However, the mechanism remains elusive, especially the source of ROS has not well investigated yet. Se, including organic-Se, inorganic Se and Se-containing proteins, are all enzymatically or non-enzymatically metabolized in the biological environment, and finally incorporated into Se-containing proteins16. our pervious studies, generation of reactive oxide varieties (ROS), oxidative stress-mediated DNA damage, mitochondrial dysfunction, imbalance of Bcl-2 family manifestation, and the dysregulation of MAPKs and AKT pathways all contributed to Se-containing compounds-induced apoptosis in several human being malignancy cells11C15. We have emphasized the significance of ROS in Se-containing compounds-induced apoptosis in our earlier reports11C15. However, the mechanism remains elusive, especially the source of ROS has not well investigated yet. Se, including organic-Se, inorganic Se and Se-containing proteins, are all enzymatically or non-enzymatically metabolized in the biological environment, and finally integrated into Se-containing proteins16. Se can function in the active sites of a large number of Se-containing enzymes, such as glutathione peroxidase (GSH-Px) and thioredoxin reductase (TrxR) 17C19. Selenocysteine mainly because the major form found in Se-containing proteins takes on important part in regulating the intracellular redox balance16. Se product either enhance the intracellular antioxidant ability by replenishing the Se-containing enzymes, or induce ROS-mediated malignancy cell apoptosis through disturbing hucep-6 the antioxidase system, which depends on the form and dose of Se-containing compounds. TrxR like a selenium-containing oxidoreductases is definitely overpressed in many human being tumors and is of significance in keeping intracellular redox balance18, 19. Hence, the TrxR offers emerged as potential target for anticancer drug design. Selenocystine (SeC) a natural available Se-containing amino acid has been proven effective in inhibiting several cancer cells growth by induction of cell cycle arrest or/and apoptosis through triggering ROS-mediated oxidative damage in our earlier studies5, 11C15. For instance, SeC can inhibit A549 human being lung adenocarcinoma cells growth through inhibition of TrxR activity and TrxR manifestation and and and through induction Glyparamide of apoptosis. Open in a separate window Number 1 SeC induces apoptosis in human being glioma cells. (A) Cell apoptosis and cell cycle distribution. U251 cells exposed to SeC were assayed by circulation cytometric analysis for cell apoptosis and cell cycle distribution. The hypodiploid DNA content (Sub-G1 peak) were considered as the apoptotic cell death. (B) Activation of caspases. U251 cells exposed to SeC were collected and total protein was extracted and incubated with specific caspase Glyparamide substrates for examination of caspase activity as explained in method section. (C) DNA fragmentation. U251 cells exposed to SeC was imaged by TUNEL-DAPI staining. Dose- (D) and time-dependent (E) effects of SeC on caspases activation and PARP manifestation. The manifestation of caspases and PARP was recognized by western blotting methods. All data and images are showed with three self-employed experiments. Bars with * or ** show the statistically different in the as an early apoptotic event was obviously observed as early as in 2?h by JC-1 probe, while depicted from the fluorescence shift from red to green in SeC-treated U251 cells (Fig.?2A). Moreover, SeC treatment also caused mitochondrial fragmentation. As demonstrated in Fig.?2B, health U251 cells showed filamentous mitochondrial network with extensively Glyparamide interconnection throughout the cytoplasm. SeC treatment dramatically caused the mitochondrial fragmentation from protonema to punctiform. These findings clearly suggested that SeC caused mitochondrial dysfunction in U251 cells. Bcl-2 family, including the pro-apoptotic and pro-survival users, has been identified as essential factors in regulating the mitochondrial permeability21, 22. Consequently, it is of great significance to detect whether the imbalance of Bcl-2 family was involved in SeC-induced mitochondrial dysfunction. As demonstrated in Fig.?2C, SeC treatment dose-dependently suppressed the Bcl-2 and Bcl-XL expression, but increased the expression of Bax and Bad. The time-course showed that SeC caused continuous down-regulation of Bcl-2 and up-regulation of Bad at the point of 12?h. These results above suggested Glyparamide that SeC induced mitochondria-mediated apoptosis by triggering mitochondrial dysfunction through influencing Bcl-2 family balance. SeC causes ROS-mediated DNA damage Previous studies possess found that SeC inhibited human being glioma cells growth in 48?h mainly by induction of S-phase arrest through triggering ROS-mediated DNA damage5. To explore the oxidative status in SeC-induced apoptosis, we consequently investigated the ROS generation and several oxidative damage markers. As display in Fig.?3A, SeC treatment resulted.