Effects Of NADPH-dependent Enzyme On Tumor Resistance And Analytical Study Of Cellular NADPH Homeostasis

Mammalian thioredoxin reductase (TrxR), one of NADPH-dependent enzymes, catalyzes the reduction of oxidized thioredoxin (Trx) by delivering electrons from NADPH, plays a central role in regulating cellular redox homeostasis, cell growth and apoptosis. Increasing evidences show that TrxR is over-expressed in many tumor cells, which is closely related to increased tumor cell growth and a resistance to chemotherapy. It might be a target for therapeutic intervetion. In this paper, we evaluated the activity of TrxR in drug sensitive parental lines (K562) and drug resistant leukemic cell sublines (K562/ADM), and found that TrxR activity was higher in K562/ADM cells compared to K562 cells. Auranofin, a gold (I) compound clinically used as an antirheumatic agent, significantly inhibited the activity of TrxR in K562/ADM cells with the treatment for 24 h. The growth inhibitory effects of auranofin and adriamycin toward K562/ADM cells were evaluated by means of MTT test. Cells were tested and analyzed with the concentrations of 0.25,0.5,1,2 and 3μM of auranofin and adriamycin for 6 h, and it showed that the proliferation of K562/ADM cells exposed to adriamycin was decreased respectively by 0%,1.1%,7.9%,3.9% and 6.6%, while the cell viability of auranofin treatment group was obviously decreased by 22.8%,34%,52.9%,67% and 81.3%, respectively. When the exposure time was increased to 24 h and 48 h, auranofin decreased the viability of K562/ADM cells in a time-dependent manner compared to adriamycin. Flow cytometry (FCM) technique was employed to detect the percentage of apoptotic cells and caspase-3 expression. The results indicated that the percentage of apoptotic cells with auranofin for 24 h was increased concentration-dependently from 2.1% to 24.6%, which was significantly higher than that in the adriamycin treated cells (from 2.1% to 4.8%, P<0.05 between auranofin and adriamycin treatment). The activation of caspase-3 was increased by 53% in k562/ADM cells after 12 h of treatment with 1μM auranofin, which was higher than in the adriamycin treatment of 4.5%(P<0.05 between auranofin and adriamycin treatment). These results clearly demonstrate that TrxR inhibitor-auranofin reduces TrxR activity, exhibiting cytotoxic effect and inducing cell apoptosis with an increased activation of caspase-3. It might be a candidate drug to overcome the drug resistance of human chronic myelogenous leukemia. NADPH is an important cofactor of many biosynthesis pathways and involves in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), and isocitrate dehydrogenase (IDH). These three enzymes are part of the hexose monophosphate shunt, pyruvate cycle, and tricarboxylic acid (TCA) cycle, respectively. To understand how these three enzymes maintain the cellular NADPH homeostasis, we generated G6PD-knockdown A549 cells in which approximately 10% residual activity of G6PD existed compared to the controls. There were no significant differences in NADPH level in control and G6PD-knockdown cells by HPLC analysis. The relative levels of three NADPH-producing proteins in both these cells were obtained by isobaric tags for relative and absolute quantitation (iTRAQ) labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). A total of 1760 proteins with the confidential level of≥95% were indentified, and among them 7 proteins were NADPH-producing enzymes. In the proteomic analysis, we found that G6PD knockdown could cause a 1.33-fold increase in expression of cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc), while the change in ME expression was not significant. Western blot analyses and enzyme activity assays confirmed that the levels of IDPc were significantly elevated in G6PD-knockdown A549 cells and its activity was proved to be 1.55-fold higher compared to control cells, while the change in the expression and activities of ME was not different. Our results suggest that IDPc rather than ME is a key player in maintaining homeostatic balance of NADPH/NADP+ in G6PD-knockdown A549 cells.