OBJECTIVE: To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs. METHODS: TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay. RESULTS: TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005). CONCLUSION We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.
Auranofin ; Cell Line, Tumor ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus/genetics* ; RNA, Messenger ; Transfection

BACKGROUND: Acute promyelocytic leukemia (APL) is distinguished from other forms of leukemia by its association with bleeding diatheses. All-trans retinoic acid (ATRA) and arsenic trioxide (As2O3), which have been effectively used in the treatment of the APL, promptly improve coagulation/bleeding syndromes by regulating the expressions of tissue factor (TF) and thrombomodulin (TM). We have previously shown a novel activity of auranofin (AF), in that it induced apoptosis and differentiation of NB4 cells. To study whether AF also possesses similar anticoagulant effects to those of ATRA and As2O3, its effects on the expressions of TM and TF were investigated. METHODS: NB4 cells derived from APL were incubated with 1 micrometer of AF. After incubation for 12, 24 and 48 hours, the AF-regulated expressions of TM and TF were analyzed by RT-PCR, Northern blot and Western blot. The assay for the TM antigen on the cell surface was performed using a flow cytometry. RESULTS: The expression of the TM gene was increased for upto 12 hours after the AF treatment, but no change was observed in the expression of the TF gene. Western blot analysis also demonstrated that AF increased the level of TM proteinin a time-dependent manner. FACS data showed the TM antigen on the cell surface to gradually increase for upto 48 hours in AF-treated cells. CONCLUSION: The results of this study indicate that AF can have an antithrombotic function via the up-regulation of the expression of TM, which suggests it may partially contribute to the improvement of coagulopathies in APL.
Apoptosis ; Arsenic ; Auranofin* ; Blotting, Northern ; Blotting, Western ; Disease Susceptibility ; Flow Cytometry ; Hemorrhage ; Leukemia ; Leukemia, Promyelocytic, Acute* ; Thrombomodulin* ; Thromboplastin ; Tretinoin ; Up-Regulation

Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells.
Annexin A5* ; Antirheumatic Agents ; Apoptosis ; Auranofin* ; Blotting, Western ; Gene Expression ; Humans* ; Leukemia, Lymphocytic, Chronic, B-Cell ; Plasminogen Activator Inhibitor 2 ; Plasminogen Activators* ; Plasminogen* ; Prostate* ; Prostatic Neoplasms* ; RNA, Small Interfering ; Statistics as Topic

Antirheumatic gold compounds have been shown to inhibit NF-kB activation by blocking IkB kinase (IKK) activity. To examine the possible inhibitory mechanism of gold compounds, we expressed wild type and mutant forms of IKk alpha and beta subunits in COS-7 cells and determined the effect of gold on the activity of these enzymes both in vivo and in vitro. Substitution of Cys-179 of IKK beta with alanine (C179A) rendered the enzyme to become resistant to inhibition by a gold compound auranofin, however, similar protective effect was not observed with an equivalent level of IKK alpha (C178A) mutant expressed in the cells. Auranofin inhibited constitutively active IKK alpha and beta and variants; IKK alpha (S176E, S180E) or IKK beta (S177E, S181E), suggesting that gold directly cause inhibition of activated enzyme. The different inhibitory effect of auranofin on IKK alpha (C178A) and IKK beta (C179A) mutants indicates that gold could inhibit the two subunits of IKK in a different mode, and the inhibition of NF- kB and IKK activation induced by inflammatory signals in gold-treated cells appears through its interaction with Cys-179 of IKK beta.
Amino Acid Substitution ; Animals ; Auranofin/*pharmacology ; COS Cells ; Cysteine/genetics/*metabolism ; Enzyme Activation/drug effects ; Gold Compounds/*pharmacology ; Protein Subunits/chemistry ; Protein-Serine-Threonine Kinases/*antagonists & inhibitors/chemistry/genetics/*metabolism ; Sulfhydryl Compounds/pharmacology