Histolopathology.

<p>Histologic features of liver PHEO lesions stained with H&E 24 hours after <i>i.p</i>. vehicle alone (left) or with <i>i.p.</i> LB1 at 1.5 mg/kg plus TMZ by gavage at 80 mg/kg (right). Exposure to a single <i>i.p.</i> injection of vehicle showed a homogeneous field of healthy appearing tumor cells, whereas combination treatment resulted in extensive necrosis of tumor cells.</p

Western blot on MPC cells.

<p>Changes in pAKT, p53, pMDM2, and pPlk-1 after 24 hours treatment on MPC cells with 5 µM of LB1, 50 µM of TMZ and combination of both drugs. (A) Western blots show that LB1 exposure increases pAKT compared to control MPC cells (untreated, only vehicle). TMZ does not noticeably change the expression of pAKT and combination of LB1 and TMZ highly increases pAKT expression. (B) A demonstration of markedly increased expression of p53 after TMZ treatment but inhibition of expression by exposure to LB1. Noticeable increase in the expression of pMDM2 in MPC cells treated with LB1 alone or in combination with TMZ. (C) Noticeable increases in expression of pPlk1 in MPC cells after exposure to the combination of drugs.</p

<i>In vivo</i> anti-tumor activity of LB1 and TMZ and histological examination.

<p>Effects of treatment upon growth and molecular changes in hepatic tumors: (A) MRI images of untreated mice at weeks 5, 6, and 7 following intravenous injection of MPC cells and a photomicrograph showing the growth hepatic lesions and Alzet minipump. Barbed arrow indicates the gall bladder. Plain arrows indicate the same tumor nodules over time. (B) Inhibition of total hepatic tumor volume by LB1 alone at 1.5 mg/kg daily for 14 days by <i>c.i.</i> administered at 5^th day after MPC cells injection; TMZ alone at 80 mg/kg for 3 doses administered at 15^th day after MPC cells injection; and, combination of both drugs. (C) Survival curve combining the data from the study depicted in (B) a total of 10 control animals, 10 animals with combination treatment LB1 and TMZ, 5 animals with TMZ alone, and 5 animals with LB1 alone. Kaplan-Meier analysis revealed that survival following LB1 plus TMZ was significantly greater than with LB1 alone and TMZ alone (log rank, <i>P</i><0.0001). (D) MRI images of mice, treated with the combination of LB1 by <i>c.i</i>. for 14 days and 3 doses of TMZ, at week 7, 9, and 12. Partial response of treatment is presented with delayed appearance of hepatic tumors compared to untreated group. Complete response presents absence of hepatic tumors after treatment. A photomicrograph of the liver of one treated animal at week 12, showing the absence of gross tumor, and the presence of fibrous scar tissue (arrows). (E) Inhibition of estimated total hepatic tumor volume by combination treatment of LB1 and TMZ. LB1 at 1.5 mg/kg daily for 14 days by <i>c.i</i>. administered at 5^th day after MPC cells injection and TMZ at 80 mg/kg every 3 days for 14 doses beginning on 15^th day after MPC cells injection (with combination or alone). (F) Survival curve combining the data described in E. Total of 12 control animals, 5 animals for TMZ and 7 animals for combination treatment LB1 and TMZ. Survival of animals with combined treatment were significantly greater compared to controls (log rank, <i>P</i><0.0001). (G) Serial MRI images of mice, treated with the combination of LB1 and 14 doses TMZ with partial and complete responses. (H) Histologic features of liver PHEO at week 12 stained with H&E receiving no treatment or LB1 by <i>c.i.</i> and three doses of TMZ as described in D. Untreated animals showed intrahepatic deposits of cancer cells whereas the liver of an animal receiving both drugs that had no gross evidence of tumor revealed normal parenchyma and fibrous tissue, believed to be scarring at former sites of tumor masses. (I) Survival curves of animals treated with LB1 and 14 doses of TMZ when administration started at the same time, on day 5 after MPC cells injection. (n = 5 treated animals with LB1 plus TMZ, n = 5 for controls; log rank, <i>P</i> = 0.0035).</p

<i>In vitro</i> anti- proliferative activity of LB1 and TMZ and their combination.

<p>Inhibition of growth of MPC cells in culture: (A and B) Exposure for 3 days to increasing concentrations of LB1 or TMZ. (C–E) Exposure to increasing concentrations of LB1 plus TMZ. (F) Synergy analysis was done based on data from (A–E) using CalsuSyn software. CI values: C = 1 as additivity; C<1 as synergy; C>1 as antagonism. Combo 1 presents combination of 5 µM of LB1 and 100, 200, 300 µM of TMZ; Combo 2 presents combination of 7.5 µM of LB1 and 100, 200, 300 µM of TMZ; and Combo 3 10 µM of LB1 and 100, 200, 300 µM of TMZ.</p

Effect of LB1 and TMZ on tumor cell cycle and apoptosis.

<p>(A) Cell cycle analysis of MPC cells in exponential growth exposed for 48 hours to vehicle alone; LB1 alone at 5 µM; TMZ at 50 µM and; LB1 at 5 µM and TMZ at 50 µM. (B) PARP expression changes in 24 hours after treatment of mice bearing hepatic tumors with vehicle, LB1 alone at 1.5 mg/kg by gavage, TMZ alone by gavage at 80 mg/kg, of both drugs at the same doses.</p

Western blot on PHEO tumors.

<p>Changes in the state of phosphorylation and abundance of small pAKT, p53, pMDM2, and pPlk-1 24 hours after treatment of mice bearing hepatic tumors with vehicle, LB1 alone at 1.5 mg/kg by gavage, TMZ alone by gavage at 80 mg/kg, of both drugs at the same doses. (A) Western blots show that LB1 exposure increases pAKT in control-untreated tumors and treated tumors. TMZ does not change the expression of pAKT and combination of LB1 plus TMZ highly increases pAKT expression. (B) Western blots demonstrate marked increased expression of p53 after TMZ but complete inhibition of this induction by exposure to LB1 accompanied by an increase in the expression of pMDM2 in tumor cells exposed to LB1 alone or in combination with TMZ (C) Expression of pPlk1 shows a marked increase in tumors after exposure to the combination of drugs.</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

Expression of selected glycolysis, oxidative phosphorylation, and oxidative stress related genes. A

<p>mRNA expression of glucose transporter 1 (GLUT1), hexokinase-1 (HK1), hexokinase-2 (HK2), pyruvate kinase isozyme M2 (PKM2), NADH-ubiquinone oxidoreductase chain 1 (ND1), ubiquinol-cytochrome-c reductase complex core protein (UQCRC1), cytochrome c oxidase subunit 1 (CO1) in SDHB- and VHL-derived PHEOs/PGLs relative to normal adrenal medulla. Significant differences are indicated where appropriate by * for p<0.05, ** for p<0.01, and *** for p<0.001. <b>B</b> Western blot of selected proteins in SDHB- and VHL-derived PHEOs/PGLs (complex I: NADH dehydrogenase 1 beta subcomplex subunit 8 (NDUFB8); complex III: ubiquinol-cytochrome-c reductase complex core protein 2 (Uqcrc2); complex IV: CO1; complex V: ATP synthase (ATPsyn) α and β, cytochrome C (CytC), superoxide dismutase (SOD) 1, and 2).</p

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

Tumor tissue levels of oxidative phosphorylation complex activity and oxidative stress.

<p><b>A</b> Oxidative phosphorylation complex complex activity in SDHB- (n = 4) and VHL-derived (n = 4) PHEO/PGL tissue. * indicates p<0.05. <b>B</b> Malondialdehyde level in SDHB (n = 5) and VHL (n = 5) tissue as a measure of lipid oxidation. <b>C</b> Integrated density of DHE fluorescence in VHL (n = 5), SDHB (n = 6).</p