Nancy K. Mizuno

Effects of adiponectin on breast cancer cell growth and signaling.

Obesity is a risk factor for postmenopausal breast cancer. Adiponectin/Acrp30 is lower in obese individuals and may be negatively regulating breast cancer growth. Here we determined that five breast cancer cell lines, MDA-MB-231, MDA-MB-361, MCF-7, T47D, and SK-BR-3, expressed one or both of the Acrp30 receptors. In addition, we found that the addition of Acrp30 to MCF-7, T47D, and SK-BR-3 cell lines inhibited growth. Oestrogen receptor (ER) positive MCF-7 and T47D cells were inhibited at lower Acrp30 concentrations than ER-negative SK-BR-3 cells. Growth inhibition may be related to apoptosis since PARP cleavage was increased by Acrp30 in the ER-positive cell lines. To investigate the role of ER in the response of breast cancer cells to Acrp30, we established the MDA-ERα7 cell line by insertion of ER-α into ER-α-negative MDA-MB-231 cells. This line readily formed tumours in athymic mice and was responsive to oestradiol in vivo. In vitro, MDA-ERα7 cells were growth inhibited by globular Acrp30 while the parental cells were not. This inhibition appeared to be due to blockage of JNK2 signalling. These results provide information on how obesity may influence breast cancer cell proliferation and establish a new model to examine interactions between ER and Acrp30.

Physical and photophysical characterization of a BODIPY phosphatidylcholine as a membrane probe.

Lipids containing the dimethyl BODIPY fluorophore are used in cell biology because their fluorescence properties change with fluorophore concentration (C.-S. Chen, O. C. Martin, and R. E. Pagano. 1997. Biophys J. 72:37-50). The miscibility and steady-state fluorescence behavior of one such lipid, 1-palmitoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (PBPC), have been characterized in mixtures with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). PBPC packs similarly to phosphatidylcholines having a cis-unsaturated acyl chain and mixes nearly ideally with SOPC, apparently without fluorophore-fluorophore aggregation. Increasing PBPC mole fraction from 0.0 to 1.0 in SOPC membranes changes the emission characteristics of the probe in a continuous manner. Analysis of these changes shows that emission from the excited dimethyl BODIPY monomer self quenches with a critical radius of 25.9 A. Fluorophores sufficiently close (< or =13.7 A) at the time of excitation can form an excited dimer, emission from which depends strongly on total lipid packing density. Overall, the data show that PBPC is a reasonable physical substitute for other phosphatidylcholines in fluid membranes. Knowledge of PBPC fluorescence in lipid monolayers has been exploited to determine the two-dimensional concentration of SOPC in unilamellar, bilayer membranes.

Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas.

Carboxylester lipase (cholesterol esterase, EC 3.1.1.13) has been purified to homogeneity from porcine pancreas. The enzyme is isolated in two molecular mass forms, a monomer of 74 kDa and a dimer of 167 kDa. The dimer consists of two catalytically-active subunits which have molecular masses approximately 9 kDa greater than the monomers. The difference in size was not attributable to carbohydrate or lipid content. The catalytic properties of the two forms are comparable on a weight basis, the amino acid compositions are quite similar, and the N-terminal sequences are nearly identical for 24 residues. These similarities suggest a possible precursor-product relationship between the two carboxylester lipase forms.

Eleostearic Acid Inhibits Breast Cancer Proliferation by Means of an Oxidation-Dependent Mechanism

Eleostearic acid (α-ESA) is a conjugated linolenic acid that makes up ∼60% of Momordica charantia (bitter melon) seed oil. Prior work found that water extract from bitter melon was able to inhibit breast cancer. Here, we investigated effects of α-ESA on both estrogen receptor (ER)–negative MDA-MB-231 (MDA-wt) and ER-positive MDA-ERα7 human breast cancer cells. We found that α-ESA inhibited proliferation of both MDA-wt and MDA-ERα7 cells, whereas conjugated linoleic acid had comparatively weak antiproliferative activity at 20 to 80 μmol/L concentrations. We also found that α-ESA (40 μmol/L) treatment led to apoptosis in the range of 70% to 90% for both cell lines, whereas conjugated linoleic acid (40 μmol/L) resulted in only 5% to 10% apoptosis, similar to results for control untreated cells. Addition of α-ESA also caused loss of mitochondrial membrane potential and translocation of apoptosis-inducing factor as well as endonuclease G from the mitochondria to the nucleus. Additionally, α-ESA caused a G2-M block in the cell cycle. We also investigated the potential for lipid peroxidation to play a role in the inhibitory action of α-ESA. We found that when the breast cancer cells were treated with α-ESA in the presence of the antioxidant α-tocotrienol (20 μmol/L), the growth inhibition and apoptosis effects of α-ESA were lost. An AMP-activated protein kinase inhibitor (Dorsomorphin) was also able to partially abrogate the effects of α-ESA, whereas a caspase inhibitor (BOC-D-FMK) did not. These results illustrate that α-ESA can block breast cancer cell proliferation and induce apoptosis through a mechanism that may be oxidation dependent.

Role of the adiponectin leptin ratio in prostate cancer.

We hypothesize that adiponectin and leptin may be capable of mediating some of the effects that body weight has on prostate cancer and that a mouse model may be effective to examine this hypothesis. We found that tumors from the TRAMP prostate cancer model expressed adiponectin and leptin receptors. TRAMP-C2 prostate cancer cell proliferation was reduced by adiponectin. Leptin was able to block the ability of adiponectin to reduce cell proliferation through altered signaling of the ERK pathway. Overall, this work suggests that adiponectin, leptin, and their receptors may play an important role in prostate cancer.

Combination of Intermittent Calorie Restriction and Eicosapentaenoic Acid for Inhibition of Mammary Tumors

There are a number of dietary interventions capable of inhibiting mammary tumorigenesis; however, the effectiveness of dietary combinations is largely unexplored. Here, we combined 2 interventions previously shown individually to inhibit mammary tumor development. The first was the use of the omega-3 fatty acid, eicosapentaenoic acid (EPA), and the second was the implementation of calorie restriction. MMTV-Her2/neu mice were used as a model for human breast cancers, which overexpress Her2/neu. Six groups of mice were enrolled. Half were fed a control (Con) diet with 10.1% fat calories from soy oil, whereas the other half consumed a diet with 72% fat calories from EPA. Within each diet, mice were further divided into ad libitum (AL), chronic calorie-restricted (CCR), or intermittent calorie-restricted (ICR) groups. Mammary tumor incidence was lowest in ICR-EPA (15%) and highest in AL-Con mice (87%), whereas AL-EPA, CCR-Con, CCR-EPA, and ICR-Con groups had mammary tumor incidence rates of 63%, 47%, 40%, and 59%, respectively. Survival was effected similarly by the interventions. Consumption of EPA dramatically reduced serum leptin (P < 0.02) and increased serum adiponectin in the AL-EPA mice compared with AL-Con mice (P < 0.001). Both CCR and ICR decreased serum leptin and insulin-like growth factor I (IGF-I) compared with AL mice but not compared with each other. These results illustrate that mammary tumor inhibition is significantly increased when ICR and EPA are combined as compared with either intervention alone. This response may be related to alterations in the balance of serum growth factors and adipokines. Cancer Prev Res; 6(6); 540–7. ©2013 AACR.

Lipid Lateral Organization in Fluid Interfaces Controls the Rate of Colipase Association

Colipase, a cofactor of pancreatic triacylglycerol lipase, binds to surfaces of lipolysis reactants, like fatty acid and diacylglycerol, but not to the nonsubstrate phosphatidylcholine. The initial rate of colipase binding to fluid, single-phase lipid monolayers was used to characterize the interfacial requirements for its adsorption. Colipase adsorption rates to phosphatidylcholine/reactant mixed monolayers depended strongly on lipid composition and packing. Paradoxically, reactants lowered colipase adsorption rates only if phosphatidylcholine was present. This suggests that interactions between phosphatidylcholine and reactants create dynamic complexes that impede colipase adsorption. Complex formation was independently verified by physical measurements. Colipase binding rate depends nonlinearly on the two-dimensional concentration of phosphatidylcholine. This suggests that binding is initiated by a cluster of nonexcluded surface sites smaller than the area occupied by a bound colipase. Binding rates are mathematically consistent with this mechanism. Moreover, for each phosphatidylcholine-reactant pair, the complex area obtained from the analysis of binding rates agrees well with the independently measured collapse area of the complex. The dynamic complexes between phosphatidylcholine and lipids, like diacylglycerols, exist independently of the presence of colipase. Thus, our results suggest that lipid complexes may regulate the fluxes of other proteins to membranes during, for example, lipid-mediated signaling events in cells.

Regulation of lipases by lipid-lipid interactions: implications for lipid-mediated signaling in cells.

Lipases are extracellular peripheral proteins that act at the surface of lipid emulsions stabilized, typically, by phospholipids. At a critical composition lipase activity toward substrates in phospholipid monolayers is discontinuously switched on by a small increase in substrate mole fraction. This occurs in part because lipase binding is inhibited by phospholipids. Binding of the lipase cofactor, colipase, is also inhibited by phospholipids. The initial rate of colipase binding increases abruptly at a substrate mole fraction that is approximately half the critical composition for lipase activity and just above that in substrate-phospholipid complexes. Moreover, complex collapse areas show an approximately 1:1 correlation with phospholipid excluded areas determined from an analysis of colipase adsorption rates. Thus, complexes inhibit colipase binding rate. Additionally, the switching of lipase activity likely occurs when uncomplexed substrate becomes the majority species in the interface. Lipase substrates, e.g. diacylglycerols, are typically the same lipids generated in the cytoplasmic surface of the plasma membrane of stimulated cells. As colipase binding is nonspecific and complexes involving lipase substrates form on the basis of lipid-lipid interactions alone, complexes should form in the plasma membrane of stimulated cells and may regulate protein translocation to the membrane.

Is intestinal villus phospholipase A2/lysophospholipase bound pancreatic carboxylester lipase?

Similarities in substrate specificity, localization and molecular weight between villus membrane phospholipase A2/lysophospholipase and carboxylester lipase of pancreatic origin suggested their possible identity. To test this, a preparation of the phospholipase A2/lysophospholipase released from brush border vesicles by papain was compared to authentic, pancreatic carboxylester lipase. Susceptibility of both activities to the inhibitor, diisopropylfluorophosphate, was consistent with their identity, but inconclusive. It also indicated that two populations of phospholipase A2 species may be present in the papain-released preparation. However, comparison of binding of the activities to Sepharose-coupled, anti-carboxylester-lipase IgG indicates that they are immunologically distinct.

Nanoscale packing differences in sphingomyelin and phosphatidylcholine revealed by bodipy fluorescence in monolayers: Physiological implications

Phosphatidycholines (PC) with two saturated acyl chains (e.g., dipalmitoyl) mimic natural sphingomyelin (SM) by promoting raft formation in model membranes. However, sphingoid-based lipids, such as SM, rather than saturated-chain PCs have been implicated as key components of lipid rafts in biomembranes. These observations raise questions about the physical packing properties of the phase states that can be formed by these two major plasma membrane lipids with identical phosphocholine headgroups. To investigate, we developed a monolayer platform capable of monitoring changes in surface fluorescence by acquiring multiple spectra during measurement of a lipid force–area isotherm. We relied on the concentration-dependent emission changes of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled PC to detect nanoscale alterations in lipid packing and phase state induced by monolayer lateral compression. The BODIPY-PC probe contained an indacene ring with four symmetrically located methyl (Me) substituents to enhance localization to the lipid hydrocarbon region. Surface fluorescence spectra indicated changes in miscibility even when force–area isotherms showed no deviation from ideal mixing behavior in the surface pressure versus cross-sectional molecular area response. We detected slightly better mixing of Me4-BODIPY-8-PC with the fluid-like, liquid expanded phase of 1-palmitoyl-2-oleoyl-PC compared to N-oleoyl-SM. Remarkably, in the gel-like, liquid condensed phase, Me4-BODIPY-8-PC mixed better with N-palmitoyl-SM than dipalmitoyl-PC, suggesting naturally abundant SMs with saturated acyl chains form gel-like lipid phase(s) with enhanced ability to accommodate deeply embedded components compared to dipalmitoyl-PC gel phase. The findings reveal a fundamental difference in the lateral packing properties of SM and PC that occurs even when their acyl chains match.