Robert R. Bowers

Nitrosative Stress–Induced S-Glutathionylation of Protein Disulfide Isomerase Leads to Activation of the Unfolded Protein Response

The rapid proliferation of cancer cells mandates a high protein turnover. The endoplasmic reticulum (ER) is intimately involved in protein processing. An accumulation of unfolded or misfolded proteins in the ER leads to a cascade of transcriptional and translational events collectively called the unfolded protein response (UPR). Protein disulfide isomerase (PDI) is one of the most abundant ER proteins and maintains a sentinel function in organizing accurate protein folding. Treatment of cells with O(2)-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino)phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO) resulted in a dose-dependent increase in intracellular nitric oxide that caused S-glutathionylation of various proteins. Within 4 h, PABA/NO activated the UPR and led to translational attenuation as measured by the phosphorylation and activation of the ER transmembrane kinase, pancreatic ER kinase, and its downstream effector eukaryotic initiation factor 2 in human leukemia (HL60) and ovarian cancer cells (SKOV3). Cleavage of the transcription factor X-box protein 1 and transcriptional activation of the ER resident proteins BiP, PDI, GRP94, and ERO1 (5- to 10-fold induction) also occurred. Immunoprecipitation of PDI showed that whereas nitrosylation was undetectable, PABA/NO treatment caused S-glutathionylation of PDI. Mass spectroscopy analysis showed that single cysteine residues within each of the catalytic sites of PDI had a mass increase [+305.3 Da] consistent with S-glutathionylation. Circular dichroism confirmed that S-glutathionylation of PDI results in alterations in the alpha-helix content of PDI and is concurrent with inhibition of its isomerase activity. Thus, it appears that S-glutathionylation of PDI is an upstream signaling event in the UPR and may be linked with the cytotoxic potential of PABA/NO.

Norepinephrine turnover in brown and white adipose tissue after partial lipectomy

Total body fat is restored after the surgical removal (i.e., partial lipectomy) of white adipose tissue (WAT), and this is accomplished via increases in the mass of nonexcised WAT pads. The underlying mechanism for this apparent regulation of total body fat is unknown. One possibility is via the sympathetic nervous system (SNS) innervation of WAT and brown adipose tissue (BAT) through the regulation of lipolysis and thermogenesis, respectively. Specifically, decreases in SNS activity might fuel lipectomy-induced body fat compensation through energy saved from decreased BAT thermogenesis and would promote lipid accretion through decreased WAT basal lipolysis. Therefore, we tested whether lipectomy triggered decreases in the SNS drive [as indicated by the norepinephrine turnover (NETO)] to nonexcised WAT or to BAT, at times before the lipectomy-induced fat pad mass compensation was complete. Siberian hamsters received either sham or bilateral epididymal WAT lipectomy, and NETO was measured in the remaining WAT and interscapular BAT (IBAT) before, and 3 and 6 weeks after surgery. Total dissected WAT, and inguinal and retroperitoneal WAT masses were significantly increased following lipectomy, whereas dorsal subcutaneous WAT and IBAT masses, as well as food intake, were unchanged. The only significant change in NETO was a marked decrease (approximately 90%) in IBAT NETO at Week 3 postlipectomy compared with the sham-lipectomized controls. These findings suggest that the lipid accretion of nonexcised WAT pads triggered by lipectomy may be partially fueled by decreased BAT thermogenesis, inasmuch as decreased IBAT NETO reflects decreased BAT heat production.

Sulfiredoxin redox-sensitive interaction with S100A4 and non-muscle myosin IIA regulates cancer cell motility

Sulfiredoxin (Srx) is a redox active protein that participates in the reduction of oxidized cysteine residues. Here we identify a novel function of Srx through its specific binding to S-glutathionylated S100A4 affecting its interaction with non-muscle myosin (NMIIA), thereby modulating the effect of S100A4 on NMIIA function and impacting cell adhesion and migration. Srx forms a complex with S100A4 (and has stronger affinity for S-glutathionylated S100A4), regulates its activity, and mediates redox regulation of the interaction of S100A4 with NMIIA. The consequence of this regulation is microfilament remodeling and altered cellular motility and adhesion. Srx-overexpressing cells had reduced levels of adhesion, decreased levels of Tyr(397)-phosphorylated focal adhesion kinase, and increased cell motility in wound healing assays. These results describe a novel redox-sensitive role for Srx in mediating complex protein interactions with plausible consequences for cancer cell motility.

Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen

Background The obesity pandemic is associated with multiple major health concerns. In addition to diet and lifestyle, there is increasing evidence that environmental exposures to chemicals known as obesogens also may promote obesity. Objectives We investigated the massive environmental contamination resulting from the Deepwater Horizon (DWH) oil spill, including the use of the oil dispersant COREXIT in remediation efforts, to determine whether obesogens were released into the environment during this incident. We also sought to improve the sensitivity of obesogen detection methods in order to guide post-toxicological chemical assessments. Methods Peroxisome proliferator–activated receptor gamma (PPARγ) transactivation assays were used to identify putative obesogens. Solid-phase extraction (SPE) was used to sub-fractionate the water-accommodated fraction generated by mixing COREXIT, cell culture media, and DWH oil (CWAF). Liquid chromatography–mass spectrometry (LC-MS) was used to identify components of fractionated CWAF. PPAR response element (PPRE) activity was measured in PPRE-luciferase transgenic mice. Ligand-binding assays were used to quantitate ligand affinity. Murine 3T3-L1 preadipocytes were used to assess adipogenic induction. Results Serum-free conditions greatly enhanced the sensitivity of PPARγ transactivation assays. CWAF and COREXIT had significant dose-dependent PPARγ transactivation activities. From SPE, the 50:50 water:ethanol volume fraction of CWAF contained this activity, and LC-MS indicated that major components of COREXIT contribute to PPARγ transactivation in the CWAF. Molecular modeling predicted several components of COREXIT might be PPARγ ligands. We classified dioctyl sodium sulfosuccinate (DOSS), a major component of COREXIT, as a probable obesogen by PPARγ transactivation assays, PPAR-driven luciferase induction in vivo, PPARγ binding assays (affinity comparable to pioglitazone and arachidonic acid), and in vitro murine adipocyte differentiation. Conclusions We conclude that DOSS is a putative obesogen worthy of further study, including epidemiological and clinical investigations into laxative prescriptions consisting of DOSS. Citation Temkin AM, Bowers RR, Magaletta ME, Holshouser S, Maggi A, Ciana P, Guillette LJ, Bowden JA, Kucklick JR, Baatz JE, Spyropoulos DD. 2016. Effects of crude oil/dispersant mixture and dispersant components on PPARγ activity in vitro and in vivo: identification of dioctyl sodium sulfosuccinate (DOSS; CAS #577-11-7) as a probable obesogen. Environ Health Perspect 124:112–119; http://dx.doi.org/10.1289/ehp.1409672

Abstract 2077: Interactions between sulfiredoxin, S100A4 and non-muscle myosin IIA regulate cancer cell adhesion and motility

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Sulfiredoxin (Srx) is an antioxidant protein that reduces over-oxidized catalytic cysteine residues of peroxiredoxins I-IV and catalyzes reduction of some S-glutathionylated proteins. We previously showed that Srx plays a role in cell migration and interacts with non-muscle myosin IIA (NM IIA; Bowers et al AACR 2010). The purpose of the present study was to extend our knowledge of the effect of Srx on cell adhesion and migration. Immunoprecipitation of Srx from non-small lung cancer (A549 cells) revealed a number of Srx-interacting proteins including NM IIA and S100A4. NM IIA plays a central role in the control of cell adhesion and migration, and S100A4 is a key regulator of NM IIA activity that is linked to cancer metastases. Adhesion assays showed that A549 cells over-expressing Srx have reduced adhesion to fibronectin-coated dishes and decreased levels of Tyr397-phosphorylated focal adhesion kinase (p-FAK). Conversely, Srx knockdown by shRNA increased A549 cell adhesion to fibronectin and p-FAK levels. Srx over-expression in A549 cells increased actin stress fiber formation and altered focal adhesion formation. Depletion of Srx in A549 cells resulted in decreased actin stress fiber formation and also altered sites of cell:extracellular matrix adhesion. In silico molecular modeling of an Srx-S100A4 homodimer complex showed close proximity (∼13A) of the single Cys99 of Srx to the Cys85 of S100A4. The calculated binding constant for the Srx-S100A4 interaction indicated reasonable affinity (KD∼ 62 nM). This affinity was not dependent upon disulfide bonding, since mutation of Srx residue Cys99 to Ser resulted in increased affinity (KD ∼22 nM). In vitro S-glutathionylation of Cys85 of S100A4 markedly increased its affinity to Srx (KD∼4 nM), but had no effect with the inactive Srx C99S mutant. Srx over-expression produced a doubling of the basal cellular level of free Ca2+ suggesting that the protein:protein interactions altered the capacity of S100A4 to bind Ca2+. Our data show that Srx may play important role in S100A4-mediated cancer cell cytoskeletal remodeling and motility and could potentially be a target for development of specific anticancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2077. doi:10.1158/1538-7445.AM2011-2077

Abstract 5136: Sulfiredoxin promotes cell migration through direct interaction with non muscle myosin IIa

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Redox homeostasis is frequently dysregulated in cancer cells. In particular, glutathione pathways can be imbalanced and impact cell proliferation and death pathways. S-glutathionylation is the addition of glutathione to basic cysteine residues in certain proteins producing significant changes in their structure/function. Because this post-translational modification is dynamic and reversible, the S-glutathionylation cycle can provide the framework for important cell signaling events in cancer cells. We have shown that sulfiredoxin (Srx) plays a role in deglutathionylation and interacts with proteins such as the phosphatase PTP1B, actin and peroxiredoxin 6 (Prdx 6). Redox regulation of actin modulates cell adhesion and focal contacts. In the present study, we examined the role of Srx in cell migration, invasion and potentially metastasis. Srx was over-expressed by transfection in lung cancer cells (A549). Phalloidin staining showed that Srx over-expression (A549-Srx) led to phenotypic changes in cell adhesion through altered focal adhesion contact points and the cells were more refractory to trypsin release. Relative to wild type, A549-Srx cells showed enhanced migration and invasion in the scratch- and Boyden chamber assays. Proteomic analysis through mass spectrometry demonstrated that Srx interacts with non-muscle myosin IIa (NMIIa) in A549 cells. Reciprocal co-immunoprecipitation of Srx and NMIIa validated this interaction under non-stressed conditions. Either through protein: protein interaction or through redox regulation (or both), Srx appears to be an important mediator of the cell motility and invasive properties of the A549 human lung cancer cell line. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5136.

Abstract 1615: The redox modulator NOV-002 inhibits proliferation of ovarian tumor cells but increases proliferation of myeloid cells

Clinical trials in solid tumor indications have shown that NOV-002 (a formulation of glutathione disulfide) combined with standard chemotherapeutic regimens results in increased efficacy and improved tolerance of standard chemotherapy (e.g. enhanced hematological recovery, immune stimulation). Non-clinical studies have interrogated the myeloproliferative and antitumor activity of the drug. NOV-002 alters redox homeostasis, at the cell surface and intracellularly, through kinase signaling culminating in differential effects on cell proliferation/survival in myeloid verses tumor cells. Thus, we previously showed that NOV-002 has positive growth effects on human myeloid lineage cells (HL60) and yet the drug leads to cell cycle arrest and apoptosis in human ovarian (SKOV3) cancer cells. In the present study, we sought to understand differences in redox-mediated signaling events that govern the opposing pharmacological properties of NOV-002. While the redox-signaling events are quite similar, the cellular consequences are distinct. Chronic treatment of cells with NOV-002 leads to enhanced proliferation in HL60 cells and growth arrest in SKOV3 cells. The alteration in growth rate occurs in both myeloid and tumor cell types in parallel with stress-induced S-glutathionylation and activation of MAP kinase pathways. In myeloid lineage cells, activation of MAP- and JAK/STAT-kinases leads to proliferation whereas activation of this pathway in SKOV3 cells leads to apoptosis. NOV-002 treatment also results in changes in plasma and mitochondrial membrane potentials in both cell models. These changes were concurrent with time- and dose-dependent increases in the accumulation of intracellular Ca2+. Interestingly, dose- and time- dependent increases in nitric oxide (NO) generation were observed in HL60 cells and shown to be mediated through eNOS. SKOV 3 cells do not express eNOS and, consequently, NO generation was not detected. Finally, redox-modulation of cell surface thiols (via S-glutathionylation) occurs in both cell types but to different extents. NOV-002 treatment leads to a ∼75% decrease in free sulfhydryls in myeloid cells whereas ovarian cancer cell surface free sulfhydryls were reduced by only ∼5%. It remains to be determined whether these observed differences in cell surface protein glutathionylation and/or in mechanism of Ca2+ flux can be linked to the opposite effects of NOV-002 on proliferation/survival in myeloid and tumor cells. Regardless of the basis of this difference, it can be speculated that the differential effects of NOV-002 on myeloid and tumor cells in vitro may relate to the unique clinical profile that NOV-002 has demonstrated to date– increased survival and anti-tumor efficacy combined with mitigation of chemotherapy-induced hematological toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1615.

Abstract C109: NOV‐002 suppresses tumor cell growth by modulating redox‐sensitive cell signaling

Regulation of cell proliferation/survival is complex, impacted by multiple endogenous and exogenous stimuli capable of triggering overlapping and competing signaling pathways whose net effects can be quite cell dependent. Changes in redox status is one regulator of cell life/death decisions under both physiologic and pathologic conditions. Here we report on the anti‐proliferative effects of a pharmacologically generated oxidative signal in human tumor cells using NOV‐002, a glutathione disulfide‐mimetic in advanced clinical trials for oncological indications. NOV‐002 has demonstrated efficacy (increased survival and/or decreased tumor growth) in non‐small cell lung, breast and ovarian cancers when combined with standard chemotherapeutic agents. In addition, treatment with NOV‐002 mitigates chemotherapy‐associated hematological toxicity. Pre‐clinical data has linked this effect to proliferation of bone marrow progenitor cells subsequent to generation of an oxidative signal, intracellularly and at the cell surface of myeloid lineage cells, leading to activation of multiple kinases known to regulate cell proliferation (e.g. MAP kinases, JAK/STAT kinases). The data presented here extend redox modulation studies with NOV‐002 to tumor cells. SKOV3 cells (a human ovarian tumor cell line) were exposed to NOV‐002 (250 uM). Within 5 min after a single treatment, cellular levels of reactive oxygen species (ROS) were significantly elevated indicating the generation of an oxidative signal by NOV‐002. This was not, however, sufficient to influence redox‐sensitive cell signaling or tumor cell proliferation rate. In contrast, more prolonged exposure to NOV‐002 (daily treatment for 5 days) resulted in a sustained elevation in ROS and dose‐dependent activation of the proliferation‐regulating MAP kinase, JNK, as evidenced by an increase in its active, phosphorylated form. Most importantly, this redox‐activated cell signaling effect was associated with a significantly decreased proliferation rate of the SKOV3 cells that persisted even after stopping treatment with NOV‐002. Thus, oxidative signal generation and MAP kinase pathway activation by NOV‐002 appears to result in cell‐type dependent effects on proliferation/survival. In myeloid lineage/bone marrow cells, chronic exposure to NOV‐002 leads to increased proliferation while in tumor cells the result is a decrease in proliferation. These dichotomous effects may contribute to the unique clinical profile that NOV‐002 has demonstrated to date — increased anti‐tumor efficacy and survival combined with enhanced recovery from chemotherapy‐induced hematological toxicity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C109.