Tumor cytotoxicity by endothelial cells. Impairment of the mitochondrial system for glutathione uptake in mouse B16 melanoma cells that survive after in vitro interaction with the hepatic sinusoidal endothelium.
High GSH content associates with high metastatic activity in B16-F10 melanoma cells cultured to low density (LD B16M). GSH homeostasis was investigated in LD B16M cells that survive after adhesion to the hepatic sinusoidal endothelium (HSE). Invasive B16M (iB16M) cells were isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting. HSE-derived NO and H(2)O(2) caused GSH depletion and a decrease in gamma-glutamylcysteine synthetase activity in iB16M cells. Overexpression of gamma-glutamylcysteine synthetase heavy and light subunits led to a rapid recovery of cytosolic GSH, whereas mitochondrial GSH (mtGSH) further decreased during the first 18 h of culture. NO and H(2)O(2) damaged the mitochondrial system for GSH uptake (rates in iB16M were approximately 75% lower than in LD B16M cells). iB16M cells also showed a decreased activity of mitochondrial complexes II, III, and IV, less O(2) consumption, lower ATP levels, higher O(2) and H(2)O(2) production, and lower mitochondrial membrane potential. In vitro growing iB16M cells maintained high viability (>98%) and repaired HSE-induced mitochondrial damages within 48 h. However, iB16M cells with low mtGSH levels were highly susceptible to TNF-alpha-induced oxidative stress and death. Therefore depletion of mtGSH levels may represent a critical target to challenge survival of invasive cancer cells.
Ortega
Angel L
AL
Departamento de Fisiologia, Universidad de Valencia, Spain.
Carretero
Julian
J
Obrador
Elena
E
Gambini
Juan
J
Asensi
Miguel
M
Rodilla
Vicente
V
Estrela
José M
JM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion; D016923:Cell Death; D002470:Cell Survival; D018920:Coculture Techniques; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D008546:Melanoma, Experimental; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology*; D009566:Nitrates / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D017382:Reactive Oxygen Species; D013997:Time Factors
D018076:DNA, Complementary; D009566:Nitrates; D009573:Nitrites; D017382:Reactive Oxygen Species; D012313:RNA; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D010100:Oxygen