Global Effects of Adriamycin Treatment on Mouse Splenic Protein Levels

Adriamycin (ADR) is a potent anticancer drug used to treat a variety of cancers. Patients treated with ADR have experienced side effects such as heart failure, cardiomyopathy, and “chemobrain”, which have been correlated to changes in protein expression in the heart and brain. In order to better understand cellular responses that are disrupted following ADR treatment in immune tissues, this work focuses on spleen. Significantly reduced spleen sizes were found in ADR-treated mice. Global isotopic labeling of tryptic peptides and nanoflow reversed-phase liquid chromatography-tandem mass spectrometry (LC–MS/MS) were employed to determine differences in the relative abundances of proteins from ADR-treated mice relative to controls. Fifty-nine proteins of the 388 unique proteins identified showed statistically significant differences in expression levels following acute ADR treatment. Differentially expressed proteins are involved in processes such as cytoskeletal structural integrity, cellular signaling and transport, transcription and translation, immune response, and Ca²⁺ binding. These are the first studies to provide insight to the downstream effects of ADR treatment in a peripheral immune organ such as spleen using proteomics

Differential effects of thiaminase on branched chain amino acid catabolism.

<p>A. Schematic diagram showing branched chain amino acid catabolism. B. Different metabolomic signatures indicating inhibition of BCKDH by thiaminase. The top three panels show accumulation of BCKDH substrates after 48-7 cells after thiaminase treatment- most notably isovalerylcarnitine, which is also reversed by rapamycin. C. An immunoblot of cytosolic and mitochondrial branched chain amino acid transferase (cBCAT and mBCAT), the enzymes that catalyzes the reactions that produce BCKDH substrates, and total and phosphorylated BCKDH subunit E1 (BCKD-E1 and pBCKD-E1, respectively) in RS4 leukemia cells treated with thiaminase, rapamycin or both. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p<0.05 and * indicates 0.05</p

Effects of thiaminase on aromatic amino acid catabolism.

<p>A. Accumulation of the products of phenylalanine (phenylpyruvate and phenyllactate) and tyrosine (hydroxyphenylpyruvate and hydroxyphenyllactate) catabolism in RS4 cells after treatment with thiaminase for 48 hours. B. Accumulation of tryptophan catabolites after thiaminase treatment, showing accumulation of indolelactate but not kynurenine. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p<0.05 and * indicates 0.05</p

Effect of thiamine antagonists on thiaminase activity.

<p>A. Growth inhibition of Reh and RS4 leukemia cells in the presence or absence of thiaminase and either N3PT or oxythiamine. B. Growth inhibition of RS4 and Reh leukemia cells incubated in normal medium, thiamine-free medium or medium containing thiaminase prior to exposure to different concentrations of N3PT. C. RS4 subcutaneous xenografts showing untreated control, N3PT alone, 1k-PEGylated thiaminase and 1k-PEGylated thiaminase followed by N3PT. The time-to-endpoint was 16.5 days for control, 23 days for N3PT, 25 days for 1k-PEG thiaminase and 55 days for 1k-PEG thiaminase (p<0.01 Log rank test). D. Oxygen consumption rate of RS4 cells treated with thiaminase, N3PT or thiaminase followed by N3PT (clear: control; thin-stripe: thiaminase; thick- stripe: N3PT; solid: thiaminase + N3PT).</p

In vivo evidence of thiaminase anticancer activity.

<p>A. A Kaplan-Meyer plot of time to pre-defined tumor volume endpoint for subcutaneous MCF-7 breast cancer xenografts treated with thiaminase 2000 units SC QOD or buffer control. The median time to endpoint was 41 days for untreated control and 59 days (p = 0.03 Log rank test). B. A Kaplan-Meyer plot of time to pre-defined tumor volume endpoint for subcutaneous RS4 leukemia subcutaneous xenografts treated with thiaminase 850 units SC BIW or buffer control. The median time to endpoint was 16.5 days for the control group and not reached after 60 days of observation in the treated group (p<0.001 Log rank test). C. Primary ALL and AML specimens were thawed and plated in triplicate in two concentrations of thiaminase 0.4 units/ml and 4 units/ml, and assessed for viability at 48 hours relative to untreated cells. The ALL specimen with the asterisk was used for the <i>in vivo</i> study shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085702#pone-0085702-g001" target="_blank">Figure 1D</a>. D. Primary ALL cells were injected IV on Day 1; three thiaminase treatments of 2000 units/kg SC were administered on days 17, 20 and 24. The animals were sacrificed on Day 33 and bone marrow was examined by flow cytometry for human ALL cells (percent human CD45); Untreated n = 4; vehicle treated n = 10, native thiaminase n = 8 (* p<0.01, Mann-Whitney test).</p

OCR and ECAR of leukemia and breast cell lines incubated for 48(clear: control; thin-stripe: thiaminase; thick- stripe: rapamycin; solid: thiaminase + rapamycin).

<p>A. Quantification of OCR parameters. The ATP-linked rate is the basal rate minus the rate measured after the addition of oligomycin. The maximal capacity is the rate measured after the addition of FCCP. The reserve capacity is the basal rate minus the FCCP rate. All data are the mean ± SEM, of triplicate samples and are representative of 3 independent experiments (†p<0.05, *p<0.01 two way ANOVA (Newman Kruskal Wallis test). B. Quantification of ECAR parameters. Glycolysis is the rate determine from subtracting the basal rate from the rate after the addition of glucose. Glycolytic reserve is the rate determined by subtracting the rate following the addition of oligomycin from the rate following the addition of glucose. All data are the mean ± SEM, of triplicate samples and are representative of 3 (†p<0.05, *p<0.01 two way ANOVA (Newman Kruskal Wallis test).</p

p62 Pathology Model in the Rat Substantia Nigra with Filamentous Inclusions and Progressive Neurodegeneration

<div><p>One of the proteins most frequently found in neuropathological lesions is the ubiquitin binding protein p62 (sequestosome 1). Post-mortem analysis of p62 is a defining diagnostic marker in several neurodegenerative diseases including amyotrophic lateral sclerosis and inclusion body myositis. Since p62 functions in protein degradation pathways including autophagy, the build-up of p62-positive inclusions suggests defects in protein clearance. p62 was expressed unilaterally in the rat substantia nigra with an adeno-associated virus vector (AAV9) in order to study p62 neuropathology. Inclusions formed within neurons from several days to several weeks after gene transfer. By electron microscopy, the inclusions were found to contain packed 10 nm thick filaments, and mitochondria cristae structure was disrupted, resulting in the formation of empty spaces. In corollary cell culture transfections, p62 clearly impaired mitochondrial function. To probe for potential effects on macroautophagy, we co-expressed p62 with a double fluorescent tagged reporter for the autophagosome protein LC3 in the rat. p62 induced a dramatic and specific dissociation of the two tags. By 12 weeks, a rotational behavior phenotype manifested, consistent with a significant loss of dopaminergic neurons analyzed post-mortem. p62 overexpression resulted in a progressive and robust pathology model with neuronal inclusions and neurodegeneration. p62 gene transfer could be a novel methodological probe to disrupt mitochondrial function or autophagy in the brain and other tissues in vivo.</p></div

Development of a turning bias over time in rats overexpressing p62 unilaterally in the substantia nigra.

<p>Amphetamine-stimulated rotations occur when there is a large side-to-side difference in dopamine levels in the nigrostriatal pathway in rats, i.e., when there is a large loss of dopamine neurons on one side. A) In the p62 group, the behavioral phenotype of ipsilateral turning bias developed by 12 weeks (N = 7, P < 0.05, t-test), but not at earlier times in the p62 group. B) Turning bias did not manifest in a group of rats expressing the control protein GFP (N = 4).</p

p62 impairs mitochondrial function in transfected cells: decreased oxidative phosphorylation and increased glycolysis.

<p>HEK 293T cells were transfected with a plasmid for p62 or control plasmids (GFP and empty). A) Basal oxygen consumption, i.e., oxidative phosphorylation, was decreased in the p62 group compared to the two control groups (ANOVA/Bonferroni, p < 0.001). There was also a small decrease in oxygen consumption in the GFP group relative to the empty group (ANOVA/Bonferroni, p < 0.001). B) Glycolysis and glycolytic reserve were increased in the p62 group compared to the two controls as evaluated by the extracellular acidification rate (ANOVA/Bonferroni, p < 0.001). C) Lactate, a by-product of glycolysis, was increased in the p62 group compared to the two controls (ANOVA/Bonferroni, p < 0.001). N is indicated in parentheses, asterisk indicates significance compared to the empty vector group.</p

p53 triggers autophagy and cell death.

<p>A. Biochemical marker for cardiac injury by autophagy. Western blot analysis of autophagy marker (LC3) expression in heart tissue homogenates from WT and p53^(−/−) mice treated with saline or DOX. Quantitative analysis represents the Mean ± SEM n = 5 in each group; ^#p<0.001 as compared to other groups. B. Western blot analysis of gamma H2AX marker for cell death in heart tissue nuclear extracts from WT and p53^(−/−) mice treated with saline or DOX. Quantitative analysis represents the Mean ± SEM n = 5 in each group. ^#p<0.001 as compared to other groups.</p