Effects of redox modulation by inhibition of thioredoxin reductase on radiosensitivity and gene expression.

The thioredoxin system is a promising target when aiming to overcome the problem of clinical radiation resistance. Altered cellular redox status and redox sensitive thiols contributing to induction of resistance strongly connect the ubiquitous redox enzyme thioredoxin reductase (TrxR) to the cellular response to ionizing radiation. To further investigate possible strategies in combating clinical radiation resistance, human radio-resistant lung cancer cells were subjected to a combination of single fractions of γ-radiation at clinically relevant doses and non-toxic levels of a well-characterized thioredoxin reductase inhibitor, the phosphine gold(I) compound [Au(SCN)(PEt(3))]. The combination of the TrxR-inhibitor and ionizing radiation reduced the surviving fractions and impaired the ability of the U1810 cells to repopulate by approximately 50%. In addition, inhibition of thioredoxin reductase caused changes in the cell cycle distribution, suggesting a disturbance of the mitotic process. Global gene expression analysis also revealed clustered genetic expression changes connected to several major cellular pathways such as cell cycle, cellular response to stress and DNA damage. Specific TrxR-inhibition as a factor behind the achieved results was confirmed by correlation of gene expression patterns between gold and siRNA treatment. These results clearly demonstrate TrxR as an important factor conferring resistance to irradiation and the use of [Au(SCN)(PEt(3))] as a promising radiosensitizing agent.

Chelation of lysosomal iron protects against ionizing radiation.

Ionizing radiation causes DNA damage and consequent apoptosis, mainly due to the production of hydroxyl radicals (HO•) that follows radiolytic splitting of water. However, superoxide (O2•-) and H2O2 also form and induce oxidative stress with resulting LMP (lysosomal membrane permeabilization) arising from iron-catalysed oxidative events. The latter will contribute significantly to radiation-induced cell death and its degree largely depends on the quantities of lysosomal redox-active iron present as a consequence of autophagy and endocytosis of iron-rich compounds. Therefore radiation sensitivity might be depressed by lysosome-targeted iron chelators. In the present study, we have shown that cells in culture are significantly protected from ionizing radiation damage if initially exposed to the lipophilic iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone), and that this effect is based on SIH-dependent lysosomal stabilization against oxidative stress. According to its dose-response-modifying effect, SIH is a most powerful radioprotector and a promising candidate for clinical application, mainly to reduce the radiation sensitivity of normal tissue. We propose, as an example, that inhalation of SIH before each irradiation session by patients undergoing treatment for lung malignancies would protect normally aerated lung tissue against life-threatening pulmonary fibrosis, whereas the sensitivity of malignant lung tumours, which usually are non-aerated, will not be affected by inhaled SIH.

Selenium and the selenoprotein thioredoxin reductase in the prevention, treatment and diagnostics of cancer.

Selenium is an essential element that is specifically incorporated as selenocystein into selenoproteins. It is a potent modulator of eukaryotic cell growth with strictly concentration-dependant effects. Lower concentrations are necessary for cell survival and growth, whereas higher concentrations inhibit growth and induce cell death. It is well established that selenium has cancer preventive effects, and several studies also have shown that it has strong anticancer effects with a selective cytotoxicity on malignant drug-resistant cells while only exerting marginal effects on normal and benign cells. This cancer-specific cytotoxicity is likely explained by high affinity selenium uptake dependent on proteins connected to multidrug resistance. One of the most studied selenoproteins in cancer is thioredoxin reductase (TrxR) that has important functions in neoplastic growth and is an important component of the resistant phenotype. Several reports have shown that TrxR is induced in tumor cells and pre-neoplastic cells, and several commonly used drugs interact with the protein. In this review, we summarize the current knowledge of selenium as a potent preventive and tumor selective anticancer drug, and we also discuss the potential of using the expression and modulation of the selenoprotein TrxR in the diagnostics and treatment of cancer.

Expression profiles of thioredoxin family proteins in human lung cancer tissue: correlation with proliferation and differentiation.

AIMS: Lung cancer is one of the most common causes of cancer lethality worldwide. Despite recent progress, long-term survival remains poor. The aim of this study was to explore the expression pattern of the thioredoxin superfamily of proteins as potential new diagnostic and/or predictive markers. METHODS AND RESULTS: The expression of thioredoxin 1 (Trx1), thioredoxin reductase 1 (TrxR1), the isoforms TrxR1-v.2,3,5, glutaredoxin 1 (Grx1) and glutaredoxin 2 (Grx2) was examined by immunohistochemistry on paraffin-embedded sections from 42 cases of non-small cell lung cancer patients. Additional cases of lung cancer from tissue microarray were examined and the immunoreactivity was compared. All proteins except TrxR1 showed a significant correlation with the degree of differentiation in adenocarcinoma. Trx1 and TrxR1-v.2,3,5 also showed a significant correlation with differentiation in squamous carcinoma. Furthermore, Grx1 and Grx2 showed a clear inverse correlation with proliferation. The proliferation rate was further analysed in vitro in stably transfected Grx2 overproducing cells, showing that the proliferative effect of Grx2 is strictly dependent on subcellular localization. CONCLUSIONS: The thioredoxin family of proteins is important for growth and differentiation of lung cancer cells. The correlation with differentiation and proliferation of these enzymes makes them promising predictive/diagnostic markers.

Treatment of lung cancer cells with cytotoxic levels of sodium selenite: effects on the thioredoxin system.

Selenium at subtoxic doses has been shown to have tumor specific cytotoxic effects. In this work, viability measurements in different lung cancer cell lines showed that selenite was more effective compared to three different conventional cytotoxic drugs. In addition, the cell line most sensitive to selenite toxicity comprised the highest level of thioredoxin reductase 1 (TrxR1). The human selenoenzyme TrxR1 is a central enzyme for cell growth, differentiation, and the protection against oxidative stress. TrxR1, which in several studies has been shown to be up-regulated in various tumor cells, is also a target for many anticancer drugs. In this study, inhibition of TrxR resulted in enhanced selenite cytotoxicity, clearly connecting the thioredoxin system to the toxic effects mediated by selenite. The complex regulation of TrxR1, involving the expression of many different transcript forms of mRNA, was investigated by real-time qPCR in lung cancer cell lines following treatment with toxic doses (2.5-10 microM) of sodium selenite. Selenium treatment resulted in increased expression of almost all TrxR1 mRNA variants with increasing concentrations of selenite. On the contrary, the TrxR protein level and activity, increased at low to moderate doses followed by a decrease at higher doses, indicating impairment of protein synthesis by selenite.

Quantification of alternative mRNA species and identification of thioredoxin reductase 1 isoforms in human tumor cells.

The human selenoenzyme thioredoxin reductase 1 (TrxR1) is a very important enzyme for cell growth, differentiation, and the defense against oxidative stress. Several studies have shown that TrxR1 is up-regulated in tumor cells. The regulation of TrxR1 is very complex and involves the expression of different transcript forms of mRNA. We have, by quantitative polymerase chain reaction, investigated the total expression of TrxR1 mRNA and quantified the expression of alternative mRNA forms (alpha1/2, alpha6, alpha7/8, alpha10/11, alpha13, gamma2-4, and beta1) in six different human malignant mesothelioma cell lines of epithelioid, sarcomatoid, or mixed phenotype. The most abundant alpha-form was surprisingly alpha1/2 and not the expected alpha7/8. Selenium treatment resulted in increased expression of all alpha-variants, except the alpha10/11, where the levels were unaffected. The expression of protein isoforms was studied and the less abundant forms TrxR1v.2, TrxR1v.3, and TrxR1v.5 were detected in cell lysates and in human tumor tissue, using specific peptide antibodies. Furthermore, TrxR1v.3 and TrxR1v.5, previously not identified in human cells, were detected by mass spectrometry. Our data show differential expression of TrxR1 mRNA forms in malignant mesothelioma of different phenotype, and investigation of alternative transcript variants of TrxR1 could be a valuable tool in the diagnostics and characterization of tumors.