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

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

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte

The Cell Culture Medium Affects Growth, Phenotype Expression and the Response to Selenium Cytotoxicity in A549 and HepG2 Cells

Selenium compounds influence cell growth and are highly interesting candidate compounds for cancer chemotherapy. Over decades an extensive number of publications have reported highly efficient growth inhibitory effects with a number of suggested mechanisms f especially for redox-active selenium compounds. However, the studies are difficult to compare due to a high degree of variations in half-maximal inhibitor concentration (IC50) dependent on cultivation conditions and methods to assess cell viability. Among other factors, the variability in culture conditions may affect the experimental outcome. To address this, we have compared the maintenance effects of four commonly used cell culture media on two cell lines, A549 and HepG2, evaluated by the toxic response to selenite and seleno-methylselenocysteine, cell growth and redox homeostasis. We found that the composition of the cell culture media greatly affected cell growth and sensitivity to selenium cytotoxicity. We also provided evidence for change of phenotype in A549 cells when maintained under different culture conditions, demonstrated by changes in cytokeratin 18 (CK18) and vimentin expression. In conclusion, our results have shown the importance of defining the cell culture medium used when comparing results from different studies

Optimised Electroporation for Loading of Extracellular Vesicles with Doxorubicin

The clinical use of chemotherapeutics is limited by several factors, including low cellular uptake, short circulation time, and severe adverse effects. Extracellular vesicles (EVs) have been suggested as a drug delivery platform with the potential to overcome these limitations. EVs are cell-derived, lipid bilayer nanoparticles, important for intercellular communication. They can transport bioactive cargo throughout the body, surmount biological barriers, and target a variety of tissues. Several small molecule drugs have been successfully incorporated into the lumen of EVs, permitting efficient transport to tumour tissue, increasing therapeutic potency, and reducing adverse effects. However, the cargo loading is often inadequate and refined methods are a prerequisite for successful utilisation of the platform. By systematically evaluating the effect of altered loading parameters for electroporation, such as total number of EVs, drug to EV ratio, buffers, pulse capacitance, and field strength, we were able to distinguish tendencies and correlations. This allowed us to design an optimised electroporation protocol for loading EVs with the chemotherapeutic drug doxorubicin. The loading technique demonstrated improved cargo loading and EV recovery, as well as drug potency, with a 190-fold increased response compared to naked doxorubicin

Stability of Sugar Solutions:A Novel Study of the Epimerization Kinetics of Lactose in Water

This article reports on the stereo chemical aspects of the chemical stability of lactose solutions stored between 25 and 60 degrees C. The lactose used for the preparation of the aqueous solutions was a-lactose monohydrate with an anomer purity of 96% alpha and 4% beta based on the supplied certificate of analysis (using a GC analytical protocol), which was further confirmed here by nuclear magnetic resonance (NMR) analysis. Aliquots of lactose solutions were collected at different time points after the solutions were prepared and freeze-dried to remove water and halt epimerization for subsequent analysis by NMR. Epimerization was also monitored by polarimetry and infrared spectroscopy using a specially adapted Fourier transform infrared attenuated total reflectance (FTIR-ATR) method. Hydrolysis was analyzed by ion chromatography. The three different analytical approaches unambiguously showed that the epimerization of lactose in aqueous solution follows first order reversible kinetics between 25 to 60 degrees C. The overall rate constant was 4.4 X 10(-4) s(-1) +/- 0.9 (+/- standard deviation (SD)) at 25 degrees C. The forward rate constant was 1.6 times greater than the reverse rate constant, leading to an equilibrium constant of 1.6 +/- 0.1 (+/- SD) at 25 degrees C. The rate of epimerization for lactose increased with temperature and an Arrhenius plot yielded an activation energy of +52.3 kJ/mol supporting the hypothesis that the mechanism of lactose epimerization involves the formation of extremely short-lived intermediate structures. The main mechanism affecting lactose stability is epimerization, as no permanent hydrolysis or chemical degradation was observed. When preparing aqueous solutions of lactose, immediate storage in an ice bath at 0 degrees C will allow approximately 3 min (180 s) of analysis time before the anomeric ratio alters significantly (greater than 196) from the solid state composition of the starting material. In contrast a controlled anomeric composition (similar to 38% a and similar to 62% beta) will be achieved if an aqueous solution is left to equilibrate for over 4 h at 25 degrees C, while increasing the temperature up to 60 degrees C rapidly reduces the required equilibration time.</p