Individual-specific variation in the respiratory activities of HMECs and their bioenergetic response to IGF1 and TNFα.

Metabolic reprograming is a hallmark of cancer cells. However, the roles of pre-existing differences in normal cells metabolism toward cancer risk is not known. In order to assess pre-existing variations in normal cell metabolism, we have quantified the inter-individual variation in oxidative metabolism of normal primary human mammary epithelial cells (HMECs). We then assessed their response to selected cytokines such as insulin growth factor 1 (IGF1) and tumor necrosis factor alpha (TNFα), which are associated with breast cancer risk. Specifically, we compared the oxidative metabolism of HMECs obtained from women with breast cancer and without cancer. Our data show considerable inter-individual variation in respiratory activities of HMECs from different women. A bioenergetic parameter called pyruvate-stimulated respiration (PySR) was identified as a key distinguishing feature of HMECs from women with breast cancer and without cancer. Samples showing PySR over 20% of basal respiration rate were considered PySR+ve and the rest as PySR-ve . By this criterion, HMECs from tumor-affected breasts (AB) and non-tumor affected breasts (NAB) of cancer patients were mostly PySR-ve (88% and 89%, respectively), while HMECs from non-cancer patients were mostly PySR+ve (57%). This suggests that PySR-ve/+ve phenotypes are individual-specific and are not caused by field effects due to the presence of tumor. The effects of IGF1 and TNFα treatments on HMECs revealed that both suppressed respiration and extracellular acidification. In addition, IGF1 altered PySR-ve/+ve phenotypes. These results reveal individual-specific differences in pyruvate metabolism of normal breast epithelial cells and its association with breast cancer risk.

The effects of diet induced obesity on breast cancer associated pathways in mice deficient in SFRP1.

BACKGROUND: Secreted frizzled-related proteins (SFRPs) are a family of proteins that block the Wnt signaling pathway and loss of Sfrp1 expression is observed in breast cancer. The molecular mechanisms by which obesity contributes to breast tumorigenesis are not well defined, but involve increased inflammation. Mice deficient in Sfrp1 show enhanced mammary gland inflammation in response to diet induced obesity (DIO). Furthermore, mammary glands from Sfrp1-/- mice exhibit increased Wnt signaling, decreased cell death responses, and excessive hyper branching. The work described here was initiated to investigate whether obesity exacerbates the aforementioned pathways, as they each play a key roles in the development of breast cancer. FINDINGS: Wnt signaling is significantly affected by DIO and Sfrp1-/- loss as revealed by analysis of Myc mRNA expression and active ß-catenin protein expression. Furthermore, Sfrp1-/- mice fed a high fat diet (HFD) exhibit an increase in mammary cell proliferation. The death response is also impaired in the mammary gland of Sfrp1-/- mice fed a normal diet (ND) as well as a HFD. In response to γ-irradiation, mammary glands from Sfrp1-/- mice express significantly less Bax and Bbc3 mRNA, caspase-3 positive cells, and p53 protein. The expression of Wnt4 and Tnfs11 are critical for normal progesterone mediated mammary gland development and in response to obesity, Sfrp1-/- mice express significantly more Wnt4 and Tnfs11 mRNA expression. Evaluation of progesterone receptor (PR) expression showed that DIO increases the number of PR positive cells. CONCLUSIONS: Our data indicate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular homeostasis in response to the onset of obesity.

Efficacy of polyMPC-DOX prodrugs in 4T1 tumor-bearing mice.

We report the in vivo efficacy, in tumor-bearing mice, of cancer prodrugs consisting of poly(methacryloyloxyethyl phosphorylcholine) (polyMPC) conjugated to doxorubicin (DOX). Our synthesis of polyMPC-DOX conjugates established prodrugs with tunable drug loading, pH sensitive release kinetics, and a maximum tolerated dose in the range of 30-50 mg/kg (DOX equivalent) in healthy mice. Here we show prolonged circulation of polyMPC-DOX, with a measured in vivo half-life (t1/2) 8 times greater than that of the free drug. We observed reduced drug uptake in healthy tissue, and 2-3 times enhanced drug accumulation in tumors for polyMPC-DOX prodrugs compared to free DOX, using BALB/c mice bearing 4T1 tumors. Prolonged survival and reduced tumor growth were observed in mice receiving the polyMPC-DOX prodrug treatment. Moreover, we evaluated immunogenicity of polyMPC-DOX prodrugs by examining complete blood count (CBC) and characteristic cytokine responses, demonstrating no apparent innate or adaptive immune system response.

PolyMPC-doxorubicin prodrugs.

We demonstrate the conjugation of the cancer drug doxorubicin (DOX) to poly(methacryloyloxyethyl phosphorylcholine) (polyMPC), linked by hydrazone groups, using (1) a one-pot ATRP/click sequence, and (2) a post-polymerization conjugation strategy. While the one-pot method gave polyMPC-DOX conjugates in a facile single step, post-polymerization conjugation gave higher-molecular-weight polymers with very high DOX loadings. DOX release from the polyMPC backbone was pH-dependent (faster at pH 5.0 than at pH 7.4) owing to the hydrazone linkage. Half-life values of DOX release ranged from 2 to 40 h at pH 5.0. Cell culture experiments showed that highly loaded polyMPC-DOX conjugates exhibited higher intracellular drug accumulation and lower half-maximal inhibitory concentration (IC(50)) values, while a polymer with 30 wt % drug loading showed a maximum tolerated dose in the range of 30-50 mg/kg DOX equivalent weight in healthy mice.

Pentafluorophenyl ester-functionalized phosphorylcholine polymers: preparation of linear, two-arm, and grafted polymer-protein conjugates.

Novel pentafluorophenyl (PFP)-ester-functionalized phosphorylcholine (PC) polymers of different architectures were prepared and conjugated to lysozyme as a model protein. Linear and two-arm poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC) structures containing PFP functionality at the chain-end were prepared by atom transfer radical polymerization (ATRP) from novel initiators. Additional conjugates were prepared from phosphorylcholine-substituted cyclooctene (PC-COE) polymers containing PFP-ester bearing comonomers. The polymer-protein conjugates were characterized by HPLC, FPLC, and DLS and were seen to retain most (~80% or greater) of their native enzymatic activity. Pharmacokinetic profiles of the polymer-protein conjugates were studied in mice and found to increase the circulation half-life compared with lysozyme alone.

Neuronal modulation of brown adipose activity through perturbation of white adipocyte lipogenesis.

OBJECTIVE: Crosstalk between adipocytes and local neurons may be an important regulatory mechanism to control energy homeostasis. We previously reported that perturbation of adipocyte de novo lipogenesis (DNL) by deletion of fatty acid synthase (FASN) expands sympathetic neurons within white adipose tissue (WAT) and stimulates the appearance of "beige" adipocytes. Here we tested whether WAT DNL activity can also influence neuronal regulation and thermogenesis in brown adipose tissue (BAT). METHODS AND RESULTS: Induced deletion of FASN in all adipocytes in mature mice (iAdFASNKO) enhanced sympathetic innervation and neuronal activity as well as UCP1 expression in both WAT and BAT. This increased sympathetic innervation could be observed at both 22 °C and 30 °C, indicating it is not a response to heat loss but rather adipocyte signaling. In contrast, selective ablation of FASN in brown adipocytes of mice (iUCP1FASNKO) failed to modulate sympathetic innervation and the thermogenic program in BAT. Surprisingly, DNL in brown adipocytes was also dispensable in maintaining euthermia when UCP1FASNKO mice were cold-exposed. CONCLUSION: These results indicate that DNL in white adipocytes influences long distance signaling to BAT, which can modify BAT sympathetic innervation and expression of genes involved in thermogenesis.

Adipocyte lipid synthesis coupled to neuronal control of thermogenic programming.

BACKGROUND: The de novo biosynthesis of fatty acids (DNL) through fatty acid synthase (FASN) in adipocytes is exquisitely regulated by nutrients, hormones, fasting, and obesity in mice and humans. However, the functions of DNL in adipocyte biology and in the regulation of systemic glucose homeostasis are not fully understood. METHODS & RESULTS: Here we show adipocyte DNL controls crosstalk to localized sympathetic neurons that mediate expansion of beige/brite adipocytes within inguinal white adipose tissue (iWAT). Induced deletion of FASN in white and brown adipocytes of mature mice (iAdFASNKO mice) enhanced glucose tolerance, UCP1 expression, and cAMP signaling in iWAT. Consistent with induction of adipose sympathetic nerve activity, iAdFASNKO mice displayed markedly increased neuronal tyrosine hydroxylase (TH) and neuropeptide Y (NPY) content in iWAT. In contrast, brown adipose tissue (BAT) of iAdFASNKO mice showed no increase in TH or NPY, nor did FASN deletion selectively in brown adipocytes (UCP1-FASNKO mice) cause these effects in iWAT. CONCLUSIONS: These results demonstrate that downregulation of fatty acid synthesis via FASN depletion in white adipocytes of mature mice can stimulate neuronal signaling to control thermogenic programming in iWAT.

Rhodiola crenulata induces an early estrogenic response and reduces proliferation and tumorsphere formation over time in MCF7 breast cancer cells.

BACKGROUND: Rhodiola crenulata is a Tibetan mountainous plant, commonly used in Eastern alternative medicine. Many phytochemicals possess estrogenic activity, a critical regulator of proliferation in mammary epithelial cells. We have previously characterized anti-cancer properties of R. crenulata in aggressive triple negative breast cancer cells, lacking the expression of estrogen receptor. Currently, it is unknown whether R. crenulata exerts estrogenic effects and as such consumption may be a concern for women with estrogen receptor positive breast cancer that use Rhodiola sp. to relieve mild to moderate depression. PURPOSE: In this study, we wished to determine whether a hydroalcoholic fraction of the R. crenulata root extract exhibits estrogenic activity in estrogen receptor positive (ER+) breast cancer cells in vitro and whether it affects normal mammary epithelial ER target gene expression in vivo. METHODS: ER transcriptional activity was analyzed in MCF7 cells expressing an ERE reporter construct and confirmed via qPCR of endogenous ER target genes. We also monitored cellular proliferation over time. Additionally, to assess stem-like properties in MCF7 cells, we performed a tumorsphere formation assay under anchorage independent conditions. We examined whether R. crenulata treatment reduced ß-catenin levels via Western blotting and measured ß-catenin transcriptional activity by a reporter assay. To examine the effects of R. crenulata on normal mammary epithelial cells, we performed immunohistochemical staining of ER and PR in the mammary glands of mice fed R. crenulata for 12 weeks. RESULTS: We show an initial activation of ER transcriptional activity by dual reporter assay, qPCR and proliferation of MCF7 ER+ cells in response to 24 h of R. crenulata treatment. However, upon longer treatment basal and R. crenulata induced transcriptional activity was suppressed. There was a decrease in cell doubling times and a decrease in tumorsphere formation. In association with these changes, ERα transcript levels were decreased and active ß-catenin levels were reduced in the cells treated for 2 weeks. Finally, we show no change in estrogen targets in normal mammary cells in vivo. CONCLUSION: These data suggest that the R. crenulata extract contains components with estrogenic activity. However, R. crenulata treatment could still be protective in ER+ breast cancer cells, as longer treatment reduced the transcriptional activity of ß-catenin and ER responses leading to reduced proliferation and tumorsphere formation. Furthermore, administration of 20 mg/kg/day R. crenulata to mice did not have an observable effect on mammary epithelial ERα target gene expression in vivo.

Comparative Analysis of the Mitochondrial Physiology of Pancreatic ß Cells.

The mitochondrial metabolism of ß cells is thought to be highly specialized. Its direct comparison with other cells using isolated mitochondria is limited by the availability of islets/ß cells in sufficient quantity. In this study, we have compared mitochondrial metabolism of INS1E/ß cells with other cells in intact and permeabilized states. To selectively permeabilize the plasma membrane, we have evaluated the use of perfringolysin-O (PFO) in conjunction with microplate-based respirometry. PFO is a protein that binds membranes based on a threshold level of active cholesterol. Therefore, unless active cholesterol reaches a threshold level in mitochondria, they are expected to remain untouched by PFO. Cytochrome c sensitivity tests showed that in PFO-permeabilized cells, the mitochondrial integrity was completely preserved. Our data show that a time-dependent decline of the oligomycin-insensitive respiration observed in INS1E cells was due to a limitation in substrate supply to the respiratory chain. We predict that it is linked with the ß cell-specific metabolism involving metabolites shuttling between the cytoplasm and mitochondria. In permeabilized ß cells, the Complex l-dependent respiration was either transient or absent because of the inefficient TCA cycle. The TCA cycle insufficiency was confirmed by analysis of the CO2 evolution. This may be linked with lower levels of NAD+, which is required as a co-factor for CO2 producing reactions of the TCA cycle. ß cells showed comparable OxPhos and respiratory capacities that were not affected by the inorganic phosphate (Pi) levels in the respiration medium. They showed lower ADP-stimulation of the respiration on different substrates. We believe that this study will significantly enhance our understanding of the ß cell mitochondrial metabolism.

Mice deficient in Sfrp1 exhibit increased adiposity, dysregulated glucose metabolism, and enhanced macrophage infiltration.

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Wnt signaling plays an important role in preadipocyte differentiation and adipogenesis. The expression of a Wnt antagonist, secreted frizzled related protein 1 (SFRP1), is increased in response to initial weight gain, then levels are reduced under conditions of extreme obesity in both humans and animals. Here we report that loss of Sfrp1 exacerbates weight gain, glucose homeostasis and inflammation in mice in response to diet induced obesity (DIO). Sfrp1(-/-) mice fed a high fat diet (HFD) exhibited an increase in body mass accompanied by increases in body fat percentage, visceral white adipose tissue (WAT) mass, and adipocyte size. Moreover, Sfrp1 deficiency increases the mRNA levels of key de novo lipid synthesis genes (Fasn, Acaca, Acly, Elovl, Scd1) and the transcription factors that regulate their expression (Lxr-α, Srebp1, Chreb, and Nr1h3) in WAT. Fasting glucose levels are elevated, glucose clearance is impaired, hepatic gluconeogenesis regulators are aberrantly upregulated (G6pc and Pck1), and glucose transporters are repressed (Slc2a2 and Slc2a4) in Sfrp1(-/-) mice fed a HFD. Additionally, we observed increased steatosis in the livers of Sfrp1(-/-) mice. When there is an expansion of adipose tissue there is a sustained inflammatory response accompanied by adipokine dysregulation, which leads to chronic subclinical inflammation. Thus, we assessed the inflammatory state of different tissues and revealed that Sfrp1(-/-) mice fed a HFD exhibited increased macrophage infiltration and expression of pro-inflammatory markers including IL-6, Nmnat, Tgf-ß2, and SerpinE1. Our findings demonstrate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular signaling in response to the onset of obesity.

Effects of shampoo and water washing on hair cortisol concentrations.

BACKGROUND: Measurement of cortisol in hair is an emerging biomarker for chronic stress in human and nonhuman primates. Currently unknown, however, is the extent of potential cortisol loss from hair that has been repeatedly exposed to shampoo and/or water. METHODS: Pooled hair samples from 20 rhesus monkeys were subjected to five treatment conditions: 10, 20, or 30 shampoo washes, 20 water-only washes, or a no-wash control. For each wash, hair was exposed to a dilute shampoo solution or tap water for 45 s, rinsed 4 times with tap water, and rapidly dried. Samples were then processed for cortisol extraction and analysis using previously published methods. RESULTS: Hair cortisol levels were significantly reduced by washing, with an inverse relationship between number of shampoo washes and the cortisol concentration. This effect was mainly due to water exposure, as cortisol levels following 20 water-only washes were similar to those following 20 shampoo treatments. CONCLUSIONS: Repeated exposure to water with or without shampoo appears to leach cortisol from hair, yielding values that underestimate the amount of chronic hormone deposition within the shaft. Collecting samples proximal to the scalp and obtaining hair washing frequency data may be valuable when conducting human hair cortisol studies.