Oxidative stress in cell and tissue damage and selenium-based therapeutics in cancer

Sammanfattning: Redox homeostasis is the balance between cellular prooxidants and antioxidants, maintained by the interplay of electrons. Reactive oxygen species (ROS) is a term for molecules with unpaired valence electrons or unstable bonds. Initially thought to predominantly produce cellular damage, ROS were found to be key mediators in several biological processes. These events are primarily facilitated by modulating free thiol groups. However, an imbalance due to an excessive generation of ROS or a dysfunction of the cellular antioxidant response leads to oxidative stress. Prolonged exposure to oxidative stress is implicated in several disorders, including ischemia and reperfusion injury, neurodegenerative disorders, cardiovascular disease, and cancer. Cancer cells in particular acquire an increased basal level of ROS, attributed to high cellular metabolism, which renders the cells vulnerable to subtle changes in redox levels. This can be harnessed in cancer therapy by inducing ROS-generation, pushing the level of oxidative stress beyond the tolerance of the cell. Several cytostatic agents used in the clinic today use this approach to target malignant cells. Inherent and acquired resistance is however a problem as many chemotherapeutic drugs exhibit single-target sites. Resistance is elicited by target site modifications, multidrug resistant efflux pumps, or upregulation of redox proteins and detoxification pathways. I. Expression patterns of redox proteins in cells and tissue with oxidative stress Upregulation of thioredoxin (TXN) and glutaredoxin (GLRX) proteins is an event seen in many tumor cells. Investigation of the expression pattern of these oxidoreductases was conducted in hepatocellular carcinoma patients in Paper I. TXN1 and TXN2, and GLRX5 were found to be upregulated compared to the surrounding liver tissue. In colorectal liver metastases tissue, TXN1 and TXN2, GLRX1, GLRX3 and GLRX5 were upregulated. These results might merit the implementation of oxidoreductases as diagnostic markers for hepatocellular carcinoma (HCC). Portal triad clamping was used in Paper II as a controlled experimental setting for investigating alterations in TXN and GLRX upon induced oxidative stress by ischemia and reperfusion. Ultrastructural changes revealed that ischemic mediated damage was borne by the liver sinusoidal endothelial cells (LSECs). At reperfusion the LSECs re-attached to some extent with signs of activation. No differences in redox protein expression could be found between the different states of oxygen tension in the tissue. This indicated that the prompted oxidative stress in the tissue by short periods of ischemia and reperfusion probably result in reversible modifications in the tissue. II. Induction of oxidative stress via ROS generation by redox active selenium compounds Redox active selenium compounds are promising candidates for the application in cancer therapy. Selenite and methylselenocysteine are two compounds with the highest therapeutic potential. Their tumoricidal effects are facilitated by their reactive metabolites. In Paper III, Modulation of the MSC metabolizing enzyme kynurenine aminotransferase (KYAT) was implemented in order to increase the growth inhibitory effects of the selenium compound. KYATs are PLP-dependent cysteine S-conjugate β-lyases, that display both transaminase and β-lyase activity. Overexpression of KYAT1 resulted in increased sensitivity towards MSC. Further modulation of the enzyme by site directed mutagenesis in the active site allowed for a phenotype that favors β-elimination over transamination. This was done in order to increase the cleavage of MSC to the reactive methylselenol. Mutant KYAT1 further sensitized the cells towards MSC to an exceptional level. Growth inhibitory effects of selenide, MSC and two novel selenium compounds, Seleno-folate and Seleno-aniline were compared between cells grown in 2D and cells grown in 3D spheroids in Paper IV. Increased resistance towards selenite and Seleno-aniline was seen in the 3D culture. Additionally, the use of an ex vivo organotypic model was used as a novel drug screening system. The culture consisted of surgical specimens of pancreatic adenocarcinoma (PDAC) grown on an insert in wells. Our results revealed that MSC could lower the metabolic activity of the tissue components as well as reduce the number of cells associated with tumorous outgrowth in the cultured section. Taking the results in part II together, MSC is found to be highly interesting for chemotherapeutic purposes, both as a single agent and in combination with conventional cytostatic drugs. Increasing the sensitivity towards MSC by modulating its metabolizing enzyme could serve to develop an increased specificity towards the compound.