Influence of statin treatment on spontaneous cell migration.

<p>MPC (A) and MTT (B) were plated in vehicle (Ctr), 5 µM fluvastatin (Fluva), 5 µM simvastatin (Simva), 5 µM lovastatin (Lova) with or without 100 µM trans, trans farnesol (FOH) and spontaneous migration was recorded for 24.</p

Combination of two different statins to evaluate potential additive effects on MPC or MTT.

<p>Relative viability of MPC (A) and MTT (B) at the indicated doses and durations for the most effective combined treatments including simvastatin relative to simvastatin alone.</p

Relative proliferation of MPC and MTT under statin treatment.

<p>MPC (A–F) and MTT (G–K) were treated with ▪ 6.25 µM, ▴ 12.50 µM, ▾ 25.00 µM, and ⧫ 50.00 µM of atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin for 24, 48, and 72 hours.</p

Expression of selected proteins in MPC and MTT after treatment with fluvastatin, simvastatin, or lovastatin.

<p>A. Western blot showing decreased levels of pMAPK1/3 in treated vs. untreated MPC and MTT relative to total MAPK1/3 and GAPDH. B. Western blot showing decreased levels of intact PARP (top bands) and increased levels of cleaved PARP (lower bands) in treated vs. untreated MPC and MTT. In accordance, cleaved CASP-3 was elevated in treated cells, indicating apoptosis. Cells were treated with 25 µM of the indicated statin for 48 hours.</p

MAPK pathway representation in SDHB-derived PHEOs/PGLs.

<p>A. Western blot of pMAPK1/3, total MAPK1/3, and GAPDH in human PHEOs/PGLs. Patient and tumor information for each sample are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097712#pone-0097712-t001" target="_blank">Table 1</a>. Abbreviations: B) SDHB, D) SDHD, N) NF1, M) normal adrenal medulla. B. Heatmap showing expression of 21 MAPK pathway genes in pseudohypoxic PHEOs/PGLs. Expression of these 21 genes was significantly elevated in SDHB compared to normal medulla (p<0.002). Each sample was assigned a number. The corresponding sample identifier from the original article <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097712#pone.0097712-Shankavaram1" target="_blank">[33]</a> is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097712#pone.0097712.s001" target="_blank">Table S1</a>. Patient information and link to deposited data are given in the original article.</p

Effect of 6.25–50 µM statin treatment for 3 days on MPC and MTT proliferation.

<p>Percent significant results over all statins for all treatment doses at the different treatment durations for MPC (A) and MTT (B). The percentage of significant results was increased in MTT compared to MPC after 48 and 72 h, suggesting that MTT are more sensitive to statin treatment.</p

Patient information.

<p>Abbreviations: A: adrenal, E: extra-adrenal, F: female, HN: head and neck, ID: identifier, M: male, MM: metastatic metastases, N: normal, P: primary non-metastatic, PM: primary metastatic.</p

Warburg Effect’s Manifestation in Aggressive Pheochromocytomas and Paragangliomas: Insights from a Mouse Cell Model Applied to Human Tumor Tissue

<div><p>A glycolytic profile unifies a group of pheochromocytomas and paragangliomas (PHEOs/PGLs) with distinct underlying gene defects, including von Hippel-Lindau (VHL) and succinate dehydrogenase B (SDHB) mutations. Nevertheless, their tumor aggressiveness is distinct: PHEOs/PGLs metastasize rarely in VHL-, but frequently in SDHB-patients. To date, the molecular mechanisms causing the more aggressive phenotype in SDHB-PHEOs/PGLs remain largely unknown. Recently, however, an excellent model to study aggressive PHEOs (mouse tumor tissue (MTT) cells) has been developed from mouse PHEO cells (MPC). We employed this model for a proteomics based approach to identify changes characteristic for tumor aggressiveness, which we then explored in a homogeneous set of human SDHB- and VHL-PHEOs/PGLs. The increase of glucose transporter 1 in VHL, and of hexokinase 2 in VHL and SDHB, confirmed their glycolytic profile. In agreement with the cell model and in support of decoupling of glycolysis, the Krebs cycle and oxidative phosphorylation (OXPHOS), SDHB tumors showed increased lactate dehydrogenase levels. In SDHB-PGLs OXPHOS complex activity was increased at complex III and, as expected, decreased at complex II. Moreover, protein and mRNA expression of all tested OXPHOS-related genes were higher in SDHB- than in VHL-derived tumors. Although there was no direct evidence for increased reactive oxygen species production, elevated superoxide dismutase 2 expression may reflect elevated oxidative stress in SDHB-derived PHEOs/PGLs. For the first time, we show that despite dysfunction in complex II and evidence for a glycolytic phenotype, the Warburg effect does not seem to fully apply to SDHB-PHEOs/PGLs with respect to decreased OXPHOS. In addition, we present evidence for increased LDHA and SOD2 expression in SDHB-PHEOs/PGLs, proteins that have been proposed as promising therapeutic targets in other cancers. This study provides new insight into pathogenic mechanisms in aggressive human PHEOs/PGLs, which may lead to identifying new diagnostic and prognostic markers in the near future.</p> </div

Patient Information.

<p>ID (identifier): B followed by a number indicates SDHB, V followed by a number indicates VHL cases. Genetic Background: SDHB^p: SDHB polymorphism, VHL^c: VHL-Chuvash. Gender: F: female, M: male. Type: bp: bilateral primary, mm: metastatic metastases, mltp: multiple primary, pm: primary metastatic, sp: solitary primary. Location: R: right, L: left. Biochem. (biochemical phenotype): A: adrenergic, DA: dopaminergic, NA: noradrenergic, nk: not known. The 4 right columns indicate which samples have been used in the experiments specified by the column headings. Subscript letters are used if more than one tumor from the same patient was available, to indicate which sample has been used (R: right, L: left, A and B: as specified under the heading “location”. OXP (oxidative phosphorylation complex activity), DHE (dihydroethidium fluorescence), MDA (malondialdehyde), WB (western blot). In the western blot column, samples used for LDHA and B and LDHB blots are indicated by . Samples used for all other proteins are indicated by *.</p

Oxidative phosphorylation complex activity, reactive oxygen species production, and expression of selected oxidative phosphorylation complex subunits in MPC and MTT. A

<p>Ratio of ADP-stimulated (S3) and baseline oxygen consumption (S4) in MPC and MTT. <b>B</b> Baseline hydrogen peroxide levels (left) and those observed after addition of substrate (glutamate/malate, complex I (center); succinate, complex II (right). ** indicates p<0.01. <b>C</b> protein expression of selected subunits of OXPHOS complexes (complex I: NADH dehydrogenase 1 beta subcomplex subunit 8 (NDUFB8); complex II: succinate dehydrogenase B (SDHB); complex III: ubiquinol-cytochrome-c reductase complex core protein 2 (Uqcrc2); complex V: ATP synthase α (ATPsynα)).</p