Proton NMR visible mobile lipid signals in sensitive and multidrug-resistant K562 cells are modulated by rafts

Background  

Most cancer cells are characterized by mobile lipids visible on proton NMR (¹H-NMR), these being comprised mainly of methyl and methylene signals from lipid acyl chains. Erythroleukemia K562 cells show narrow signals at 1.3 and 0.9 ppm, corresponding to mobile lipids (methylene and methyl, respectively), which are reduced when K562 cells are multidrug resistant (MDR). While the significance of the mobile lipids is unknown, their subcellular localization is still a matter of debate and may lie in the membrane or the cytoplasm. In this study, we investigate the role of cholesterol in the generation of mobile lipid signals.     

Spontaneous mitochondrial membrane potential change during apoptotic induction by quercetin in K562 and K562/adr cells

Natural products from plants such as flavonoids are potential drugs to overcome multidrug resistance (MDR) in cancer treatments. However, their modes of action are still unclear. In this study, the effects of quercetin on mitochondrial membrane potential (DeltaPsim) change as well as quercetin's ability to induce apoptosis and inhibit Pgp-mediated efflux of 99mTc-MIBI in K562/adr cells were investigated. Quercetin exhibits cytotoxicity against erythroleukemic cells: IC50 are 11.0 +/- 2.0 micromol/L and 5.0 +/- 0.4 micromol/L for K562 and K562/adr, respectively. Quercetin induces cell death via apoptosis in both K562 and K562/adr cells and does not inhibit Pgp-mediated efflux of 99mTc-MIBI. Quercetin (10 micromol/L, 3 h) and etoposide (100 micromol/L, 24 h) induce similar levels of apoptosis in K562 and K562/adr cells. Quercetin induces an increase followed by a decrease in |DeltaPsim| value depending on its concentration. A decrease in the |DeltaPsim| value is associated with an increase in the percentage of early apoptotic cells. It is clearly shown that quercetin results in a spontaneous DeltaPsim change during apoptotic induction. Therefore, quercetin is potentially an apoptotic-inducing agent, which reacts at the mitochondrial level.     

High-resolution proton NMR measures mobile lipids associated with Triton-resistant membrane domains in haematopoietic K562 cells lacking or expressing caveolin-1

High-resolution proton NMR spectra of intact tumour cells generally exhibit intense signals due to isotropically mobile lipids (MLs) of still uncertain nature and origin. NMR studies performed on intact wild-type and caveolin-1-infected haematopoietic K562 cells showed that, under our experimental conditions, part of the ML signals are due to lipid complexes resistant to extraction in Triton X-100 at 4 degrees C. This evidence suggests that a portion of NMR-visible lipid structures are compatible with Triton-resistant membrane rafts and therefore biophysically distinct from NMR-visible Triton-soluble lipid bodies. Similarly to lipid rafts and caveolae, the organization of the Triton-insoluble ML domains could be compromised by treatment with beta-octylglucoside or methyl-beta-cyclodextrin. Exposure to exogenous sphingomyelinase caused an increase in ML NMR visibility, indicating the possible involvement of ceramides in ML formation. The mobility of these lipids was found to be temperature sensitive, suggesting a transition in cells going from 4 degrees C to 25-37 degrees C. These new results are here discussed in the light of possible contributions of plasma membrane microdomains to NMR-visible ML signals.     

The surface of lipid droplets is a phospholipid monolayer with a unique Fatty Acid composition

We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079-1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2beta and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain.     

Down-regulation of P-glycoprotein expression by sustained intracellular acidification in K562/Dox cells

We have investigated the involvement of intracellular pH (pH_i) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na⁺/H⁺ exchanger1 (NHE1) inhibitor cariporide and the “high K⁺” buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pH_i than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pH_i and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pH_i recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pH_i and P-gp in K562/DOX cells.     

Synthesis and evaluation of substituted dibenzo[c,e]azepine-5-ones as P-glycoprotein-mediated multidrug resistance reversal agents

A series of substituted dibenzo[c,e]azepine-5-ones (7a-h) were synthesized and evaluated as P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) reversal agents. The most potent compound 7h could significantly and selectively enhance the chemo-sensitivity of drug-resistant K562/A02 cells to the cytotoxic effect of adriamycin (ADR) in a dose-dependent manner. Further studies indicated that 7h could markedly increase intracellular accumulation of both rhodamine 123 and ADR in K562/A02 cells and inhibit their efflux from the cells. And 7h had little effect on the levels of P-gp mRNA and protein in K562/A02 cells. These results suggest that the anti-MDR effect of 7h might be attributed to the inhibition of drug efflux function of P-gp, leading to the increased drug accumulation in K562/A02 cells, and thus the compound could be served as a lead for developing P-gp-mediated MDR reversal agents.     

The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.     

Detergent-resistant membrane fractions contribute to the total 1H NMR-visible lipid signal in cells.

Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR-visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and monocytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was significant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs contained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospholipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR-visible lipid in DRMs is therefore unlikely to be a contaminant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intracellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains.     

Lysolipids do not inhibit influenza virus fusion by interaction with hemagglutinin

The interaction of a spin-labeled lysophosphatidylcholine analog with intact and bromelain-treated influenza viruses as well as with the bromelain-solubilized hemagglutinin ectodomain has been studied. The inhibition of fusion of influenza viruses with erythrocytes by the lysophosphatidylcholine analog was similar to that observed for non-labeled lysophosphatidylcholine. Only a weak interaction of the lysophosphatidylcholine analog with the hemagglutinin ectodomain was observed even upon triggering the conformational change of the ectodomain at a low pH. A significant interaction of spin-labeled lysophosphatidylcholine with the hemagglutinin ectodomain of intact viruses was observed neither at neutral nor at low pH, whereas a strong interaction of the lipid analog with the viral lipid bilayer was evident. We suggest that the high number of lipid binding sites of the virus bilayer and their affinity compete efficiently with binding sites of the hemagglutinin ectodomain. We conclude that the inhibition of influenza virus fusion by lysolipids is not mediated by binding to the hemagglutinin ectodomain, preventing its interaction with the target membrane. The results unambiguously argue for an inhibition mechanism based on the action of lysolipid inserted into the lipid bilayer.     

On the effect of lysophosphatidylcholine, platelet activating factor and other surfactants on calcium permeability in sarcoplasmic reticulum vesicles.

The effect of low concentrations of lysophosphatidylcholine (LPC), platelet-activating factor (PAF) and other surfactants (Triton X-100, C12E8, sodium dodecyl sulfate, sodium cholate and sodium deoxycholate) on membrane permeability of native sarcoplasmic reticulum vesicles and sarcoplasmic reticulum lipid vesicles, has been studied. Triton X-100, C12E8, sodium dodecyl sulfate, sodium cholate and sodium deoxycholate were all able to permeabilize membranes at concentrations of surfactants below their critical micellar concentration (CMC) in both lipid and native vesicles, being the K0.5 of calcium release from native vesicles lower than that from lipid vesicles. The values of these K0.5 were well correlated with the corresponding CMC values for each type of membrane. However, both LPC and PAF behaved in a different way since, although they induced permeabilization of the native vesicles at values of K0.5 close to their CMC, their K0.5 values for permeabilizing vesicles, prepared by using lipids extracted from sarcoplasmic reticulum, were much higher than their corresponding CMC.     

Reversal in multidrug resistance by magnetic nanoparticle of Fe3O4 loaded with adriamycin and tetrandrine in K562/A02 leukemic cells

Drug resistance is a primary hindrance for efficiency of chemotherapy. To investigate whether Fe3O4-magnetic nanoparticles (Fe3O4-MNPs) loaded with adriamycin (ADM) and tetrandrine (Tet) would play a synergetic reverse role in multidrug resistant cell, we prepared the drug-loaded nanoparticles by mechanical absorption polymerization to act with K562 and one of its resistant cell line K562/A02. The survival of cells which were cultured with these conjugates for 48 h was observed by MTT assay. Using cells under the same condition described before, we took use of fluorescence microscope to measure fluorescence intensity of intracellular ADM at an excitation wavelength of488 nm. P-glycoprotein (P-gp) was analyzed with flow cytometer. The expression ofmdrl mRNA was measured by RT-PCR. The results showed that the growth inhibition efficacy of both the two cells increased with augmenting concentrations of Fe3O4-MNPs which were loaded with drugs. No linear correlation was found between fluorescence intensity of intracellular adriamycin and augmenting concentration of Fe3O4-MNPs. Tet could downregulate the level of mdr-1 gene and decrease the expression of P-gp. Furthermore, Tet polymerized with Fe3O4-MNPs reinforced this downregulation, causing a 100-fold more decrease in mdrl mRNA level, but did not reduce total P-gp content. Our results suggest that Fe3O4-MNPs loaded with ADM or Tet can enhance the effective accumulation of the drugs in K562/A02. We propose that Fe3O4-MNPs loaded with ADM and Tet probably have synergetic effect on reversal in multidrug resistance.     

Analysis of membrane lipids by 500 MHz 1H NMR

A nondestructive method has been developed for rapid analysis of lipid content of membrane extracts based on high field proton NMR spectroscopy. Lipid extraction is done by stepwise sonication of purified membranes into chloroform:methanol:water mixtures, and 1H spectra are recorded at 35 degrees C on final preparations consisting of at least 1 mg dried lipid solubilized in 2:1 CD3OD:CDCl3. Spectral peaks of lipid mixtures are assigned to lipid classes using a data base of standard lipid characteristic resonances derived from purified single membrane lipids and known mixtures of them. Peak intensities of characteristic peaks yield ratios of various lipids such as cholesterol:phospholipid and phosphatidylcholine:phosphatidylethanolamine, degree of unsaturation, average acyl chain length, total glycerol lipid content, and presence or absence of particular lipids, such as glycolipids or lysolipids. This procedure of membrane lipid analysis has been verified using known mixtures of purified standard lipids.     

Assessment of Thiosemicarbazone-Containing Compounds as Potential Antileukemia Agents against P-gp Overexpressing Drug Resistant K562/A02 Cells

P-Glycoprotein (P-gp) overexpression is considered to be the leading cause of multidrug resistance (MDR) and failure of chemotherapy for leukemia. In this study, seventeen thiosemicarbazone-containing compounds were prepared and evaluated as potential antileukemia agents against drug resistant K562/A02 cell overexpressing P-gp. Among them, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could significantly inhibit K562/A02 cells proliferation with an IC₅₀ value of 0.96 μM. Interestingly, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could dose-dependently increase ROS levels of drug resistant K562/A02 cells, thus displaying a potential collateral sensitivity (CS)-inducing effect and selectively killing K562/A02 cells. Furthermore, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide possessed potent inhibitory effect on HDAC1 and HDAC6, and could promote K562/A02 cells apoptosis via dose-dependently increasing Bax expression, reducing Bcl-2 protein level, and inducing the cleavage of PARP and caspase3. These present findings suggest that N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide might be a promising lead to discover novel antileukemia agents against P-gp overexpressing leukemic cells.     

Autologous lymphocyte-monocyte co-culture increases NMR-visible and cytoplasmic lipids in the absence of increased markers of lymphocyte activation.

Alterations in nuclear magnetic resonance (NMR)-visible lipid, morphometric lipid volume fraction, distribution of subcellular lipid droplets and activation antigen expression were examined in human peripheral blood lymphocytes, activated using phorbol myristate acetate (PMA) and ionomycin or by co-culture with autologous monocytes. PMA/Ionomycin treatment caused significant time-dependent increases in mobile lipid and in oil red O-positive lipid droplets that were accompanied by lymphocyte proliferation and increases in activation antigens, CD25, CD69 and CD71. Co-culture of lymphocytes and monocytes also induced significant increases in NMR-visible lipid signals and cytoplasmic lipid droplets, but in contrast, no correspondent increases in activation antigens were observed. Strong correlations were observed between the intensity of the NMR signal and the percentage of total cells containing lipid droplets (r=0.95) and the morphometric lymphocyte lipid volume fraction (r=0.80), indicating that the droplets were the source of the mobile lipid signal. Lipid droplets in PMA/Ionomycin-treated cells were evenly distributed throughout the population, but in co-cultures, only lymphocytes in close proximity to monocytes with lipid droplets contained oil red O-positive lipid. This data shows that the NMR-visible mobile lipid signal observed in lymphocytes co-cultured with monocytes is not directly dependent on either proliferation or the upregulation of activation antigens, similar to the previously observed response of T cells exposed to antibodies to the T cell receptor.     

Lysophosphatidylcholine biosynthesis in developing barley

Acetate-2-[¹⁴C] and choline-Me-[ ¹⁴C], absorbed through the stems of isolated barley heads, were used to label lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) of the endosperm tissue. Labelling of LPC occurred in barley heads at almost all stages of development but was at a maximum when the fr. wt of the seeds had attained ca 60–70% of their maximum wt. In time-course experiments labelling of PC from each substrate reached a maximum after 50 hr and then declined. Label in LPC, however, continued to accumulate throughout 72 hr. Stimulation of labelling of LPC from choline-Me-[¹⁴C] by sucrose was observed. A bound form of LPC (starch lipid) and a free form were distinguished by differential solvent extraction.     

Lysophospholipids do not directly modulate Na+-H+ exchange

Lysophosphatidylcholine (LPC) has been reported to stimulate Na⁺-H⁺ exchange in rat cardiomyocytes. This action may be important in pathological conditions like ischemic injury where LPC is generated and Na⁺-H⁺ exchange activation is an important determinant of cardiac damage and dysfunction. It is unclear, however, if this stimulation of Na⁺-H⁺ exchange by LPC occurs through a direct action on the exchanger or through stimulation of a second messenger pathway. The purpose of the present investigation was to determine if lysolipids could directly affect Na⁺-H⁺ exchange. Purified cardiac sarcolemmal membranes were isolated and Na⁺-H⁺ exchange was measured by radioisotopic methods following addition of LPC. There were no effects of LPC on Na⁺-H⁺ exchange at LPC concentrations of 100 M at all reaction times examined. Lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI) and lysoplasmenylcholine (LP_EC) also did not alter Na⁺-H⁺ exchange at all concentrations and reaction times examined. We conclude that any stimulatory effects of lysolipids on Na⁺-H⁺ exchange do not occur through a direct action on the exchanger or its membrane lipid environment and must occur through a second messenger pathway.