chaetomellic-acid-a and Disease-Models--Animal

In previous studies we found that the GTPase p21 Harvey-Ras (Ha-Ras) stimulates the production of reactive oxygen species and induces apoptosis by oxidative stress; this effect was reversed by farnesyl transferase inhibitors (FTIs). In this study we investigated whether FTIs reduce rat brain damage induced by an excitotoxic stimulus, and the signalling pathway(s) underlying the neuroprotection by FTIs. In brain tissue, protein levels of Ha-Ras and farnesylation inhibition were assayed by Western blot, and superoxide production was measured by hydroethidine. The excitotoxic lesion was induced by intrastriatal injection of N-methyl-d-aspartate (NMDA). The survival of mouse neuronal cortical cells was assessed by 3-(4,5 dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In brain tissue, NMDA increased the protein levels of Ha-Ras, FTIs caused the accumulation of non-prenylated inactive Ras in the cytosolic fraction, and significantly reduced superoxide production and necrotic volume after excitotoxicity. FTIs increased the viability of mouse neuronal cortical cells following oxidative stress. In conclusion, FTIs inhibited Ha-Ras, decreased oxidative stress and reduced necrotic volume by partly acting on neuronal cells. Thus, Ha-Ras inhibition plays a role in the pathology of neuroprotection, suggesting a potential role of FTIs in the treatment of cerebrovascular diseases.

Topics: Animals; Brain; Cell Survival; Cells, Cultured; Disease Models, Animal; Embryo, Mammalian; Excitatory Amino Acid Agonists; Farnesyltranstransferase; Male; Maleates; Mice; N-Methylaspartate; Neurons; Neuroprotective Agents; Neurotoxicity Syndromes; Proto-Oncogene Proteins p21(ras); Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Tetrazolium Salts; Thiazoles

The small GTPase p21 Ras and its downstream effectors play a central role in the control of cell survival and apoptosis. We studied the effects of Ras/ERK1/2 signaling inhibition on oxidative damage in cultured renal and endothelial cells and on renal ischemia-reperfusion injury in the rat. Primary human renal tubular and human endothelial ECV304 cells underwent significant cell death when subjected to oxidative stress. This type of stress induced robustly ERK1/2 and phosphoinositide 3-kinase (PI3-kinase) signaling. Inhibition of Ras/ERK1/2 with a farnesyl transferase inhibitor, chaetomellic acid A (S-FTI), or with PD-98059, an inhibitor of MEK, a kinase upstream ERK1/2, significantly reduced the fraction of dead cells. The inhibitor of the PI3-kinase/Akt pathway, LY-294002, failed to exert a protective effect. We have translated these data in a rat model of renal ischemic injury in vivo. In uninephrectomized animals, anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg i.p.), 24 h after an acute ischemic renal insult (45-min occlusion of left renal artery) a significant fraction of kidney cells succumbed to cell death resulting in renal failure [glomerular filtration rate (GFR) 0.17 +/- 0.1 vs. 0.90 +/- 0.4 ml x min(-1) x 100 g body wt(-1) in normal rats]. Rats treated with S-FTI maintained the renal function (GFR 0.50 +/- 0.1 ml x min(-1) x 100 g body wt(-1)), and the kidneys showed a significant reduction of tubular necrosis. Reduction of ischemic damage in kidney and tubular cells paralleled Ha-Ras inhibition, assayed by cytosolic translocation of the protein. These data demonstrate that inhibition of farnesylation and consequently of Ras/ERK1/2 signaling significantly reduces acute postischemic renal injury.

Topics: Animals; Cell Death; Cell Line; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Flow Cytometry; Humans; Kidney; Kidney Tubules; Male; Maleates; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oxidative Stress; Phosphatidylinositol 3-Kinases; Protein Prenylation; Proto-Oncogene Proteins p21(ras); Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Umbilical Veins