evaluation-lung-metastasis-mouse-mammary-tumor-models-quantitative

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

FirstString Research, Inc.

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.

Characterization of alveolar epithelial cells cultured in semipermeable hollow fibers.

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.

Hypoxia up-regulates expression of hemoglobin in alveolar epithelial cells.

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.

Redox metabolism and malignancy.

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.

The impact of redox and thiol status on the bone marrow: Pharmacological intervention strategies.

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.

S-glutathionylated serine proteinase inhibitors as plasma biomarkers in assessing response to redox-modulating drugs.

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.

Causes and consequences of cysteine S-glutathionylation.

Gap junctions and their connexin components are indispensable in mediating the cellular coordination required for tissue and organ homeostasis. The critical nature of their existence mandates a connection to disease while at the same time offering therapeutic potential. Therapeutic intervention may be offered through the pharmacological and molecular disruption of the pathways involved in connexin biosynthesis, gap junction assembly, stabilization, or degradation. Chemical inhibitors aimed at closing connexin channels, peptide mimetics corresponding to short connexin sequences, and gene therapy approaches have been incredibly useful molecular tools in deciphering the complexities associated with connexin biology. Recently, therapeutic potential in targeting connexins has evolved from basic research in cell-based models to clinical opportunity in the form of human trials. Clinical promise is particularly evident with regards to targeting connexin43 in the context of wound healing. The following review is aimed at highlighting novel advances where the pharmacological manipulation of connexin biology has proven beneficial in animals or humans.

Cardiac to cancer: connecting connexins to clinical opportunity.

The gap junction protein, connexin43 (Cx43), has critical roles in the inflammatory, edematous, and fibrotic processes following dermal injury and during wound healing, and is abnormally upregulated at the epidermal wound margins of venous leg ulcers (VLUs). Targeting Cx43 with ACT1, a peptide mimetic of the carboxyl-terminus of Cx43, accelerates fibroblast migration and proliferation, and wound reepithelialization. In a prospective, multicenter clinical trial conducted in India, adults with chronic VLUs were randomized to treatment with an ACT1 gel formulation plus conventional standard-of-care (SOC) protocols, involving maintaining wound moisture and four-layer compression bandage therapy, or SOC protocols alone. The primary end point was mean percent ulcer reepithelialization from baseline to 12 weeks. A significantly greater reduction in mean percent ulcer area from baseline to 12 weeks was associated with the incorporation of ACT1 therapy (79% (SD 50.4)) as compared with compression bandage therapy alone (36% (SD 179.8); P=0.02). Evaluation of secondary efficacy end points indicated a reduced median time to 50 and 100% ulcer reepithelialization for ACT1-treated ulcers. Incorporation of ACT1 in SOC protocols may represent a well-tolerated, highly effective therapeutic strategy that expedites chronic venous ulcer healing by treating the underlying ulcer pathophysiology through Cx43-mediated pathways.

The effect of a connexin43-based Peptide on the healing of chronic venous leg ulcers: a multicenter, randomized trial.

Nonhealing neuropathic foot ulcers remain a significant problem in individuals with diabetes. The gap-junctional protein connexin43 (Cx43) has roles in dermal wound healing and targeting Cx43 signalling accelerates wound reepithelialization. In a prospective, randomized, multicenter clinical trial we evaluated the efficacy and safety of a peptide mimetic of the C-terminus of Cx43, alpha connexin carboxy-terminal (ACT1), in accelerating the healing of chronic diabetic foot ulcers (DFUs) when incorporated into standard of care (SOC) protocols. Adults with DFUs of at least four weeks duration were randomized to receive SOC with or without topical application of ACT1. Primary outcome was mean percent ulcer reepithelialization and safety variables included incidence of treatment related adverse events (AEs) and detection of ACT1 immunogenicity. ACT1 treatment was associated with a significantly greater reduction in mean percent ulcer area from baseline to 12 weeks (72.1% vs. 57.1%; p = 0.03). Analysis of incidence and median time-to-complete-ulcer closure revealed that ACT1 treatment was associated with a greater percentage of participants that reached 100% ulcer reepitheliazation and a reduced median time-to-complete-ulcer closure. No AEs reported were treatment related, and ACT1 was not immunogenic. Treatment protocols that incorporate ACT1 may present a therapeutic strategy that safely augments the reepithelialization of chronic DFUs.

Topical administration of a connexin43-based peptide augments healing of chronic neuropathic diabetic foot ulcers: A multicenter, randomized trial.

Treatment 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.

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.

Exogenous or endogenous hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that can lead to oxidation of cellular nucleophiles, particularly cysteines in proteins. Commercial mouthwashes containing H2O2 provide the opportunity to determine clinically whether changes in S-glutathionylation of susceptible proteins in buccal mucosa cells can be used as biomarkers of ROS exposure.

Article	Year
Characterization of alveolar epithelial cells cultured in semipermeable hollow fibers. Experimental lung research\| PubMed ID: 19263283	2009
Hypoxia up-regulates expression of hemoglobin in alveolar epithelial cells. American journal of respiratory cell and molecular biology\| PubMed ID: 20508070	2011
Redox metabolism and malignancy. Current opinion in pharmacology\| PubMed ID: 20627682	2010
The impact of redox and thiol status on the bone marrow: Pharmacological intervention strategies. Pharmacology & therapeutics\| PubMed ID: 20951732	2011
S-glutathionylated serine proteinase inhibitors as plasma biomarkers in assessing response to redox-modulating drugs. Cancer research\| PubMed ID: 22406622	2012
Causes and consequences of cysteine S-glutathionylation. The Journal of biological chemistry\| PubMed ID: 23861399	2013
Cardiac to cancer: connecting connexins to clinical opportunity. FEBS letters\| PubMed ID: 24607540	2014
The effect of a connexin43-based Peptide on the healing of chronic venous leg ulcers: a multicenter, randomized trial. The Journal of investigative dermatology\| PubMed ID: 25072595	2015
Topical administration of a connexin43-based peptide augments healing of chronic neuropathic diabetic foot ulcers: A multicenter, randomized trial. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society\| PubMed ID: 25703647	2015
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 A... BMC cancer\| PubMed ID: 25881004	2015
S-glutathionylation of buccal cell proteins as biomarkers of exposure to hydrogen peroxide. BBA clinical\| PubMed ID: 26673080	2014

Christina L. Grek

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