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Objective Drug resistance is the main cause of chemotherapy failure in hepatocellular carcinoma (HCC), and thioredoxin reductase 1 (TXNRD1), as a major influencing factor for reactive oxygen species (ROS) metabolism, has been proven to be associated with the poor prognosis of patients with HCC. This study aims to explore the role of TXNRD1 in the mechanism of multidrug resistance in HCC. Methods BEL/FU cells in BEL-7402 cell line were selected as the multidrug-resistant cell line. The siRNA was used for the intervention of TXNRD1 expression; quantitative real-time PCR and Western blotting were used to measure the expression of TXNRD1; CCK-8 assay and flow cytometry were used to evaluate the effect of TXNRD1 on hepatocyte ROS accumulation, resistance to 5-fluorouracil (5-Fu) and doxorubicin (DOX), and apoptosis in vitro; a xenograft tumor model was established to investigate the effect of auranofin (AUR) on drug resistance in vivo. The two-independent-samples t test was used for comparison of continuous data between two groups. Results As a multidrug-resistant HCC cell line, BEL/Fu showed high mRNA and protein expression levels of TXNRD1 (both P < 0.05). Compared with 5-Fu or DOX treatment alone, the TXNRD1 inhibitor AUR combined with 5-Fu or DOX had had a significant reduction in the number of colony formation ( P < 0.01) and a significant increase in apoptosis ratio ( P < 0.001). The ROS scavenger N-acetylcysteine (NAC) significantly weakened the effect of TXNRD1 knockdown by siRNA on the drug resistance of BEL/Fu cells, and the application of NAC effectively reduced the apoptosis ratio of cells after siRNA interference ( P < 0.001). Animal experiments also confirmed that compared with the nude mice treated with 5-Fu alone, the nude mice treated with 5-Fu and AUR had a significantly lower tumor mass ( P < 0.001) and a significantly smaller tumor volume ( P < 0.001). Conclusion TXNRD1 plays an important role in the drug resistance of HCC, and inhibition of its level in cells can effectively improve drug resistance. As a TXNRD1 inhibitor, AUR has great application prospects in the multimodality therapy for HCC.
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Objective:To investigate the effects of thioredoxin reductase 1(TR1) overexpression on hippocampus in ovariectomized SD rats.Methods:Totally 54 female SD rats were divided into normal group, ovariectomized group and ovariectomized over-expressioned TR1 group (ovariectomy-TR1 group) according to the random number table method with 18 in each group. The overexpressed TR1 vector was constructed by lentivirus, and the recombinant lentivirus was injected into the hippocampus by a brain stereotactic instrument.The mRNA levels and protein levels of TR1, Bcl-2, p53 and p21 in the hippocampus of SD rats were detected by qRT-PCR and Western blot.The expression of Caspase-3 in the hippocampus of SD rats was detected by Western blot. The activity of SOD was measured by the WST-1 cell proliferation and cytotoxicity method, the content of GSH was measured by the microplate method, and the content of MDA in the hippocampus of SD rats was measured by the TBA method. The behavioral changes of SD rats were detected by the open field test and water maze test. GraphPad Prism 9.0 was used for statistical analysis.One-way analysis of variance was used for comparisons among the three groups, and the LSD test was used for further pairwise comparisons, the t-test was used to compare the mean number of samples between the two groups. Results:(1) The mRNA and protein levels of TR1 in hippocampus of ovariectomized rats were lower than those of normal rats ( t=3.125, 4.402, both P<0.05). The mRNA and protein levels of TR1 in hippocampus of ovariectomized-TR1 rats were higher than those of ovariectomized rats ( t=4.945, 4.845, both P<0.05). (2) There were significant differences among the three groups in the escape latency in water maze test, the movement distance and the stay time in central area in the open field test ( F=44.73, 33.67, 6.51, all P<0.05), the movement distance in the open field test of ovariectomized rats was more than that of the normal group ((4 700±141) mm, (3 967±163) mm, P<0.05), the stay time in the central area was longer than that of the normal group ((87.33±3.93) s, (80.83±2.48) s, P<0.05), the movement distance ((4 267±150) mm) and the stay time in the central area ((82.17±3.43) s) of ovariectomized-TR1 group were lower than that of ovariectomized group ( P<0.05). In the water maze test, the escape latency of ovariectomized rats was longer than that of the normal group ((28.67±2.50) s, (19.50±2.59) s, P<0.05), and the escape latency in the ovariectomy-TR1 group((25.00±1.67) s) was shorter than that of ovariectomized TR1 group ( P<0.05). (3)There were significant differences in the levels of MDA, SOD and GSH in the hippocampus oxidative stress injury indexes among the three groups ( F=87.41, 91.38, 28.69, all P<0.01). The level of MDA in hippocampus of ovariectomized group was higher than that of normal group, and that in the ovariectomized-TR1 group was lower than that of ovariectomized rats group ( P<0.05). And what's more the levels of SOD and GSH in ovariectomized group were lower than those of normal group ( P<0.05), and the ovariectomized-TR1 group was higher than that of ovariectomized group ( P<0.05). (4) The results of Western blot and RT-PCR showed that the levels of p21 and p53 in hippocampal tissue of ovariectomized group were higher than those of normal group ( P<0.05), while the level of aging-related protein p21 and p53 in ovariectomized-TR1 group were significantly lower than those in ovariectomized group ( P<0.05). The level of apoptotic protein Caspase-3 in the hippocampus of ovariectomized rats was higher than that in the normal group ( P<0.05), while the level of Caspase-3 in ovariectomized-TR1 group was significantly lower than that in ovariectomized rats ( P<0.05). The level of anti-apoptotic protein Bcl-2 in hippocampus of ovariectomized group was lower than that of normal rats ( P<0.05), while the level of Bcl-2 in ovariectomized-TR1 group was significantly higher than that in ovariectomized rats ( P<0.05). Conclusion:Overexpression of TR1 can reduce apoptosis of hippocampal cells by regulating oxidative damage and reducing cell senescence.
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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.
Subject(s)
Humans , Auranofin , Cell Line, Tumor , Genetic Vectors , HEK293 Cells , Lentivirus/genetics , RNA, Messenger , TransfectionABSTRACT
Objective To study the expression and enzyme activity of thioredoxin reductase 1 (TrxR1) in liver and peripheral blood of human and rats exposed to airborne arsenic through coal-burning as well as its role in liver injury of coal-burning-borne arsenic poisoning.Methods This study was divided into 2 parts.Part 1 was a population study:133 local residents exposed to airborne arsenic through coal-burning were selected as arsenic exposure groups including a non-patient group (25 cases),no obvious hepatopathy group (38 cases),mild (43 cases) and moderate to severe hepatopathy groups (27 cases) from areas affected by endemic arsenism in Guizhou Province.Thirty-four healthy residents from arsenic not affected areas were selected as controls.Peripheral blood samples were collected from all these people.The expression of TrxR1 mRNA was determined by real-time fluorescence quantitative PCR (qPCR),and enzyme activity of TrxR was tested by visible spectrophotometry.Part 2 was an animal experiment study:Thirty Wistar rats,weighing about 80-100 g,were divided into control group,drinking-waterborne arsenic poisoning group and coal-burning-borne arsenic poisoning group (including low,medium and high arsenic contaminated grain groups) by means of a table of random number according to body mass,6 rats in each group.The control group was fed with normal diet for 3 months; drinking-water-borne arsenic poisoning group and coal-burning-borne arsenic poisoning group were fed with 10 mg/kg As2O3 solution and different concentrations(25,50,100 mg/kg) of arsenic-containing feed,respectively,for 3 months.The expression of TrxR1 mRNA was determined by qPCR; protein expression level of TrxR1 in liver tissue was detected by immunohistochemistry,and enzyme activity of TrxR in serum and liver tissue was tested by visible spectrophotometry.Results The mRNA expressions of TrxR1 in peripheral blood were 1.599 8 (1.128 9-2.156 8),1.469 3 (1.146 1-1.976 3),1.203 6 (0.463 1-1.816 2) and 0.912 3(0.631 8-1.535 0),respectively,among non-patient group,no obvious hepatopathy group,mild and moderate to severe hepatopathy groups.Compared to the control group[1.649 7(1.161 1-2.380 2)],the differences were significant statistically in mild and moderate to severe hepatopathy groups (all P ＜ 0.05).The enzyme activity of TrxR in peripheral blood was (3.12 ± 0.76),(2.81 ± 0.84),(2.52 ± 0.73),(2.42 ± 0.76)U/ml,respectively,in those corresponding groups.Compared to the control group [(3.02 ± 0.70)U/ml],the differences were significant statistically in mild and moderate to severe hepatopathy groups (all P ＜ 0.05).The mRNA expressions of TrxR1 in peripheral blood were 1.05 ± 0.14,1.18 ± 0.18,1.04 ± 0.10 and 0.97 ± 0.13,respectively,among drinking-water-borne arsenic poisoning group,low,medium and high arsenic contaminated grain groups; all of which were lower than that in the control group (1.23 ± 0.15,all P ＜ 0.05) except that of the low arsenic contaminated grain group.The mRNA expressions of TrxR1 in liver tissue were 0.78± 0.10,0.83 ± 0.10,0.79 ± 0.09 and 0.77 ± 0.11,respectively; all of which were lower than that in the control group (0.94 ± 0.12,all P ＜ 0.05).The protein expression of TrxR1 in liver tissue was 310.33 ± 38.81,312.50 ± 23.36,305.67 ± 20.57 and 298.17 ± 23.52,respectively,among the arsenic poisoning groups; all of which were lower than that in the control group (348.50 ± 32.35,all P ＜ 0.05).The enzyme activity of TrxR in serum was (4.22 ± 0.73),(4.86 ± 0.63),(4.04 ± 0.57),(3.73 ± 0.64)U/ml,respectively; all of which were lower than that in the control group [(9.52 ± 1.08)U/ml,all P ＜ 0.05].The enzyme activity of TrxR in liver tissue was (14.82 ± 1.67),(18.76 ± 2.76),(14.90 ± 2.17),(11.55 ± 1.74) U/mg,respectively; all of which were lower than that in the control group [(23.71 ± 3.05)U/mg,all P ＜ 0.05].Conclusion Arsenic aggravates liver injury of coal-burning arsenic poisoning through down-regulating the expressions of TrxR1 mRNA and protein and reducing its enzyme activity as well.
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Purpose To explore the expression of thioredoxin reductase-1 ( TrxR1 ) in non-small cell lung cancer and to observe the effects of TrxR1 on non-small cell lung cancer ( NSCLC) . Methods Immunohistochemistry was used to determine the expression of TrxR1 in 118 NSCLC tissues and 27 normal lung tissues, and to analyze its relationships with clinical pathological features. TrxR1 mR-NA levels were measured by quantitative RT-PCR in 13 cases of NSCLC and 4 cases of normal lung tissue. TrxR1 protein and its mR-NA levels were assessed by Western blotting and quantitative RT-PCR in normal lung epithelial cells and different lung cancer cell lines. After the silence TrxR1 expression in A549 cells, the changes of proliferation were compared. Results The data of immunohis-tochemistry and quantitative RT-PCR showed the overexpression of TrxR1 in NSCLC ( P <0. 05 ) . After using shRNA silencing of TrxR1 in lung cancer cells, cell proliferation changed significantly. Conclusion There are a high mRNA and protein levels of TrxR1 in NSCLC. TrxR1 may be a critical therapeutic target for the treatment in NSCLC.
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PURPOSE: Drug-induced liver injury (DILI) is the most common adverse drug reaction; however, it is not easily predicted. We hypothesize that DILI has a common genetic basis. Based on the findings of previous animal studies on toxic hepatitis, we selected the thioredoxin reductase 1 gene (TXNRD1) as a candidate marker of DILI for this genetic association study. METHODS: Records from 118 patients with DILI were extracted from the database of the Adverse Drug Reaction Research Group in South Korea. Causative drugs included antituberculosis drugs (n=68, 57.6%), antibiotics (n=22, 18.6%), antiepileptic drugs (n=7, 5.9%), non-steroidal anti-inflammatory drugs (n=5, 4.2%), and others (n=16, 13.7%). Seven single nucleotide polymorphisms (SNPs) in TXNRD1 (rs10735393, rs4964287, rs4595619, rs10861201, rs11111997, rs4246270, and rs4246271) were scored in 118 DILI patients and in 120 drug-matched controls without liver injury. RESULTS: No differences were found between the frequencies of any of the 7 SNPs in the cases and controls; however, a significant association was found between a TTA haplotype composed of rs10735393, rs4964287, and rs4595619 and DILI using an allele model (odds ratio, 1.79; 95% confidence interval, 1.18-2.73; P=0.008; Bonferroni corrected P=0.024). CONCLUSIONS: These results suggest that genetic variations in TXNRD1 favor the development of DILI, although a larger confirmative study is needed.
Subject(s)
Animals , Humans , Alleles , Anti-Bacterial Agents , Anticonvulsants , Drug-Related Side Effects and Adverse Reactions , Chemical and Drug Induced Liver Injury , Genetic Association Studies , Genetic Variation , Haplotypes , Liver , Polymorphism, Genetic , Polymorphism, Single Nucleotide , Republic of Korea , Thioredoxin Reductase 1ABSTRACT
This study investigated the effects of hyperoxia on dynamic changes of thioredoxin-1 (Trx1)and thioredoxin reductase-1 (TrxR1) in alveolar type Ⅱ epithelial cells (AECⅡ) of premature rats.Pregnant Sprague-Dawley rats were sacrificed on day 19 of gestation.AEC Ⅱ were isolated and purified from the lungs of premature rats.When cultured to 80％ confluence,in vitro cells were randomly divided into air group and hyperoxia group.Cells in the hyperoxia group were continuously exposed to 95％ O2/5％ CO2 and those in the air group to 95％ air/5％ CO2.After 12,24 and 48 h,cells in the two groups were harvested to detect their reactive oxygen species (ROS),apoptosis,TrxR1 activity and the expressions of Trx1 and TrxR1 by corresponding protocols,respectively.The results showed that AEC Ⅱ exposed to hyperoxia generated excessive ROS and the apoptosis percentage in the hyperoxia group was increased significantly at each time points as compared with that in the air group (P＜0.001).Moreover,TrxR1 activity was found to be markedly depressed in the hyperoxia group in comparison to that in the air group (P＜0.001).RT-PCR showed the expressions of both Trx1 and TrxR1 mRNA were significantly increased in AEC Ⅱ exposed to hyperoxia for 12 and 24 h (P＜0.01),respectively.At 48 h,the level of Trx1 mRNA as well as that of TrxR1 mRNA in the hyperoxia group was reduced and showed no significant difference from that in the air group (P＞0.05).Western blotting showed the changes of Trx 1 protein expressions in the hyperoxia group paralleled those of Trx1 mRNA expressions revealed by RT-PCR.It was concluded that hyperoxia can up-regulate the protective Trx1/TrxR1 expressed by AEC Ⅱ in a certain period,however,also cause dysfunction of the cytoplasmic thioredoxin system by decreasing TrxR1 activity,which may contribute to the progression of oxidative stress and cell apoptosis and finally result in lung injury.
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Thioredoxin reductase(TrxR) is an important component of thiol regulating system and contains a reactive and solvent accessible selenocysteine residue which is located on a flexible C-terminal arm of the protein and can combine various substrates and inhibitors. Many tumor cells have elevated TrxR levels and TrxR has also been shown to play a major role in drug resistance. A great number of effective natural and synthetic TrxR inhibitors are now availably possessing antitumor potential through induction of oxidative stress, cell cycle arrest and apoptosis. Inhibitors of TrxR may thus contribute to a successfully single, combinatory or adjuvant cancer therapy.
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Thioredoxin reductase 1 (TrxR) is a homodimeric selenoenzyme catalyzing thioredoxin (Trx) in an NADPHdependent manner. With regard to carcinogenesis, these redox proteins have been implicated in cell proliferation, transformation and anti-apoptosis. In the present study, using a hamster cholangiocarcinoma (ChC) model, we evaluated the immunohistochemical expression pattern of TrxR in precancerous lesions and ChCs as well as in normal bile ducts. The goal of this study was to determine the potential role and importance of TrxR in cholangiocarcinogenesis. For the ChC model, we obtained liver tissue specimens with dysplastic bile ducts prior to the development of ChC 8 weeks after initiation of the experiment and ChC samples at 27 weeks. The immunohistochemical analysis showed diffuse cytoplasmic overexpression of TrxR in the dysplastic bile duct epithelial cells as well as in cholangiocarcinoma; this was comparable to the negative or weakly positive in normal and type 1 hyperplastic bile ducts. However, TrxR appeared to be considerably down-regulated in the ChCs when compared to the higher expression observed in the dysplastic bile ducts. Therefore, these results suggest that TrxR overexpression followed by down-regulation might be an important event in cholangiocarcinogenesis, especially at early stages including the cellular transformation of candidate bile ducts. Further studies are however required to determine whether TrxR may be a potential target molecule for chemoprevention against cholangiocarcinogenesis. In addition, the molecular mechanism as well as the importance of the loss of TrxR in the development of cholangiocarcinoma, following dysplastic transformation of bile duct cells, also remains to be clarified.