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Biological Products↗
Biomedical subjects
Emily Shacter
Publications and source records attributed to Emily Shacter.
Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues.
Human pharmacokinetics data indicate that i.v. ascorbic acid (ascorbate) in pharmacologic concentrations could have an unanticipated role in cancer treatment. Our goals here were to test whether ascorbate killed cancer cells selectively, and if so, to determine mechanisms, using clinically relevant conditions. Cell death in 10 cancer and 4 normal cell types was measured by using 1-h exposures. Normal cells were unaffected by 20 mM ascorbate, whereas 5 cancer lines had EC(50) values of <4 mM, a concentration easily achievable i.v. Human lymphoma cells were studied in detail because of their sensitivity to ascorbate (EC(50) of 0.5 mM) and suitability for addressing mechanisms. Extracellular but not intracellular ascorbate mediated cell death, which occurred by apoptosis and pyknosis/necrosis. Cell death was independent of metal chelators and absolutely dependent on H(2)O(2) formation. Cell death from H(2)O(2) added to cells was identical to that found when H(2)O(2) was generated by ascorbate treatment. H(2)O(2) generation was dependent on ascorbate concentration, incubation time, and the presence of 0.5-10% serum, and displayed a linear relationship with ascorbate radical formation. Although ascorbate addition to medium generated H(2)O(2), ascorbate addition to blood generated no detectable H(2)O(2) and only trace detectable ascorbate radical. Taken together, these data indicate that ascorbate at concentrations achieved only by i.v. administration may be a pro-drug for formation of H(2)O(2), and that blood can be a delivery system of the pro-drug to tissues. These findings give plausibility to i.v. ascorbic acid in cancer treatment, and have unexpected implications for treatment of infections where H(2)O(2) may be beneficial.
Antioxidants↗
Rho GDP dissociation inhibitor protects cancer cells against drug-induced apoptosis.
Rho GDP dissociation inhibitor (RhoGDI) plays an essential role in control of a variety of cellular functions through interactions with Rho family GTPases, including Rac1, Cdc42, and RhoA. RhoGDI is frequently overexpressed in human tumors and chemo-resistant cancer cell lines, raising the possibility that RhoGDI might play a role in the development of drug resistance in cancer cells. We found that overexpression of RhoGDI increased resistance of cancer cells (MDA-MB-231 human breast cancer cells and JLP-119 lymphoma cells) to the induction of apoptosis by two chemotherapeutic agents: etoposide and doxorubicin. Conversely, silencing of RhoGDI expression by DNA vector-mediated RNA interference (small interfering RNA) sensitized MDA-MB-231 cells to drug-induced apoptosis. Resistance to apoptosis was restored by reintroduction of RhoGDI protein expression. The mechanism for the anti-apoptotic activity of RhoGDI may derive from its ability to inhibit caspase-mediated cleavage of Rac1 GTPase, which is required for maximal apoptosis to occur in response to cytotoxic drugs. Taken together, the data show that RhoGDI is an anti-apoptotic molecule that mediates cellular resistance to these chemotherapy agents.
Antibiotics, Antineoplastic↗
Desferal inhibits breast tumor growth and does not interfere with the tumoricidal activity of doxorubicin.
Desferal is a clinically approved iron chelator used to treat iron overload. Doxorubicin is an anthracycline cancer chemotherapy drug used in the treatment of breast cancer. It can undergo redox cycling in the presence of iron to produce reactive oxygen species. The oxidant-generating activity of doxorubicin is thought to be responsible for the cardiotoxic side effects of the drug, but it is unclear whether it is also required for its anti-tumor activity. To test whether an iron-chelating antioxidant would interfere with the tumor-killing activity of doxorubicin, nude mice were transplanted with xenografts of human breast cancer MDA-MB 231 cells and then treated with doxorubicin and/or desferal. Not only did desferal not interfere with the anti-tumor activity of doxorubicin, it inhibited tumor growth on its own. In vitro studies confirmed that desferal inhibits breast tumor growth. However, it did not induce apoptosis, nor did it induce cell cycle arrest. Instead, desferal caused cytostasis, apparently through iron depletion. The cytostatic activity of desferal was partially ameliorated by pretreatment with iron-saturated transferrin, and transferrin receptor expression on breast cancer cells nearly doubled after exposure to desferal. In contrast to its effect on tumor cells, desferal did not inhibit growth of normal breast epithelial cells. The data indicate that the anti-tumor activity of doxorubicin is not dependent on iron-mediated ROS production. Furthermore, desferal may have utility as an adjunctive chemotherapy due to its ability to inhibit breast tumor growth and cardiotoxic side effects without compromising the tumor-killing activity of an anthracycline chemotherapy drug.
Animals↗
Taurine chloramine, an oxidant derived from neutrophils, induces apoptosis in human B lymphoma cells through mitochondrial damage.
Taurine chloramine (TN-Cl) is one of the most abundant compounds generated by activated neutrophils. In contrast to HOCl, which causes necrosis, TN-Cl is a potent inducer of apoptosis in tumor cells. Here we show that the apoptosis induced by TN-Cl in human B lymphoma cells is dependent upon oxidant-mediated mitochondrial damage, a decrease in mitochondrial membrane potential, and caspase-9 activation. Further, we show that TN-Cl is taken up into the cells and is concentrated in the mitochondria, where it induces opening of the permeability transition pore and mitochondrial swelling. Identical activity is seen upon treatment of isolated mitochondria with TN-Cl and is blocked by the permeability transition pore inhibitors bongkrekic acid and cyclosporin A, as well as by the sulfhydryl-reducing agent tris(2-carboxyethyl)-phosphine. The data suggest that TN-Cl causes apoptosis through direct damage to the mitochondria.
Anti-Bacterial Agents↗
Mechanism of the guanine nucleotide exchange reaction of Ras GTPase--evidence for a GTP/GDP displacement model.
Ras GTPases function as binary switches in the signaling pathways controlling cell growth and differentiation by cycling between the inactive GDP-bound and the active GTP-bound states. They are activated through interaction with guanine nucleotide exchange factors (GEFs) that catalyze the exchange of bound GDP with cytosolic GTP. In a conventional scheme, the biochemical roles of GEFs are postulated as stimulating the release of the bound GDP and stabilizing a nucleotide-free transition state of Ras. Herein we have examined in detail the catalyzed GDP/GTP exchange reaction mechanism by a Ras specific GEF, GRF1. In the absence of free nucleotide, GRF1 could not efficiently stimulate GDP dissociation from Ras. The release of the Ras-bound GDP was dependent upon the concentration and the structure of the incoming nucleotide, in particular, the hydrophobicity of the beta and gamma phosphate groups, suggesting that the GTP binding step is a prerequisite for GDP dissociation, is the rate-limiting step in the GEF reaction, or both. Using a pair of fluorescent guanine nucleotides (N-methylanthraniloyl GDP and 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-GTP) as donor and acceptor probes, we were able to detect fluorescence resonance energy transfer between the incoming GTP and the departing GDP on Ras under controlled kinetic conditions, providing evidence that there may exist a novel intermediate of the GEF-Ras complex that transiently binds to two nucleotides simultaneously. Furthermore, we found that Ras was capable of binding pyrophosphate (PPi) with a dissociation constant of 26 microM and that PPi and GMP, but neither alone, synergistically potentiated the GRF1-stimulated GDP dissociation from Ras. These results strongly support a GEF reaction mechanism by which nucleotide exchange occurs on Ras through a direct GTP/GDP displacement model.
Binding Sites↗
Rac1 inhibits apoptosis in human lymphoma cells by stimulating Bad phosphorylation on Ser-75.
The small GTPase Rac1 has emerged as an important regulator of cell survival and apoptosis, but the mechanisms involved are not completely understood. In this report, constitutively active Rac1 is shown to stimulate the phosphorylation of the Bcl-2 family member Bad, thereby suppressing drug-induced caspase activation and apoptosis in human lymphoma cells. Rac1 activation leads to human Bad phosphorylation specifically at serine-75 (corresponding to murine serine-112) both in vivo and in vitro. Inhibition of constitutive and activated Rac1-induced Bad phosphorylation by a cell-permeable competitive peptide inhibitor representing this Bad phosphorylation site sensitizes lymphoma cells to drug-induced apoptosis. The data show further that endogenous protein kinase A is a primary catalyst of cellular Bad phosphorylation in response to Rac activation, while Akt is not involved. These findings define a mechanism by which active Rac1 promotes lymphoma cell survival and inhibits apoptosis in response to cancer chemotherapy drugs.
Animals↗
Natural anticoagulant proteins in the regulation of autoimmunity: potential role of protein S.
Autoimmunity results when the immune system fails to distinguish between self and non-self factors in the body. The cellular and biochemical mechanisms that underlie development of autoimmunity are only partly understood. One current theory is that autoimmunity can result when there is a failure to clear dying cells from a tissue before they undergo lysis of the plasma membrane. That is, cells that die by apoptosis are thought to be cleared from a tissue by neighboring phagocytic cells, such as macrophages, before the cells have lost their plasma membrane integrity. This rapid removal of early apoptotic cells is thought to prevent induction of an inflammatory response to intracellular macromolecules, thereby allowing for an immunologically silent removal of the dying cells. Hence, any factor or condition that inhibits phagocytosis of early apoptotic cells may trigger or promote an autoimmune response to intracellular components. Depletion of factors required for the efficient phagocytosis of dying cells would have a similar outcome. The recent discovery that the natural anticoagulant protein S is required for efficient uptake of apoptotic cells (Anderson, H.A., Maylock, C.A., Williams, J.A., Paweletz, C.P., Shu, H., and Shacter, E. (2003) Nature Immunology 4, 87-91) reveals a potential new linkage between autoimmunity and coagulation systems. This article will review the dual roles of protein S as an anticoagulant and in regulating phagocytosis of apoptotic cells, with emphasis on exposing a possible novel role in regulating autoimmunity.
Apoptosis↗
Caspase 3-mediated inactivation of rac GTPases promotes drug-induced apoptosis in human lymphoma cells.
The Rac members of the Rho family GTPases control signaling pathways that regulate diverse cellular activities, including cytoskeletal organization, gene transcription, and cell transformation. Rac is implicated in apoptosis, but little is known about the mechanism by which it responds to apoptotic stimuli. Here we demonstrate that endogenous Rac GTPases are caspase 3 substrates that are cleaved in human lymphoma cells during drug-induced apoptosis. Cleavage of Rac1 occurs at two unconventional caspase 3 sites, VVGD11/G and VMVD47/G, and results in inactivation of the GTPase and effector functions of the protein (binding to the p21-activated protein kinase PAK1). Expression of caspase 3-resistant Rac1 mutants in the cells suppresses drug-induced apoptosis. Thus, proteolytic inactivation of Rac GTPases represents a novel, irreversible mechanism of Rac downregulation that allows maximal cell death following drug treatment.
Apoptosis↗
Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells.
Rapid phagocytosis of apoptotic cells is thought to limit the development of inflammation and autoimmune disease. Serum enhances macrophage phagocytosis of apoptotic cells. Here we identified protein S as the factor responsible for serum-stimulated phagocytosis of apoptotic cells. Protein S is best known for its anti-thrombotic activity, serving as a cofactor for protein C. Purified protein S was equivalent to serum in its ability to stimulate macrophage phagocytosis of apoptotic lymphoma cells, and immunodepletion of protein S eliminated the prophagocytic activity of serum. Protein S acted by binding to phosphatidylserine expressed on the apoptotic cell surface. Protein S is thus a multifunctional protein that can facilitate clearance of early apoptotic cells in addition to regulating blood coagulation.
Apoptosis↗
Distinct modes of cell death induced by different reactive oxygen species: amino acyl chloramines mediate hypochlorous acid-induced apoptosis.
Oxidants derived from inflammatory phagocytes compose a key element of the host immune defense system and can kill mammalian cells by one of several different mechanisms. In this report, we compare mechanisms of cell death induced in human B lymphoma cells by the inflammatory oxidants superoxide, H(2)O(2), and HOCl. The results indicate that the mode of cell death induced depends on the nature of the oxidant involved and the medium in which the cells are treated. When human Burkitt's lymphoma cells are exposed to superoxide anion, generated as a flux from xanthine and xanthine oxidase, the cells die by a non-apoptotic mechanism (pyknosis/necrosis) identical to that seen when cells are treated with a bolus of reagent H(2)O(2). Addition of superoxide dismutase has no effect, whereas catalase is completely protective, indicating that exogenously generated superoxide kills cells entirely through its dismutation into H(2)O(2). In contrast, cells treated in culture media with reagent HOCl die largely by apoptosis. HOCl-induced apoptosis is mediated by aminoacyl chloramines generated in the culture media and can be mimicked by treatment of cells with taurine chloramine or with long lived chloramines generated from modified Lys or Arg. The results suggest that in a physiological milieu in which O(2)(-) and H(2)O(2) are the main oxidants being formed, the principal form of cell death may be necrotic, and under inflammatory conditions in which HOCl is generated, apoptotic cell death may predominate.
Adenosine Triphosphate↗
Induction of apoptosis by chemotherapeutic drugs without generation of reactive oxygen species.
Studies in a variety of cell types have suggested that cancer chemotherapy drugs induce tumor cell apoptosis in part by inducing formation of reactive oxygen species (ROS). Using human B lymphoma cells as the targets, we have found that apoptosis can be induced in the absence of any detectable oxidative stress. Apoptosis was induced with the chemotherapy drugs VP-16 and cisplatin. To determine whether oxidants are formed as part of the drug-induced apoptotic process, intracellular markers of oxidative stress were examined. These included measurement of (1) protein carbonyl groups by Western blot immunoassay, (2) protein methionine sulfoxide residues by amino acid analysis, (3) protein sulfhydryl oxidation by Western blot immunoassay, (4) F2-isoprostanes by GC/MS, and (5) intracellular ROS production using the oxidant-sensitive dyes DCFDA and dihydrorhodamine 123. Apoptosis was quantified using fluorescence microscopy to assess nuclear morphology. The results show that VP-16 and cisplatin induce extensive apoptosis in the absence of any detectable protein or lipid oxidation, measured in both the cytosolic and mitochondrial compartments of the cell. In contrast, H2O2, which kills the cells by nonapoptotic pathways, caused increases in both protein and lipid oxidation. Three different antioxidant compounds (N-acetyl cysteine, Tempol, and MnTBAP) failed to inhibit VP-16-induced apoptosis, while inhibiting H2O2-induced cell death. Only N-acetyl cysteine inhibited cisplatin-induced cell death and this is attributed to its known ability to react directly with and inactivate cisplatin before it enters the cell. The results demonstrate that, at least in B lymphoma cells, chemotherapy-induced apoptosis occurs using a mechanism that does not involve oxidants.
Acetylcysteine↗
Chronic inflammation and cancer.
A substantial body of evidence supports the conclusion that chronic inflammation can predispose an individual to cancer, as demonstrated by the association between chronic inflammatory bowel diseases and the increased risk of colon carcinoma. Chronic inflammation is caused by a variety of factors, including bacterial, viral, and parasitic infections, chemical irritants, and nondigestible particles. The longer the inflammation persists, the higher the risk of associated carcinogenesis. This review describes some of the underlying causes of the association between chronic inflammation and cancer. Inflammatory mediators contribute to neoplasia by inducing proneoplastic mutations, adaptive responses, resistance to apoptosis, and environmental changes such as stimulation of angiogenesis. All these changes confer a survival advantage to a susceptible cell. In this article, we discuss the contribution of reactive oxygen and nitrogen intermediates, prostaglandins, and inflammatory cytokines to carcinogenesis. A thorough understanding of the molecular basis of inflammation-associated neoplasia and progression can lead to novel approaches to the prevention and treatment of cancer.
Animals↗