Kristina A. Trujillo

Conjugated linoleic acid or omega 3 fatty acids increase mitochondrial biosynthesis and metabolism in skeletal muscle cells

BackgroundPolyunsaturated fatty acids are popular dietary supplements advertised to contribute to weight loss by increasing fat metabolism in liver, but the effects on overall muscle metabolism are less established. We evaluated the effects of conjugated linoleic acid (CLA) or combination omega 3 on metabolic characteristics in muscle cells.MethodsHuman rhabdomyosarcoma cells were treated with either DMSO control, or CLA or combination omega 3 for 24 or 48 hours. RNA was determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was determined using flow cytometry and immunohistochemistry. Metabolism was quantified by measuring extracellular acidification and oxygen consumption rates.ResultsOmega 3 significantly induced metabolic genes as well as oxidative metabolism (oxygen consumption), glycolytic capacity (extracellular acidification), and metabolic rate compared with control. Both treatments significantly increased mitochondrial content.ConclusionOmega 3 fatty acids appear to enhance glycolytic, oxidative, and total metabolism. Moreover, both omega 3 and CLA treatment significantly increase mitochondrial content compared with control.

Characterization of the metabolic effects of irisin on skeletal muscle in vitro

This work explored the effects of irisin on metabolism, gene expression and mitochondrial content in cultured myocytes.

Markers of fibrosis and epithelial to mesenchymal transition demonstrate field cancerization in histologically normal tissue adjacent to breast tumors.

Previous studies have shown that a field of genetically altered but histologically normal tissue extends 1 cm or more from the margins of human breast tumors. The extent, composition and biological significance of this field are only partially understood, but the molecular alterations in affected cells could provide mechanisms for limitless replicative capacity, genomic instability and a microenvironment that supports tumor initiation and progression. We demonstrate by microarray, qRT‐PCR and immunohistochemistry a signature of differential gene expression that discriminates between patient‐matched, tumor‐adjacent histologically normal breast tissues located 1 cm and 5 cm from the margins of breast adenocarcinomas (TAHN‐1 and TAHN‐5, respectively). The signature includes genes involved in extracellular matrix remodeling, wound healing, fibrosis and epithelial to mesenchymal transition (EMT). Myofibroblasts, which are mediators of wound healing and fibrosis, and intra‐lobular fibroblasts expressing MMP2, SPARC, TGF‐β3, which are inducers of EMT, were both prevalent in TAHN‐1 tissues, sparse in TAHN‐5 tissues, and absent in normal tissues from reduction mammoplasty. Accordingly, EMT markers S100A4 and vimentin were elevated in both luminal and myoepithelial cells, and EMT markers α‐smooth muscle actin and SNAIL were elevated in luminal epithelial cells of TAHN‐1 tissues. These results identify cellular processes that are differentially activated between TAHN‐1 and TAHN‐5 breast tissues, implicate myofibroblasts as likely mediators of these processes, provide evidence that EMT is occurring in histologically normal tissues within the affected field and identify candidate biomarkers to investigate whether or how field cancerization contributes to the development of primary or recurrent breast tumors.

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.

Inhibitors of vacuolar ATPase proton pumps inhibit human prostate cancer cell invasion and prostate-specific antigen expression and secretion.

Vacuolar ATPases (V‐ATPases) comprise specialized and ubiquitously distributed pumps that acidify intracellular compartments and energize membranes. To gain new insights into the roles of V‐ATPases in prostate cancer (PCa), we studied the effects of inhibiting V‐ATPase pumps in androgen‐dependent (LNCaP) and androgen‐independent (C4‐2B) cells of a human PCa progression model. Treatment with nanomolar concentrations of the V‐ATPase inhibitors bafilomycin A or concanamycin A reduced the in vitro invasion in both cell types by 80%, regardless that V‐ATPase was prominent at the plasma membrane of C4‐2B cells and only traces were detected in the low‐metastatic LNCaP parental cells. In both cell types, intracellular V‐ATPase was excessive and co‐localized with prostate‐specific antigen (PSA) in the Golgi compartment. V‐ATPase inhibitors reversibly excluded PSA from the Golgi and led to the accumulation of largely dispersed PSA‐loaded vesicles of lysosomal composition. Inhibition of acridine orange staining and transferrin receptor recycling suggested defective endosomal and lysosomal acidification. The inhibitors, additionally, interfered with the AR‐PSA axis under conditions that reduced invasion. Bafilomycin A significantly reduced steady‐state and R1881‐induced PSA mRNA expression and secretion in the LNCaP cells which are androgen‐dependent, but not in the C4‐2B cells which are androgen ablation‐resistant. In the C4‐2B cells, an increased susceptibility to V‐ATPase inhibitors was detected after longer treatments, as proliferation was reduced and reversibility of bafilomycin‐induced responses impaired. These findings make V‐ATPases attractive targets against early and advanced PCa tumors.

Markers of Field Cancerization: Proposed Clinical Applications in Prostate Biopsies

Field cancerization denotes the occurrence of genetic, epigenetic, and biochemical aberrations in structurally intact cells in histologically normal tissues adjacent to cancerous lesions. This paper tabulates markers of prostate field cancerization known to date and discusses their potential clinical value in the analysis of prostate biopsies, including diagnosis, monitoring progression during active surveillance, and assessing efficacy of presurgical neoadjuvant and focal therapeutic interventions.

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.

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

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.

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.

Breast Field Cancerization: Isolation and Comparison of Telomerase-Expressing Cells in Tumor and Tumor Adjacent, Histologically Normal Breast Tissue

Telomerase stabilizes chromosomes by maintaining telomere length, immortalizes mammalian cells, and is expressed in more than 90% of human tumors. However, the expression of human telomerase reverse transcriptase (hTERT) is not restricted to tumor cells. We have previously shown that a subpopulation of human mammary epithelial cells (HMEC) in tumor-adjacent, histologically normal (TAHN) breast tissues expresses hTERT mRNA at levels comparable with levels in breast tumors. In the current study, we first validated a reporter for measuring levels of hTERT promoter activity in early-passage HMECs and then used this reporter to compare hTERT promoter activity in HMECs derived from tumor and paired TAHN tissues 1, 3, and 5 cm from the tumor (TAHN-1, TAHN-3, and TAHN-5, respectively). Cell sorting, quantitative real-time PCR, and microarray analyses showed that the 10% of HMECs with the highest hTERT promoter activity in both tumor and TAHN-1 tissues contain more than 95% of hTERT mRNA and overexpress many genes involved in cell cycle and mitosis. The percentage of HMECs within this subpopulation showing high hTERT promoter activity was significantly reduced or absent in TAHN-3 and TAHN-5 tissues. We conclude that the field of “normal tissue” proximal to the breast tumors contains a population of HMECs similar in hTERT expression levels and in gene expression to the HMECs within the tumor mass and that this population is significantly reduced in tissues more distal to the tumor. Mol Cancer Res; 9(9); 1209–21. ©2011 AACR.