As we can see from Figure 2(F,G), the total Trx1 protein expression showed no significant difference between the treated groups and untreated groups, so total Trx1 of the HepG2 cells were not affected by treatment of the CPUL1; however, the monomer of Trx1 ratio was decreased and Trx1 dimer ratio was increased after treated with CPUL1 in both dose- and time-dependent fashion against negative and positive control groups (Figure 2(F,G)), respectively. 1(B)), which was confirmed by laser scanning confocal microscopy (LSCM). This freakishly phenomenon was distinguishing from typical topoisomerase I/II inhibitors, such as doxorubicin12, etoposide13 and 10-hydroxycamptothecin14, which were reported as locating at nucleus in cancer cell lines by LSCM methods. The discrepant results of CPUL1 between the LSCM and topoisomerase I/II inhibition experiments aroused a suspicion that the CPUL1 might not targeting to the topoisomerase I/II in Hep G2 cell lines. Considering the controversial role of the CPUL1 against Hep G2 cells, the target of CPUL1 against Hep G2 cells becomes the crux of the scene to be unveiled. Thus, we attempted to discover and identify the anticancer target of CPUL1 in this study. Open in a separate window Figure 1. The preliminary experiment, including design strategy, LSCM and time course of the redox related key factor for investigating the target of CPUL1. (A) Design of ROS inducer molecule CPUL1 with molecular hybridization strategy. (B) The distribution of CPUL1 in the Hep G2 cells. Hep G2 cells were stained with 2?M CPUL1, 0.1?M Mito Tracker Red CMXROS, and 0.1? Dihydrochloride (DAPI) for 30?min. (i) Ex = 488?nm for CPUL1. (ii) Ex = 580?nm for Mito Tracker Red CMXROS. (iii) Ex = 360?nm for DAPI. (iv) Merged images of (i) and (iii) in dark field. (v) Merged images of (i) and (iii) in bright field. (C) A summary plot displays the time relationships between the Trx1red/Trx1total ratio, ROS levels, GSH/GSSG ratio, NADPH lifetimes and ATP contents in Hep G2 cells treated with 2?M of CPUL1. Materials and methods The general procedures, the details concerning the experiment steps and the analytical data are provided in the Supplementary Material. Results and discussion Since we observed GABOB (beta-hydroxy-GABA) visible apoptosis of Hep G2 cells after treated with CPUL1, we sought to find clues from the process of redox status. We GABOB (beta-hydroxy-GABA) tested the time courses of redox related key factors in Hep G2 cells, among them ROS levels, GSH/GSSG ratios, NAPDH levels and ATP levels before and after treated with CPUL1 at different time, respectively (Figure 1(C) and Figures S1CS4, see Supplementary Material). In these results, most unexpectedly, the ROS levels were Rabbit Polyclonal to POU4F3 dramatically increased at the first 15?min (Listed in Figure 1(C) and Figure S1). GABOB (beta-hydroxy-GABA) However, NADPH (Figure S4) and ATP levels (Figure S2) did not show significant differences with control groups before 18?h, respectively. It is widely recognized that the depletion of NADPH and ATP is associated with the pace of apoptosis15,16. However, the stable NADPH and ATP levels in the first 4?h after treated with CPUL1 can deduce a result that ATP mediating the ROS produce process did rather not take place in HepG2 cells after treated by CPUL1. Combined the results of the redox related key factors time-course study, a conjectural apoptosis process was hypothesized as following: (1) CPUL1 could trigger apoptosis mainly through elevating the ROS level rather than inhibiting the topoisomerase I/II; and (2) deleting ROS function instead of accelerating ROS production might be inhibited by CPUL1 in apoptosis GABOB (beta-hydroxy-GABA) cells. In mammalian cells, there are two major thiol-dependent antioxidant systems, the thioredoxin- (Trx) and the glutathione- (GSH) dependent enzyme systems which may act in concert17,18. In the next experiment, we tried to verify if there were significant differences between Trx1red/Trx1total and GSH/GSSG levels under treatment of CPUL1 in Hep G2 cell lines. Amazingly, Trx1red/Trx1total levels decreased to 57% at 0.25?h GABOB (beta-hydroxy-GABA) and 43% at 0.5?h (Figure 2(G)), whereas, GSH/GSSG ratios are markedly decreasing after 2?h (Figure S3), respectively. These results can be elucidated that the reductive Trx1 level decreased dramatically at the first 0.5?h, and the ROS level simultaneously increased by 3.4-folds, then the GSH compensation mechanism had come into force and decreased to 24% after 2?h. Harris18 and Mandal19 have also demonstrated homoplastically standpoint that the Trx1 and GSH can work synergistically as antioxidant.