Altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning.

Altered expression of mitochondrial electron transport proteins has been shown in early preconditioned myocardial tissue. We wished to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated during subsequent ischemia. Fourteen pigs underwent a SWOP protocol with ten 2-minute balloon inflations in the LAD artery, each separated by 2 minutes reperfusion. Twenty-four hours later, mitochondria were isolated from SWOP and SHAM pig hearts and analyzed for uncoupling protein (UCP)-2 content by western blot analysis, proteomic changes by iTRAQ(®) and respiration by an oxygen electrode. In parallel in vivo studies, high-energy nucleotides were obtained by transmural biopsy from anesthetized SWOP and SHAM pigs at baseline and during sustained low-flow ischemia. Compared with SHAM mitochondria, ex vivo SWOP heart tissue demonstrated increased expression of UCP-2, Complex IV (cytochrome c oxidase) and Complex V (ATPase) proteins. In comparison with SHAM pigs during in vivo conditions, transmural energetics in SWOP hearts, as estimated by the free energy of ATP hydrolysis (ΔG(0)), were similar at baseline but had decreased by the end of low-flow ischemia (-57.0 ± 2.1 versus -51.1 ± 1.4 kJ/mol; P < 0.05). In conclusion, within isolated mitochondria from preconditioned SWOP hearts, UCP-2 is increased and in concert with enhanced Complex IV and V proteins, imparts a favorable energetic state during low-flow ischemia. These data support the notion that mitochondrial adaptations that may reduce oxidant damage do not reduce the overall efficiency of energetics during sustained oxygen deprivation.

Mouse prostate proteomes are differentially altered by supranutritional intake of four selenium compounds.

We have shown that, in contrast to selenomethionine (SeMet) or selenite, methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) exert prostate cancer (PCa) inhibitory effect in preclinical models. Here we investigated the prostate proteome signatures of mice treated with each selenium (Se) form for hypothesis generation concerning their potential in vivo molecular targets and cancer risk modification. Nude mice bearing subcutaneous PC-3 xenografts were treated daily with each Se form (3 mg Se/kg) orally for 45 days. Five prostates were pooled from each group. Their proteomes were profiled by LC-MS/MS with iTRAQ labeling. Of the 1,088 proteins identified, 72 were significantly modulated by one or more Se forms. MSeA and MSeC each induced separate sets of tumor suppressor proteins and suppressed different onco-proteins. Proteins induced by selenite and shared with MSeC were related to energy metabolism (e.g., fatty-acid synthase), and those induced by SeMet included vimentin and heat-shock protein-70, favoring cancer growth. While proteome changes induced by MSeA were associated with PCa risk reduction, desirable risk-reducing signatures induced by MSeC were counterbalanced by risk-promoting patterns shared with selenite and SeMet. We propose that the balance of oncogenic vs. suppressor protein patterns in the prostate may impact the direction of PCa risk modification by a given selenium.

Continued depression of maximal oxygen consumption and mitochondrial proteomic expression despite successful coronary artery bypass grafting in a swine model of hibernation.

OBJECTIVE: Clinical studies indicate incomplete functional recovery of hibernating myocardium after coronary artery bypass grafting. We hypothesized that persistent contractile abnormalities after coronary artery bypass grafting are associated with decreased mitochondrial proteins involving electron transport chain that might limit maximal oxygen consumption. METHODS: Seven pigs with hibernating myocardium underwent off-pump revascularization with left internal thoracic artery to mid left anterior descending artery. At 4 weeks, left internal thoracic artery anastomosis was patent by multidetector computed tomography. Regional function (transthoracic echocardiography) and blood flow (microspheres) were assessed at rest and during high-dose dobutamine (40 µg/[kg · min]). Expression of electron transport chain proteins was analyzed with isobaric tags for relative and absolute quantification. RESULTS: After revascularization, multidetector computed tomography confirmed severe left anterior descending stenosis and patent left internal thoracic artery graft. Regional function and blood flow normalized at rest; however, function in left anterior descending distribution remained depressed relative to remote regions, and myocardial blood flow in that region did not increase normally when challenged with high-work state. Concomitant with reduced maximal blood flow response in left anterior descending region was more than 40% reduction in electron transport chain proteins essential to adenosine triphosphate production. CONCLUSIONS: Despite successful revascularization of hibernating myocardium, regional function and blood flow remained depressed during catecholamine stress. Electron transport chain proteins known to be downregulated during adaptive process within hibernating myocardium did not normalize after revascularization. These data demonstrate a potential bioenergetic cause of persistent dysfunction and heart failure within successfully revascularized hibernating myocardium.

Proteomic profiling of potential molecular targets of methyl-selenium compounds in the transgenic adenocarcinoma of mouse prostate model.

Because the Selenium (Se) and Vitamin E Cancer Prevention Trial (SELECT) failed to show the efficacy of selenomethionine for prostate cancer prevention, there is a critical need to identify safe and efficacious Se forms for future trials. We have recently shown significant preventive benefit of methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) in the transgenic adenocarcinoma mouse prostate (TRAMP) model by oral administration. The present work applied iTRAQ proteomic approach to profile protein changes of the TRAMP prostate and to characterize their modulation by MSeA and MSeC to identify their potential molecular targets. Dorsolateral prostates from wild-type mice at 18 weeks of age and TRAMP mice treated with water (control), MSeA, or MSeC (3 mg Se/kg) from 8 to 18 weeks of age were pooled (9-10 mice per group) and subjected to protein extraction, followed by protein denaturation, reduction, and alkylation. After tryptic digestion, the peptides were labeled with iTRAQ reagents, mixed together, and analyzed by two-dimensional liquid chromatography/tandem mass spectrometry. Of 342 proteins identified with >95% confidence, the expression of 75 proteins was significantly different between TRAMP and wild-type mice. MSeA mainly affected proteins related to prostate functional differentiation, androgen receptor signaling, protein (mis)folding, and endoplasmic reticulum-stress responses, whereas MSeC affected proteins involved in phase II detoxification or cytoprotection, and in stromal cells. Although MSeA and MSeC are presumed precursors of methylselenol and were equally effective against the TRAMP model, their distinct affected protein profiles suggest biological differences in their molecular targets outweigh similarities.

Quantitative proteomic analysis by iTRAQ(R) for the identification of candidate biomarkers in ovarian cancer serum.

BACKGROUND: Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ(R). RESULTS: Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ(R) to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified. CONCLUSIONS: This study provides the first analysis of immunodepleted serum in combination with iTRAQ(R) to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.

Identification of candidate biomarkers in ovarian cancer serum by depletion of highly abundant proteins and differential in-gel electrophoresis.

Ovarian cancer is the fifth leading cause of cancer death for women in the US, yet survival rates are over 90% when it is diagnosed at an early stage, highlighting the need for biomarkers for early detection. To enhance the discovery of tumor-specific proteins that could represent novel serum biomarkers for ovarian cancer, we depleted serum of highly abundant proteins which can mask the detection of proteins present in serum at low concentrations. Three commercial immunoaffinity columns were used in parallel to deplete the highly abundant proteins in serum from 60 patients with serous ovarian carcinoma and 60 non-cancer controls. Medium and low abundance serum proteins from each serum pool were then evaluated by the quantitative proteomic technique of differential in-gel electrophoresis. The number of protein spots that were elevated in ovarian cancer sera by at least twofold ranged from 36 to 248, depending upon the depletion and separation methods. From the 33 spots picked for MS analysis, nine different proteins were identified, including the novel candidate ovarian cancer biomarkers leucine-rich alpha2 glycoprotein-1 and ficolin 3. Western blotting validated the relative increases in serum protein levels for three of the proteins identified, demonstrating the utility of this approach for the identification of novel serum biomarkers for ovarian cancer.

Proteomic analysis uncovers novel actions of the neurosecretory protein VGF in nociceptive processing.

Peripheral tissue injury is associated with changes in protein expression in sensory neurons that may contribute to abnormal nociceptive processing. We used cultured dorsal root ganglion (DRG) neurons as a model of axotomized neurons to investigate early changes in protein expression after nerve injury. Comparing protein levels immediately after DRG dissociation and 24 h later by proteomic differential expression analysis, we found a substantial increase in the levels of the neurotrophin-inducible protein VGF (nonacronymic), a putative neuropeptide precursor. In a rodent model of nerve injury, VGF levels were increased within 24 h in both injured and uninjured DRG neurons, and the increase persisted for at least 7 d. VGF was also upregulated 24 h after hindpaw inflammation. To determine whether peptides derived from proteolytic processing of VGF participate in nociceptive signaling, we examined the spinal effects of AQEE-30 and LQEQ-19, potential proteolytic products shown previously to be bioactive. Each peptide evoked dose-dependent thermal hyperalgesia that required activation of the mitogen-activated protein kinase p38. In addition, LQEQ-19 induced p38 phosphorylation in spinal microglia when injected intrathecally and in the BV-2 microglial cell line when applied in vitro. In summary, our results demonstrate rapid upregulation of VGF in sensory neurons after nerve injury and inflammation and activation of microglial p38 by VGF peptides. Therefore, VGF peptides released from sensory neurons may participate in activation of spinal microglia after peripheral tissue injury.

Chemopreventive agents modulate the protein expression profile of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone plus benzo[a]pyrene-induced lung tumors in A/J mice.

We used isobaric tag labeling coupled with mass spectrometry to compare the relative abundance of proteins in lung tumors from A/J mice treated with a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene versus normal mouse lung tissues. Levels of 59 proteins changed-30 increased and 29 decreased-in tumor tissues versus normal tissues. Among proteins that showed increased levels in tumor tissues versus normal tissues were glycolytic enzymes, ribosomal proteins, fatty acid synthase, cathepsins D and H and carbonic anhydrase 2. On the other hand, the levels of cytochrome P450 enzymes 2B10 and 2F2, glutathione S-transferases mu-1, procollagen VI, Clara cell 10 kDA (CC10) protein, histones, receptor advanced glycation end product, and lung carbonyl reductase were lower in tumor tissues versus normal lung tissues. Upon dietary administration of a combination of N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine plus myo-inositol or indole-3-carbinol to carcinogen-treated mice, the relative abundance of 60S ribosomal protein L4 and carbonic anhydrase in tumor tissues decreased whereas that of histones, glutathione S-transferases mu, receptor advanced glycation end product, transglutaminase, and procollagen VI increased. Western assays with lung tissue homogenates not only verified the proteomics results for selected proteins but also showed differential expression of hypoxia inducible factor-1alpha, a transcription factor for most of the proteins that showed changes in relative abundance. This is the first report on the application of quantitative proteomics to study the relative abundance of proteins in a mouse model of lung carcinogenesis. These proteins may have utility for development of candidate lung cancer biomarkers and as targets of chemopreventive/chemotherapeutic agents.

Indole-3-carbinol inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone plus benzo(a)pyrene-induced lung tumorigenesis in A/J mice and modulates carcinogen-induced alterations in protein levels.

We tested the chemopreventive efficacy of indole-3-carbinol (I3C), a constituent of Brassica vegetables, and its major condensation product, 3,3'-diindolylmethane (DIM), against lung tumorigenesis induced by a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) in A/J mice. The mixture of NNK plus BaP (2 micromol each) was administered by gavage as eight weekly doses, whereas I3C (112 micromol/g diet) and DIM (2 and 30 micromol/g diet in experiments 1 and 2, respectively) were given in the diet for 23 weeks beginning at 50% of carcinogen treatment. I3C reduced NNK plus BaP-induced tumor multiplicity by 78% in experiment 1 and 86% in experiment 2; the respective reductions in tumor multiplicity by DIM were 5% and 66%. Using a quantitative proteomics method, isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry, we identified and quantified at least 250 proteins in lung tissues. Of these proteins, nine showed differences in relative abundance in lung tissues of carcinogen-treated versus untreated mice: fatty acid synthase, transketolase, pulmonary surfactant-associated protein C (SP-C), L-plastin, annexin A1, and haptoglobin increased, whereas transferrin, alpha-1-antitrypsin, and apolipoprotein A-1 decreased. Supplementation of the diet of carcinogen-treated mice with I3C reduced the level of SP-C, L-plastin, annexin A1, and haptoglobin to that of untreated controls. These results were verified using immunoblotting. We show here that tumor-associated signature proteins are increased during NNK plus BaP-induced lung carcinogenesis, and I3C inhibits this effect, suggesting that the lung tumor chemopreventive activity of I3C might be related to modulation of carcinogen-induced alterations in protein levels.