肺腺癌A549/DDP细胞周期变化及其多药耐药性

用Fura-2/AM标记药物敏感的肺腺癌细胞A₅₄₉和抗顺铂药物的肺腺癌细胞A₅₄₉/DDP两种细胞胞内游离Ca²⁺,用碘化丙锭(PI)标记细胞DNA,检测其胞内Ca²⁺的变化及两种细胞增殖能力和细胞周期.实验结果表明,抗药性细胞株A₅₄₉/DDP胞浆内游离Ca²⁺的浓度仅为药物敏感细胞株A₅₄₉的1/3左右,同时前者的细胞增殖能力较后者明显增强,而且细胞周期也明显缩短.当用BAPTA-AM和EGTA或A₂₃₁₈₇和Thapsigargin处理细胞以降低或升高其胞内自由Ca²⁺浓度时可改变细胞的生长周期,二者也呈现明显差别.这些结果表明,对顺铂产生耐药性的人肺腺癌A₅₄₉/DDP细胞胞内Ca²⁺浓度的降低,可能影响细胞的增殖,缩短细胞的生长周期,特别是影响起决定作用的G1期,从而有利于肿瘤细胞多药耐药特性的维持.     

肺腺癌A549/DDP细胞周期变化及其多药耐药性

用Fura 2 /AM标记药物敏感的肺腺癌细胞A54 9和抗顺铂药物的肺腺癌细胞A54 9/DDP两种细胞胞内游离Ca2 ,用碘化丙锭 (PI)标记细胞DNA ,检测其胞内Ca2 的变化及两种细胞增殖能力和细胞周期 .实验结果表明 ,抗药性细胞株A54 9/DDP胞浆内游离Ca2 的浓度仅为药物敏感细胞株A54 9的 1/ 3左右 ,同时前者的细胞增殖能力较后者明显增强 ,而且细胞周期也明显缩短 .当用BAPTA AM和EGTA或A2 3187和Thapsigargin处理细胞以降低或升高其胞内自由Ca2 浓度时可改变细胞的生长周期 ,二者也呈现明显差别 .这些结果表明 ,对顺铂产生耐药性的人肺腺癌A54 9/DDP细胞胞内Ca2 浓度的降低 ,可能影响细胞的增殖 ,缩短细胞的生长周期 ,特别是影响起决定作用的G1期 ,从而有利于肿瘤细胞多药耐药特性的维持     

Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

Alterations of membrane lipid biophysical properties of sensitive A₅₄₉ and resistant A₅₄₉/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Merocyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A₅₄₉ cell and 0.194 for the resistant A₅₄₉/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A₅₄₉/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that alterations of the membrane fluidity of both cells were mainly located on the surface and middle of the lipid bilayer. In addition, the packing density of phospholipid molecules in the membrane of the two cell lines detected by MC540 probe indicated that lipid packing of A₅₄₉ cell membranes was looser than that of the A₅₄₉/DDP cells. And unsaturation degree of plasma membrane fatty acids of the A₅₄₉/DDP cells was also lower than that of A₅₄₉ cells. Taken together, it was proposed that the alteration of membrane lipid biophysical state may be involved in the resistance of A₅₄₉/DDP cells to cisplatin.     

Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

Alterations of membrane lipid biophysical properties of sensitive A₅₄₉ and resistant A₅₄₉/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Merocyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A₅₄₉ cell and 0.194 for the resistant A₅₄₉/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A₅₄₉/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that alterations of the membrane fluidity of both cells were mainly located on the surface and middle of the lipid bilayer. In addition, the packing density of phospholipid molecules in the membrane of the two cell lines detected by MC540 probe indicated that lipid packing of A₅₄₉ cell membranes was looser than that of the A₅₄₉/DDP cells. And unsaturation degree of plasma membrane fatty acids of the A₅₄₉/DDP cells was also lower than that of A₅₄₉ cells. Taken together, it was proposed that the alteration of membrane lipid biophysical state may be involved in the resistance of A₅₄₉/DDP cells to cisplatin.     

1α,25-Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in rat osteosarcoma cells

1α,25-Dihydroxyvitamin D₃ increases intracellular calcium in rat osteoblast-like cells that possess the classic receptor (ROS 17/2.8) as well as those that lack the classic receptor (ROS 24/1), indicating that a separate signalling system mediates this rapid nongenomic action. To determine the intracellular sites of this calcium increase, cytosolic and nuclear fluorescence (340 nm/380 nm ratio) were measured in Fura 2AM loaded ROS 17/2.8 cells using digital microscopy. Within 5 min, cytosolic fluorescence increased by 29% (P < 0.05) and nuclear fluorescence by 30% (P < 0.01) after exposure to 1α,25-dihydroxyvitamin D₃ (20 nM). This effect was blocked by the inactive epimer 1β,25-dihydroxyvitamin D₃. In an individual cell, cytosolic and nuclear fluorescence increased gradually after 1, 3, and 5 min exposure to vitamin D. Nuclei were then isolated from ROS 17/2.8 cells to directly measure the hormone's effect on nuclear calcium. The calcium content of Fura 2AM loaded nuclei was not affected by increasing the calcium concentration in the incubation buffer from 50 nM to 200 nM. After 5 min, 1α,25-dihydroxyvitamin D₃, 20 nM, increased the calcium of isolated nuclei in medium containing 50 nM calcium and 200 nM calcium. 1β,25-dihydroxyvitamin D₃, 20 nM, had no effect on nuclear calcium but blocked the 1α,25-dihydroxyvitamin D₃ induced rise in the isolated nuclei. The results indicate that the nuclear membrane of the ROS 17/2.8 cells contain calcium permeability barriers and transport systems that are sensitive to and specific for 1α,25-dihydroxyvitamin D₃. 1α,25-Dihydroxyvitamin D₃ rapidly increases nuclear calcium levels in both intact cells and isolated nuclei suggesting that rapid nongenomic activation of nuclear calcium may play a functional role in osteoblastic activity.     

Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A₂ activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A₂ activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A₂ activities and arachidonic acid release in cells pretreated with nominal Ca²⁺ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A₂ activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A₂ activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A₂ activity (ii) in addition to the presence of extracellular Ca²⁺, release of Ca²⁺ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A₂ activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A₂ activities does not appear to require new RNA or protein synthesis.Abbreviations A23187 calcium ionophore - AA arachidonic acid - PMSF phenylmethyl sulfonylfuoride - DFP diisopropyl-fluorophosphate - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - PBS phosphate buffered saline - HBPS Hank's buffered physiological saline - PLA₂ phospholipase A₂     

NMR studies of the relationship between the changes of membrane lipids and the cisplatin-resistance of A549/DDP cells

Changes of membrane lipids in cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells during the apoptotic process induced by a clinical dose of cisplatin (30 μM) were detected by ¹H and ³¹P-NMR spectroscopy and by membrane fluidity measurement. The apoptotic phenotypes of the two cell lines were monitored with flow cytometry. The assays of apoptosis showed that significant apoptotic characteristics of the A549 cells were induced when the cells were cultured for 24 hours after treatment with cisplatin, while no apoptotic characteristic could be detected for the resistant A549/DDP cells even after 48 hours. The results of ¹H-NMR spectroscopy demonstrated that the CH₂/CH₃ and Glu/Ct ratios of the membrane of A549 cells increased significantly, but those in A549/DDP cell membranes decreased. In addition, the Chol/CH₃ and Eth/Ct ratios decreased for the former but increased for the latter cells under the same conditions. ³¹P-NMR spectroscopy indicated levels of phosphomonoesters (PME) and ATP decreased in A549 but increased in A549/DDP cells after being treated with cisplatin. These results were supported with the data obtained from ¹H-NMR measurements. The results clearly indicated that components and properties of membrane phospholipids of the two cell lines were significantly different during the apoptotic process when they were treated with a clinical dose of cisplatin. Plasma membrane fluidity changes during cisplatin treatment as detected with the fluorescence probe TMA-DPH also indicate marked difference between the two cell lines. We provided evidence that there are significant differences in plasma membrane changes during treatment of cisplatin sensitive A549 and resistant A549/DDP cells.     

Determination of intracellular Ca2+ in cells of intact wheat roots: loading of acetoxymethyl ester of Fluo‐3 under low temperature

Loading of Ca²⁺-sensitive fluorescent probes into plant cells is an essential step to measuring activities of cytoplasmic free Ca²⁺ ions with a fluorescent imaging technique. A major barrier to the loading of the fluorescent probes into plant cells using the acetoxymethyl (AM) esters of the Ca²⁺-sensitive dyes is the presence of cell-wall associated esterases. These esterases hydrolyse the esterified form of the fluorescent probes, rendering the probes membrane-impermeable. A novel non-invasive loading protocol was described in this paper to load the Ca²⁺-sensitive fluorescent probe Fluo-3/AM ester into apical cells of intact wheat roots by incubating the roots in Fluo-3/AM ester solution at 4°C for 2 h followed by 2-h incubation in the dye-free solution at 20°C. The incubation at low temperature inhibited extracellular hydrolysis of Fluo-3/AM ester but had less effect on diffusion of membrane-permeable Fluo-3/AM ester across the plasma membrane, because hydrolysis of Fluo-3/AM ester by extracellular esterases is a chemical process (high Q₁₀), while diffusion of Fluo-3/AM across the plasma membrane is a physical process (low Q₁₀). The Fluo-3/AM ester, accumulated in the root cells during the low temperature incubation, was then cleaved by intracellular esterases during the incubation at 20°C, releasing the membrane-impermeable Ca²⁺-sensitive Fluo-3 in the cytoplasm. The root cells loaded with Fluo-3 showed strong intracellular fluorescence under confocal microscopy. The fluorescence from the root cells was sensitive to the Ca²⁺ ionophore and hydrogen peroxide, indicating that the intracellular fluorescence was due to intracellular Ca²⁺ ions.     

Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines

Background

Insulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293–297).

Methodology/Principal Findings

We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway.

Conclusions

Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and complexity of the IGF-independent actions of these IGF binding proteins.     

PGE2 attenuates PGF2α-induced increases in free intracellular calcium in ovine large luteal cells

When ovine large luteal cells are placed in culture and exposed to PGF_2α, there is a rapid and sustained increase in the concentration of free intracellular calcium which is believed to play a major role in the luteolytic and cytotoxic effects of PGF_2α. Since administration of exogenous PGE₂ can prevent spontaneous and PGF_2α-induced luteolysis in vivo, and the cytotoxic effects of PGF_2α on large luteal cells in vitro, the objective of this study was to determine if one mechanism by which PGE₂ acts is to attenuate increases in free intracellular calcium induced by PGF_2α. At concentrations of 10 nM or greater, PGF_2α caused a significant and sustained increase in free intracellular calcium in large luteal cells. Similarly, PGE₂ also induced increases in free intracellular calcium but required doses 20-fold greater than PGF_2α. When PGE₂ (1, 10 or 100 nM) was incubated with PGF_2α (100 nM) increases in free intracellular calcium induced by PGF_2α were attenuated (P<0.05) when measured 5 min, but not at 30 min, after initiation of treatment. The observed decrease in the concentration of free intracellular calcium at 5 min in response to PGF_2α was the result of fewer cells responding to PGF_2α. In addition, the concentrations of free intracellular calcium attained in the cells that did respond was reduced 25% compared to cells treated with PGF_2α alone. Thus, part of the luteal protective actions of PGE₂ appears to involve an inhibition of the early (5 min) increase in free intracellular calcium induced by PGF_2α.     

Physiological and Biochemical Analysis of the Transformants of Aerobic Methylobacteria Expressing the dcmA Gene of Dichloromethane Dehalogenase

Transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr^–/pME 8220 and DM4-2cr^–/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl^– ions, CH₂O, or HCOOH, nor to the impairment of glutathione synthesis or to the disturbance of intracellular pH homeostasis. The (exo^–) Klenow fragment–mediated incorporation of [-³²P]dATP into the DNA of the transformants DM4-2cr^–/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH₂Cl₂ than when they were incubated with CH₃OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH₂Cl₂ showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH₂Cl₂ degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.     

Polarization of Migrating Monocytic Cells Is Independent of PI 3-Kinase Activity

Background

Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization.

Methodology/Principal Findings

We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the α_2A-adrenoceptor (α_2AAR) display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homlogue UK 14''304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microcopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET) of α_2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinse activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes – in contrast to neutrophils – rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation.

Conclusions/Significance

Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process appears to be independent of PI 3-kinase activity.     

Inositol 1,4,5-trisphosphate 3-kinase activity in frog skeletal muscle

Frog skeletal muscle contains a kinase activity that phosphorylates inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. The inositol 1,4,5-trisphosphate 3-kinase activity was mainly recovered in the soluble fraction, where it presented a marked dependency on free calcium concentration in the physiological range in the presence of endogenous calmodulin. At pCa 5, where the activity was highest, the soluble 3-kinase activity displayed a K_m for inositol 1,4,5-trisphosphate of 1.6 μM and a V_max value of 25.1 pmol mg⁻¹ min⁻¹. The removal rates of inositol 1,4,5-trisphosphate by 3-kinase and 5-phosphatase activities of the total homogenate under physiological ionic conditions were very similar, suggesting that both routes are equally important in metabolizing inositol 1,4,5-trisphosphate in frog skeletal muscle.     

Lymphocyte calcium extrusion: kinetic and thermodynamic measurements using ratiometric dual-emission spectrofluorometry

A method is described for monitoring intracellular ionized calcium (Ca²⁺) and determining kinetic and thermodynamic parameters of Ca⁺-extrusion from intact lymphocytes. The method uses ratiometric spectrofluorometry and the fluorescent Ca²⁺ dye indo-1. Lymphocytes were loaded with calcium and placed in a low calcium medium. A novel formula for calculation of intracellular Ca^2– that corrects for background fluorescence and fluorescence quenching was used. Calcium extrusion resulted in exponential decrease in cytoplasmic Ca²⁺ with a rate constant of 0.031 ± 0.003 sec^–1, maximal rate of 23 ± 7 nM/sec, dissociation constant of 366 ± 63 nM, Hill coefficient of 2.3 ± 0.4, Q₁₀ of 2.58 ± 0.28, and activation energy of 18.3 Kcal/mol. This method should allow for characterization of the Ca²⁺-extrusion system of lymphocytes and may be applicable to other blood cell types.Abbreviations DMSO dimethyl sulfoxide - HEPES [N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid],sodium salt - Indo-1/AM acetoxymethyl ester of indo-1 - IP₃ inositol 1,4,5-triphosphate - IP₄ inositol 1,3,4,5-tetrakisphosphate - RPMI Roswell Park Memorial Institute — 1640 culture medium - TPEN tetrakis-[2-pyridylmethyl]-ethylenediamine     

Physical state changes of membrane lipids in human lung adenocarcinoma A(549) cells and their resistance to cisplatin

The properties of membrane lipids in sensitive A(549) and resistant A(549)/DDP cells to cis-dichlorodiammine platinum[II] (cisplatin) were examined by combining different approaches. The results showed that fluorescence intensity (deltaF) of Merocyanine 540 (MC540) was 93.5 +/- 21.8 for the sensitive A(549) cells and 49.5 +/- 11.2 for the resistive A(549)/DDP cells, monitored by flow cytometry, which may indicate that membrane lipid packing of the sensitive A(549) cells were looser than that of the resistant A(549)/DDP cells. Diffusion rate of N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-1,2-hexadecanoyl-Sn-glycero-3-phosphatidyl-ethanolamine (NBD-PE) was slower in A(549)/DDP cells than in A(549) cells as detected by fluorescence recovery after photobleaching (FRAP) technique. Fatty acid analysis of the membrane lipids showed 21.6, 27.0 and 31.8% increase in the amount of C(18:1), C(18:2) and C(18:3) fatty acid, respectively, in A(549) cells as compared to A(549)/DDP cells. The total amount of unsaturated fatty acids in the plasma membrane lipid is 69.13% +/- 2.2% for A(549), and 55.08% +/- 1.8% for A(549)/DDP cells, respectively. The resistance to cisplatin in A(549)/DDP cells was confirmed by the measurements of the transmembrane influx of Rhodamine-123, cisplatin or Bodipy-cisplatin by fluorescence assay and inductively coupled plasma mass spectrometry (ICP-MS). From the results described previously, it is concluded that changes in the membrane lipids "composition" cause a change in the physical state of the plasma membrane lipids and that this may be associated with the resistance of A(549)/DDP cells to cisplatin.     

Silence of lncRNA UCA1 rescues drug resistance of cisplatin to non–small-cell lung cancer cells

The aim of this study was to investigate the effect of long noncoding RNA (lncRNA) urogenital carcinoma antigen 1 (UCA1) on drug resistance in A549/DDP cell and explore its underlying mechanism. The inhibition rate and IC ₅₀ of DDP were detected in A549 and A549/DDP cells by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay. The expression of lncRNA UCA1 was measured in A549 and A549/DDP cells by quantitative real-time polymerase chain reaction. The expressions of N-cadherin, E-cadherin, vimentin, and Snail were detected in A549 and A549/DDP cells by Western blot analysis. Results showed that the IC ₅₀ of DDP was 16.20 ± 2.27 μmol/L and 69.72 ± 4.83 μmol/L in A549 and A549/ DDP cells, respectively. Compared with the A549 group, the expressions of N-cadherin, vimentin, and Snail was significantly upregulated in A549/DDP group, but E-cadherin was significantly downregulated. Compared with the shCon group, the abundance of N-cadherin, vimentin, and Snail was significantly downregulated in short hairpin RNA UCA1 (shUCA1) group, while E-cadherin was significantly upregulated. Cell migration and invasion were significantly suppressed and IC ₅₀ was reversed to 16.20 ± 2.27 μmol/L in the shUCA1 group. Silencing lncRNA UCA1 inhibited the migration and invasion of A549/DDP cells and reversed the resistance of A549/DDP cells to DDP. The mechanism might be related to downregulation of epithelial-mesenchymal transition, which will provide a new direction for the treatment of non–small-cell lung cancer with cisplatin.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Activation of the cytosolic calcium-independent phospholipase A2 β isoform contributes to TRPC6 externalization via release of arachidonic acid

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A₂ (PLA₂), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA₂ in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA₂ enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA₂ involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA₂α, cPLA₂γ, iPLA₂β, or iPLA₂γ. Downregulation of the β isoform of iPLA₂ blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.     

Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A_(549) cells to cisplatin

Alterations of membrane lipid biophysical properties of sensitive A549 and resistant A549/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Mero-cyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A549 cell and 0.194 for the resistant A549/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A549/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that alterations of the membrane fluidity of both     

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A₂ (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219–225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced a significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA₂ and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA₂ activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMLP was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMLP-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA₂ rather than secretory PLA₂. Inhibition of PLA₂ with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA₂-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.