of glucocorticoid receptor knockdown on the rates of GSH synthesis and efflux in iB16 melanoma cells.

<p>(A–C) Melanoma cells were isolated from the liver or lungs 7 days after inoculation and from subcutaneous tumors 14 days after inoculation for culture. Glutathione efflux corresponded to GSH because GSSG was, in all conditions, 1–2% of the total glutathione found in the extracellular space (not shown). To prevent degradation of the GSH accumulated in the extracellular space, γ-GT was blocked by adding 10 µM acivicin to the culture medium 2 h before measuring efflux. Enzyme activities were measured 22 h after seeding. Results obtained in iB16 cells transfected with lentiviral vector not harboring any gene (negative control) were not different from control values (not shown). Data are mean values ± S.D. (n = 9–10 in all cases). *p<0.05,**p<0.01<i>versus</i> iB16 controls. +p<0.05, ++p<0.01 <i>versus</i> melanoma cells isolated from liver metastases. (D) γ-GCS-HS and γ-GCS-LS expression was determined in cells cultured for 24h (previously isolated from <i>in vivo</i> tumors). Data, expressed as a fold change, show mean values ± S.D. from 5 to 6 different experiments. *p<0.01 <i>versus</i> iB16 cells.</p

Glucocorticoid receptor knockdown and GSH content in B16 melanoma cell subsets; and plasma corticosterone, ACTH, and IL-6 levels during melanoma growth <i>in vivo</i>.

<p>(A) GCR levels were measured by Western blot in control metastatic iB16 melanoma cells isolated from the liver and their equivalents stably expressing GCR-shRNA. Similar blots were run for B16-F10 and B16-F10-shGCR growing <i>in vitro</i>. Each lane in the blots corresponds to an individual representative animal in the indicated group. The relative density of each band was normalized against the internal standard (β-actin) on each blot (n = 4–5 in all cases) and expressed as relative changes in arbitrary densitometry units. Results obtained in cells transfected with lentiviral vector not harboring any gene (negative control) were not different from control values (not shown). *p<0.01 <i>versus</i> iB16 cells. <i>In vivo</i> experiments show data obtained after 7 days of inoculation. <i>In vitro</i> experiments show results obtained in cells cultured for 72h. (B–D)Blood was collected from the tail vein during a 24-h period starting 7 days after tumor inoculation, and peak plasma levels of corticosterone and ACTH (6 h and 12 h, circadian time, respectively) measured. Melanoma cells were isolated before GSH determination. Tumor volume and GSH levels were measured 8 days after inoculation. Data are mean values ± S.D. of 7–8 different animals. *p<0.05, **p<0.01 <i>versus</i> controls.</p

ROS, Nrf2 and GSH levels, and γ-GCS activity in iB16 and iB16-shGCR cells isolated from metastatic foci.

<p>Melanoma cells were isolated from the liver 7 days after inoculation, cultured, and transfected with anti-Nrf2-siRNA. H₂O₂ and O₂⁻generation, γ-GCS activity, and GSH levels were measured 48 h after seeding. Nrf2 levels (Western blotting) were measured 24 h after seeding. AU, arbitrary units. Data are mean values ± S.D. (n = 6–7 in all cases). *p<0.05,**p<0.01 <i>versus</i> iB16 controls. Results obtained in cells transfected with control Nrf2 sense or scrambled oligonucleotides were not significantly different from those obtained in cells cultured in the absence of anti-Nrf2-siRNA (not shown).</p

Effect of AS101 and anti-p53 antisense oligonucleotides on γ-GCS activity and expression and on GSH levels in metastatic melanoma cell subsets.

<p>Measurements and treatments were performed in isolated metastatic cells as indicated in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096466#pone-0096466-g005" target="_blank">Fig. 5</a>. Control experiments on p53 and Nrf2 levels were similar to those obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096466#pone-0096466-g005" target="_blank">Fig. 5</a> A (not shown). Results obtained in iB16 cells transfected with p53 sense or scrambled oligonucleotides were not significantly different from those obtained in controls or cells incubated with AS101 alone (not shown). Data are mean values ± S.D. (n = 4–5 in all cases). *p<0.01 <i>versus</i> controls.</p

Effect of AS101 and anti-p53 antisense oligonucleotides on nuclear p53 and Nrf2 levels, and expression of oxidative stress-related enzymes in metastatic melanoma cell subsets.

<p>(A) and (B) Melanoma cells isolated 7 days after inoculation were cultured for 48 h. Western blot (A), protein band quantification (B), and data pooling (n = 5–6 in all cases) were performed as in Fig. 1. AS101 (0.1 µg/ml) was added to the culture medium 2 h after seeding. Oligonucleotides (50 nM) were added 2h and 24 h after seeding as 1∶1 complexes with the Lipofectamine RNAiMAX reagent. Data are mean values ± S.D. (n = 4–5 in all cases). *p<0.01 <i>versus</i> controls.(C) and (D)Melanoma cells isolated from liver or lung metastatic foci 7 days after inoculation were cultured for 48 h. Data from quantitative RT-PCR are expressed as mean fold change ± S.D. (n = 5–6 in all cases). *p<0.05, **p<0.01 <i>versus</i> controls.(A–D) Results obtained in iB16 cells transfected with p53 sense or scrambled oligonucleotides were not significantly different from those obtained in controls or cells incubated with AS101 alone (not shown).</p

Effect of glucocorticoid receptor knockdown and GSH depletion on the invasive activity of B16 melanoma cells in the liver.

<p>(A) <i>In vivo</i> video microscopic study of the viability of intraportally injected B16 melanoma cell subsets arrested in the mouse liver microvasculature. B16-F10 (○), B16-F10 pre-cultured for 24 h in the presence of 0.5 mM BSO (•), iB16-shGCR isolated from solid tumors growing in the foot pad (□), iB16-shGCR pre-cultured for 24 h in the presence of 0.5 mM BSO (▪), and iB16-shGCR pre-cultured for 24 h in the presence of 1.0 mM GSH ester (Δ). The average number of arrested B16 cells per hepatic lobule was similar independently of the cell subset considered. Results obtained in iB16 cells transfected with lentiviral vector not harboring any gene (negative control) were not different from control values (not shown). Data are mean values ± S.D. from 4 to 5 different experiments. *p<0.01 <i>versus</i> B16-F10 controls. (B) In a first step, metastatic B16 cells establish a weak molecular bridge (docking) with the vascular endothelium. Metastatic growth factors induce endothelial cytokine release and, consequently, generation of high ROS and RNS levels that, in cooperation with the immune system, cause tumor cytoxicity in up to 90% of all attached B16-shGCR cells. Subsequent rolling facilitates locking through very late antigen 4 (VLA4) and intercellular adhesion molecule 1 (VCAM1). Cancer cells attached to the endothelium of pre-capillary arterioles or capillaries may follow two mechanisms of extravasation: a) migration through vessel fenestrae and/or b) intravascular proliferation followed by vessel rupture and microinflammation. Invading cancer cells will form micrometastases within the normal lobular hepatic architecture via a mechanism regulated by cross-talk with the stroma and multiple microenvironment-related, and possibly also systemic, molecular signals. Activation of angiogenesis will facilitate metastatic growth and spread. The result of conventional/targeted therapy on the small percent of surviving metastatic cells or whether they adapt during invasion, generating more resistant cell subsets, are unanswered questions. VEGF, vascular endothelial growth factor; SC, stellate cell; KC, Kupffer cell.</p

Glucocorticoid receptor knockdown is associated with a decrease in nuclear Nrf2.

<p>iB16 or iB16-shGCR cells were isolated from metastatic foci growing in the liver or lung and nuclear accumulation of Nrf1 and Nrf2 measured by Western blotting. Results obtained in iB16 cells transfected with lentiviral vector not harboring any gene (negative control) were not different from control values (not shown). Data show mean values ± S.D. from 5 to 6 different experiments. *p<0.01 <i>versus</i> iB16 cells.</p