Therese Karlenius

Thioredoxin and Cancer: A Role for Thioredoxin in all States of Tumor Oxygenation

Thioredoxin is a small redox-regulating protein, which plays crucial roles in maintaining cellular redox homeostasis and cell survival and is highly expressed in many cancers. The tumor environment is usually under either oxidative or hypoxic stress and both stresses are known up-regulators of thioredoxin expression. These environments exist in tumors because their abnormal vascular networks result in an unstable oxygen delivery. Therefore, the oxygenation patterns in human tumors are complex, leading to hypoxia/re-oxygenation cycling. During carcinogenesis, tumor cells often become more resistant to hypoxia or oxidative stress-induced cell death and most studies on tumor oxygenation have focused on these two tumor environments. However, recent investigations suggest that the hypoxic cycling occurring within tumors plays a larger role in the contribution to tumor cell survival than either oxidative stress or hypoxia alone. Thioredoxin is known to have important roles in both these cellular responses and several studies implicate thioredoxin as a contributor to cancer progression. However, only a few studies exist that investigate the regulation of thioredoxin in the hypoxic and cycling hypoxic response in cancers. This review focuses on the role of thioredoxin in the various states of tumor oxygenation.

Regulation of the human thioredoxin gene promoter and its key substrates: a study of functional and putative regulatory elements

BACKGROUND The thioredoxin system maintains redox balance through the action of thioredoxin and thioredoxin reductase. Thioredoxin regulates the activity of various substrates, including those that function to counteract cellular oxidative stress. These include the peroxiredoxins, methionine sulfoxide reductase A and specific transcription factors. Of particular relevance is Redox Factor-1, which in turn activates other redox-regulated transcription factors. SCOPE OF REVIEW Experimentally defined transcription factor binding sites in the human thioredoxin and thioredoxin reductase gene promoters together with promoters of the major thioredoxin system substrates involved in regulating cellular redox status are discussed. An in silico approach was used to identify potential putative binding sites for these transcription factors in all of these promoters. MAJOR CONCLUSIONS Our analysis reveals that many redox gene promoters contain the same transcription factor binding sites. Several of these transcription factors are in turn redox regulated. The ARE is present in several of these promoters and is bound by Nrf2 during various oxidative stress stimuli to upregulate gene expression. Other transcription factors also bind to these promoters during the same oxidative stress stimuli, with this redundancy supporting the importance of the antioxidant response. Putative transcription factor sites were identified in silico, which in combination with specific regulatory knowledge for that gene promoter may inform future experiments. GENERAL SIGNIFICANCE Redox proteins are involved in many cellular signalling pathways and aberrant expression can lead to disease or other pathological conditions. Therefore understanding how their expression is regulated is relevant for developing therapeutic agents that target these pathways.

Cycling hypoxia up-regulates thioredoxin levels in human MDA-MB-231 breast cancer cells

The thioredoxin system is a key cellular antioxidant system and is highly expressed in cancer cells, especially in more aggressive and therapeutic resistant tumors. We analysed the expression of the thioredoxin system in the MDA-MB-231 breast cancer cell line under conditions mimicking the tumor oxygen microenvironment. We grew breast cancer cells in either prolonged hypoxia or hypoxia followed by various lengths of reoxygenation and in each case cells were cultured with or without a hypoxic cycling preconditioning (PC) phase preceding the hypoxic growth. Flow cytometry-based assays were used to measure reactive oxygen species (ROS) levels. Cells grown in hypoxia showed a significant decrease in ROS levels compared to normoxic cells, while a significant increase in ROS levels over normoxic cells was observed after 4 h of reoxygenation. The PC pre-treatment did not have a significant effect on ROS levels. Thioredoxin levels were also highest after 4 h of reoxygenation, however cells subjected to PC pre-treatment displayed even higher thioredoxin levels. The high level of intracellular thioredoxin was also reflected on the cell surface. Reporter assays showed that activity of the thioredoxin and thioredoxin reductase gene promoters was also highest in the reoxygenation phase, although PC pre-treatment did not result in a significant increase over non-PC treated cells. The use of a dominant negative Nrf-2 negated the increased thioredoxin promoter activity during reoxygenation. This data suggests that the high levels of thioredoxin observed in tumors may arise due to cycling between hypoxia and reoxygenation.

The selenium content of cell culture serum influences redox-regulated gene expression.

Cell Culture The MDA-MB-231 breast cancer cell line (see Reference 24 in the main text) was cultured in RPMI 1640 medium (Invitrogen, Melbourne, Australia), supplemented with either 10% FBS [either Invitrogen, Lonza (Melbourne, Australia), Quantum (Brisbane, Australia), or 10% serum supreme (Lonza) and 100 μg/mL penicillin (Invitrogen) and 100 μg/mL streptomycin (Invitrogen) in 5% CO2 at 37°C. Media used in transfections did not contain penicillin or streptomycin. All chemicals were purchased from SigmaAldrich (Castle Hill, Australia) unless otherwise specified.

The Interaction Between Redox and Hypoxic Signalling Pathways in the Dynamic Oxygen Environment of Cancer Cells

Oxygen is essential for the survival of all living beings. A balanced oxygen environment is required since both lower and higher than the required oxygen levels can be detrimental to the cells (Figure 1). The oxygen state of a tissue results fr om the relative contributions of oxygen consumption and delivery. Different organs in the body exist under different oxygen environments, depending on the location and function of the cells in an organ. Most healthy organs reside in 3-6% oxygen [1] while conditions lower than 3% oxygen are described as hypoxia. Cells also survive in hypoxic environments during normal development [2]. However, hypoxia is mostly detrimental to the cells by disrupting the oxygen homeostasis.