agents including etoposide, doxorubicin, and mitoxantrone are Topo II inhibitors, which induce DNA strand breaks, thereby causing apoptosis. Reactive oxygen species (ROS), such as the free radical superoxide radical (), the hydroxyl free radical (HO), and the non-radical hydrogen peroxide (H2O2), are unstable metabolites of molecular oxygen and associated with nearly all bodily processes4. Under physiological conditions, a certain level ROS acts as an important signaling molecule, which plays a role in the regulation of other important processes through modulation of signal pathways, thereby influencing the synthesis of antioxidant enzymes, repair processes, inflammation, and cell proliferation5,6. However, high levels of ROS lead to apoptosis and necrosis, which are implicated in cancer, aging, and neurodegenerative disorders7,8. Myelin Basic Protein (68-82), guinea pig IGFBP6 Although ROS are maintained at low levels via various enzyme systems participating in thein vivoredox homeostasis, many factors, including endogenous biological or exogenous environmental factors, can induce an imbalance between the pro-oxidants and antioxidants or alter enzyme activity, as observed with chemical exposure, drugs, pollution, or radiation4. ROS have also been implicated in the antitumor effects of some anticancer drugs9,10. A number of anticancer drugs produce ROS at various cellular sitesin vivo. Epipolythiodioxopiperazines (ETPs) are produced by fungi, and recent studies have highlighted the potential of ETPs as anticancer agents11. Many studies have shown that ETPs exert anti-tumor effects through inhibition of cancer cell growth, induction of apoptosis, and autophagy12, inhibition of DNA topoisomerase activity13, antiangiogenesis14, and induction of ROS11. G226 is a novel ETP that was synthesized by replacing methyl with acetyl at point 13 of 11-deoxyverticillin A (Figure 1). In previous studies, we have reported that G226 exhibits potent cytotoxic activity against breast cancer cells. This agent also displays remarkable apoptosis-inducing effects, which were shown to be caspase 8-dependent. Additionally, G226 triggers autophagy. Upregulated LC3 and p62 were co-localized with caspase 8, which mediated caspase 8 activation and apoptosis induction. Our group has continued to study the anti-tumor mechanism of this compound. In the current study, we investigated whether G226 altered ROS and inhibited Topo II activity, analyzed the function of ROS in G226-induced DNA damage, and determined the role that ROS play in the anti-tumor activity of G226. == Figure 1. == Chemical structure of G226. == Materials and methods == == Chemicals == G226 was kindly Myelin Basic Protein (68-82), guinea pig Myelin Basic Protein (68-82), guinea pig provided by Prof Yong-sheng CHE Myelin Basic Protein (68-82), guinea pig (Beijing Institute of Pharmacology & Toxicology, Beijing, China). The purity of the compound was greater than 98%. Adriamycin (ADR), etoposide (VP16), camptothecin (CPT), 7-ethyl-10-hydroxycamptothecin (SN38), 2,7-dichlorodihydro fluorescein diacetate (DCFH-DA), and hydroethidine (HE) were purchased from Sigma (St Louis, MO, USA). All of the above compounds were dissolved in DMSO at 10 mmol/L (usedin vitro) as a stock solution and stored at -20 C. Glutathione (GSH), N-acetylcysteine (NAC), and dithiothreitol (DTT) were purchased from Beyotime (Nantong, China), dissolved in sterile water at 100 mmol/L as a stock solution and stored at 4 C. All compounds were diluted to.
Sigma1 Receptors