Tumor Cytotoxicity by Endothelial Cells

Ortega, Ángel; Carretero, Julián; Obrador, Elena; Gambini, Juan; Asensi, Miguel; Rodilla, Vicente; Estrela, José M.

doi:10.1074/jbc.m207140200

“…These results appear to be in agreement with a recent report showing that GSH depletion enforces the mitochondrial permeability transition and causes cell death in HL60 cells that overexpress Bcl-2 (20). Furthermore, when BSO-and Bcl-2-AS-pretreated B16M-F10 cells where inoculated intravascularly into mice, the number of intact arrested cells on the HSE decreased by ϳ98% and the very small number of metastatic cell survivors (probably bearing molecular damages) (29) did not form detectable colonies (Table IV). Moreover, using HMB45 or S100 monoclonal antibodies (51) as markers of melanocytic tumors, no B16M-F10 cells could be detected within the microvasculature or the hepatic parenchyma 5 or 10 days after inoculation (not shown), which suggests that probably intravascular granulocytes and/or the Kupffer cells, present in the metastatic microenvironment (not shown), eliminate those few survivors.…”

Section: Table V Bcl-2-induced Inhibition Of Gsh Efflux From B16m Cellssupporting

confidence: 80%

“…This effect was similar in B16M-F10 cells treated with DEM plus TNF-␣, Bcl-2-AS, and GSH ester even when only cytGSH levels where maintained low (ϳ10 -15% of control values) by using monochlorobimane as in Ref. 29 (not shown). This proves, as previously indicated (29), that the GSH pool relevant for cell survival is the mitochondrial one.…”

Section: Table III Effect Of In Vitro Bso-induced Gsh Depletion and Tmentioning

confidence: 54%

“…Bcl-2 itself is an anti-death protein, and its overexpression has been linked to cancer development, metastatic growth, and chemotherapy resistance (9 -12, 40). GSH protects highly metastatic B16M cells against nitrosative and oxidative stress within blood vessels (4,6,29) and, in addition, promotes metastatic growth (8). Nevertheless we found that different B16M cell lines, containing similar GSH levels, showed different rates of survival after in vivo interaction with the HSE (Table I).…”

Section: Discussionmentioning

confidence: 99%

“…29 (not shown). This proves, as previously indicated (29), that the GSH pool relevant for cell survival is the mitochondrial one.…”

Section: Table III Effect Of In Vitro Bso-induced Gsh Depletion and Tmentioning

confidence: 99%

“…29 and references therein), can cause a fall in mitochondrial membrane potential, opening of the permeability transition pore complex, and the release of proapoptotic molecular signals. Bcl-2 overexpression prevented the GSH depletion-associated increase in mitochondrial membrane permeability, although only when mtGSH levels remained above a critical threshold …”

Section: Mitochondrial Gsh Depletion and The Molecular Activation Of mentioning

confidence: 99%

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Down-regulation of Glutathione and Bcl-2 Synthesis in Mouse B16 Melanoma Cells Avoids Their Survival during Interaction with the Vascular Endothelium

Ortega

¹

,

Ferrer

²

,

Carretero

³

et al. 2003

Journal of Biological Chemistry

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B16 melanoma (B16M) cells with high GSH contentshow high metastatic activity. However, the molecular mechanisms linking GSH to metastatic cell survival are unclear. The possible relationship between GSH and the ability of Bcl-2 to prevent cell death was studied in B16M cells with high (F10) and low (F1) metastatic potential. Analysis of a Bcl-2 family of genes revealed that B16M-F10 cells, as compared with B16M-F1 cells, overexpressed preferentially Bcl-2 (ϳ5.7-fold). Hepatic sinusoidal endothelium-induced B16M-F10 cytotoxicity in vitro increased from ϳ19% (controls) to ϳ97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucleotide (Bcl-2-AS). L-Buthionine (S,R)-sulfoximine-induced GSH depletion or Bcl-2-AS decreased the metastatic growth of B16M-F10 cells in the liver. However, the combination of L-buthionine (S,R)-sulfoximine and Bcl-2-AS abolished metastatic invasion. Bcl-2-overexpressing B16M-F1/Tet-Bcl-2 and B16M-F10/TetBcl-2 cells, as compared with controls, showed an increase in GSH content, no change in the rate of GSH synthesis, and a decrease in GSH efflux. Thus, Bcl-2 overexpression may increase metastatic cell resistance against oxidative/nitrosative stress by inhibiting release of GSH. In addition, Bcl-2 availability regulates the mitochondrial GSH (mtGSH)-dependent opening of the permeability transition pore complex. Death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of controls values. However, this critical threshold increased to ϳ60% of control values in Bcl-2-AS-treated B16M-F10 cells. GSH ester-induced replenishment of mtGSH levels (even under conditions of cytosolic GSH depletion) prevented cell death. Our results indicate that survival of B16M cells with high metastatic potential can be challenged by inhibiting their GSH and Bcl-2 synthesis.The majority of metastatic cells entering microvascular beds are killed within the first hours and do not generate colonies. This failure, termed "metastatic inefficiency" (1), is due to mechanical trauma produced by blood flow (2), the inability of cancer cells to withstand deformation (3), cytotoxicity of locally released reactive oxygen and nitrogen species (4), and the lytic action of lymphocytes and macrophages (5). The B16 melanoma (B16M) 1 is a model widely used to study metastatic spread and tissue invasion (6). The liver is a common site for metastasis development, and we recently reported that GSH (␥-glutamylcysteinyl-glycine) protects B16M-F10 cells (with high metastatic potential) against nitrosative and oxidative stress in the hepatic microvasculature (4, 6). In fact, multidrug and/or radiation resistance, which are characteristic features of malignant tumors, frequently associate with high GSH content in the cancer cells (7). B16M-F10 cell resistance to the HSE-induced cytotoxicity is highly dependent on GSH and GSH peroxidase (4). However, B16M-F10 cells cultured to low density (LD), with high GSH content, were more resistant to NO and H 2 O 2 than B16M cultured to high density (with ϳ25% of th...

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A role for the 2-oxoglutarate carrier in glutathione transport into hepatocyte mitochondria?

Estrela¹,

Ortega²,

Carretero³

2004

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We read with great interest the recent article by Kugelmas et al., 1 reporting plasma cytokine levels in patients with NASH and the effect of diet and vitamin E on this disease. They found that plasma tumor necrosis factor (TNF), interleukin 8, and interleukin 6 concentrations were significantly elevated compared with control values, and only plasma interleukin 6 levels significantly decreased with diet therapy. This article further reported that vitamin E therapy did not independently influence the biochemical data and plasma cytokine levels in patients with NASH. We previously investigated the plasma transforming growth factor-␤1 (TGF-␤1) level and the efficacy of vitamin E in patients with NASH. 2 We enrolled 12 patients with NASH and 10 with fatty liver. All patients were given dietary instruction for 6 months, and thereafter vitamin E (␣-tocopherol acetate, Juvera™, Eisai Pharmaceutical Co., Tokyo, Japan, 300 mg/day; 100 mg is equivalent to 100 IU) was given for 1 year. In consequence, the plasma TGF-␤1 level in patients with NASH (37 Ϯ 5 ng/ml) was significantly higher than that in patients with fatty liver (10 Ϯ 4 ng/ml) and healthy subjects (9 Ϯ 5 ng/ml). Moreover, this elevated plasma TGF-␤1 level decreased after a 1-year vitamin E treatment, together with the improvement of biochemical markers and hepatic pathological findings (Table 1). In 5 out of 9 NSAH patients in whom liver biopsy was performed after vitamin E treatment, inflammation and fibrosis were improved. On the other hand, diet therapy improved biochemical data only in patients with fatty liver, but not in those with NASH. We conducted our previous study to assess the efficacy of a long-term vitamin E treatment (a 1-year vitamin E administration after a 6-month diet). However, in the studies performed by Kugelmas et al., 1 vitamin E was given for only 12 weeks and the patients were given diet therapy at the same time. In fact, Kugelmas et al. 1 mentioned that longer study duration might have shown the efficacy of vitamin E on NASH. However, we had already reported the efficacy of a 12-month treatment of vitamin E on NASH. Moreover, long-term treatment with vitamin E for patients with NASH was tried for obese children and Japanese adults, and the beneficial effect of this vitamin has already been confirmed. 3,4 In addition, the NIH plans to conduct randomized controlled trials of insulin-sensitizing agents and vitamin E therapy for NASH. 5 Although the role of tumor necrosis factor is currently debated in the literature, 6,7 elevated blood level of TGF-␤ has recently been ascertained. 8 The role of TGF-␤1 in hepatic inflammation and fibrosis has been well documented in both animal and clinical studies. 9,10 Therefore, for better understanding of the cytokine network in NASH, we believe that the investigation of plasma TGF-␤1 is essential, as well as long-term observation of vitamin E treatment.

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Reply:

Fernández-Checa

¹

,

Garcı́a-Ruiz

²

2004

Hepatology

0

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We read with great interest the recent article by Kugelmas et al., 1 reporting plasma cytokine levels in patients with NASH and the effect of diet and vitamin E on this disease. They found that plasma tumor necrosis factor (TNF), interleukin 8, and interleukin 6 concentrations were significantly elevated compared with control values, and only plasma interleukin 6 levels significantly decreased with diet therapy. This article further reported that vitamin E therapy did not independently influence the biochemical data and plasma cytokine levels in patients with NASH. We previously investigated the plasma transforming growth factor-␤1 (TGF-␤1) level and the efficacy of vitamin E in patients with NASH. 2 We enrolled 12 patients with NASH and 10 with fatty liver. All patients were given dietary instruction for 6 months, and thereafter vitamin E (␣-tocopherol acetate, Juvera™, Eisai Pharmaceutical Co., Tokyo, Japan, 300 mg/day; 100 mg is equivalent to 100 IU) was given for 1 year. In consequence, the plasma TGF-␤1 level in patients with NASH (37 Ϯ 5 ng/ml) was significantly higher than that in patients with fatty liver (10 Ϯ 4 ng/ml) and healthy subjects (9 Ϯ 5 ng/ml). Moreover, this elevated plasma TGF-␤1 level decreased after a 1-year vitamin E treatment, together with the improvement of biochemical markers and hepatic pathological findings (Table 1). In 5 out of 9 NSAH patients in whom liver biopsy was performed after vitamin E treatment, inflammation and fibrosis were improved. On the other hand, diet therapy improved biochemical data only in patients with fatty liver, but not in those with NASH. We conducted our previous study to assess the efficacy of a long-term vitamin E treatment (a 1-year vitamin E administration after a 6-month diet). However, in the studies performed by Kugelmas et al., 1 vitamin E was given for only 12 weeks and the patients were given diet therapy at the same time. In fact, Kugelmas et al. 1 mentioned that longer study duration might have shown the efficacy of vitamin E on NASH. However, we had already reported the efficacy of a 12-month treatment of vitamin E on NASH. Moreover, long-term treatment with vitamin E for patients with NASH was tried for obese children and Japanese adults, and the beneficial effect of this vitamin has already been confirmed. 3,4 In addition, the NIH plans to conduct randomized controlled trials of insulin-sensitizing agents and vitamin E therapy for NASH. 5 Although the role of tumor necrosis factor is currently debated in the literature, 6,7 elevated blood level of TGF-␤ has recently been ascertained. 8 The role of TGF-␤1 in hepatic inflammation and fibrosis has been well documented in both animal and clinical studies. 9,10 Therefore, for better understanding of the cytokine network in NASH, we believe that the investigation of plasma TGF-␤1 is essential, as well as long-term observation of vitamin E treatment.

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Tumor Cytotoxicity by Endothelial Cells

Cited by 44 publications

References 72 publications

Down-regulation of Glutathione and Bcl-2 Synthesis in Mouse B16 Melanoma Cells Avoids Their Survival during Interaction with the Vascular Endothelium

Down-regulation of Glutathione and Bcl-2 Synthesis in Mouse B16 Melanoma Cells Avoids Their Survival during Interaction with the Vascular Endothelium

A role for the 2-oxoglutarate carrier in glutathione transport into hepatocyte mitochondria?

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