The Receptor for Advanced Glycation Endproducts (RAGE) and Its Ligands S100A8/A9 and High Mobility Group Box Protein 1 (HMGB1) Are Key Regulators of Myeloid-Derived Suppressor Cells.

Immunotherapies including checkpoint blockade immunotherapy (CBI) and chimeric antigen receptor T cells (CAR-T) have revolutionized cancer treatment for patients with certain cancers. However, these treatments are not effective for all cancers, and even for those cancers that do respond, not all patients benefit. Most cancer patients have elevated levels of myeloid-derived suppressor cells (MDSCs) that are potent inhibitors of antitumor immunity, and clinical and animal studies have demonstrated that neutralization of MDSCs may restore immune reactivity and enhance CBI and CAR-T immunotherapies. MDSCs are homeostatically regulated in that elimination of mature circulating and intratumoral MDSCs results in increased production of MDSCs from bone marrow progenitor cells. Therefore, targeting MDSC development may provide therapeutic benefit. The pro-inflammatory molecules S100A8/A9 and high mobility group box protein 1 (HMGB1) and their receptor RAGE are strongly associated with the initiation and progression of most cancers. This article summarizes the literature demonstrating that these molecules are integrally involved in the early development, accumulation, and suppressive activity of MDSCs, and postulates that S100A8/A9 and HMGB1 serve as early biomarkers of disease and in conjunction with RAGE are potential targets for reducing MDSC levels and enhancing CBI and CAR-T immunotherapies.

Steady-state estradiol triggers a unique innate immune response to allergen resulting in increased airway resistance.

RATIONALE: Asthma is a chronic airway condition that occurs more often in women than men during reproductive years. Population studies have collectively shown that long-term use of oral contraceptives decreased the onset of asthma in women of reproductive age. In the current study, we hypothesized that steady-state levels of estrogen would reduce airway inflammation and airway hyperresponsiveness to methacholine challenge. METHODS: Ovariectomized BALB/c mice (Ovx) were implanted with subcutaneous hormone pellets (estrogen, OVX-E2) that deliver consistent levels of estrogen [68 ± 2 pg/mL], or placebo pellets (OVX-Placebo), followed by ovalbumin sensitization and challenge. In conjunction with methacholine challenge, immune phenotyping was performed to correlate inflammatory proteins and immune populations with better or worse pulmonary outcomes measured by invasive pulmonary mechanics techniques. RESULTS: Histologic analysis showed an increase in total cell infiltration and mucus staining around the airways leading to an increased inflammatory score in ovarectomized (OVX) animals with steady-state estrogen pellets (OVX-E2-OVA) as compared to other groups including female-sham operated (F-INTACT-OVA) and OVX implanted with a placebo pellet (OVX-Pl-OVA). Airway resistance (Rrs) and lung elastance (Ers) were increased in OVX-E2-OVA in comparison to F-INTACT-OVA following aerosolized intratracheal methacholine challenges. Immune phenotyping revealed that steady-state estrogen reduced CD3+ T cells, CD19+ B cells, ILC2 and eosinophils in the BAL across all experiments. While these commonly described allergic cells were reduced in the BAL, or airways, we found no changes in neutrophils, CD3+ T cells or CD19+ B cells in the remaining lung tissue. Similarly, inflammatory cytokines (IL-5 and IL-13) were also decreased in OVX-E2-OVA-treated animals in comparison to Female-INTACT-OVA mice in the BAL, but in the lung tissue IL-5, IL-13 and IL-33 were comparable in OVX-E2-OVA and F-INTACT OVA mice. ILC2 were sorted from the lungs and stimulated with exogenous IL-33. These ILC2 had reduced cytokine and chemokine expression when they were isolated from OVX-E2-OVA animals, indicating that steady-state estrogen suppresses IL-33-mediated activation of ILC2. CONCLUSIONS: Therapeutically targeting estrogen receptors may have a limiting effect on eosinophils, ILC2 and potentially other immune populations that may improve asthma symptoms in those females that experience perimenstrual worsening of asthma, with the caveat, that long-term use of estrogens or hormone receptor modulators may be detrimental to the lung microenvironment over time.

The receptor for advanced glycation end products (RAGE) is involved in mitochondrial function and cigarette smoke-induced oxidative stress.

The mechanisms underlying muscle dysfunction with Chronic Obstructive Pulmonary Disease (COPD) are poorly understood. Indirect evidence has recently suggested a role of Advanced Glycation End Products (AGEs) and their receptor (RAGE) in the pathophysiology of COPD. Accordingly, this study aimed to examine the redox balance and mitochondrial alterations in the skeletal muscle of a mouse model deficient in the receptor for AGE (RAGE-KO) and wild-type C57BL/6 exposed to cigarette smoke for 8-months using immunoblotting, spectrophotometry, and high-resolution respirometry. Cigarette smoke exposure increased by two-fold 4-HNE levels (P < 0.001), a marker of oxidative stress, and markedly downregulated contractile proteins, mitochondrial respiratory complexes, and uncoupling proteins levels (P < 0.001). Functional alterations with cigarette smoke exposure included a greater reliance on complex-I supported respiration (P < 0.01) and lower relative respiratory capacity for fatty acid (P < 0.05). RAGE knockout resulted in 47% lower 4-HNE protein levels than the corresponding WT control mice exposed to cigarette smoke (P < 0.05), which was partly attributed to increased Complex III protein levels. Independent of cigarette smoke exposure, RAGE KO decreased mitochondrial specific maximal respiration (P < 0.05), resulting in a compensatory increase in mitochondrial content measured by citrate synthase activity (P < 0.001) such that muscle respiratory capacity remained unaltered. Together, these findings suggest that knockout of RAGE protected the skeletal muscle against oxidative damage induced by 8 months of cigarette smoke exposure. In addition, this study supports a role for RAGE in regulating mitochondrial content and function and can thus serve as a potential therapeutic target.

IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17ß-estradiol.

Asthmatic women tend to develop severe airway disease in their reproductive years, and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells, or ILC2, are readily detected in allergic conditions, such as rhinosinusitis, in individuals that develop nasal polyps do to allergen exposures, and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33, an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in naïve and ovalbumin (OVA) challenged, female BALB/c mice in comparison to male mice following stimulation with IL-33. Here, we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-κB signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated, and had reduced NF-κB activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2, and Nfkb1 was found among top-ranked regulatory interactions. Together, these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases.

Skeletal muscle mitochondrial adaptations induced by long-term cigarette smoke exposure.

Because patients with chronic obstructive pulmonary disease (COPD) are often physically inactive, it is still unclear whether the lower respiratory capacity in the locomotor muscles of these patients is due to cigarette smoking per se or is secondary to physical deconditioning. Accordingly, the purpose of this study was to examine mitochondrial alterations in the quadriceps muscle of 10 mice exposed to 8 mo of cigarette smoke, a sedentary mouse model of emphysema, and 9 control mice, using immunoblotting, spectrophotometry, and high-resolution respirometry in permeabilized muscle fibers. Mice exposed to smoke displayed a twofold increase in the oxidative stress marker, 4-HNE, (P < 0.05) compared with control mice. This was accompanied by significant decrease in protein expression of UCP3 (65%), ANT (58%), and mitochondrial complexes II-V (∼60%-75%). In contrast, maximal ADP-stimulated respiration with complex I and II substrates (CON: 23.6 ± 6.6 and SMO: 19.2 ± 8.2 ρM·mg-1·s-1) or octanoylcarnitine (CON: 21.8 ± 9.0 and SMO: 16.5 ± 6.6 ρM·mg-1·s-1) measured in permeabilized muscle fibers, as well as citrate synthase activity, were not significantly different between groups. Collectively, our findings revealed that sedentary mice exposed to cigarette smoke for 8 mo, which is typically associated with pulmonary inflammation and emphysema, exhibited a preserved mitochondrial respiratory capacity for various substrates, including fatty acid, in the skeletal muscle. However, the mitochondrial adaptations induced by cigarette smoke favored the development of chronic oxidative stress, which can indirectly contribute to augment the susceptibility to muscle fatigue and exercise intolerance.NEW & NOTEWORTHY It is unclear whether the exercise intolerance and skeletal muscle mitochondrial dysfunction observed in patients with COPD is due to cigarette smoke exposure, per se, or if they are secondary consequences to inactivity. Herein, while long-term exposure to cigarette smoke induces oxidative stress and an altered skeletal muscle phenotype, cigarette smoke does not directly contribute to mitochondrial dysfunction. With this evidence, we demonstrate the critical role of physical inactivity in cigarette smoke-related skeletal muscle dysfunction.

The receptor for advanced glycation endproducts (RAGE) decreases survival of tumor-bearing mice by enhancing the generation of lung metastasis-associated myeloid-derived suppressor cells.

Metastatic cancer has a poor prognosis. Novel pharmacologic targets need to be identified. The receptor for advanced glycation endproducts (RAGE) is a pattern recognition receptor constitutively expressed in the lungs. Absence of overt disease in RAGE null mice suggests that RAGE is unnecessary or redundant in health. We report that RAGE null tumor-bearing mice have reduced lung metastasis and improved survival. Bone marrow chimera studies suggest that hematopoietic cell RAGE is an important contributor to these effects. Deletion of RAGE reduces both the quantity and suppressive activity of tumor-induced MDSC. Protein and mRNA studies suggest that RAGE contributes to the generation and function of MDSC including expression of the alarmins S100A8/A9 and activity of inducible nitric oxide synthase, arginase-1, and NF-κB. These findings demonstrate the important role of RAGE in determining the quantity and function of tumor-associated MDSC and suggest RAGE as a pharmacologic target for patients with metastatic disease.

A 73-Year-Old Man With Progressive Whole Body Subcutaneous Gas After Pleural Catheter Removal.

CASE PRESENTATION: A 73-year-old man presented to the ED of an outside hospital with asymptomatic chest wall swelling 10 h after discharge from our hospital. Four days earlier, he had presented to our hospital with increased dyspnea, cough, and sputum production. His history was notable for severe COPD with bullous emphysema. Chest imaging demonstrated bilateral opacities and a collection of gas and liquid in the major fissure of the left lung. A catheter was placed into the collection of gas and liquid under imaging guidance. After 4 days, the catheter was removed without event and the patient was discharged from the hospital with an extended course of antibiotics. Imaging performed in the ED revealed gas in the tissues of the chest wall and no evidence of a pneumothorax. He was transported back to our hospital by helicopter.

RAGE is a Critical Mediator of Pulmonary Oxidative Stress, Alveolar Macrophage Activation and Emphysema in Response to Cigarette Smoke.

The receptor for advanced glycation end products (RAGE), a cell membrane receptor, recognizes ligands produced by cigarette smoke (CS) and has been implicated in the pathogenesis of COPD. We demonstrate that deletion or pharmacologic inhibition of RAGE prevents development of CS-induced emphysema. To identify molecular pathways by which RAGE mediates smoking related lung injury we performed unbiased gene expression profiling of alveolar macrophages (AM) obtained from RAGE null and C57BL/6 WT mice exposed to CS for one week or four months. Pathway analysis of RNA expression identified a number of genes integral to the pathogenesis of COPD impacted by the absence of RAGE. Altered expression of antioxidant response genes and lung protein 4-HNE immunostaining suggest attenuated oxidative stress in the RAGE null mice despite comparable CS exposure and lung leukocyte burden as the WT mice. Reduced endoplasmic reticulum stress in response to CS exposure also was observed in the AM from RAGE null mice. These findings provide novel insight into the sources of oxidative stress, macrophage activation, and the pathogenesis of lung disease due to CS exposure.

Consequences of Hypoxia for the Pulmonary Alveolar Epithelial Cell Innate Immune Response.

Pulmonary innate immune responses involve a highly regulated multicellular network to defend the enormous surface area of the lung. Disruption of these responses renders the host susceptible to pneumonia. Alveolar epithelial cells (AEC) are a critical source of innate immune molecules such as GM-CSF, which determine the functional maturation of alveolar macrophages. In many pulmonary diseases, heterogeneous ventilation leads to regional hypoxia in the lung. The effect of hypoxia on AEC innate immune function is unknown. We now report that exposure of primary murine AEC to hypoxia (1% oxygen) for 24 h results in significant suppression of key innate immune molecules, including GM-CSF, CCL2, and IL-6. This exposure did not cause toxicity but did induce stabilization of hypoxia-inducible factor 1α protein (HIF-1α) and shift to glycolytic metabolism. Focusing on GM-CSF, we found that hypoxia greatly decreased the rate of GM-CSF transcription. Hypoxia both decreased NF-κB signaling in AEC and induced chromosomal changes, resulting in decreased accessibility in the GM-CSF proximal promoter of target sequences for NF-κB binding. In mice exposed to hypoxia in vivo (12% oxygen for 2 d), lung GM-CSF protein expression was reduced. In vivo phagocytosis of fluorescent beads by alveolar macrophages was also suppressed, but this effect was reversed by treatment with GM-CSF. These studies suggest that in critically ill patients, local hypoxia may contribute to the susceptibility of poorly ventilated lung units to infection through complementary effects on several pathways, reducing AEC expression of GM-CSF and other key innate immune molecules.

Receptors for advanced glycation end-products targeting protect against hyperoxia-induced lung injury in mice.

Patients with acute lung injury almost always require supplemental oxygen during treatment; however, elevated oxygen itself is toxic. Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors predominantly localized to alveolar type I cells that influence development and cigarette smoke-induced inflammation, but studies that address the role of RAGE in acute lung injury are insufficient. In the present investigation, we test the hypothesis that RAGE signaling functions in hyperoxia-induced inflammation. RAGE-null mice exposed to hyperoxia survived 3 days longer than age-matched wild-type mice. After 4 days in hyperoxia, RAGE-null mice had less total cell infiltration into the airway, decreased total protein leak, diminished alveolar damage in hematoxylin and eosin-stained lung sections, and a lower lung wet-to-dry weight ratio. An inflammatory cytokine antibody array revealed decreased secretion of several proinflammatory molecules in lavage fluid obtained from RAGE knockout mice when compared with wild-type control animals. Real-time RT-PCR and immunoblotting revealed that hyperoxia induced RAGE expression in primary alveolar epithelial cells, and immunohistochemistry identified increased RAGE expression in the lungs of mice after exposure to hyperoxia. These data reveal that RAGE targeting leads to a diminished hyperoxia-induced pulmonary inflammatory response. Further research into the role of RAGE signaling in the lung should identify novel targets likely to be important in the therapeutic alleviation of lung injury and associated persistent inflammation.

NOX4 mediates hypoxia-induced proliferation of human pulmonary artery smooth muscle cells: the role of autocrine production of transforming growth factor-{beta}1 and insulin-like growth factor binding protein-3.

Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-beta1 (TGF-beta1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-beta1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-beta1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-beta1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-beta1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.

Cell type-- dependent effects of Polo-like kinase 1 inhibition compared with targeted polo box interference in cancer cell lines.

Multiple critical roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. Plk1 contains two domains amenable for targeted interference: a kinase domain responsible for the enzymatic function and a polo box domain necessary for substrate recognition and subcellular localization. Here, we compare two approaches for targeted interference with Plk1 function, either by a Plk1 small-molecule enzyme inhibitor or by inducible overexpression of the polo box in human cancer cell lines. Inducible expression of the Plk1 polo box resulted in growth inhibition of RKOp27 human colon adenocarcinoma cells without obvious signs of mitotic abnormalities. A Plk1 kinase inhibitor in the same cell line arrested cells in mitosis with subsequent onset of apoptosis. Similarly, PC-3 human prostate cancer cells were growth inhibited on expression of the polo box. Prolonged expression of the polo box in these cells resulted in the occurrence of binucleated or multinucleated cells. In contrast, U2OS human osteosarcoma cells responded to overexpression of the polo box with a massive mitotic accumulation coinciding with the onset of apoptosis. Comparison of spindle formation revealed very similar mitotic abnormalities in polo box-overexpressing U2OS cells compared with U2OS cells treated with the Plk1 kinase inhibitor. We conclude that interference with polo box function and inhibition of Plk1 kinase activity can exert very similar phenotypic effects in certain cell lines but highly contrasting effects in others. This may point to subtle differences in the molecular machinery of mitosis regulation in cancer cells.

Distinct pharmacological properties of second generation HDAC inhibitors with the benzamide or hydroxamate head group.

Advanced second generation inhibitors of histone deacetylases (HDAC) are currently used in clinical development. This study aimed at comparing the pharmacological properties of selected second generation HDAC inhibitors with the hydroxamate and benzamide head group, namely SAHA, LAQ824/LBH589, CI994, MS275 and MGCD0103. In biochemical assays using recombinant HDAC1, 3, 6 and 8 isoenzymes, SAHA and LAQ824/LBH589 behave as quite unselective HDAC inhibitors. In contrast, the benzamides CI994, MS275 and MGCD0103 are more selective, potent inhibitors of at least HDAC1 and HDAC3. All HDAC inhibitors induce histone H3 hyperacetylation, correlating with inhibition of proliferation, induction of cell differentiation and apoptosis. A broad cytotoxicity is seen across cell lines from different tumor entities with LAQ824/LBH589 being the most potent agents. The apoptosis inducing activity is evident in arrested and proliferating RKO colon cancer cells with inducible, heterologous p21(waf1) expression, indicative for a cell-cycle independent mode-of-action. Differentiation of MDA-MB468 breast cancer cells is induced by benzamide and hydroxamate analogs. The reversibility of drug action was evaluated by pulse treatment of A549 lung cancer cells. Whereas paclitaxel induced irreversible cell cycle alterations already after 6 hr treatment, HDAC inhibitor action was retarded and irreversible after >16 hr treatment. Interestingly, pulse treatment was equally effective as continous treatment. Finally, the efficacy of LAQ824, SAHA and MS275 in A549 nude mice xenografts was comparable to that of paclitaxel at well tolerated doses. We conclude that despite a different HDAC isoenzyme inhibition profile, hydroxamate and benzamide analogs as studied display similar cellular profiles.

Nox4 mediates TGF-beta1-induced retinoblastoma protein phosphorylation, proliferation, and hypertrophy in human airway smooth muscle cells.

Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in increasing airway smooth muscle mass in severe asthma by inducing proliferation and hypertrophy of human airway smooth muscle. The mechanism(s) for these effects of TGF-beta1 have not been fully elucidated. In this study, we demonstrate that TGF-beta1 is a potent inducer of expression of the nonphagocyte NAD(P)H oxidase catalytic homolog Nox4, diphenylene iodonium-inhibitable reactive oxygen species production, proliferation, and hypertrophy in cultured human airway smooth muscle cells. By confocal microscopy, TGF-beta1-induced Nox4 was localized with the endoplasmic reticulum and the nucleus, implying a role for Nox4 in regulation of both the cell cycle and protein synthesis. Consistent with this hypothesis, TGF-beta1 increased retinoblastoma protein phosphorylation at both Ser807/811 and Ser780. Silencing Nox4 prevented TGF-beta1-mediated retinoblastoma protein phosphorylation, proliferation, and cell hypertrophy. TGF-beta1 also increased phosphorylation of eukaryotic translation initiation factor 4E binding protein-1 at Thr37/46, and this was likewise blocked by silencing Nox4. This is the first report to suggest a functional role for Nox4 in cell cycle transition and to demonstrate that Nox4 influences the pathobiochemistry of asthma by generating reactive oxygen species that promote TGF-beta1-induced proliferation and hypertrophy of human airway smooth muscle.

Molecular alterations after Polo-like kinase 1 mRNA suppression versus pharmacologic inhibition in cancer cells.

Multiple roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. We have employed chimeric antisense oligonucleotides to investigate the molecular alterations after targeted interference with Plk1 in RKO human colon adenocarcinoma and PC3 prostate cancer cells. Suppression of Plk1 mRNA resulted in a dramatic increase of the mitotic index followed by the onset of apoptosis. Mitotically arrested cells displayed randomly separated condensed chromosomes and the occurrence of multiple spindle poles with well-formed asters. Induction of apoptosis was strictly dependent on cell cycle progression: Genetically engineered RKO cells with inducible expression of the cyclin-dependent kinase inhibitor p27(Kip1) were completely refractory to Plk1 depletion-induced apoptosis when they were arrested in the G1 phase of the cell cycle. Various mitotic markers, including MPM-2, cdc25c, cyclin B1, or phosphorylated histone H3, were investigated to explore the molecular consequences of Plk1 depletion. Whereas most marker proteins showed similar alterations compared with treatment with paclitaxel, cdc25c was fully phosphorylated solely in paclitaxel-treated cells but only partially phosphorylated in Plk1-depleted cells, although both treatments caused a profound mitotic arrest. This differential phosphorylation of cdc25c was used to test whether a pharmacologic inhibitor of Plk1 would exert the same cellular effects as interference with Plk1 on a mRNA level. It was found that the differential electrophoretic mobility of cdc25c can serve as a reliable molecular marker to track inhibition of Plk1 by small-molecule inhibitors within a cell.

G3139 and other CpG-containing immunostimulatory phosphorothioate oligodeoxynucleotides are potent suppressors of the growth of human tumor xenografts in nude mice.

Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.

Hypertonic induction of COX-2 in collecting duct cells by reactive oxygen species of mitochondrial origin.

Our previous studies have documented MAPK mediation of the hypertonicity-induced stimulation of COX-2 expression in cultured renal medullary epithelial cells. The present study extends this observation by examining the role of reactive oxygen species (ROSs). ROS levels, determined using dichlorodihydrofluorescence diacetate and cytochrome c, were rapidly and significantly increased following exposure of mIMCD-K2 cells to media made hypertonic by adding NaCl. Hypertonic treatment (550 mosmol/kg) for 16 h induced a 5.6-fold increase in COX-2 protein levels and comparable increases in prostaglandin E(2) release, both of which were completely abolished by the NADPH oxidase inhibitor diphenyleneiodonium (25-50 microM). The general antioxidant N-acetyl-l-cysteine (6 mM), and the superoxide dismutase mimetic TEMPO (2.0 mm) reduced COX-2 levels by 75.6 and 79.8%, respectively. Exposure of mIMCD-K2 cells to exogenous O(2)(-.) generated by the xanthine/xanthine oxidase system mimicked the effect of hypertonicity on COX-2 expression and prostaglandin E(2) release. The increases in phosphorylation of ERK1/2 and p38 were detected 20 min following the hypertonic treatment and were both prevented by N-acetyl-l-cysteine. The increases in ROSs in response to hypertonic treatment were completely blocked by any one of the mitochondrial inhibitors tested, such as rotenone, thenoyltrifluoroacetone, or carbonyl cyanide m-chlorophenylhydrazone, associated with remarkable inhibition of COX-2 expression. In contrast, the increases in ROSs were not significantly altered in IMCD cells deficient in either gp91(phox) or p47(phox), nor were the increases in COX-2 expression. We conclude that ROSs derived from mitochondria, but not NADPH oxidase, mediate the hypertonicity-induced phosphorylation of MAPK and the stimulation of COX-2 expression.

The role of endogenous NADPH oxidases in airway and pulmonary vascular smooth muscle function.

Reactive oxygen species generated from NADPH oxidase(s) in airway smooth muscle cells and pulmonary artery smooth muscle cells are important signaling intermediates. Nox4 appears to be the predominant gp91 homologue in these cells. However, expression of NADPH oxidase components is dependent on phenotype, and different homologues may be expressed during different functional states of the cell. NADPH oxidase(s) appear to be important not only for mitogenesis by these cells, but also for O(2) sensing. The regulation of NADPH oxidase(s) in airway and pulmonary artery smooth muscle cells has important implications for the pathobiochemistry of asthma and pulmonary vascular diseases.