Identification and characterization of epicuticular proteins of nematodes sharing motifs with cuticular proteins of arthropods.

Specific collagens and insoluble proteins called cuticlins are major constituents of the nematode cuticles. The epicuticle, which forms the outermost electron-dense layer of the cuticle, is composed of another category of insoluble proteins called epicuticlins. It is distinct from the insoluble cuticlins localized in the cortical layer and the fibrous ribbon underneath lateral alae. Our objective was to identify and characterize genes and their encoded proteins forming the epicuticle. The combination between previously obtained laboratory results and recently made available data through the whole-genome shotgun contigs (WGS) and the transcriptome Shotgun Assembly (TSA) sequencing projects of Ascaris suum allowed us to identify the first epicuticlin gene, Asu-epic-1, on the chromosome VI. This gene is formed of exon1 (55 bp) and exon2 (1067 bp), separated by an intron of 1593 bp. Exon 2 is formed of tandem repeats (TR) whose number varies in different cDNA and genomic clones of Asu-epic-1. These variations could be due to slippage of the polymerases during DNA replication and RNA transcription leading to insertions and deletions (Indels). The deduced protein, Asu-EPIC-1, consists of a signal peptide of 20 amino acids followed by 353 amino acids composed of seven TR of 49 or 51 amino acids each. Three highly conserved tyrosine motifs characterize each repeat. The GYR motif is the Pfam motif PF02756 present in several cuticular proteins of arthropods. Asu-EPIC-1 is an intrinsically disordered protein (IDP) containing seven predicted molecular recognition features (MoRFs). This type of protein undergoes a disorder-to-order transition upon binding protein partners. Three epicuticular sequences have been identified in A. suum, Ascaris lumbricoides, and Toxocara canis. Homologous epicuticular proteins were identified in over 50 other nematode species. The potential of this new category of proteins in forming the nematode cuticle through covalent interactions with other cuticular components, particularly with collagens, is discussed. Their localization in the outermost layer of the nematode body and their unique structure render them crucial candidates for biochemical and molecular interaction studies and targets for new biotechnological and biomedical applications.

Prenatal exposure to maternal psychological distress and telomere length in childhood.

Telomere length (TL) is a biological marker of cellular aging, and shorter TL in adulthood is associated with increased morbidity and mortality risk. It is likely that these differences in TL are established long before adulthood, and there is growing evidence that TL can reflect prenatal experiences. Although maternal prenatal distress predicts newborn TL, it is unknown whether the relation between prenatal exposure to maternal distress and child TL persists through childhood. The purpose of the current longitudinal, prospective study is to examine the relation between prenatal exposure to maternal distress (perceived stress, depressive symptoms, pregnancy-related anxiety) and TL in childhood. Participants included 102 children (54 girls) and their mothers. Mothers' distress was assessed five times during pregnancy, at 12 weeks postpartum, and at the time of child telomere measurement between 6 and 16 years of age. Maternal distress during pregnancy predicted shorter offspring TL in childhood, even after accounting for postnatal exposure to maternal distress and other covariates. These findings indicate that maternal mental health predicts offspring TL biology later in childhood than previously observed. This study bolsters claims that telomere biology is subject to fetal programming and highlights the importance of supporting maternal mental health during pregnancy.

Prostate field cancerization and exosomes: Association between CD9, early growth response 1 and fatty acid synthase.

Intracapsular and well­defined adenocarcinomas of the prostate are often surrounded by tissue areas that harbor molecular aberrations, including those of genetic, epigenetic and biochemical nature. This is known as field cancerization, or a field effect and denotes a state of pre­malignancy. Such alterations in histologically normal tumor­adjacent prostatic tissues have been recognized as clinically important and are potentially exploitable as biomarkers of disease and/or targets for preventative/therapeutic intervention. The authors have previously identified and validated two protein markers of field cancerization: The expressional upregulation of the transcription factor early growth response 1 (EGR­1) and the lipogenic enzyme fatty acid synthase (FASN). However, the molecular etiology of prostate field cancerization, including EGR­1 and FASN upregulation, remains largely unknown. It was thus hypothesized that extracellular vesicles, notably exosomes, released by tumor lesions may induce molecular alterations in the surrounding tissues, resulting in field cancerization, priming the tissue, and ultimately promoting multifocal tumorigenesis, which is often observed in prostate cancer. Towards testing this hypothesis, the current study, to the best of our knowledge, for the first time, presents correlative protein expression data, generated in disease­free, tumor­adjacent and cancerous human prostate tissues by quantitative immunofluorescence, between the exosomal marker CD9, and EGR­1 and FASN. Despite the pilot character of the present study, and the static nature and heterogeneity of human tissues, the data suggest that CD9 expression itself is part of a field effect. In support of this hypothesis, the results suggest a possible contribution of exosomes to the induction of field cancerization in the prostate, particularly for EGR­1. These findings were corroborated in established cell models of cancerous (LNCaP) and non­cancerous (RWPE­1) human prostate epithelial cells. The findings of this study warrant further investigation into the functional interface between exosomes and field cancerization, as a detailed understanding of this characterization may lead to the development of clinical applications related to diagnosis and/or prognosis and targeted intervention to prevent progression from pre­malignancy to cancer.

EDB-FN Targeted Peptide-Drug Conjugates for Use against Prostate Cancer.

Prostate cancer (PCa) is the most common malignancy in men and is the leading cause of cancer-related male mortality. A disulfide cyclic peptide ligand [CTVRTSADC] 1 has been previously found to target extra domain B of fibronectin (EDB-FN) in the extracellular matrix that can differentiate aggressive PCa from benign prostatic hyperplasia. We synthesized and optimized the stability of ligand 1 by amide cyclization to obtain [KTVRTSADE] 8 using Fmoc/tBu solid-phase chemistry. Optimized targeting ligand 8 was found to be stable in phosphate buffered saline (PBS, pH 6.5, 7.0, and 7.5) and under redox conditions, with a half-life longer than 8 h. Confocal microscopy studies demonstrated increased binding of ligand 8 to EDB-FN compared to ligand 1. Therefore, we hypothesized that the EDB-FN targeted peptides (1 and 8) conjugated with an anticancer drug via a hydrolyzable linker would provide selective cytotoxicity to the cancer cells. To test our hypothesis, we selected both the normal prostate cell line, RWPE-1, and the cancerous prostate cell lines, PC3, DU-145, LNCaP, and C4-2, to evaluate the anticancer activity of synthesized peptide-drug conjugates. Docetaxel (Doce) and doxorubicin (Dox) were used as anticancer drugs. Dox conjugate 13 containing disulfide linkage showed comparable cytotoxicity versus Dox after 72 h incubation in all the cancer cell lines, whereas it was found to be less cytotoxic on RWPE-1, suggesting that it can act as a Dox prodrug. Doce conjugate 14 was found to be less cytotoxic in all the cell lines as compared to drug alone.

Effect of novel dietary supplement on metabolism in vitro and in vivo.

Obesity is an increasingly prevalent and preventable morbidity with multiple behavioral, surgical and pharmacological interventions currently available. Commercial dietary supplements are often advertised to stimulate metabolism and cause rapid weight and/or fat loss, although few well-controlled studies have demonstrated such effects. We describe a commercially available dietary supplement (purportedly containing caffeine, catechins, and other metabolic stimulators) on resting metabolic rate in humans, and on metabolism, mitochondrial content, and related gene expression in vitro. Human males ingested either a placebo or commercially available supplement (RF) in a randomized double-blind placebo-controlled cross-over fashion. Metabolic rate, respiratory exchange ratio, and blood pressure were measured hourly for 3 h post-ingestion. To investigate molecular effects, human rhabdomyosarcoma cells (RD) and mouse myocytes (C2C12) were treated with various doses of RF for various durations. RF enhanced energy expenditure and systolic blood pressure in human males without altering substrate utilization. In myocytes, RF enhanced metabolism, metabolic gene expression, and mitochondrial content suggesting RF may target common energetic pathways which control mitochondrial biogenesis. RF appears to increase metabolism immediately following ingestion, although it is unclear if RF provides benefits beyond those provided by caffeine alone. Additional research is needed to examine safety and efficacy for human weight loss.

Towards a personalized surgical margin for breast conserving surgery-Implications of field cancerization in local recurrence.

The amount of normal tissue that should be excised during breast conserving surgery is widely debated. Tissue adjacent to breast tumors, although histologically normal, possesses many of the molecular abnormalities found in tumor tissues. Here, we propose that the ideal physical distance for a surgical margin may not be universal. Rather, an adequate surgical margin likely varies from patient to patient, depending on the biology of the tissue that remains after surgery. J. Surg. Oncol. 2017;115:109-115. © 2017 Wiley Periodicals, Inc.

Single and Combinational siRNA Therapy of Cancer Cells: Probing Changes in Targeted and Nontargeted Mediators after siRNA Treatment.

Cancer cells are known to be heterogeneous and plastic, which imparts innate and acquired abilities to resist molecular targeting by short interfering RNA (siRNA). Not all cancer cells in a population would show a similar responsiveness to targeting of genes critical for their survival and even the responders could quickly transform and switch to alternative mechanism(s) for their survival. This study was designed to look at this phenomenon by analyzing the effect of siRNA silencing of selected protein mRNAs involved in cell survival and proliferation on other protein mRNAs that could contribute to cell survival. We compared the gene expression profile of the initial population after siRNA silencing to the subpopulation that survived the siRNA silencing, to identify potential overexpressions that might explain the cell survival. Our studies show that silencing well-selected protein mRNAs simultaneously could offer advantages compared to individual siRNA silencing due to an additional impact on the expression level of certain protein mRNAs. We also demonstrate that overexpression of certain protein mRNAs could explain the innate unresponsiveness of a subpopulation of cells. These observations could be a stepping stone for further investigation of the possibility of significant synergistic effect for this combinational RNA interference strategy.

Association and regulation of protein factors of field effect in prostate tissues.

Field effect or field cancerization denotes the presence of molecular aberrations in structurally intact cells residing in histologically normal tissues adjacent to solid tumors. Currently, the etiology of prostate field­effect formation is unknown and there is a prominent lack of knowledge of the underlying cellular and molecular pathways. We have previously identified an upregulated expression of several protein factors representative of prostate field effect, i.e., early growth response-1 (EGR­1), platelet-derived growth factor­A (PDGF­A), macrophage inhibitory cytokine­1 (MIC­1), and fatty acid synthase (FASN) in tissues at a distance of 1 cm from the visible margin of intracapsule prostate adenocarcinomas. We have hypothesized that the transcription factor EGR­1 could be a key regulator of prostate field­effect formation by controlling the expression of PDGF­A, MIC­1, and FASN. Taking advantage of our extensive quantitative immunofluorescence data specific for EGR­1, PDGF­A, MIC­1, and FASN generated in disease­free, tumor­adjacent, and cancerous human prostate tissues, we chose comprehensive correlation as our major approach to test this hypothesis. Despite the static nature and sample heterogeneity of association studies, we show here that sophisticated data generation, such as by spectral image acquisition, linear unmixing, and digital quantitative imaging, can provide meaningful indications of molecular regulations in a physiologically relevant in situ environment. Our data suggest that EGR­1 acts as a key regulator of prostate field effect through induction of pro­proliferative (PDGF­A and FASN), and suppression of pro­apoptotic (MIC­1) factors. These findings were corroborated by computational promoter analyses and cell transfection experiments in non­cancerous prostate epithelial cells with ectopically induced and suppressed EGR­1 expression. Among several clinical applications, a detailed knowledge of pathways of field effect may lead to the development of targeted intervention strategies preventing progression from pre-malignancy to cancer.

Prostate field cancerization: deregulated expression of macrophage inhibitory cytokine 1 (MIC-1) and platelet derived growth factor A (PDGF-A) in tumor adjacent tissue.

Prostate field cancerization denotes molecular alterations in histologically normal tissues adjacent to tumors. Such alterations include deregulated protein expression, as we have previously shown for the key transcription factor early growth response 1 (EGR-1) and the lipogenic enzyme fatty acid synthase (FAS). Here we add the two secreted factors macrophage inhibitory cytokine 1 (MIC-1) and platelet derived growth factor A (PDGF-A) to the growing list of protein markers of prostate field cancerization. Expression of MIC-1 and PDGF-A was measured quantitatively by immunofluorescence and comprehensively analyzed using two methods of signal capture and several groupings of data generated in human cancerous (n = 25), histologically normal adjacent (n = 22), and disease-free (n = 6) prostate tissues. A total of 208 digitized images were analyzed. MIC-1 and PDGF-A expression in tumor tissues were elevated 7.1x to 23.4x and 1.7x to 3.7x compared to disease-free tissues, respectively (p<0.0001 to p = 0.08 and p<0.01 to p = 0.23, respectively). In support of field cancerization, MIC-1 and PDGF-A expression in adjacent tissues were elevated 7.4x to 38.4x and 1.4x to 2.7x, respectively (p<0.0001 to p<0.05 and p<0.05 to p = 0.51, respectively). Also, MIC-1 and PDGF-A expression were similar in tumor and adjacent tissues (0.3x to 1.0x; p<0.001 to p = 0.98 for MIC-1; 0.9x to 2.6x; p<0.01 to p = 1.00 for PDGF-A). All analyses indicated a high level of inter- and intra-tissue heterogeneity across all types of tissues (mean coefficient of variation of 86.0%). Our data shows that MIC-1 and PDGF-A expression is elevated in both prostate tumors and structurally intact adjacent tissues when compared to disease-free specimens, defining field cancerization. These secreted factors could promote tumorigenesis in histologically normal tissues and lead to tumor multifocality. Among several clinical applications, they could also be exploited as indicators of disease in false negative biopsies, identify areas of repeat biopsy, and add molecular information to surgical margins.

Effects of the exercise-inducible myokine irisin on malignant and non-malignant breast epithelial cell behavior in vitro.

Exercise has been shown to reduce risk and improve prognosis of several types of cancers. Irisin is a myokine linked to exercise and lean body mass, which is thought to favorably alter metabolism systemically, potentially providing benefit for metabolic disease (including cancer). We evaluated the effects of various concentrations of irisin (with and without post-translational modifications) on malignant and non-malignant breast epithelial cell number, migration and viability. Irisin significantly decreased cell number, migration and viability in malignant MDA-MB-231 cells, without affecting non-malignant MCF-10a cells. Moreover, irisin enhanced the cytotoxic effect of doxorubicin (Dox) when added to a wide spectrum of irisin concentrations in the malignant cell type (with simultaneous reduction in Dox uptake), which was not observed in non-malignant MCF-10a cells. Additionally, we found that irisin decreases malignant cell viability in part through stimulation of caspase activity leading to apoptotic death. Interestingly, we found that irisin suppresses NFκB activation, an opposite effect of other myokines such as tumor necrosis factor alpha (TNF-α). Our observations suggest that irisin may offer therapeutic benefits for breast cancer prevention and treatment possibly through an anti-inflammatory response, induction of apoptotic cell death, or through enhanced tumor sensitivity to common antineoplastic agents such as Dox.

Enhanced cellular uptake of short polyarginine peptides through fatty acylation and cyclization.

Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, R5 and R6. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F'-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F'-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. Dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4-2.5-fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids.

Dietary stimulators of the PGC-1 superfamily and mitochondrial biosynthesis in skeletal muscle. A mini-review

Mitochondrial dysfunction has been linked to many diseases including metabolic diseases such as diabetes. Peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1) is a superfamily of transcriptional co-activators which are important precursors to mitochondrial biosynthesis found in most cells including skeletal muscle. The PGC-1 superfamily consists of three variants all of which are directly involved in controlling metabolic gene expression including those regulating fatty acid oxidation and mitochondrial proteins. In contrast to previous reviews on PGC-1, this mini-review summarizes the current knowledge of many known dietary stimulators of PGC-1 and the subsequent mitochondrial biosynthesis with associated metabolic benefit in skeletal muscle

Optical and SPION-enhanced MR imaging shows that trans-stilbene inhibitors of NF-κB concomitantly lower Alzheimer's disease plaque formation and microglial activation in AßPP/PS-1 transgenic mouse brain.

Alzheimer's disease (AD) is associated with a microglia-dependent neuroinflammatory response against plaques containing the fibrous protein amyloid-ß (Aß). Activation of microglia, which closely associate with Aß plaques, engenders the release of pro-inflammatory cytokines and the internalization of Aß fibrils. Since the pro-inflammatory transcription factor NF-κB is one of the major regulators of Aß-induced inflammation, we treated transgenic amyloid-ß protein protein/presenilin-1 (AßPP/PS1) mice for one year with a low dose (0.01% by weight in the diet) of either of two trans-stilbene NF-κB inhibitors, resveratrol or a synthetic analog LD55. The 3D distribution of Aß plaques was measured ex vivo in intact brains at 60 µm resolution by quantitative magnetic resonance imaging (MRI) using blood-brain barrier-permeable, anti-AßPP-conjugated superparamagentic iron oxide nanoparticles (SPIONs). The MRI measurements were confirmed by optical microscopy of thioflavin-stained brain tissue sections and indicated that supplementation with either of the two trans-stilbenes lowered Aß plaque density in the cortex, caudoputamen, and hippocampus by 1.4 to 2-fold. The optical measurements also included the hippocampus and indicated that resveratrol and LD55 reduced average Aß plaque density by 2.3-fold and 3.1-fold, respectively. Ex vivo measurements of the regional distribution of microglial activation by Iba-1 immunofluorescence of brain tissue sections showed that resveratrol and LD55 reduced average microglial activation by 4.2- fold and 3.5-fold, respectively. Since LD55 lacked hydroxyl groups but both resveratrol and LD55 concomitantly reduced both Aß plaque burden and neuroinflammation to a similar extent, it appears that the antioxidant potential of resveratrol is not an important factor in plaque reduction.

ß-alanine suppresses malignant breast epithelial cell aggressiveness through alterations in metabolism and cellular acidity in vitro.

BACKGROUND: Deregulated energetics is a property of most cancer cells. This phenomenon, known as the Warburg Effect or aerobic glycolysis, is characterized by increased glucose uptake, lactate export and extracellular acidification, even in the presence of oxygen. ß-alanine is a non-essential amino acid that has previously been shown to be metabolized into carnosine, which functions as an intracellular buffer. Because of this buffering capacity, we investigated the effects of ß-alanine on the metabolic cancerous phenotype. METHODS: Non-malignant MCF-10a and malignant MCF-7 breast epithelial cells were treated with ß-alanine at 100 mM for 24 hours. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate (OCR). mRNA of metabolism-related genes was quantified by qRT-PCR with corresponding protein expression quantified by immunoblotting, or by flow cytometry which was verified by confocal microscopy. Mitochondrial content was quantified using a mitochondria-specific dye and measured by flow cytometry. RESULTS: Cells treated with ß-alanine displayed significantly suppressed basal and peak ECAR (aerobic glycolysis), with simultaneous increase in glucose transporter 1 (GLUT1). Additionally, cells treated with ß-alanine exhibited significantly reduced basal and peak OCR (oxidative metabolism), which was accompanied by reduction in mitochondrial content with subsequent suppression of genes which promote mitochondrial biosynthesis. Suppression of glycolytic and oxidative metabolism by ß-alanine resulted in the reduction of total metabolic rate, although cell viability was not affected. Because ß-alanine treatment reduces extracellular acidity, a constituent of the invasive microenvironment that promotes progression, we investigated the effect of ß-alanine on breast cell viability and migration. ß-alanine was shown to reduce both cell migration and proliferation without acting in a cytotoxic fashion. Moreover, ß-alanine significantly increased malignant cell sensitivity to doxorubicin, suggesting a potential role as a co-therapeutic agent. CONCLUSION: Taken together, our results suggest that ß-alanine may elicit several anti-tumor effects. Our observations support the need for further investigation into the mechanism(s) of action and specificity of ß-alanine as a co-therapeutic agent in the treatment of breast tumors.

Dietary stimulators of the PGC-1 superfamily and mitochondrial biosynthesis in skeletal muscle. A mini-review.

Mitochondrial dysfunction has been linked to many diseases including metabolic diseases such as diabetes. Peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1) is a superfamily of transcriptional co-activators which are important precursors to mitochondrial biosynthesis found in most cells including skeletal muscle. The PGC-1 superfamily consists of three variants all of which are directly involved in controlling metabolic gene expression including those regulating fatty acid oxidation and mitochondrial proteins. In contrast to previous reviews on PGC-1, this mini-review summarizes the current knowledge of many known dietary stimulators of PGC-1 and the subsequent mitochondrial biosynthesis with associated metabolic benefit in skeletal muscle.

Leucine treatment enhances oxidative capacity through complete carbohydrate oxidation and increased mitochondrial density in skeletal muscle cells.

Leucine has been largely implicated for increasing muscle protein synthesis in addition to stimulating mitochondrial biosynthesis. Limited evidence is currently available on the effects and potential benefits of leucine treatment on skeletal muscle cell glycolytic and oxidative metabolism. This work identified the effects of leucine treatment on oxidative and glycolytic metabolism as well as metabolic rate of human and murine skeletal muscle cells. Human rhabdomyosarcoma cells (RD) and mouse myoblast cells (C2C12) were treated with leucine at either 100 or 500 µM for 24 or 48 h. Glycolytic metabolism was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate. Peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α), an important stimulator of mitochondrial biosynthesis, was quantified using flow cytometry and verified by immunofluorescent confocal microscopy. Mitochondrial content was quantified using mitochondrial and cytochrome C staining measured by flow cytometry and confirmed with confocal microscopy. Treatment with leucine significantly increased both basal and peak oxidative metabolism in both cell models. Leucine treated cells also exhibited significantly greater mitochondrial proton leak, which is associated with heightened energy expenditure. Basal ECAR was significantly reduced in both cell models following leucine treatment, evidence of reduced lactate export and more complete carbohydrate oxidation. In addition, both PGC-1α and cytochrome C expression were significantly elevated in addition to mitochondrial content following 48 h of leucine treatment. Our observations demonstrated few dose-dependent responses induced by leucine; however, leucine treatment did induce a significant dose-dependent expression of PGC-1α in both cell models. Interestingly, C2C12 cells treated with leucine exhibited dose-dependently reduced ATP content, while RD ATP content remain unchanged. Leucine presents a potent dietary constituent with low lethality with numerous beneficial effects for increasing oxidative preference and capacity in skeletal muscle. Our observations demonstrate that leucine can enhance oxidative capacity and carbohydrate oxidation efficiency, as well as verify previous observations of increased mitochondrial content.

Tumor necrosis factor alpha increases aerobic glycolysis and reduces oxidative metabolism in prostate epithelial cells.

BACKGROUND: Chronic inflammation promotes prostate cancer formation and progression. Furthermore, alterations in energy metabolism are a hallmark of prostate cancer cells. However, the actions of inflammatory factors on the energy metabolism of prostate epithelial cells have not been previously investigated. This is the first study to report on the effect of the inflammatory cytokine tumor necrosis factor alpha (TNFα) on the glycolytic and oxidative metabolism, and the mitochondrial function of widely used prostate epithelial cells. METHODS: Pre-malignant RWPE-1 and cancerous LNCaP and PC-3 cells were treated with low-dose TNFα. Glycolytic and oxidative metabolism was quantified by measuring extracellular acidification and oxygen consumption rates, respectively. ATP content and lactate export were measured by luminescence and fluorescence, respectively. Mitochondrial content and the expression of glucose transporter 1 (GLUT1), peroxisome proliferator-activated receptor co-activator 1 alpha (PGC-1α), and Cytochrome C were measured by flow cytometry. RESULTS: Our data suggest that TNFα increases glycolysis, ATP production, and lactate export, while it reduces oxidative metabolism and mitochondrial function in prostate epithelial cells. The highly aggressive PC-3 cells tend to be less responsive to the actions of TNFα than the pre-malignant RWPE-1 and the non-aggressive LNCaP cells. CONCLUSIONS: Cellular energetics, that is, glycolytic and oxidative metabolism is significantly influenced by low-level inflammation in prostate epithelial cells. In widely used prostate epithelial cell models, the micro-environmental inflammatory cytokine TNFα induces aerobic glycolysis while inhibiting oxidative metabolism. This supports the hypothesis that low-level inflammation can induce Warburg metabolism in prostate epithelial cells, which may promote cancer formation and progression.

Tumor necrosis factor alpha induces Warburg-like metabolism and is reversed by anti-inflammatory curcumin in breast epithelial cells.

The reprogramming of cellular metabolism in cancer cells is a well-documented effect. It has previously been shown that common oncogene expression can induce aerobic glycolysis in cancer cells. However, the direct effect of an inflammatory microenvironment on cancer cell metabolism is not known. Here, we illustrate that treatment of nonmalignant (MCF-10a) and malignant (MCF-7) breast epithelial cells with low-level (10 ng/ml) tumor necrosis factor alpha (TNF-α) significantly increased glycolytic reliance, lactate export and expression of the glucose transporter 1 (GLUT1). TNF-α decreased total mitochondrial content; however, oxygen consumption rate was not significantly altered, suggesting that overall mitochondrial function was increased. Upon glucose starvation, MCF7 cells treated with TNF-α demonstrated significantly lower viability than nontreated cells. Interestingly, these properties can be partially reversed by coincubation with the anti-inflammatory agent curcumin in a dose-dependent manner. This work demonstrates that aerobic glycolysis can be directly induced by an inflammatory microenvironment independent of additional genetic mutations and signals from adjacent cells. Furthermore, we have identified that a natural dietary compound can reverse this effect.

Ubiquinol rescues simvastatin-suppression of mitochondrial content, function and metabolism: implications for statin-induced rhabdomyolysis.

Statin medications diminish cholesterol biosynthesis and are commonly prescribed to reduce cardiovascular disease. Statins also reduce production of ubiquinol, a vital component of mitochondrial energy production; ubiquinol reduction may contribute to rhabdomyolysis. Human rhabdomyosarcoma cells were treated with either ethanol and dimethyl sulfoxide (DMSO) control, or simvastatin at 5 µM or 10 µM, or simvastatin at 5 µM with ubiquinol at 0.5 µM or 1.0 µM for 24 h or 48 h. PGC-1α RNA levels were determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was determined using flow cytometry and immunocytochemistry. Metabolism was determined by quantification of extracellular acidification rate and oxygen consumption rate. Treatment of human rhabdomyosarcoma cells with simvastatin significantly reduced oxidative, total metabolism, and cellular ATP content in a time- and dose-dependent manner which was rescued by concurrent treatment with ubiquinol. Treatment with simvastatin significantly reduced mitochondrial content as well as cell viability which were both rescued by simultaneous treatment with ubiquinol. This work demonstrates that the addition of ubiquinol to current statin treatment regimens may protect muscle cells from myopathies.