6 ± 0.13 fold) associated with decreased ROS activity (0.38 ± 0.06 fold), and unchanged TXNIP RNA level in MC/CAR cells (Figure 1A-C). These results clearly show that TXNIP RNA regulation by hyperglycemia varies among multiple myeloma cell lines with a grading in response ARH77 > NCIH929 > U266B1 as compared to non-responder MC/CAR cells (Figure 1A-C). This effect translates in a consequent grading of reduced TRX activity and increased ROS level by the same order in these cell lines. On the other hand, hyperglycemia seems to have a protective effect by increasing TRX activity and reducing ROS level in MC/CAR cells, the ones not responding to glucose-TXNIP
regulation. This effect hampers ROS production in the same cell line. Figure 1 Txnip -ROS- TRX axis regulation by hyperglycemia varies among cell lines. Selleck HM781-36B Cells were grown chronically in RPMI 5 or 20 mM glucose (GLC). Data is represented as fold change over 5 mM baseline, with > 1 fold change indicating an increase over baseline and < 1 a decrease
over baseline levels. Multiple myeloma-derived ARH77, NCIH929 and U266B1, which showed glucose response, were grouped and the mean value ± SD for the group presented above.. A. Thioredoxin-interacting protein (TXNIP) RNA levels. B. Reactive l oxygen species (ROS)-levels. C.Thioredoxin (TRX) activity. Black star represents p-value compared to 5 mM, cross indicates p- value of MC/CAR compared to grouped value. Response of the TXNIP-ROS-TRX axis to DEX in conditions of hyperglycemia DEX induces hyperglycemia by itself as adverse event in some patients. Furthermore, selleck kinase inhibitor recent studies have demonstrated that TXNIP gene contains glucocorticoid-responsive Demeclocycline elements (GC-RE) and it has been described as prednisolone-responsive gene in acute lymphoblastic leukemia cells [11, 12]. We decided to study the response of TXNIP-ROS-TRX axis in vitro as
a mimicker of the in vivo situation involving a patient who either experiences GC-induced hyperglycemia or uses DEX in a condition of existing frank diabetes. Our expectations were that DEX would have had an additive effect on the axis amplifying the ROS production and the oxidative stress. When DEX was added to cells grown in condition of hyperglycemia, no additive effect was seen in NCIH929, ARH77 and U266B1 cell lines. The mean TXNIP response was similar with DEX (mean 1.29 ± 0.17) or without it (mean 1.37 ± 0.19) in the same three cell lines (e.g., compare Figure 1A and 2A). ROS levels were significantly lower as compared to isolated hyperglycemia in NCIH929 and ARH77 cells but unchanged in U266B1 (Figure 1B and 2B). TRX activity was not different compared to isolated hyperglycemia in all three-cell lines (Figure 1C and 2C). Paradoxically, the data suggested that DEX was hampering the effect of TXNIP on ROS level in NCIH929 and ARH77 cells, but not in U266B1 cells that were less sensitive to TXNIP-ROS-TRX axis regulation in the first place.