Renal glomerular and tubular responses to glutaraldehyde- polymerized human hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and volume replacement therapeutics, however, their molecular and cellular effects on the vasculature and different organ systems are not fully defined. Using a guinea pig transfusion model, we examined the renal glomerular and tubular responses to PolyHeme, a highly characterized glutaraldehyde-polymerized human hemoglobin with low tetrameric hemoglobin content. PolyHeme-infused animals showed no major changes in glomerular histology or loss of specific markers of glomerular podocytes (Wilms tumor 1 protein, podocin, and podocalyxin) or endothelial cells (ETS-related gene and claudin-5) after 4, 24, and 72 h. Relative to sham controls, PolyHeme-infused animals also showed similar expression and subcellular distribution of N-cadherin and E-cadherin, two key epithelial junctional proteins of proximal and distal tubules, respectively. In terms of heme catabolism and iron-handling responses, PolyHeme induced a moderate but transient expression of heme oxygenase-1 in proximal tubular epithelium and tubulointerstitial macrophages that was accompanied by increased iron deposition in tubular epithelium. Contrary to previous findings with other modified or acellular hemoglobins, the present data show that PolyHeme does not disrupt the junctional integrity of the renal glomerulus and tubular epithelium, and triggers moderate activation of heme catabolic and iron sequestration systems likely as part of a renal adaptive response.

Ferroportin inhibition attenuates plasma iron, oxidant stress, and renal injury following red blood cell transfusion in guinea pigs.

BACKGROUND: Red blood cell (RBC) transfusions result in the sequestration and metabolism of storage-damaged RBCs within the spleen and liver. These events are followed by increased plasma iron concentrations that can contribute to oxidant stress and cellular injury. We hypothesized that administration of a ferroportin inhibitor (FPN-INH) immediately after acute RBC exchange transfusion could attenuate posttransfusion circulatory compartment iron exposure, by retaining iron in spleen and hepatic macrophages. STUDY DESIGN AND METHODS: Donor guinea pig blood was leukoreduced, and RBCs were preserved at 4°C. Recipient guinea pigs (n = 5/group) were exchange transfused with donor RBCs after refrigerator preservation and dosed intravenously with a small-molecule FPN-INH. Groups included transfusion with vehicle (saline), 5 mg/kg or 25 mg/kg FPN-INH. A time course of RBC morphology, plasma non-transferrin-bound iron (NTBI) and plasma hemoglobin (Hb) were evaluated. End-study spleen, liver, and kidney organ iron levels, as well as renal tissue oxidation and injury, were measured acutely (24-hr after transfusion). RESULTS: RBC transfusion increased plasma NTBI, with maximal concentrations occurring 8 hours after transfusion. Posttransfusion iron accumulation resulted in tubule oxidation and acute kidney injury. FPN inhibition increased spleen and liver parenchymal/macrophage iron accumulation, but attenuated plasma NTBI, and subsequent renal tissue oxidation/injury. CONCLUSION: In situations of acute RBC transfusion, minimizing circulatory NTBI exposure by FPN inhibition may attenuate organ-specific adverse consequences of iron exposure.

An Hb-mediated circulating macrophage contributing to pulmonary vascular remodeling in sickle cell disease.

Circulating macrophages recruited to the lung contribute to pulmonary vascular remodeling in various forms of pulmonary hypertension (PH). In this study we investigated a macrophage phenotype characterized by intracellular iron accumulation and expression of antioxidant (HO-1), vasoactive (ET-1), and proinflammatory (IL-6) mediators observed in the lung tissue of deceased sickle cell disease (SCD) patients with diagnosed PH. To this end, we evaluated an established rat model of group 5 PH that is simultaneously exposed to free hemoglobin (Hb) and hypobaric hypoxia (HX). Here, we tested the hypothesis that pulmonary vascular remodeling observed in human SCD with concomitant PH could be replicated and mechanistically driven in our rat model by a similar macrophage phenotype with iron accumulation and expression of a similar mixture of antioxidant (HO-1), vasoactive (ET-1), and inflammatory (IL-6) proteins. Our data suggest phenotypic similarities between pulmonary perivascular macrophages in our rat model and human SCD with PH, indicating a potentially novel maladaptive immune response to concomitant bouts of Hb and HX exposure. Moreover, by knocking out circulating macrophages with gadolinium trichloride (GdCl3), the response to combined Hb and hypobaric HX was significantly attenuated in rats, suggesting a critical role for macrophages in the exacerbation of SCD PH.

Attenuation of human hypoxic pulmonary vasoconstriction by acetazolamide and methazolamide.

Acetazolamide (AZ), a carbonic anhydrase inhibitor used for preventing altitude illness attenuates hypoxic pulmonary vasoconstriction (HPV) while improving oxygenation. Methazolamide (MZ), an analog of acetazolamide, is more lipophilic, has a longer half-life, and activates a major antioxidant transcription factor. However, its influence on the hypoxic pulmonary response in humans is unknown. The aim of this study was to determine whether a clinically relevant dosing of MZ improves oxygenation, attenuates HPV, and augments plasma antioxidant capacity in men exposed to hypoxia compared with an established dosing of AZ known to suppress HPV. In this double-blind, placebo-controlled crossover trial, 11 participants were randomized to treatments with MZ (100 mg 2× daily) and AZ (250 mg 3× daily) for 2 days before 60 min of hypoxia (FIO2 ≈0.12). Pulmonary artery systolic pressure (PASP), alveolar ventilation (V̇A), blood gases, and markers of redox status were measured. Pulmonary vascular sensitivity to hypoxia was determined by indexing PASP to alveolar PO2. AZ caused greater metabolic acidosis than MZ, but the augmented V̇A and improved oxygenation with hypoxia were similar. The rise in PASP with hypoxia was lower with MZ (9.0 ± 0.9 mmHg) and AZ (8.0 ± 0.7 mmHg) vs. placebo (14.1 ± 1.3 mmHg, P < 0.05). Pulmonary vascular sensitivity to hypoxia (ΔPASP/ΔPAO2) was reduced equally by both drugs. Only AZ improved the nonenzymatic plasma antioxidant capacity. Although AZ had only plasma antioxidant properties, MZ led to similar improvements in oxygenation and reduction in HPV at a dose causing less metabolic acidosis than AZ in humans.NEW & NOTEWORTHY Both acetazolamide and methazolamide are effective in the prevention of acute mountain sickness by inducing an increase in ventilation and oxygenation. Acetazolamide attenuates hypoxic pulmonary vasoconstriction; however, it was previously unknown whether methazolamide has the same effect in humans. This study shows that a dosing of methazolamide causing less metabolic acidosis improves oxygenation while attenuating hypoxic pulmonary vasoconstriction and pulmonary vascular sensitivity to hypoxia. Acetazolamide improved plasma antioxidant capacity better than methazolamide.

Renal Toxicodynamic Effects of Extracellular Hemoglobin After Acute Exposure.

Plasma hemoglobin (Hb) is elevated in some hematologic disease states, during exposures to certain toxicants, and with the use of some medical devices. Exposure to free Hb can precipitate oxidative reactions within tissues and alter the normal physiological function of critical organ systems. As kidney structures can be highly sensitive to Hb exposures, we evaluated the acute dose dependent renal toxicologic response to purified Hb isolated from RBCs. Male Hartley guinea pigs (n = 5 per group) were dosed with 0.9% saline (2 ml), 15, 75, 150, or 300 mg of purified Hb, infused over a 2-h period. The primary endpoints of this study were to define toxicokinetic parameters after increasing doses of purified Hb, identify clinically recognized and experimental markers of acute kidney injury (AKI), and determine relevant toxicological parameters and potential causes of renal toxicity in this model. Experimental findings demonstrated a dose dependent increase in Cmax after a 2-h infusion, which correlated with an elevation in serum creatinine, renal Kim-1 mRNA expression and increased urinary Kim-1. Renal NGAL mRNA expression and urinary NGAL excretion were also increased in several groups, but these parameters did not correlate with exposure. Iron increased in the renal cortex as Hb exposure increased and its deposition colocalized with 4-hydroxy-nonenal and 8-Oxo-2-deoxyguanosine immune reactivity, suggesting oxidative stressors may contribute to AKI in animals exposed to Hb. The results presented here suggest that Cmax may effectively predict the risk of AKI in normal healthy animals exposed to Hb.

Phenotype-specific recombinant haptoglobin polymers co-expressed with C1r-like protein as optimized hemoglobin-binding therapeutics.

BACKGROUND: Preclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1-1 versus Hp 2-2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200-900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions. RESULTS: In the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies. CONCLUSIONS: We present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions.

Evaluation of renal oxygen homeostasis in a preclinical animal model to elucidate difference in blood quality after transfusion.

BACKGROUND: Red blood cell (RBC) oxygen (O2 ) delivery may be impacted at the tissue, cellular, and molecular levels after storage duration, preservation strategies, and pathogen reduction. Collectively, the preclinical measurement of arterial and venous PO2 , systemic blood flow, tissue hypoxia-inducible factors (HIFs), pimonidazole adduction, and erythropoietin (EPO) regulation can serve to elucidate differential RBC quality after storage and processing. STUDY DESIGN AND METHODS: Donor guinea pig blood was collected, leukoreduced, and stored at 4°C in AS-3 for 1 (fresh) or 14 (stored) days. RBC variables-2,3-diphosphoglycerate, adenosine triphosphate, hemoglobin, morphology, deformability, and in vivo recovery at 24 hours-were measured at each storage duration. Recipient guinea pigs were exchange transfused until 80% volume replacement was achieved. Arterial and venous blood gases, systemic blood flow, renal HIF-1α and HIF-2α, renal EPO mRNA, plasma EPO, and renal tissue pimonidazole adduction were measured after transfusion. RESULTS: RBC variables declined significantly with storage; however, hemolysis and in vivo recovery remained within the allowable limits for human blood storage. Posttransfusion arterial and venous PO2 and systemic blood flow decreased, and renal HIFs, EPO mRNA, and pimonidazole adducts increased. Subsequently, EPO accumulated in plasma indicating decreased O2 availability in the kidneys. Conversely, all variables remained at basal levels in the fresh blood group. CONCLUSION: The evaluation of renal O2 homeostasis after transfusion represents an effective approach to defining RBC quality between predicate and novel processing. Methods are adapted from standardized techniques and ideal for preclinical evaluation.

The quaternary state of polymerized human hemoglobin regulates oxygenation of breast cancer solid tumors: A theoretical and experimental study.

A major constraint in the treatment of cancer is inadequate oxygenation of the tumor mass, which can reduce chemotherapeutic efficacy. We hypothesize that polymerized human hemoglobin (PolyhHb) can be transfused into the systemic circulation to increase solid tumor oxygenation, and improve chemotherapeutic outcomes. By locking PolyhHb in the relaxed (R) quaternary state, oxygen (O2) offloading at low O2 tensions (<20 mm Hg) may be increased, while O2 offloading at high O2 tensions (>20 mm Hg) is facilitated with tense (T) state PolyhHb. Therefore, R-state PolyhHb may deliver significantly more O2 to hypoxic tissues. Biophysical parameters of T and R-state PolyhHb were used to populate a modified Krogh tissue cylinder model to assess O2 transport in a tumor. In general, we found that increasing the volume of transfused PolyhHb decreased the apparent viscosity of blood in the arteriole. In addition, we found that PolyhHb transfusion decreased the wall shear stress at large arteriole diameters (>20 µm), but increased wall shear stress for small arteriole diameters (<10 µm). Therefore, transfusion of PolyhHb may lead to elevated O2 delivery at low pO2. In addition, transfusion of R-state PolyhHb may be more effective than T-state PolyhHb for O2 delivery at similar transfusion volumes. Reduction in the apparent viscosity resulting from PolyhHb transfusion may result in significant changes in flow distributions throughout the tumor microcirculatory network. The difference in wall shear stress implies that PolyhHb may have a more significant effect in capillary beds through mechano-transduction. Periodic top-load transfusions of PolyhHb into mice bearing breast tumors confirmed the oxygenation potential of both PolyhHbs via reduced hypoxic volume, vascular density, tumor growth, and increased expression of hypoxia inducible genes. Tissue section analysis demonstrated primary PolyhHb clearance occurred in the liver and spleen indicating a minimal risk for renal damage.

Iron accelerates hemoglobin oxidation increasing mortality in vascular diseased guinea pigs following transfusion of stored blood.

Non-transferrin-bound iron (NTBI) and free hemoglobin (Hb) accumulate in circulation following stored RBC transfusions. This study investigated transfusion, vascular disease, and mortality in guinea pigs after stored RBC transfusion alone and following cotransfusion with apo-transferrin (apo-Tf) and haptoglobin (Hp). The effects of RBC exchange transfusion dose (1, 3, and 9 units), storage period (14 days), and mortality were evaluated in guinea pigs with a vascular disease phenotype. Seven-day mortality and the interaction between iron and Hb as cocontributors to adverse outcome were studied. Concentrations of iron and free Hb were greatest after transfusion with 9 units of stored RBCs compared with fresh RBCs or stored RBCs at 1- and 3-unit volumes. Nine units of stored RBCs led to mortality in vascular diseased animals, but not normal animals. One and 3 units of stored RBCs did not cause a mortality effect, suggesting the concomitant relevance of NTBI and Hb on outcome. Cotransfusion with apo-Tf or Hp restored survival to 100% following 9-unit RBC transfusions in vascular diseased animals. Our data suggest that increases in plasma NTBI and Hb contribute to vascular disease-associated mortality through iron-enhanced Hb oxidation and enhanced tissue injury.

A mechanistic investigation of thrombotic microangiopathy associated with IV abuse of Opana ER.

Since 2012, a number of case reports have described the occurrence of thrombotic microangiopathy (TMA) following IV abuse of extended-release oxymorphone hydrochloride (Opana ER), an oral opioid for long-term treatment of chronic pain. Here, we present unique clinical features of 3 patients and investigate IV exposure to the tablet's inert ingredients as a possible causal mechanism. Guinea pigs were used as an animal model to understand the hematopathologic and nephrotoxic potential of the inert ingredient mixture (termed here as PEO+) which primarily contains high-molecular-weight polyethylene oxide (HMW PEO). Microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury were found in a group of 3 patients following recent injection of adulterated extended-release oxymorphone tablets. Varying degrees of cardiac involvement and retinal ischemia occurred, with TMA evident on kidney biopsy. A TMA-like state also developed in guinea pigs IV administered PEO+. Acute tubular and glomerular renal injury was accompanied by nonheme iron deposition and hypoxia-inducible factor-1α upregulation in the renal cortex. Similar outcomes were observed following dosing with HMW PEO alone. IV exposure to the inert ingredients in reformulated extended-release oxymorphone can elicit TMA. Although prescription opioid abuse shows geographic variation, all physicians should be highly inquisitive of IV drug abuse when presented with cases of TMA.

Extracellular Hb enhances cardiac toxicity in endotoxemic guinea pigs: protective role of haptoglobin.

Endotoxemia plays a major causative role in the myocardial injury and dysfunction associated with sepsis. Extracellular hemoglobin (Hb) has been shown to enhance the pathophysiology of endotoxemia. In the present study, we examined the myocardial pathophysiology in guinea pigs infused with lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, and purified Hb. We also examined whether the administration of the Hb scavenger haptoglobin (Hp) could protect against the effects observed. Here, we show that Hb infusion following LPS administration, but not either insult alone, increased myocardial iron deposition, heme oxygenase-1 expression, phagocyte activation and infiltration, as well as oxidative DNA damage and apoptosis assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) immunostaining, respectively. Co-administration of Hp significantly attenuated the myocardial events induced by the combination of LPS and Hb. These findings may have relevant therapeutic implications for the management of sepsis during concomitant disease or clinical interventions associated with the increased co-exposures to LPS and Hb, such as trauma, surgery or massive blood transfusions.

Isolated Hb Providence ß82Asn and ß82Asp fractions are more stable than native HbA(0) under oxidative stress conditions.

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the ß-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or ß-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two ß-subunit variants with single amino acid mutations at ßLys82→Asp (ßK82D) and at ßLys82→Asn (ßK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its ß-mutant fractions with HbAo. Relative to HbAo, both ßK82N and ßK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of ß-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that ßCys93 and ßCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in ßK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.

Induction of hypoxia inducible factor (HIF-1α) in rat kidneys by iron chelation with the hydroxypyridinone, CP94.

Hypoxia inducible factor (HIF-1α) is a master regulator of tissue adaptive responses to hypoxia whose stability is controlled by an iron containing prolyl hydroxylase domain (PHD) protein. A catalytic redox cycle in the PHD's iron center that results in the formation of a ferryl (Fe(+4)) intermediate has been reported to be responsible for the hydroxylation and subsequent degradation of HIF-1α under normoxia. We show that induction of HIF-1α in rat kidneys can be achieved by iron reduction by the hydroxypyridin-4 one (CP94), an iron chelator administered intraperitoneally in rats. The extent of HIF protein stabilization as well as the expression of HIF target genes, including erythropoietin (EPO), in kidney tissues was comparable to those induced by known inhibitors of the PHD enzyme, such as desferrioxamine (DFO) and cobalt chloride (CoCl(2)). In human kidney cells and in vitro PHD activity assay, we were able to show that the HIF-1α protein can be stabilized by addition of CP94. This appears to inactivate PHD; and thus prevents the hydroxylation of HIF-1α. In conclusion, we have identified the inhibition of iron-binding pocket of PHD as an underlying mechanism of HIF induction in vivo and in vitro by a bidentate hydroxypyridinone.

Overexpression of AQP5, a putative oncogene, promotes cell growth and transformation.

Overexpression of several aquaporins has been reported in different types of human cancer but the role of AQPs in human carcinogenesis has not yet been clearly defined. Here, we demonstrate that ectopic expression of human AQP5 (hAQP5), a water channel expressed in lung, salivary glands, and kidney, induces many phenotypic changes characteristic of transformation both in vitro and in vivo. Furthermore, the cell proliferative ability of AQP5 appears to be dependent upon the phosphorylation of a cAMP-protein kinase (PKA) consensus site located in a cytoplasmic loop of AQP5. In addition, phosphorylation of the PKA consensus site was found to be phosphorylated preferentially in tumors. These findings altogether indicate that hAQP5 plays an important role in human carcinogenesis and, furthermore, provide an attractive therapeutic target.

DCC promoter hypermethylation in esophageal squamous cell carcinoma.

Deleted in Colorectal Cancer (DCC) is a putative tumor suppressor gene, whose loss has been implicated in colorectal tumorigenesis. Decreased or loss of DCC expression has been demonstrated in a number of human cancers, including esophageal cancer. In this study, we analyzed esophageal squamous cell carcinoma (ESCC) cell lines and primary ESCCs as well as normal esophageal tissues for DCC methylation by bisulfite sequencing, methylation-specific PCR (MSP) and/or quantitative methylation-specific PCR (qMSP). When a qMSP cut-off value for positivity was set to 1.0, DCC methylation was detected in 10 of 12 ESCC cell lines tested, 74% of primary ESCCs (n = 70), 0% of corresponding normal esophageal tissues (n = 20) and 0% of normal esophagus from healthy individuals (n = 19). DCC expression was undetectable in the majority of ESCC cell lines, and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reactivated gene expression. DCC overexpression suppressed colony formation in ESCC cell lines, suggesting that DCC may function as a tumor suppressor gene in the esophagus. However, DCC methylation was not associated with any clinical or pathologic parameters measured. We have demonstrated that DCC methylation is a frequent and cancer-specific event in primary ESCCs, suggesting that DCC and associated pathways may represent a new diagnostical therapeutic target.

Membrane trafficking of AQP5 and cAMP dependent phosphorylation in bronchial epithelium.

Phosphorylation pathway has been identified as an important step in membrane trafficking for AQP5. We generated stably transfected BEAS-2B human bronchial epithelial cells with various over-expression constructs on permeable support. In stable cells with wild-type AQP5 and S156A (AQP5 mutant targeting PKA consensus sequence), AQP5 expression was predominantly polarized to the apical membrane, whereas stable cells with N185D (AQP5 mutant targeting second NPA motif), mainly localized to the cytoplasm. Treatment with H89 and/or chlorophenylthio-cAMP (cpt-cAMP) did not affect membrane expression of AQP5 in any of three stable cells. In cells with wild-type AQP5 and N185D, AQP5s were phosphorylated by PKA, while phosphorylation of AQP5 was not detected in cells with S156A. These results indicate that, in AQP5, serine156 may be phosphorylated by PKA, but membrane expression of AQP5 may not be regulated by PKA phosphorylation. We conclude that AQP5 membrane targeting can include more than one mechanism besides cAMP dependent phosphorylation.

Spermidine/spermine-N1-acetyltransferase 2 is an essential component of the ubiquitin ligase complex that regulates hypoxia-inducible factor 1alpha.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that functions as a master regulator of oxygen homeostasis. The HIF-1alpha subunit is subjected to O(2)-dependent prolyl hydroxylation leading to ubiquitination by the von Hippel-Lindau protein (VHL)-Elongin C ubiquitin-ligase complex and degradation by the 26 S proteasome. In this study, we demonstrate that spermidine/spermine-N(1)-acetyltransferase (SSAT) 2 plays an essential role in this process. SSAT2 binds to HIF-1alpha, VHL, and Elongin C and promotes ubiquitination of hydroxylated HIF-1alpha by stabilizing the interaction of VHL and Elongin C. Multivalent interactions by SSAT2 provide a mechanism to ensure efficient complex formation, which is necessary for the extremely rapid ubiquitination and degradation of HIF-1alpha that is observed in oxygenated cells.

N-methyl-D-aspartate receptor type 2B is epigenetically inactivated and exhibits tumor-suppressive activity in human esophageal cancer.

Promoter hypermethylation accompanied by gene silencing is a common feature of human cancers. We identified previously several new tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of reexpression on microarray analysis. In this study, we modified the selection of candidates from our previous microarray data by excluding genes that showed basal expression in cancer cell lines. With the new method, we found novel methylated genes with 90% accuracy. Among these 33 novel methylated genes that we identified in esophageal squamous cell carcinoma (ESCC) cell lines, N-methyl-D-aspartate receptor type 2B (NMDAR2B) was of particular interest. NMDAR2B was methylated in 95% of primary human ESCC tissue specimens and 12 ESCC cell lines by sequence analysis. NMDAR2B expression was silenced in all 12 ESCC cell lines and was reactivated by the demethylating agent 5-aza-2'-deoxycytidine. Moreover, reintroduction of the gene was accompanied by marked Ca(2+)-independent apoptosis in ESCC cell lines, suggesting that NMDAR2B can suppress tumor growth. Thus, NMDAR2B promoter methylation is common in ESCC, abrogating gene transcription and leading to cellular resistance to apoptosis.

Response of non-small cell lung cancer cells to the inhibitors of phosphatidylinositol 3-kinase/Akt- and MAPK kinase 4/c-Jun NH2-terminal kinase pathways: an effective therapeutic strategy for lung cancer.

PURPOSE: We previously showed that phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways cooperate to promote non-small cell lung cancer (NSCLC) cell proliferation in vitro. This study was designed to explore whether inhibition of these pathways effectively inhibits NSCLC tumor growth in vivo. EXPERIMENTAL DESIGN: The effects of PI3K/Akt inhibitors {LY294002, adenoviruses expressing dominant-negative mutant of the p85alpha adaptor subunit of PI3K (Ad-dnp85alpha), dominant-negative Akt [Ad-HA-Akt(KM)], or PTEN (Ad-PTEN)}, MKK4/c-jun NH2-terminal kinase (JNK) inhibitor [SP600215, adenovirus expressing dominant-negative MKK4, Ad-MKK4(KR)], and their combinations on proliferation and apoptosis in NSCLC cells were tested in vitro and in vivo using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a flow cytometry-based terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, Western blot and immunohistochemical analyses, and an NSCLC xenograft tumor model. RESULTS: Ad-dnp85alpha significantly inhibited proliferation of a subset of NSCLC cell lines used in our study. Intratumoral injection of Ad-dnp85alpha induced a significant decrease in the growth of H1299 NSCLC xenograft tumors. Concurrent inhibition of the PI3K/Akt and MKK4/JNK pathways showed enhanced antiproliferative effects on H1299 cells in vitro and in vivo by increasing apoptosis. CONCLUSIONS: PI3K/Akt and MKK4/JNK pathways cooperate to stimulate NSCLC cell proliferation by maintaining cell survival, suggesting that simultaneously targeting these two pathways might be an effective therapeutic strategy against NSCLC.

Wilfoside K1N isolated from Cynanchum wilfordii inhibits angiogenesis and tumor cell invasion.

Wilfoside K1N is a polyoxypregnane glycoside isolated from Cynanchum wilfordii (Asclepiadaceae). Polyoxypregnane glycosides are associated with cellular immunity and anti-tumor activity, and increase the cytotoxicity of many anti-cancer drugs showing multidrug resistant activity on tumor cells. In the present study, we investigated the anti-angiogenic and anti-invasive activities of wilfoside K1N. In in vivo Matrigel plug assay using C57BL/6 mice, wilfoside K1N strongly inhibited basic fibroblast growth factor-induced microvessel formation. Exposure of wilfoside K1N to human umbilical vein endothelial cells (HUVEC) suppressed in vitro tube formation at a concentration not affecting cell viability. Moreover, wilfoside K1N significantly reduced the proliferation of HUVEC and calf pulmonary artery endothelial cells. In addition, wilfoside K1N decreased in vitro invasion of HT1080 human fibrosarcoma cells, and the inhibition might be through down-regulation of activity as well as quantity of matrix metalloproteinase-9. Therefore, our present study suggests that wilfoside K1N may have a potential to have strong anti-angiogenic and anti-invasive activities both in vitro and in vivo.