guanosine-triphosphate and Necrosis

Benzamide riboside (BR) after anabolism to an analogue of NAD, was shown to inhibit the activity of NAD-dependent enzymes such as inosine 5'-monophosphate dehydrogenase (IMPDH), the rate limiting enzyme in de novo guanylate biosynthesis, and malate dehydrogenase which is involved in the citric cycle and respiratory chain. BR exhibits strong anti-carcinogenic effects due to growth retardation and due to induction of apoptosis and necrosis. Apoptosis is ascribed to the inhibition of IMPDH because cell death can be blocked by restoring intracellular guanylate metabolism by the addition of guanosine. It is shown here, however, that also survival-relevant genes such as cdc25A, akt, bcl-2 and transferrin receptor become repressed by BR, whereas the expression level of the apoptosis enforcing gene c-myc persists. Even though BR-mediated growth retardation still allows BR to induce apoptosis, rapamycin-mediated cell cycle block and cell contact inhibition prevent cell death, it strongly suggests that BR induces a type of c-Myc-dependent apoptosis. At high concentrations BR induces DNA double strand breaks by yet to be determined mechanisms that occur hours before necrosis can be detected. This is accompanied by a dramatic decrease of intracellular ATP. The artificial restoration of ATP by addition of adenosine or sufficient provision of an energy source such as glucose prevents BR-promoted necrosis and favors apoptosis. This observation may be of clinical relevance.

Topics: Apoptosis; Cell Division; DNA Damage; Enzyme Inhibitors; Female; Gene Expression; Glucose; Guanosine Triphosphate; HL-60 Cells; Humans; IMP Dehydrogenase; Necrosis; Neoplasms; Nucleosides; Tumor Cells, Cultured

Renal tubular cells die by apoptosis as well as necrosis in experimental models of ischemic and toxic acute renal failure as well as in humans with acute tubular necrosis. It is not yet possible, however, to determine the relative contribution of these two forms of cell death to loss of renal tubular cells in acute tubular necrosis. The beneficial effect of administering growth factors to animals with acute tubular necrosis is probably related to the potent antiapoptotic (survival) effects of growth factors as well as to their proliferative effects. Rapamycin inhibits both of these effects of growth factors and delays the recovery of renal function after acute tubular necrosis by inhibiting renal tubular cell regeneration and by increasing renal tubular cell loss by apoptosis. The administration of caspase inhibitors ameliorates ischemia-reperfusion injury in multiple organs including the kidney. However, the extent to which this protective effect of caspase inhibition is caused by reduced intrarenal inflammation, or by amelioration of renal tubular cell loss due to apoptosis, remains uncertain. In addition to caspase inhibition, the apoptotic pathway offers many potential targets for therapeutic interventions to prevent renal tubular cell apoptosis.

Topics: Acute Kidney Injury; Apoptosis; Caspase Inhibitors; Cell Adhesion; Cisplatin; Growth Substances; Guanosine Triphosphate; Humans; Ischemia; Kidney Tubules; Necrosis; Sirolimus

The purpose of this study was to prepare targeted cancer therapy formulation against insulinoma INS-1 cells and to study its effect on cell death with related mechanisms in vitro.. Polylactide-co-glycolide (PLGA) nano-micelles were used for preparation of esculetin nano-formulation (nano-esculetin). The cells were treated with nano-esculetin and free esculetin. Apoptotic and necrotic cell death percentages, cell proliferation, ATP and GTP reductions and insulin levels were investigated on insulinoma INS-1 cells for both free and nano-esculetin formulations.. About 50 mg of PLGA was able to carry 20 mg esculetin in 20 ml of formulation. The obtained optimized formulation was 150 nm, with 92% encapsulation efficiency and a slow-release behaviour was observed during release studies. Nano-esculetin bearing 25, 50 and 100 μg esculetin and free esculetin in equivalent doses successfully decreased cell viability. The prevailing cell death mechanism was necrosis. Along with cell proliferation, intracellular insulin and the ratio of ATP and GTP were decreased even with 12.5, 25 and 50 μg esculetin bearing nano-formulation and its equivalent free esculetin.. The results revealed that esculetin is able to show its anti-tumor afficacy after loading to PLGA nano-micelles and nano-encapsulation intensifies its cytotoxic activity in vitro. Current study shows that esculetin and its nano formulations are promising agents in treatment of insulinoma.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Cell Death; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Carriers; Guanosine Triphosphate; Insulin; Insulinoma; Micelles; Nanoparticles; Nanotechnology; Necrosis; Particle Size; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Umbelliferones

The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42.. Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA-mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL-overexpressing cells). All tested cells remained sensitive to MPA-mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers.. These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells.

Topics: Apoptosis; Blotting, Western; cdc42 GTP-Binding Protein; Cell Line; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Green Fluorescent Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Jurkat Cells; K562 Cells; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Microscopy, Electron; Mutation; Mycophenolic Acid; Necrosis; RNA, Small Interfering; Signal Transduction; Transfection; Tumor Cells, Cultured

Mycophenolate mofetil (MMF) is an immunosuppressive agent used in transplantation. Over the last decade, MMF has also emerged as an alternative therapeutic regimen for autoimmune diseases, mainly for patients refractory to other therapies. The active compound of MMF, mycophenolic acid (MPA), depletes the intracellular pool of guanosine tri-phosphate through inosine monophosphate dehydrogenase blockade. The molecular mechanism involved in the elimination of T and B lymphocytes upon inhibition of inosine monophosphate dehydrogenase remains elusive. In this study, we showed that in contrast to the immunosuppressors azathioprine, cyclosporin A, and tacrolimus, MPA killed lymphocytes through the activation of a caspase-independent necrotic signal. Furthermore, the MPA-mediated necrotic signal relied on the transmission of a novel intracellular signal involving Rho-GTPase Cdc42 activity and actin polymerization. In addition to its medical interest, this study sheds light on a novel and atypical molecular mechanism leading to necrotic cell death.

Topics: Actins; Adenosine Triphosphatases; B-Lymphocytes; cdc42 GTP-Binding Protein; Enzyme Inhibitors; Guanosine Triphosphate; Humans; Immunosuppressive Agents; IMP Dehydrogenase; Jurkat Cells; Lymphocyte Activation; Mycophenolic Acid; Necrosis; Organ Transplantation; Signal Transduction; T-Lymphocytes

The rapid and efficient phagocytosis of apoptotic cells plays a critical role in preventing secondary necrosis, inflammation as well as in tissue remodeling and regulating immune responses. However, the molecular details of engulfment are just beginning to be elucidated. Among the Rho family GTPases, previous studies have implicated a role for Rac and Cdc42 in the uptake of apoptotic cells by phagocytes, yet the role of Rho has remained unclear. Here, we present evidence that Rho-GTP levels decrease during engulfment. RhoA seems to negatively affect basal engulfment, such that inhibition of Rho-mediated signaling in phagocytes enhanced the uptake of apoptotic targets. Activation of endogenous Rho or overexpression of constitutively active forms of Rho also inhibited engulfment. By testing mutants of RhoA that selectively activate downstream effectors, the Rho-kinase seemed to be primarily responsible for this inhibitory effect. Taken together, these data suggest that inhibition of Rho- and Rho-kinase-mediated signaling might be important during engulfment, which could have important implications for several clinical trials involving inhibition of the Rho kinase.

Topics: Amides; Animals; Apoptosis; Azepines; cdc42 GTP-Binding Protein; Cell Line; Cricetinae; Enzyme Inhibitors; Gene Expression Regulation; Green Fluorescent Proteins; Guanosine Triphosphate; Immunoblotting; Luminescent Proteins; Mice; Microscopy, Fluorescence; Mutation; Naphthalenes; Necrosis; Phagocytes; Phagocytosis; Plasmids; Pyridines; rac GTP-Binding Proteins; rho GTP-Binding Proteins; Signal Transduction; Time Factors; Transfection

Intracellular ATP depletion is a hallmark event in ischemic injury. It has been extensively characterized in models of chemical anoxia in vitro. In contrast, the fate of GTP during ischemia remains unknown. We used LLC-PK proximal tubular cells to measure GTP and ATP changes during anoxia. In 45 min, antimycin A decreased ATP and GTP to 8% and 2% of controls, respectively. Ischemia in vivo resulted in comparable reductions in GTP and ATP. After 2 h of recovery, GTP levels in LLC-PK cells increased to 65% while ATP increased to 29%. We also investigated steady-state models of selective ATP or GTP depletion. Combinations of antimycin A and mycophenolic acid selectively reduced GTP to 51% or 25% of control. Similarly, alanosine selectively reduced ATP to 61% or 26% of control. Selective GTP depletion resulted in significant apoptosis. Selective ATP depletion caused mostly necrosis. These models of ATP or GTP depletion can prove useful in dissecting the relative contribution of the two nucleotides to the ischemic phenotype.

Topics: Adenosine Triphosphate; Alanine; Animals; Antibiotics, Antineoplastic; Antimycin A; Apoptosis; Cell Hypoxia; Cells, Cultured; Deoxyglucose; Enzyme Inhibitors; Guanosine; Guanosine Triphosphate; Ischemia; Kidney Cortex; Kidney Tubules, Proximal; LLC-PK1 Cells; Male; Models, Biological; Mycophenolic Acid; Necrosis; Oxidative Phosphorylation; Rats; Rats, Sprague-Dawley; Swine

We have examined the effect of killing of host monocytes infected with bacillus Calmette-Guérin (BCG) on the viability of the intracellular mycobacteria. Peripheral blood monocytes were infected in vitro with a single bacillus per cell and maintained in culture for 6-8 d to allow the bacilli to replicate. Replicating viable BCG were found singly in perinuclear vacuoles bounded by tightly apposed lipid bilayers. Monocytes were then exposed to toxic mediators that induced killing of cells as evaluated by 51Cr release into the culture medium. Both hydrogen peroxide (H2O2) (an inducer of cell necrosis) and adenosine triphosphate (ATP4-) (an inducer of cell apoptosis) treatment killed infected monocytes. H2O2-induced killing had no effect on BCG viability. ATP-induced cell death was accompanied by DNA fragmentation and nuclear condensation. Apoptosis was associated with a swelling of the phagocytic vacuoles which became multibacillary and with a reduction of BCG viability as enumerated by colony-forming units.

Topics: Adenosine Triphosphate; Apoptosis; Blood Bactericidal Activity; CD4-Positive T-Lymphocytes; Cells, Cultured; DNA; Guanosine Triphosphate; Humans; Hydrogen Peroxide; Monocytes; Mycobacterium bovis; Necrosis; Vacuoles