Pterostilbene induces lysosomal membrane permeabilization.

<p>A) Flow cytometry analysis of acridine orange staining after 24 h incubation with indicated concentrations of Pter. Increases in red fluorescence (x-axis), observed in treated cells, indicated changes at the lysosomal membrane. B) Microscopic images of Lysotracker red-stained cells untreated or treated with indicated concentrations of Pter for 24 h. C) Cytosolic cysteine cathepsin (zFRase) activity expressed as percentage of total activity (n = 7) was measured 24 h after Pter addition. D) Cytosolic aspartyl cathepsin activity expressed as percentage of total activity (n = 7) after 24 h of Pter treatment. The results were statistically analyzed by ANOVA and the Tukey test (*p<0.05; **p<0.01; ***p<0.001 vs. control). E) Cathepsins B and D inhibitors, Leupeptin and Pepstatin A, were added 1 h prior to the addition of 50 µM Pter. Cell number was analyzed 24 h later.</p

HSP70 depletion sensitizes cells to Pter.

<p>A) Representative HSP70 and GAPDH (loading control) immunoblots of proteins extracted from indicated cells. Densitometric analysis of the immunoblots (n = 3) are shown below. B) Inhibition of HSP70 expression by siRNA. Cells were transfected with HSP70 siRNA or GAPDH SiRNA (positive control). Protein levels were measured by western blotting. C) HT29 and MCF7 cells were seeded and transfected with HSP70 siRNA 24 h later. Pter was added at 24 h post-transfection. Cell viability was performed by sulforhodamine B colorimetric assay 48 h later. The number of replicates per experiment was at least six (n = 3). The results were statistically analyzed by ANOVA and the Tukey test (*p<0.05; ***p<0.001 vs. control). D) Cysteine and aspartyl cathepsins were determined in HT29 and MCF7 cells 72 h post- HSP70 siRNA transfection and 24 h after Pter treatment (n = 6). The results were statistically analyzed by ANOVA and the Tukey test (***p<0.001 vs. Pter 50µM).</p

Effect of pterostilbene on necrosis induction.

<p>A) Release of LDH activity to the extracellular medium. Cells were exposed to Pter for 24, 48 and 72 h. Results are expressed as relative LDH activities ± SD (n = 3) where control was set to 100%. B) Propidium iodide fluorescence was plotted against annexin-V fluorescence 24 h after treatment. Results are expressed as total percentage of necrotic cells ± SD (n = 3). C) Effect of Pter on tumor cell number in the presence of the necroptosis inhibitor 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin (Nec-1, Calbiochem). Nec-1 25 µM was added 1 h prior to the addition of 50 µM Pter. Cell number was analyzed 24 and 48 h later. Results are expressed as relative proliferation index ± SD (n = 3) where control is 100%. The results were statistically analyzed by ANOVA and contrasted by the Tukey test (***p<0.001; **p<0.01; *p<0.05 Vs. Control).</p

Effects of pterostilbene on autophagic flux.

<p>Proteins were detected by western blot with LC3A/B (A) or SQSTM1/p62 antibodies (B). Graphic shows the percentage of induction of eGFP-LC3 punctuation/aggregation in control and Pter (50 µM) conditions after 24 h of treatment (A). Densitometric analysis of SQSTM1/p62 correlated with GAPDH levels. The ratio in untreated control cells was set to 100% (B). MCF7-eGFP-LC3 cells were visualized by confocal and transmission electronic microscopy after treatment with 50 µM Pter for 24 h or 100 nM Rapamycin for 1 h (C).</p

Effect of pterostilbene on cell cycle distribution and DNA synthesis.

<p>A) Tumor cells were seeded in six well-plates and treated 24 h later with indicated concentrations of Pter. After 24 h, cells were fixed and stained with propidium iodide as explained under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044524#s2" target="_blank">material and methods</a>. The DNA content of the cells was analyzed by flow cytometry (10,000 cell events were collected per sample). Representative cell cycle histograms for Control and Pter-treated cells are shown. Results are expressed as % of total cells ± SD (n = 3) (*p<0.05 Vs. Control). B) DNA synthesis was evaluated by BrdU incorporation after 4 h of incubation with Pter followed by 2 h with Pter and 10 µM BrdU.</p

Effect of pterostilbene and resveratrol on tumor cell growth <i>in vitro</i>.

<p>A375, A549, HT29 and MCF7 cells were incubated for 24, 48 or 72 h with indicated concentrations of Pter or Res. Actual cell number was analyzed using the Countess Automated Cell Counter. Results were expressed as relative proliferation index ± SD (n = 4) where control is 100% (*P<0.05).</p

Pter induces caspase-independent cell death.

<p>A) Caspase-3/7 (DEVDase) activities were evaluated 24 and 48 h after addition of Pter. Results are expressed as a relative index of fluorescence ± SD (n = 3) when compared with control (100%). The results were statistically analyzed by ANOVA and contrasted by the Tukey test (*p<0.001; **p<0.01 Vs. Control). B) Induction of apoptosis was assayed by staining with Annexin V. The total percentage of Annexin V-positive cells was determined by flow cytometry 48 h after treatment with DMSO or with increasing concentrations of Pter (*p<0.001 Vs. Control). C) Effect of Pter on tumor cell number in the presence of 20 µM pancaspase inhibitor zVAD fmk, which was added 1 h prior to the addition of 50 µM Pter or vehicle (1.5 µl/ml DMSO). Cell number was analyzed 24 and 48 h later. Results are expressed as relative proliferation index ± SD (n = 3) where control is 100%.</p

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

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 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