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

Ortega, Ángel; Ferrer, Paula; Carretero, Julián; Obrador, Elena; Asensi, Miguel; Pellicer, José A.; Estrela, José M.

doi:10.1074/jbc.m303753200

Cited by 45 publications

(51 citation statements)

References 57 publications

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“…Indeed, in B16M-F10-Gln + -bearing mice treated with physiologic saline or G3139 + verapamil + acivicin, 8 days after tumor inoculation, mtGSH in the metastatic cells decreases to 3.1 F 0.5 and 2.7 F 0.5 nmol/10 6 cells, respectively (n = 5-6, P < 0.01; f45% and 39% of the mtGSH levels measured in metastatic B16M-F10 cells isolated from the liver of mice fed a standard diet). We found previously that death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of control values (21), which is critical because TNF-a -induced cytotoxicity is facilitated in B16M-F10 cells with low mtGSH levels (9,25).…”

Section: Resultsmentioning

confidence: 94%

“…Anti-S100 monoclonal antibodies (DAKO) were used for immunohistochemical detection of metastatic B16 melanoma cells (21). For that purpose, the livers were fixed and paraffin embedded as described above under Experimental Metastases.…”

Section: Methodsmentioning

confidence: 99%

“…Analysis of the bcl-2 family of genes revealed that B16M-F10 cells (high metastatic potential) overexpressed bcl-2 relative to B16M-F1 cells (low metastatic potential; ref. 21). B16M-F10 cells have higher intracellular GSH levels than B16M-F1 cells due to faster GSH synthesis and slower GSH efflux (8,22).…”

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confidence: 99%

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Bcl-2 and Glutathione Depletion Sensitizes B16 Melanoma to Combination Therapy and Eliminates Metastatic Disease

Mena

Benlloch

Ortega

et al. 2007

Clinical Cancer Research

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Purpose: Advanced melanoma resists all current therapies, and metastases in the liver are particularly problematic. Prevalent resistance factors include elevated glutathione (GSH) and increased expression of bcl-2 in melanoma cells. GSH has pleiotropic effects promoting cell growth and broad resistance to therapy, whereas Bcl-2 inhibits the activation of apoptosis and contributes to elevation of GSH. This study determined the in vivo efficacy of combination therapies administered while GSH and Bcl-2 were individually and simultaneously decreased in metastatic melanoma lesions. Experimental Design: Highly metastatic murine B16 melanoma (B16M-F10) cells have elevated levels of both GSH and Bcl-2. B16M-F10 cells were injected i.v. to establish metastatic lesions in vivo. GSH was decreased using an L-glutamine^enriched diet and administration of verapamil and acivicin, whereas Bcl-2 was reduced using oligodeoxynucleotide G3139. Paclitaxel, X-rays, tumor necrosis factor-a, and IFN-g were administered as a combination therapy. Results: Metastatic cells were isolated from liver to confirm the depletion of GSH and Bcl-2 in vivo. Reduction of Bcl-2 and GSH, combined with partial therapies, decreased the number and volume of invasive B16M-F10 foci in liver by up to 99% (P < 0.01). The full combination of paclitaxel, X-rays, and cytokines eliminated B16M-F10 cells from liver and all other systemic disease, leading to long-term survival (>120 days) without recurrence in 90% of mice receiving the full therapy.Toxicity was manageable; the mice recovered quickly, and hematology and clinical chemistry data were representative of accepted clinical toxicities. Conclusions: Our results suggest a new strategy to induce regression of late-stage metastatic melanoma.

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Section: Resultsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

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Bcl-2 and Glutathione Depletion Sensitizes B16 Melanoma to Combination Therapy and Eliminates Metastatic Disease

Mena

Benlloch

Ortega

et al. 2007

Clinical Cancer Research

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“…In a B16 model, WAVE expression levels and in vitro invasiveness of B16F1 cells are similar to those of B16F10 cells, even though there is great difference in metastatic capacity between these two cell lines. This inconsistency might reflect the fact that successful metastasis depends upon numerous genetic and biochemical requirements in addition to activation of WAVEs (Yu and Schultz, 1990;Clark et al, 2000;Hanahan and Weinberg, 2000;Ortega et al, 2003). WAVE-promoted cellular invasiveness is one of the requirements, which is demonstrated by the fact that WAVE2-RNAi impaired lung metastasis of B16F10 cells ( Figure 6) and that transduction of dominantnegative Rac in B16F10 cells inhibits lung metastasis (Yamaguchi et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells

et al. 2004

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WAVEs (WASP-family verprolin-homologous proteins) regulate the actin cytoskeleton through activation of Arp2/3 complex. As cell motility is regulated by actin cytoskeleton rearrangement and is required for tumor invasion and metastasis, blocking actin polymerization may be an effective strategy to prevent tumor dissemination. We show that WAVEs, especially WAVE2, are essential for invasion and metastasis of melanoma cells. Malignant B16F10 mouse melanoma cells expressed more WAVE1 and WAVE2 proteins and showed higher Rac activity than B16 parental cells, which are neither invasive nor metastatic. The effect of WAVE2 silencing by RNA interference (RNAi) on the highly invasive nature of B16F10 cells was more dramatic than that of WAVE1 RNAi. Membrane ruffling, cell motility, invasion into the extracellular matrix, and pulmonary metastasis of B16F10 cells were suppressed by WAVE2 RNAi. WAVE2 RNAi also had a profound effect on invasion induced by a constitutively active form of Rac (RacCA). In addition, ectopic expression of both RacCA and WAVE2 in B16 cells resulted in further increase in the invasiveness than that observed in B16 cells expressing only RacCA. Thus, WAVE2 acts as the primary effector downstream of Rac to achieve invasion and metastasis, suggesting that suppression of WAVE2 activity holds a promise for preventing cancer invasion and metastasis.

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“…32 The depletion in GSx content also correlated with the loss of metastatic activity of B16 melanoma and cell arrest. 33 Thus, PE and GSx alterations were a common denominator of a ''controlled'' tumor cell proliferation pattern resulting from direct CENU targeting or bystander factor impact, in vivo and in vitro. Their values as tumor response biomarkers for ''controlled'' tumor cell proliferation are under investigation.…”

Section: Phenotype Changes Associated With Chemotherapy-induced Bystamentioning

confidence: 99%

Bystander effects are induced by CENU treatment and associated with altered protein secretory activity of treated tumor cells A relay for chemotherapy?

Demidem

Morvan

Madelmont

2006

Intl Journal of Cancer

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In a previous study, it was reported that secondary untreated melanoma tumors implanted several weeks after and at distance from primary chloroethylnitrosourea (CENU)-treated tumors underwent differentiation and growth inhibition. To see whether the primary treated tumor released soluble factors that mediated the secondary tumor response, serum transfer experiments were performed in vivo. Administration of serum from CENU-treated tumor-bearing donors arrested tumor proliferation, decreased vessel formation and induced tumor metabolite alterations encompassing glutathione decrease and polyunsaturated fatty acid and phosphoethanolamine increase. These changes mimicked secondary tumor phenotype. To reproduce the model in vitro, cell culture supernatant transfer experiments were performed. CENU-treated cell cultures showed polyploidy and reactive oxygen species (ROS) production. Cell cultures challenged by a conditioned medium of CENU-treated cells underwent growth inhibition, cytoskeleton disorders, cytokinesis retardation, metabolite alterations, glutathione decrease and phosphoethanolamine increase, without ROS elicitation. Proteomics of CENU-treated cell conditioned media revealed altered protein secretion activity by CENU-treated cells. Among de novo secreted proteins, the most expressed were phosphatidylethanolamine-binding protein (PEBP), cardiovascular heat shock protein (cHsp), Rho-associated coiled-coil forming kinase 2 (ROCK) and actin fragments. These proteins testified of cytoskeleton disorders, growth inhibition and metabolite alterations. This article demonstrates the release by CENU-treated tumors of growth inhibitory differentiation-inducing soluble factors. These factors mediate remote bystander effects and attest persistent biological activity of residual tumors after chemotherapy. ' 2006 Wiley-Liss, Inc.Key words: chloroethylnitrosoureas; distant double tumor models; growth inhibition; bystander effects; proteomics There is increasing experimental evidence that therapy-induced tumor cell modification (cell-based gene and ionizing radiation therapies) may promote the immune system or induce the expression of cytokines or other factors that may be inhibitory for local or distant unmodified tumor cells. [1][2][3][4][5][6][7][8] Cell-based gene therapy after cytokine or suicide gene transfer has been investigated for autologous and allogeneic vaccination in cancer therapy. Tumor cells engineered to produce cytokines such as interleukin-12 (IL-12) induced antitumor immunity capable of inhibiting a challenge with unmodified parental tumor cells.1 Recently, in a model of allogeneic tumor cells transduced with IL-12 mixed with autologous lymphoma cells, IL-12 induced microenvironmental changes that, through the induction of an immune response, yielded eradication of distant preestablished lymphoma.2 Cells transfected with a suicide gene such as that of thymidine kinase from herpes simplex virus 3-6 or of cytosine desaminase, 7-8 enzymes that activate a drug or prodrug, inhibited distant tumors and provok...

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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

Cited by 45 publications

References 57 publications

Bcl-2 and Glutathione Depletion Sensitizes B16 Melanoma to Combination Therapy and Eliminates Metastatic Disease

Bcl-2 and Glutathione Depletion Sensitizes B16 Melanoma to Combination Therapy and Eliminates Metastatic Disease

Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells

Bystander effects are induced by CENU treatment and associated with altered protein secretory activity of treated tumor cells A relay for chemotherapy?

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