Selenite-induced apoptosis in doxorubicin-resistant cells and effects on the thioredoxin system

Selenium treatment of the doxorubicin-resistant cell line, U-1285dox, derived from human small cell carcinoma of the lung, resulted in massive apoptosis. This effect appeared maximal at 2 days after addition of selenite. The apoptosis was caspase-3 independent as revealed by Western blot analysis, activity measurement and by using caspase inhibitors. Induction of apoptosis was significantly more pronounced and occurred after addition of lower concentrations of selenite in the doxorubicin-resistant cells compared to the parental doxorubicin-sensitive cells. High levels of selenite caused necrosis in the doxorubicin-sensitive cells. Analysis of enzymatic activity (insulin reduction) of thioredoxin reductase (TrxR) and TrxR protein concentration, measured by ELISA, revealed increasing activity and protein levels after treatment with increasing concentrations of selenium. Maximum relative increase was induced up to 1 μM in both sublines and at this selenium level the concentrations of TrxR measured as insulin reducing activity or ELISA immunoreactivity were nearly identical. Increasing concentrations of selenite up to 10 μM resulted in increased activity and concentration of TrxR in the sensitive subline but decreasing levels in the resistant subline. The level of truncated Trx (tTrx) was higher in the resistant U-1285dox cells but the level did not change with increasing selenite concentrations. Our results demonstrate pronounced selective selenium-mediated apoptosis in therapy-resistant cells and suggest that redox regulation through the thioredoxin system is an important target for cancer therapy.     

Drug-resistant human lung cancer cells are more sensitive to selenium cytotoxicity. Effects on thioredoxin reductase and glutathione reductase

The human U-1285 and GLC(4) cell lines, both derived from small cell carcinoma of the lung, are present in doxorubicin-sensitive (U-1285 and GLC(4)) and doxorubicin-resistant MRP-expressing (U-1285dox and GLC(4)/ADR) variants. These sublines were examined here with respect to their susceptibilities to the toxic effects of selenite and compared to the toxic effects of selenite on the promyelocytic leukemia cell line HL-60 and its doxorubicin-resistant P-glycoprotein expressing variant. The drug-resistant U-1285dox and GLC(4)/ADR sublines proved to be 3- and 4-fold, respectively, more sensitive to the cytotoxicity of selenite than the drug-sensitive U-1285 and GLC(4) sublines, whereas no difference was observed between the HL-60 sublines. The presence of doxorubicin at a concentration equal to the IC(10) did not significantly potentiate the toxic effects of selenite. The presence of selenite did not significantly affect the expression of the multi-drug resistant proteins (MRP1, LRP and topoisomerase IIalpha) in the drug-resistant cells. The activities of thioredoxin reductase (TrxR) were higher (50 and 25%, respectively) in the drug resistant cell sublines U-1285dox and GLC(4)/ADR compared to the drug-sensitive parental lines. The activity of glutathione reductase (GR) was essentially the same in the drug-sensitive and -resistant cell lines. Exposure to selenite resulted in a 4-fold increase in both TrxR and GR activities in U-1285 cells, an effect, which was less pronounced in the presence of doxorubicin. Under similar conditions the increase in the TrxR activity in the resistant U-1285dox cell line, was only 30% and the activity of GR was unaltered. Different responses in the activity of the key enzymes in selenium metabolism are one possible mechanism explaining the differential cytotoxicity of selenium in these cells.     

Susceptibility of the antioxidant selenoenyzmes thioredoxin reductase and glutathione peroxidase to alkylation-mediated inhibition by anticancer acylfulvenes

Selenium, in the form of selenocysteine, is a critical component of some major redox-regulating enzymes, including thioredoxin reductase (TrxR) and glutathione peroxidase (Gpx). TrxR has emerged as an anticancer target for drug development due to its elevated expression level in many aggressive human tumors. Acylfulvenes (AFs) are semisynthetic derivatives of the natural product illudin S and display improved cytotoxic selectivity profiles. AF and illudin S alkylate cellular macromolecules. Compared to AFs, illudin S more readily reacts with thiol-containing small molecules such as cysteine, glutathione, and cysteine-containing peptides. However, a previous study indicates that the reactivity of AFs and illudin S with glutathione reductase, a thiol-containing enzyme, is inversely correlated with the reactivity toward small molecule thiols. In this study, we investigate mechanistic aspects underlying the enzymatic and cellular effects of the AFs and illudin S on thioredoxin reductase. Both AF and HMAF were found to inhibit mammalian TrxR in the low- to submicromolar range, but illudin S was significantly less potent. TrxR inhibition by AFs was shown to be irreversible, concentration- and time-dependent, and mediated by alkylation of C-terminus active site Sec/Cys residues. In contrast, neither AFs nor illudin S inhibits Gpx, demonstrating that enzyme structure-specific small molecule interactions have a significant influence over the inherent reactivity of the Sec residue. In human cancer cells, TrxR activity can be inhibited by low micromolar concentrations of all three drugs. Finally, it was demonstrated that preconditioning cells by the addition of selenite to the cell culture media results in an enhancement in cell sensitivity toward AFs. These data suggest potential strategies for increasing drug activity by combination treatments that promote selenium enzyme activity.     

The effect of sodium selenite on liver growth and thioredoxin reductase expression in regenerative and neoplastic liver cell proliferation

Selenium in supra-nutritional doses is tumour-preventative in animal models and in humans. In this work, we have compared the effect of sodium selenite on tumour growth in a rat hepatocarcinogenesis model with the effect of sodium selenite on the regeneration of liver mass after partial hepatectomy. In the tumour model, 5 μg/mL sodium selenite in the drinking water reduced the rate of tumour growth for up to 12 months after initiation; the volume fraction of liver cancers was 14±4% with a mean bromodeoxyuridine-index of 19±11% in the treated rats compared to a volume fraction of 26±7% with a mean bromodeoxyuridine-index of 42±27% in the control rats. Despite its efficacy in reducing tumour growth, 5 μg/mL sodium selenite treatment did not affect the gain of liver mass or the rate of cell proliferation after partial hepatectomy. In the regenerating livers, the activity of the cytosolic selenoenzyme thioredoxin reductase (TrxR1) was briefly and transiently increased, an increase further potentiated by sodium selenite. TrxR1 was selectively over expressed in proliferating liver tumours in relation to the surrounding liver tissue in the tumour model, as shown using immunohistochemistry analyses. We suggest that sodium selenite is a suitable candidate for liver cancer prevention in patients with chronic liver diseases that are dependent on sustained liver regeneration due to its differential effects on neoplastic and regenerative cell proliferation. Furthermore, the over expression of TrxR1 in liver neoplasia can only partly be explained by increased growth.     

Further insight into the impact of sodium selenite on selenoenzymes: high-dose selenite enhances hepatic thioredoxin reductase 1 activity as a consequence of liver injury

Selenium (Se) at supranutritional levels can enhance the activity of glutathione S-transferase (GST), whose gene is a target of nuclear factor erythroid-2 related factor 2 (Nrf2). Recent studies indicated that the thioredoxin reductase 1 (TrxR1) gene could also be targeted by Nrf2. Thus, high-dose Se may stimulate TrxR1 provided it enhances GST activity. Indeed, one study found that Se at supranutritional levels transiently increased hepatic TrxR1 activity. However, another study reported that supranutritional Se had no such effect on hepatic TrxR1 activity. In view of this discrepancy, the present research investigated whether high-dose Se has any impact on hepatic TrxR1. Moreover, we investigated whether Se preferentially activates GST over TrxR1. We observed that when sodium selenite (SS) caused liver injury, both hepatic TrxR1 activity and hepatic GST activity increased. Further experiments indicated that SS increased hepatic GST activity at either toxic or high but non-toxic dose levels; however, increase in hepatic TrxR1 activity occurred only at toxic levels, suggesting that enhanced TrxR1 activity correlates with liver injury. To corroborate this, we showed that hepatotoxic agents, thioacetamide or carbon tetrachloride, caused marked increases in hepatic TrxR1 activity. In conclusion, high-dose SS indeed can enhance hepatic TrxR1 activity, but only on the condition that it causes liver injury. High-dose SS affects hepatic GST more readily than hepatic TrxR1. Thus, the cancer-preventive mechanism of Se at non-toxic supranutritional levels relies more on its modulation of GST rather than TrxR1, at least in liver tissue.     

The role of thioredoxin reductase activity in selenium-induced cytotoxicity

The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1 microM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50 microM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50 microM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1 microM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.     

Gold(I) carbene complexes causing thioredoxin 1 and thioredoxin 2 oxidation as potential anticancer agents

Gold(I) complexes with 1,3-substituted imidazole-2-ylidene and benzimidazole-2-ylidene ligands of the type NHC-Au-L (NHC = N-heterocyclic carbene L = Cl or 2-mercapto-pyrimidine) have been synthesized and structurally characterized. The compounds were evaluated for their antiproliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin (A2780S/R), as well in the nontumorigenic human embryonic kidney cell line (HEK-293T), showing in some cases important cytotoxic effects. Some of the complexes were comparatively tested as thioredoxin reductase (TrxR) and glutathione reductase (GR) inhibitors, directly against the purified proteins or in cell extracts. The compounds showed potent and selective TrxR inhibition properties in particular in cancer cell lines. Remarkably, the most effective TrxR inhibitors induced extensive oxidation of thioredoxins (Trxs), which was more relevant in the cancerous cells than in HEK-293T cells. Additional biochemical assays on glutathione systems and reactive oxygen species formation evidenced important differences with respect to the classical cytotoxic Au(I)-phosphine compound auranofin.     

Inhibition of thioredoxin reductase by auranofin induces apoptosis in adriamycin-resistant human K562 chronic myeloid leukemia cells

Mammalian thioredoxin reductase (TrxR) catalyzes the NADPH-dependent reduction of oxidized thioredoxin (Trx) and plays a central role in regulating cellular redox homeostasis, cell growth and apoptosis. Increasing evidence shows that TrxR is over-expressed or constitutively active in many tumor cells. Moreover, TrxR appears to contribute to increased tumor cell growth and a resistance to chemotherapy. In this study, we evaluated the activity of TrxR in adriamycin-resistant leukemic cells (K562/ADM) and adriamycin-sensitive parental lines (K562), and found that TrxR activity was higher in the drug resistant cell sublines K562/ADM than in K562 drug sensitive parental cells. Auranofin, a gold(I) compound clinically used as an antirheumatic agent, reduced TrxR activity and was more effective than adriamycin in decreasing cell viability in K562/ADM cells. In addition, auranofin induced apoptosis in dose-dependent manners, accompanied by caspase-3 activation in K562/ADM cells. Our results demonstrate that inhibition of TrxR and induction of apoptosis by auranofin provides its ability in overcoming adriamycin resistance in K562/ADM cells.     

Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, ‘auranofin-like’ gold(I) complexes all containing the [Au(PEt₃)]⁺ synthon and the ligands: Cl⁻, Br⁻, cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.     

Exposure to monomethylarsonous acid (MMA) leads to altered selenoprotein synthesis in a primary human lung cell model

Monomethylarsonous acid (MMA^III), a trivalent metabolite of arsenic, is highly cytotoxic and recent cell culture studies suggest that it might act as a carcinogen. The general consensus of studies indicates that the cytotoxicity of MMA^III is a result of increased levels of reactive oxygen species (ROS). A longstanding relationship between arsenic and selenium metabolism has led to the use of selenium as a supplement in arsenic exposed populations, however the impact of organic arsenicals (methylated metabolites) on selenium metabolism is still poorly understood. In this study we determined the impact of exposure to MMA^III on the regulation of expression of TrxR1 and its activity using a primary lung fibroblast line, WI-38. The promoter region of the gene encoding the selenoprotein thioredoxin reductase 1 (TrxR1) contains an antioxidant responsive element (ARE) that has been shown to be activated in the presence of electrophilic compounds. Results from radiolabeled selenoproteins indicate that exposure to low concentrations of MMA^III resulted in increased synthesis of TrxR1 in WI-38 cells, and lower incorporation of selenium into other selenoproteins. MMA^III treatment led to increased mRNA encoding TrxR1 in WI-38 cells, while lower levels of mRNA coding for cellular glutathione peroxidase (cGpx) were detected in exposed cells. Luciferase activity of TrxR1 promoter fusions increased with addition of MMA^III, as did expression of a rat quinone reductase (QR) promoter fusion construct. However, MMA^III induction of the TRX1 promoter fusion was abrogated when the ARE was mutated, suggesting that this regulation is mediated via the ARE. These results indicate that MMA^III alters the expression of selenoproteins based on a selective induction of TrxR1, and this response to exposure to organic arsenicals that requires the ARE element.     

Augmented antitumor effects of combination therapy of cisplatin with ethaselen as a novel thioredoxin reductase inhibitor on human A549 cell in vivo

Ethaselen (1, 2-[bis (1, 2-Benzisoselenazolone-3 (2H) -ketone)] ethane, BBSKE), as a novel organoselenium compound targeting thioredoxin reductase (TrxR), has been reported to inhibit tumor growth and TrxR activity in several human tumor cell lines. It has now entered Phase I clinical trails. Here we report the effects of ethaselen and cisplatin (cis-diamminedichloroplatinum II, DDP) combination therapy (ethaselen 36 mg/kg, i.g., o.d. × 10 d and cisplatin 1 mg/kg, i.p., single at day 0) on human A549-grafted nude mouse model (female, BALB/c nude mouse, n = 5, treatment after tumor volume reached 100 mm³). Compared to single drug administration (either ethaselen: 36 mg/kg, i.g., o.d. × 10 d or cisplatin: 1.0 mg/kg, i.p., single at day 0), the combination therapy showed significantly reduced tumor size (presumably due to a synergistic effect) and no obvious toxic damage (both in terms of body weight maintenance and liver/kidney damage). These results will be significant in the development of novel anti-tumoral therapeutic strategies directed to non-small cell lung cancer (NSCLC).     

硫氧还蛋白还原酶作为新型生物标志物在乳腺癌诊断和治疗中的价值

已有研究证明硫氧还蛋白还原酶在多种人原发肿瘤中过度表达,其活性在肿瘤人群和健康人群中有明显差异。本义研究了健康人群和乳腺癌患者中TrxR活性的不同,并探讨了硫氧还蛋白还原酶作为乳腺癌治疗中的一个新型治疗靶点的可能性。通过研究．我们第一次发现未经治疗的肿瘤患者的TrxR活性值水平明显高于健康人群。存乳腺癌患者中,TrxR活性值在不同的年龄组和癌症分期组没有明显的差异。当TrxR活性值小于10U／mL时,TrxR活性值和肿瘤影像学评价结果有很高的一致性。这些发现证明硫氧还蛋白还原酶可以作为一个有效的新型生物标志物用于对乳腺痛临床治疗效果的监测和评价,并能成为乳腺癌早期检测的一种方法。     

Effect of selenium-containing compounds on hepatic chemoprotective enzymes in mice

Selenite and organoselenium compounds have been examined at supranutritional levels for their ability to influence the activity and mRNA levels of chemoprotective enzymes in the livers of selenium-sufficient mice and the changes compared to those elicited by oltipraz. Compounds investigated included novel selenocysteine prodrugs that have previously been evaluated for their ability to reduce the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. Following seven daily doses (i.g.), all compounds except 2-methylselenazolidine-4(R)-carboxylic acid (MSCA) increased thioredoxin reductase activity (43–92%) but only for 2-oxoselenazolidine-4(R)-carboxylic acid (OSCA) was there an accompanying increase in mRNA. No compound enhanced glutathione peroxidase activity, although sodium selenite significantly elevated the mRNA of this enzyme. Oltipraz was an efficacious inducer of both thioredoxin reductase and glutathione peroxidase mRNAs. Sodium selenite, selenazolidine-4(R)-carboxylic acid (SCA), and OSCA elevated NAD(P)H-quinone oxidoreductase mRNA but only for OSCA was the elevation in mRNA accompanied by an increase in enzyme activity. l-Selenocystine significantly increased this activity without increasing mRNA levels. Sodium selenite, l-selenocystine, l-selenomethionine, and Se-methyl-l-selenocysteine all enhanced glutathione S-transferase activity. The increased activity with sodium selenite was accompanied by increases in mRNAs of Gst , Gst μ and Gst π classes, while for l-selenocystine and Se-methyl-l-selenocysteine, only an elevation in the mRNA for the Gst class was observed. Gst and Gst μ class mRNAs were elevated by OSCA without a significant elevation in enzyme activity. SCA and MSCA both elevated a Gst π mRNA and MSCA elevated Gst μ in addition. By comparison, oltipraz only significantly elevated the mRNA of Gst μ, adding to the conclusion that across the entire study, no selenium compound appears to be acting purely through the antioxidant response typified by oltipraz. Despite their chemical similarity, the three cysteine prodrugs, SCA, MSCA, and OSCA, each produced its own unique pattern of effects on protective enzymes and none was identical to the pattern elicited by sodium selenite, l-selenocystine, l-selenomethionine, and Se-methyl-l-selenocysteine. The study also shows that after 7 days of administration, there was only occasional concordance between elevations in mRNA and enzyme activity for any selenium compound and for any protective enzyme, there was no response in common for all selenium compounds.     

Correlations between the activities of 19 standard anticancer agents, antioxidative enzyme activities and the expression of ATP-binding cassette transporters: comparison with the National Cancer Institute data

The aim of this work was to determine the functional activities of four different antioxidative enzymes (glutathione reductase, glutathione-S-transferase, glutathione peroxidase, thioredoxin reductase) and the protein expression of three ATP-binding cassette transporters (P-glycoprotein, multidrug resistance protein 1, multidrug resistance protein 2) in a panel of 14 human cancer cell lines. Enzyme activities and transporter expression were then correlated with the in-vitro cytotoxic activities (GI50 values) of 19 standard antitumor drugs. Analogous data from the National Cancer Institute were used for comparison. The GI50 values of the platinum complexes, alkylating agents, antimetabolites, topoisomerase inhibitors and antimitotic drugs were determined by crystal violet or 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay. Standard enzymatic assays employed to measure the glutathione peroxidase, glutathione-S-transferase, glutathione reductase and thioredoxin reductase activities. The protein expression of the ATP-binding cassette transporter proteins was investigated by the Western-blot method. The delta method was used to normalize the data before bivariant correlation analysis. Only a few correlations between enzyme and cytotoxic activities of the antitumor agents were found. The GI50 values for melphalan and camptothecin correlated positively with the activity of glutathione-S-transferase, whereas GI50 values for methotrexate correlated positively with the cellular activities of both glutathione reductase and thioredoxin reductase. A significant correlation between glutathione reductase and thioredoxin reductase activities was found in our panel of cell lines. Neither P-glycoprotein nor multidrug resistance protein 2 expression could be detected by Western blot analysis in any cell lines investigated, but multidrug resistance protein 1 was consistently observed in all but four lines. Multidrug resistance protein 1 expression correlates positively with the GI50 values of several drugs, e.g. vinblastine and etoposide, and negatively with the GI50 values of 5-fluorouracil. The results confirm the complexity of resistance to antitumor agents and show that the GSH-thioredoxin system alone is not a good indication of intrinsic resistance for many of these anticancer drugs.     

Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.     

丙泊酚对布比卡因诱导的PC12细胞毒性和硫氧还蛋白系统的影响

目的：探讨丙泊酚对布比卡因诱导的PC12细胞毒性的保护作用及内源性硫氧还蛋白（Trx）系统在其中的作用。方法：培养的PC12细胞分成四组，正常对照组、丙泊酚组、布比卡因组、丙泊酚＋布比卡因（PB）组，每组6孔。培养6h和24h后，用MTT比色微量分析细胞存活率，测定上清液乳酸脱氢酶（LDH）活性和细胞内硫氧还蛋白还原酶（TrxR）、活性氧（ROS）活性，RT-PCR检测Trx-1 mRNA和TrxR-1 mRNA表达。结果：与正常PC12细胞相比，布比卡因可显著降低细胞存活率（P〈0．01）和细胞内TrxR活性（P〈0．05），增加上清液中LDH活性和细胞内ROS活性（P〈0．05，P〈0．01），明显降低Trx mRAN和Trx mRAN表达（P〈0．05）；丙泊酚对正常PC12细胞无明显影响；与布比卡因组相比，PB组细胞存活率（P〈0．01）和细胞内Trx活性（P〈0．05）明显增加，上清液中LDH活性和细胞内ROS活性显著降低（P〈0．05，P〈0．01），Trx mRAN和Trx mRAN表达明显增加（P〈0．05）。结论：布比卡因对PC12细胞具有毒性作用可能与降低细胞内TrxR活性、增加ROS活性有关，丙泊酚通过保护细胞内Trx系统的活性及清除ROS来减轻布比卡因诱导的PC12细胞毒性。     

Induction of apoptosis and autophagy by sodium selenite in A549 human lung carcinoma cells through generation of reactive oxygen species

Selenium in the form of sodium selenite has been reported to exert anti-tumor effects in several cancer cell types by inducing autophagic cell death and apoptosis mediated by reactive oxygen species (ROS). However, the exact molecular pathways underlying these effects have not been fully established. The present study used A549 human lung carcinoma cells for further investigation of the anti-cancer mechanism of sodium selenite. We showed that sodium selenite modulated both the extrinsic and intrinsic apoptotic pathways, which were interconnected by Bid truncation. We used z-VAD-fmk, a pan-caspase inhibitor, to demonstrate that sodium selenite-induced apoptosis was dependent on the activation of caspases. Sodium selenite also increased autophagy, as indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and elevation of autophagic flux. Pretreatment with bafilomycin A1 enhanced sodium selenite-induced apoptosis, indicating that sodium selenite-induced autophagy functioned as a survival mechanism. Sodium selenite treatment also resulted in generation of ROS, which abrogated mitochondrial membrane potential (MMP) and regulated both apoptosis and autophagy. Phospho-nuclear factor erythroid 2-related factor 2 (p-Nrf2) showed a ROS-dependent translocation to the nucleus, which suggested that Nrf2 might increase cell survival by suppressing ROS accumulation and apoptosis mediated by oxidative stress. Sodium selenite treatment of A549 cells therefore appeared to trigger both apoptosis and cytoprotective autophagy, which were both mediated by ROS. The data suggest that regulation of ROS generation and autophagy can be a potential strategy for treating lung cancer that is resistant to pro-apoptotic therapeutics.     

Inhibition of cell colony formation by selenite: involvement of glutathione.

Selenium is an essential trace element that has been shown to have anticarcinogenic activity. One mechanism that has been proposed for this activity is a cytotoxic effect of selenium on tumor cells. As a means of assessing its cytotoxicity, we have examined the effect of selenite on tumor cell viability, using as an assay the ability of the cells to form colonies. We have found that brief exposure of HeLa cells to micromolar concentrations of selenite resulted in significant inhibition of colony formation, indicating that this is an assay for selenite cytotoxicity that is more sensitive than those that have been employed previously. In order to investigate the involvement of cellular glutathione in selenite cytotoxicity, we treated cells with buthionine sulfoximine (BSO) before selenite exposure. This treatment, which resulted in a 7-fold reduction in the level of intracellular glutathione, also caused a significant decrease in the inhibitory effect of selenite on colony formation. However, when cells were exposed to selenite that had previously been reacted with glutathione, the BSO-induced decrease in cytotoxicity was eliminated. In contrast, reaction of selenite with other sulfhydryl compounds, such as cysteine and mercaptoethylamine, did not restore its potency in BSO-treated cells. The simplest explanation for these results is that, for selenite to exert its inhibitory effect, it must react with intracellular glutathione to form the selenodiglutathione derivative.     

Hexavalent chromium causes the oxidation of thioredoxin in human bronchial epithelial cells

Hexavalent chromium [Cr(VI)] species such as chromates are cytotoxic. Inhalational exposure is a primary concern in many Cr-related industries and their immediate environments, and bronchial epithelial cells are directly exposed to inhaled Cr(VI). Chromates are readily taken up by cells and are reduced to reactive Cr species which may also result in the generation of reactive oxygen species (ROS). The thioredoxin (Trx) system has a key role in the maintenance of cellular thiol redox balance and is essential for cell survival. Cells normally maintain the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins largely in the reduced state. Redox Western blots were used to assess the redox status of the thioredoxins in normal human bronchial epithelial cells (BEAS-2B) incubated with soluble Na2CrO4 or insoluble ZnCrO4 for different periods of time. Both chromates caused a dose- and time-dependent oxidation of Trx2 and Trx1. Trx2 was more susceptible in that it could all be converted to the oxidized form, whereas a small amount of reduced Trx1 remained even after prolonged treatment with higher Cr concentrations. Only one of the dithiols, presumably the active site, of Trx1 was oxidized by Cr(VI). Cr(VI) did not cause significant GSH depletion or oxidation indicating that Trx oxidation does not result from a general oxidation of cellular thiols. With purified Trx and thioredoxin reductase (TrxR) in vitro, Cr(VI) also resulted in Trx oxidation. It was determined that purified TrxR has pronounced Cr(VI) reducing activity, so competition for electron flow from TrxR might impair its ability to reduce Trx. The in vitro data also suggested some direct redox interaction between Cr(VI) and Trx. The ability of Cr(VI) to cause Trx oxidation in cells could contribute to its cytotoxic effects, and could have important implications for cell survival, redox-sensitive cell signaling, and the cells' tolerance of other oxidant insults.