stilbenes and Anemia--Sickle-Cell

Hypoxic tumor niches are chief causes of treatment resistance and tumor recurrence. Sickle erythrocytes' (SSRBCs') intrinsic oxygen-sensing functionality empowers them to access such hypoxic niches wherein they form microaggregates that induce focal vessel closure. In search of measures to augment the scale of SSRBC-mediated tumor vaso-occlusion, we turned to the vascular disrupting agent, combretastatin A-4 (CA-4). CA-4 induces selective tumor endothelial injury, blood stasis, and hypoxia but fails to eliminate peripheral tumor foci. In this article, we show that introducing deoxygenated SSRBCs into tumor microvessels treated with CA-4 and sublethal radiation (SR) produces a massive surge of tumor vaso-occlusion and broadly propagated tumor infarctions that engulfs treatment-resistant hypoxic niches and eradicates established lung tumors. Tumor regression was histologically corroborated by significant treatment effect. Treated tumors displayed disseminated microvessels occluded by tightly packed SSRBCs along with widely distributed pimidazole-positive hypoxic tumor cells. Humanized HbS-knockin mice (SSKI) but not HbA-knockin mice (AAKI) showed a similar treatment response underscoring SSRBCs as the paramount tumoricidal effectors. Thus, CA-4-SR-remodeled tumor vessels license SSRBCs to produce an unprecedented surge of tumor vaso-occlusion and infarction that envelops treatment-resistant tumor niches resulting in complete tumor regression. Strategically deployed, these innovative tools constitute a major conceptual advance with compelling translational potential.

Topics: Anemia, Sickle Cell; Animals; Antineoplastic Agents, Phytogenic; Cell Adhesion; Cell Hypoxia; Cell Line, Tumor; Combined Modality Therapy; Erythrocytes, Abnormal; Female; Gene Knock-In Techniques; Hemoglobin, Sickle; Humans; Lung; Lung Neoplasms; Male; Mice; Mice, Transgenic; Microvessels; Neoplasm Recurrence, Local; Stilbenes; Transplantation, Heterologous; Tumor Burden; Xenograft Model Antitumor Assays

Resveratrol, a natural dietary polyphenol, has been postulated to be implicated in the cardioprotective effect of red wine and the low incidence of breast and prostate cancers among vegetarians and Orientals respectively. This compound inhibits ribonucleotide reductase as does hydroxyurea, the first therapeutic agent used in the treatment of sickle cell disease. Using the human erythroleukaemic K562 cell line as an in vitro model, we show here that 50 micromol/l of resveratrol induced a higher haemoglobin production (sevenfold) in K562 cells than 500 micromol/l of hydroxyurea (3.5-fold). This erythroid differentiation was linked to a dose- and time-dependent inhibition of cell proliferation associated with an equivalent increased expression of p21 mRNA, but with a higher increased level of p21 protein (sixfold) for cells treated with resveratrol than for those treated with hydroxyurea (1.5-fold). We also show that 50 micromol/l of resveratrol and 25 micromol/l of hydroxyurea induced variable but similar enhancements of fetal haemoglobin synthesis in cultured erythroid progenitors for the majority of the sickle cell patients studied. These inductions were linked to, but not correlated with, a variable decrease in erythroid burst-forming unit clone number. Taken together, these results show that resveratrol merits further investigations in sickle cell disease therapy.

Topics: Anemia, Sickle Cell; Antioxidants; Blotting, Western; Cell Differentiation; Cell Division; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Erythroid Precursor Cells; Fetal Hemoglobin; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Hemoglobins; Humans; Hydroxyurea; Leukemia, Erythroblastic, Acute; Models, Biological; Proto-Oncogene Proteins p21(ras); Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleotide Reductases; Stem Cells; Stilbenes; Time Factors

Peripheral human red blood cells (RBCs) are not generally known to become activated and adhesive in response to cell signaling. We show, however, that soluble thrombospondin via integrin-associated protein (IAP; CD47) increases the adhesiveness of sickle RBCs (SS RBCs) by activating signal transduction in the SS RBC. This stimulated adhesion requires occupancy of IAP and shear stress and is mediated by the activation of large G proteins and tyrosine kinases. Reticulocyte-enriched RBCs derived from sickle-cell disease (SCD) patients are most responsive to IAP-induced activation. These studies therefore establish peripheral SS RBCs as signaling cells that respond to a novel synergy between IAP-induced signal transduction and shear stress, suggesting new therapeutic targets in SCD.

Topics: Anemia, Sickle Cell; Antigens, CD; Carrier Proteins; CD47 Antigen; Cell Adhesion; Cells, Cultured; Enzyme Inhibitors; Erythrocytes, Abnormal; Genistein; Humans; Models, Biological; Oligopeptides; Phosphotyrosine; Signal Transduction; Stilbenes; Stress, Physiological; Thrombospondins; Virulence Factors, Bordetella

Deoxygenation-induced cation movements in sickle cells were inhibited 80% to 85% by the anion transport inhibitor, 4,4'-diisothiocyano-2,2'disulfostilbene (DIDS). Morphologic sickling was not altered by DIDS treatment, demonstrating that morphologic sickling was not sufficient to produce cation leaks in sickle cells. DIDS inhibition of deoxygenation-induced cation flux was not affected when l- replaced Cl-, indicating that conductive anion movements did not limit cation flux in deoxygenated cells treated with DIDS. Inhibition was irreversible after preincubation with DIDS at 37 degrees C for 20 minutes, and was not affected by the oxygenation state of cells at the time of drug exposure. Sulfate self-exchange was inhibited at lower DIDS concentrations than was deoxygenation-induced flux. Incubation of cells with DIDS at 4 degrees C produced progressive blockade of sulfate exchange, but did not alter deoxygenation-induced cation fluxes. Other stilbene disulfonates, including compounds incapable of covalent reactions, also inhibited deoxygenation-induced cation movements, although several other inhibitors of anion exchange did not. Dissociation of the inhibition of anion exchange and deoxygenation-induced cation flux indicates that the DIDS effect on deoxygenation-induced cation movements does not involve the well-characterized stilbene binding site of the anion exchanger. These data provide evidence for a membrane constituent on the external surface of oxygenated sickle cells capable of interacting with DIDS to prevent the increase in cation permeability associated with sickling.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anemia, Sickle Cell; Anion Exchange Resins; Benzenesulfonates; Cations; Cell Membrane Permeability; Erythrocytes, Abnormal; Heterozygote; Humans; Isothiocyanates; Oxidation-Reduction; Phloretin; Stilbenes; Thiocyanates; Time Factors

Sickle cells exhibit a striking increase in monovalent cation content upon deoxygenation. These deoxy cation fluxes are reversible upon reoxygenation, balanced (Na flux = K flux), activated under physiologic conditions of pH and PO2, and insensitive to ouabain and furosemide. Our recent evidence indicates that deoxy cation fluxes and the Na/K pump, acting in concert, contribute to cation depletion in sickle cells in vitro. The resultant dehydration worsens their rigidity and increases their tendency to sickle upon deoxygenation. Here we report our finding that deoxy cation fluxes are inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). This irreversible inhibitor of anion transport was effective at concentrations less than 1 microM. The reversible, competitive inhibitor, 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) also blocked deoxy cation fluxes, at 100 microM. Niflumic acid, which exhibits non-competitive inhibitory kinetics against anion transport, also inhibited deoxy cation fluxes at 50 microM. Phloretin (a non-competitive anion transport inhibitor), however, had no effect on deoxy fluxes. On the basis of these data, we propose the working hypothesis that deoxygenation of sickle cells causes a conformational change in the anion exchange protein which allows for the passage of cations and alters its sensitivity to inhibitors.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anemia, Sickle Cell; Anion Transport Proteins; Carrier Proteins; Cell Membrane Permeability; Erythrocyte Membrane; Hemoglobin, Sickle; Humans; In Vitro Techniques; Niflumic Acid; Phloretin; Potassium; Sodium; Stilbenes

Topics: Adult; Anemia, Sickle Cell; Antisickling Agents; Azepines; Calcium; Erythrocytes, Abnormal; Humans; Imines; In Vitro Techniques; Ionophores; Oxygen; Stilbenes