Christina L. Grek

Causes and Consequences of Cysteine S-Glutathionylation

Post-translational S-glutathionylation occurs through the reversible addition of a proximal donor of glutathione to thiolate anions of cysteines in target proteins, where the modification alters molecular mass, charge, and structure/function and/or prevents degradation from sulfhydryl overoxidation or proteolysis. Catalysis of both the forward (glutathione S-transferase P) and reverse (glutaredoxin) reactions creates a functional cycle that can also regulate certain protein functional clusters, including those involved in redox-dependent cell signaling events. For translational application, S-glutathionylated serum proteins may be useful as biomarkers in individuals (who may also have polymorphic expression of glutathione S-transferase P) exposed to agents that cause oxidative or nitrosative stress.

The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer

Glutathione S-transferase P is abundantly expressed in some mammalian tissues, particularly those associated with malignancies. While the enzyme can catalyze thioether bond formation between some electrophilic chemicals and GSH, novel nondetoxification functions are now ascribed to it. This review summarizes recent material that implicates GSTP in mediating S-glutathionylation of specific clusters of target proteins and in reactions that define a negative regulatory role in some kinase pathways through ligand or protein:protein interactions. It is becoming apparent that GSTP participates in the maintenance of cellular redox homeostasis through a number of convergent and divergent mechanisms. Moreover, drug platforms that have GSTP as a target have produced some interesting preclinical and clinical candidates.

Redox Metabolism and Malignancy

Redox balance underlies cellular homeostasis. Cancer initiation and progression has been linked to the disruption of redox balance and oxidative stress. Recent findings exemplify the distinctive roles of intracellular and extracellular redox state in the etiology and maintenance of oxidative stress associated with malignancy and metastasis. Within these compartments, redox sensitive cysteines play a crucial role in regulating cell signaling events that act to promote the malignant phenotype via the activation of survival pathways, disruption of cell-death signaling, and increases in cell proliferation. New approaches that aim to accurately evaluate subcellular and microenvironment redox potential may be useful in developing cancer diagnostics and therapeutics.

Hypoxia Up-Regulates Expression of Hemoglobin in Alveolar Epithelial Cells

Alveolar epithelial cells are directly exposed to acute and chronic fluctuations in alveolar oxygen tension. Previously, we found that the oxygen-binding protein hemoglobin is expressed in alveolar Type II cells (ATII). Here, we report that ATII cells also express a number of highly specific transcription factors and other genes normally associated with hemoglobin biosynthesis in erythroid precursors. Because hypoxia-inducible factors (HIFs) were shown to play a role in hypoxia-induced changes in ATII homeostasis, we hypothesized that the hypoxia-induced increase in intracellular HIF exerts a concomitant effect on ATII hemoglobin expression. Treatment of cells from the ATII-like immortalized mouse lung epithelial cell line-15 (MLE-15) with hypoxia for 20 hours resulted in dramatic increases in cellular levels of HIF-2α protein and parallel significant increases in hemoglobin messenger RNA (mRNA) and protein expression, as compared with that of control cells cultured in normoxia. Significant increases in the mRNA of globin-associated transcription factors were also observed, and RNA interference (RNAi) experiments demonstrated that the expression of hemoglobin is at least partially dependent on the cellular levels of globin-associated transcription factor isoform 1 (GATA-1). Conversely, levels of prosurfactant proteins B and C significantly decreased in the same cells after exposure to hypoxia. The treatment of MLE-15 cells cultured in normoxia with prolyl 4-hydroxylase inhibitors, which mimic the effects of hypoxia, resulted in increases of hemoglobin and decreases of surfactant proteins. Taken together, these results suggest a relationship between hypoxia, HIFs, and the expression of hemoglobin, and imply that hemoglobin may be involved in the oxygen-sensing pathway in alveolar epithelial cells.

The Impact of Redox and Thiol Status on the Bone Marrow: Pharmacological Intervention Strategies

Imbalances in cancer cell redox homeostasis provide a platform for new opportunities in the development of anticancer drugs. The control of severe dose-limiting toxicities associated with redox regulation, including myelosuppression and immunosuppression, remains a challenge. Recent evidence implicates a critical role for redox regulation and thiol balance in pathways that control myeloproliferation, hematopoietic progenitor cell mobilization, and immune response. Hematopoietic stem cell (HSC) self-renewal and differentiation are dependent upon levels of intracellular reactive oxygen species (ROS) and niche microenvironments. Redox status and the equilibrium of free thiol:disulfide couples are important in modulating immune response and lymphocyte activation, proliferation and differentiation. This subject matter is the focus of the present review. The potential of redox modulating chemotherapeutics as myeloproliferative and immunomodulatory agents is also covered.

Targeting connexin 43 with α–connexin carboxyl-terminal (ACT1) peptide enhances the activity of the targeted inhibitors, tamoxifen and lapatinib, in breast cancer: clinical implication for ACT1

BackgroundTreatment failure is a critical issue in breast cancer and identifying useful interventions that optimize current cancer therapies remains a critical unmet need. Expression and functional studies have identified connexins (Cxs), a family of gap junction proteins, as potential tumor suppressors. Studies suggest that Cx43 has a role in breast cancer cell proliferation, differentiation, and migration. Although pan-gap junction drugs are available, the lack of specificity of these agents increases the opportunity for off target effects. Consequently, a therapeutic agent that specifically modulates Cx43 would be beneficial and has not been tested in breast cancer. In this study, we now test an agent that specifically targets Cx43, called ACT1, in breast cancer.MethodsWe evaluated whether direct modulation of Cx43 using a Cx43-directed therapeutic peptide, called ACT1, enhances Cx43 gap junctional activity in breast cancer cells, impairs breast cancer cell proliferation or survival, and enhances the activity of the targeted inhibitors tamoxifen and lapatinib.ResultsOur results show that therapeutic modulation of Cx43 by ACT1 maintains Cx43 at gap junction sites between cell-cell membrane borders of breast cancer cells and augments gap junction activity in functional assays. The increase in Cx43 gap junctional activity achieved by ACT1 treatment impairs proliferation or survival of breast cancer cells but ACT1 has no effect on non-transformed MCF10A cells. Furthermore, treating ER+ breast cancer cells with a combination of ACT1 and tamoxifen or HER2+ breast cancer cells with ACT1 and lapatinib augments the activity of these targeted inhibitors.ConclusionsBased on our findings, we conclude that modulation of Cx43 activity in breast cancer can be effectively achieved with the agent ACT1 to sustain Cx43-mediated gap junctional activity resulting in impaired malignant progression and enhanced activity of lapatinib and tamoxifen, implicating ACT1 as part of a combination regimen in breast cancer.

Pleiotropic functions of glutathione S-transferase P.

Glutathione S-transferase P (GSTP) is one member of the GST superfamily that is prevalently expressed in mammals. Known to possess catalytic activity through deprotonating glutathione allowing formation of thioether bonds with electrophilic substrates, more recent discoveries have broadened our understanding of the biological roles of this protein. In addition to catalytic detoxification, other properties so far ascribed to GSTP include chaperone functions, regulation of nitric oxide pathways, regulation of a variety of kinase signaling pathways, and participation in the forward reaction of protein S-glutathionylation. The expression of GSTP has been linked with cancer and other human pathologies and more recently even with drug addiction. With respect to human health, polymorphic variants of GSTP may determine individual susceptibility to oxidative stress and/or be critical in the design and development of drugs that have used redox pathways as a discovery platform.

CHARACTERIZATION OF ALVEOLAR EPITHELIAL CELLS CULTURED IN SEMIPERMEABLE HOLLOW FIBERS

Cell culture methods commonly used to represent alveolar epithelial cells in vivo have lacked airflow, a 3-dimensional air-liquid interface, and dynamic stretching characteristics of native lung tissue—physiological parameters critical for normal phenotypic gene expression and cellular function. Here the authors report the development of a selectively semipermeable hollow fiber culture system that more accurately mimics the in vivo microenvironment experienced by mammalian distal airway cells than in conventional or standard air-liquid interface culture. Murine lung epithelial cells (MLE-15) were cultured within semipermeable polyurethane hollow fibers and introduced to controlled airflow through the microfiber interior. Under these conditions, MLE-15 cells formed confluent monolayers, demonstrated a cuboidal morphology, formed tight junctions, and produced and secreted surfactant proteins. Numerous lamellar bodies and microvilli were present in MLE-15 cells grown in hollow fiber culture. Conversely, these alveolar type II cell characteristics were reduced in MLE-15 cells cultured in conventional 2D static culture systems. These data support the hypothesis that MLE-15 cells grown within our microfiber culture system in the presence of airflow maintain the phenotypic characteristics of type II cells to a higher degree than those grown in standard in vitro cell culture models. Application of our novel model system may prove advantageous for future studies of specific gene and protein expression involving alveolar epithelial or bronchiolar epithelial cells.

Connexin 43, breast cancer tumor suppressor: Missed connections?

Connexins are a family of transmembrane proteins that are characterized by their capacity to form intercellular channels called gap junctions that directly link the cytoplasm of adjacent cells. The formation of gap junctions by connexin proteins facilitates intercellular communication between neighboring cells by allowing for the transfer of ions and small signaling molecules. Communication through gap junctions is key to cellular equilibrium, where connexins, and the gap junction intercellular communication that connexins propagate, have roles in cellular processes such as cell growth, differentiation, and tissue homeostasis. Due to their importance in maintaining cellular functions, the disruption of connexin expression and function underlies the etiology and progression of numerous pathologies, including cancer. Over the past half a century, the role of connexins and gap junction intercellular communication have been highlighted as critical areas of research in cellular malignancies, and much research effort has been geared toward understanding their dysfunction in human cancers. Although ample evidence supports the role of connexins in a variety of human cancers, detailed examination in specific cancers, such as breast cancer, is still lacking. This review highlights the most abundant gap junction connexin isoform in higher vertebrate organisms, Connexin 43, and its role in breast cancer.

S-Glutathionylated Serine Proteinase Inhibitors as Plasma Biomarkers in Assessing Response to Redox-Modulating Drugs

Many cancer drugs impact cancer cell redox regulatory mechanisms and disrupt redox homeostasis. Pharmacodynamic biomarkers that measure therapeutic efficacy or toxicity could improve patient management. Using immunoblot analyses and mass spectrometry, we identified that serpins A1 and A3 were S-glutathionylated in a dose- and time-dependent manner following treatment of mice with drugs that alter reactive oxygen or nitrogen species. Tandem mass spectrometry analyses identified Cys(256) of serpin A1 and Cys(263) of serpin A3 as the S-glutathionylated residues. In human plasma from cancer patients, there were higher levels of unmodified serpin A1 and A3, but following treatments with redox active drugs, relative S-glutathionylation of these serpins was higher in plasma from normal individuals. There is potential for S-glutathionylated serpins A1 and A3 to act as pharmacodynamic biomarkers for evaluation of patient response to drugs that target redox pathways.