Molecular Mechanism of the Synergistic Effects of Vitrification Solutions on the Stability of Phospholipid Bilayers

The vitrification solutions used in the cryopreservation of biological samples aim to minimize the deleterious formation of ice by dehydrating cells and promoting the formation of the glassy state of water. They contain a mixture of different cryoprotective agents (CPAs) in water, typically polyhydroxylated alcohols and/or dimethyl sulfoxide (DMSO), which can damage cell membranes. Molecular dynamics simulations have been used to investigate the behavior of pure DPPC, pure DOPC, and mixed DOPC-β-sitosterol bilayers solvated in a vitrification solution containing glycerol, ethylene glycol, and DMSO at concentrations that approximate the widely used plant vitrification solution 2. As in the case of solutions containing a single CPA, the vitrification solution causes the bilayer to thin and become disordered, and pores form in the case of some bilayers. Importantly, the degree of thinning is, however, substantially reduced compared to solutions of DMSO containing the same total CPA concentration. The reduction in the damage done to the bilayers is a result of the ability of the polyhydroxylated species (especially glycerol) to form hydrogen bonds to the lipid and sterol molecules of the bilayer. A decrease in the amount of DMSO in the vitrification solution with a corresponding increase in the amount of glycerol or ethylene glycol diminishes further its damaging effect due to increased hydrogen bonding of the polyol species to the bilayer headgroups. These findings rationalize, to our knowledge for the first time, the synergistic effects of combining different CPAs, and form the basis for the optimization of vitrification solutions.     

Molecular Dynamics Simulations of Phospholipid Bilayers

Abstract

Molecular dynamics (MD) simulations at 37°C have been performed on three phospholipid bilayer systems composed of the lipids DLPE, DOPE, and DOPC. The model used included 24 explicit lipid molecules and explicit waters of solvation in the polar head group regions, together with constant-pressure periodic boundary conditions in three dimensions. Using this model, a MD simulation samples part of an infinite planar lipid bilayer. The lipid dynamics and packing behavior were characterized. Furthermore, using the results of the simulations, a number of diverse properties including bilayer structural parameters, hydrocarbon chain order parameters, dihedral conformations, electron density profile, hydration per lipid, and water distribution along the bilayer normal were calculated. Many of these properties are available for the three lipid systems chosen, making them well suited for evaluating the model and protocols used in these simulations by direct comparisons with experimental data. The calculated MD behavior, chain disorder, and lipid packing parameter, i.e. the ratio of the effective areas of hydrocarbon tails and head group per lipid (a_t/a_h), correctly predict the aggregation preferences of the three lipids observed experimentally at 37°C, namely: a gel bilayer for DLPE, a hexagonal tube for DOPE, and a liquid crystalline bilayer for DOPC. In addition, the model and conditions used in the MD simulations led to good agreement of the calculated properties of the bilayers with available experimental results, demonstrating the reliability of the simulations. The effects of the cis unsaturation in the hydrocarbon chains of DOPE and DOPC, compared to the fully saturated one in DLPE, as well as the effects of the different polar head groups of PC and PE with the same unsaturated chains on the lipid packing and bilayer structure have been investigated. The results of these studies indicate the ability of MD methods to provide molecular-level insights into the structure and dynamics of lipid assemblies.     

Molecular Dynamics Simulations of Mixed Acidic/Zwitterionic Phospholipid Bilayers

Anionic lipids are key components in the cell membranes. Many cell-regulatory and signaling mechanisms depend upon a complicated interplay between them and membrane-bound proteins. Phospholipid bilayers are commonly used as model systems in experimental or theoretical studies to gain insight into the structure and dynamics of biological membranes. We report here 200-ns-long MD simulations of pure (DMPC and DMPG) and mixed equimolar (DMPC/DMPG, DMPC/DMPS, and DMPC/DMPA) bilayers that each contain 256 lipids. The intra- and intermolecular interaction patterns in pure and mixed bilayers are analyzed and compared. The effect of monovalent ions (Na⁺) on the formation of salt-bridges is investigated. In particular, the number of Na⁺-mediated clusters in the presence of DMPS is higher than with DMPG and DMPA. We observe a preferential clustering of DMPS (and to some extent DMPA) lipids together rather than with DMPC molecules, which can explain the phase separation observed experimentally for DMPC/DMPS and DMPC/DMPA bilayers.     

Distribution of Pentachlorophenol in Phospholipid Bilayers: A Molecular Dynamics Study

Molecular dynamics computer simulations of pentachlorophenol (PCP) in palmitoyl-oleoyl-phosphatidylethanolamine and palmitoyl-oleoyl-phosphatidylcholine lipid bilayers were carried out to investigate the distribution of PCP and the effects of PCP on the phospholipid bilayer structure. Starting from two extreme starting structures, including PCP molecules outside the lipid bilayer, the PCP distribution converges in simulations of up to 50 ns. PCP preferentially occupies the region between the carbonyl groups and the double bonds in the acyl chains of the lipid molecules in the bilayer. In the presence of PCP, the lipid chain order increases somewhat in both chains, and the average tilt angle of the lipid chains decreases. The increase in the lipid chain order in the presence of PCP was more pronounced in the palmitoyl-oleoyl-phosphatidylcholine bilayer compared to the palmitoyl-oleoyl-phosphatidylethanolamine bilayer. The number of trans conformations of lipid chain dihedrals does not change significantly. PCP aligns parallel to the alkyl chains of the lipid to optimize the packing in the dense ordered chain region of the bilayer. The hydroxyl group of PCP forms hydrogen bonds with both water and lipid oxygen atoms in the water/lipid interface region.     

Interaction of Paclitaxel with Phospholipid Bilayers

Abstract

Paclitaxel is an effective anticancer drug. Recently, paclitaxel encapsulated in liposomes was promoted as a better tolerated pharmaceutical formulation than that currently in use. The data presented in this study show the effects of paclitaxel on phospholipid bilayers. Experiments involving the phospholipid head group probe CAT-16 show significant disordering of the interfacial region. The pretransition was abolished and the main phase transition temperature in paclitaxel loaded liposomes was reduced. 2T II values of 7-NSA and 16-NSA spin probes reporting from the middle and from the core of the phospholipid bilayer, respectively, show that the presence of paclitaxel eliminated the pretransition (from Lβ/ to Pβ/) while inducing a slight reduction in the main (Pβ/ to Lα) phase transition temperature; in the same temperature interval, the central resonance line width δ H O displayed a greater rate of spin label reorientation in paclitaxel loaded bilayers. Further data are presented clearly demonstrating that the presence of paclitaxel in liposomal membrane increases the solubility of hydrophobic compounds. Differential scanning calorimetry was used to confirm that the presence of paclitaxel stabilized the lamellar structure of the bilayer and increased the transition temperature from lamellar Lα phase to hexagonal H II phase of TPE liposomes. The encapsulation of paclitaxel in liposomes depends on phospholipid characteristics; more drug is contained in the bilayer of liposomes containing unsaturated fatty acid chains and phosphorylcholine headgroups, such as DEPC and egg PC.     

Molecular Mechanism for the Interaction of Phospholipid with Cholesterol

PREVIOUS models^1–3 for the interaction of phospholipid with cholesterol have been based on the idea of highly specific one-to-one complexes of these molecules. But on the basis of our interpretation of recent nuclear magnetic resonance (NMR)⁴, electron spin resonance (ESR)⁵, calorimetric⁶ and X-ray⁷ data we propose a model in which steric interaction of the phospholipid hydrocarbon chains with the cholesterol molecule is the dominant feature. The validity of the model does not depend on a stoichiometric ratio, such as one-to-one, although it is consistent with such a ratio. The shape of the cholesterol molecule is such that variations in the length and degree of saturation of the phospholipid hydrocarbon chains can be readily accommodated within a stable bilayer structure.     

Lipid Membrane Structure and Dynamics Studied by All-Atom Molecular Dynamics Simulations of Hydrated Phospholipid Bilayers

Abstract

The structure and dynamics of phosphatidylcholine bilayers are examined by reviewing the results of several nanoseconds of molecular dynamics simulations on a number of bilayer and monolayer models. The lengths of these simulations, the longest single one of which was 2 nanoseconds, were sufficiently long to effectively sample many of the longer-scale motions governing the behaviour of biomembranes. These simulations reproduce many experimental observables well and provide a degree of resolution currently unavailable experimentally.     

Transmembrane Peptides Influence the Affinity of Sterols for Phospholipid Bilayers

Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mβCD can be measured. Comparison of how cholesterol and CTL partitioning between mβCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mβCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distribution within the bilayer and thereby the lateral organization in biomembranes.     

Influence of Electrolytes on the Thicknesses of the Phospholipid Bilayers of Lamellar Lecithin Mesophases

Over a wide range of water contents, aqueous lecithin-water mixtures are mesophases in which lecithin bilayers alternate with water layers. This paper reports on low-angle X-ray diffraction measurements of the effects of electrolytes, at 1.0 N concentration, on the thicknesses of the bilayers in mesophases formed by the synthetic lecithin: 1-octadec-9-enyl-2-hexadecylglycerophosphocholine. With solutions of LiCl, NaCl, Na₂SO₄, KCl, and CsCl, the bilayer thicknesses are less than with pure water. The maximum reduction in bilayer thickness with these electrolytes is about 10% and occurs with mesophases of high content of KCl and CsCl solutions. With HCl solutions the bilayer thicknesses are about 5% greater than with pure water, and with CaCl₂ solutions the bilayer thicknesses are about the same as with pure water. The maximum amount of solution which can be mixed with lecithin before a second, purely aqueous phase is formed is also affected by electrolytes, the order for the various 1.0 N solutions being CsCl = KCl > NaCl > Na₂SO₄ > (pure water) = LiCl > CaCl₂.     

Analysis of the Phospholipid Profile of Metaphase II Mouse Oocytes Undergoing Vitrification

Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18∶2/16∶0} [M−H]− and phosphatidylglycerol (PG) {14∶0/18∶2} [M−H]− were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38∶3} [M+H]+ and SM {d34∶0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation.     

Strong Isotope Effects on Melting Dynamics and Ice Crystallisation Processes in Cryo Vitrification Solutions

The nucleation and growth of crystalline ice during cooling, and further crystallization processes during re-warming are considered to be key processes determining the success of low temperature storage of biological objects, as used in medical, agricultural and nature conservation applications. To avoid these problems a method, termed vitrification, is being developed to inhibit ice formation by use of high concentration of cryoprotectants and ultra-rapid cooling, but this is only successful across a limited number of biological objects and in small volume applications. This study explores physical processes of ice crystal formation in a model cryoprotective solution used previously in trials on vitrification of complex biological systems, to improve our understanding of the process and identify limiting biophysical factors. Here we present results of neutron scattering experiments which show that even if ice crystal formation has been suppressed during quench cooling, the water molecules, mobilised during warming, can crystallise as detectable ice. The crystallisation happens right after melting of the glass phase formed during quench cooling, whilst the sample is still transiting deep cryogenic temperatures. We also observe strong water isotope effects on ice crystallisation processes in the cryoprotectant mixture. In the neutron scattering experiment with a fully protiated water component, we observe ready crystallisation occurring just after the glass melting transition. On the contrary with a fully deuteriated water component, the process of crystallisation is either completely or substantially supressed. This behaviour might be explained by nuclear quantum effects in water. The strong isotope effect, observed here, may play an important role in development of new cryopreservation strategies.     

Reconstitution of Cholesterol-Dependent Vaginolysin into Tethered Phospholipid Bilayers: Implications for Bioanalysis

Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from Gardnerella vaginalis into artificial tethered bilayer membranes (tBLMs) has been accomplished. The reconstitution of VLY was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to VLY solutions were consistent with the formation of water-filled pores in the membranes. It was found that reconstitution of VLY is a strictly cholesterol-dependent, irreversible process. At a constant cholesterol concentration reconstitution of VLY occurred in a concentration-dependent manner, thus allowing the monitoring of VLY concentration and activity in vitro and opening possibilities for tBLM utilization in bioanalysis. EIS methodology allowed us to detect VLY down to 0.5 nM (28 ng/mL) concentration. Inactivation of VLY by certain amino acid substitutions led to noticeably lesser tBLM damage. Pre-incubation of VLY with the neutralizing monoclonal antibody 9B4 inactivated the VLY membrane damage in a concentration-dependent manner, while the non-neutralizing antibody 21A5 exhibited no effect. These findings demonstrate the biological relevance of the interaction between VLY and the tBLM. The membrane-damaging interaction between VLY and tBLM was observed in the absence of the human CD59 receptor, known to strongly facilitate the hemolytic activity of VLY. Taken together, our study demonstrates the applicability of tBLMs as a bioanalytical platform for the detection of the activity of VLY and possibly other cholesterol-dependent cytolysins.     

DPPC Bilayers in Solutions of High Sucrose Content

The properties of lipid bilayers in sucrose solutions have been intensely scrutinized over recent decades because of the importance of sugars in the field of biopreservation. However, a consensus has not yet been formed on the mechanisms of sugar-lipid interaction. Here, we present a study on the effect of sucrose on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers that combines calorimetry, spectral fluorimetry, and optical microscopy. Intriguingly, our results show a significant decrease in the transition enthalpy but only a minor shift in the transition temperature. Our observations can be quantitatively accounted for by a thermodynamic model that assumes partial delayed melting induced by sucrose adsorption at the membrane interface.     

Effects of Dietary n-3 Fatty Acids on the Phospholipid Molecular Species of Monkey Brain

Y-79 human retinoblastoma cells grown in serum-free medium in monolayer culture have previously been shown to undergo differentiation in response to dibutyryl cyclic AMP (Bt2cAMP). We report here that Y-79 cells treated in this manner also express very high levels of functional D2 dopamine receptors. In control Y-79 cells, cultured in suspension, D2 dopamine receptors, quantified via saturation analysis with the D2 antagonist [3H]methylspiperone, are expressed at a level of approximately 3 fmol/10(6) cells (approximately 1,800 receptor sites/cell). Differentiation is initiated by attachment of the cells to the culture dish with poly-D-lysine and fibronectin and continued culture in serum-free medium. After 8 days in serum-free culture, differentiation is further induced with continuous Bt2cAMP treatment. Using this differentiation protocol, D2 receptor levels increase up to a maximum of 30 fmol/10(6) cells (18,000 receptors/cell) on day 20, the limit of culture viability. Cultures of 15-17 days (7-9 days of Bt2cAMP treatment) expressing receptor levels of 15-20 fmol/10(6) cells are used for pharmacological and functional characterization of D2 dopamine receptors. The pharmacology of competition for [3H]methylspiperone binding to differentiated Y-79 (dY-79) cell membranes by a series of dopaminergic antagonists verifies the D2 receptor nature of this site, exhibiting appropriate affinities and the following rank order of potency: YM-09151-2 approximately spiperone greater than domperidone approximately (+)-butaclamol approximately fluphenazine greater than chlorpromazine greater than (-)-sulpiride greater than (+)-sulpiride greater than promethazine greater than (+)-SCH 23390 much greater than (-)-butaclamol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Different Maturation Systems on Bovine Oocyte Quality,Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming

The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]⁺), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.     

油菜试管苗玻璃化发生机理的研究初探

研究了影响甘蓝型油菜离体下胚轴再生过程中试管苗玻璃化产生的几个因子,试验发现:调整激素6-BA与NAA的浓度、增加蔗糖的浓度提高渗透势对玻璃化无影响;降低培养基中NH4 浓度不仅没有有效地防止玻璃苗的发生反而降低了芽的诱导率;采用牛皮纸代替聚乙烯塑料膜作为封口材料的措施受到了地区的限制,没有得到有效的统计数据;选择1%的琼脂浓度可使玻璃化率显著降低55%,但诱芽率在一定程度上降低了6.9%。     

Cholesterol-Dependent Nanomechanical Stability of Phase-Segregated Multicomponent Lipid Bilayers

Cholesterol is involved in endocytosis, exocytosis, and the assembly of sphingolipid/cholesterol-enriched domains, as has been demonstrated in both model membranes and living cells. In this work, we explored the influence of different cholesterol levels (5-40 mol %) on the morphology and nanomechanical stability of phase-segregated lipid bilayers consisting of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/Chol) by means of atomic force microscopy (AFM) imaging and force mapping. Breakthrough forces were consistently higher in the SM/Chol-enriched liquid-ordered domains (L_o) than in the DOPC-enriched fluid-disordered phase (L_d) at a series of loading rates. We also report the activation energies (ΔE_a) for the formation of an AFM-tip-induced fracture, calculated by a model for the rupture of molecular thin films. The obtained ΔE_a values agree remarkably well with reported values for fusion-related processes using other techniques. Furthermore, we observed that within the Chol range studied, the lateral organization of bilayers can be categorized into three distinct groups. The results are rationalized by fracture nanomechanics of a ternary phospholipid/sphingolipid/cholesterol mixture using correlated AFM-based imaging and force mapping, which demonstrates the influence of a wide range of cholesterol content on the morphology and nanomechanical stability of model bilayers. This provides fundamental insights into the role of cholesterol in the formation and stability of sphingolipid/cholesterol-enriched domains, as well as in membrane fusion.     

The Localization of Phenolic Compounds in Liposomal Bilayers and Their Effects on Surface Characteristics and Colloidal Stability

The interactions with and effects of five chemically distinct, bioactive phenolic compounds on the lipid bilayers of model dipalmitoylphosphatidylcholine (DPPC) liposomes were investigated. Complementary analytical techniques, including differential scanning calorimetry (DSC) and phosphorus and proton nuclear magnetic resonance spectroscopy (NMR), were employed in order to determine the location of the compounds within the bilayer and to correlate location with their effects on bilayer characteristics and liposomal stability. As compared to the phenolic compounds localized in the glycerol region of the DPPC head group within the bilayer, which enhanced the colloidal stability of the liposomes, compounds located closer to the center of the bilayer reduced vesicle stability as a function of time. Molecules present in the upper region of liposomal DPPC acyl chains (C₁–C₁₀) inhibited liposomal aggregation and size increase, perhaps due to tighter packing of adjoining DPPC molecules and increased surface exposure of DPPC phosphate head groups. These data may be useful for designing liposomal systems containing hydrophobic phenols and other small molecules, selecting appropriate analytical methods for determining their location within liposomal bilayers, and predicting their effects on liposome characteristics early in the liposome formulation development process.     

Synergistic Effect and Molecular Mechanism of Homoharringtonine and Bortezomib on SKM-1 Cell Apoptosis

Background

Myelodysplastic syndromes (MDS) are clonal marrow stem-cell disorders with a high risk of progression to acute myeloid leukemia (AML). Treatment options are limited and targeted therapies are not available for MDS. In the present study, we investigated the cytotoxicity and the molecular mechanism of Homoharringtonine (HHT) and Bortezomib towards high-risk MDS cell line SKM-1 in vitro and the role of miR-3151 was first evaluated in SKM-1 cells.

Methods

SKM-1 cells were treated with different concentrations of HHT or Bortezomib, and cell viability was analyzed with CCK-8 assay. The influence on cell proliferation, cell cycle distribution and the percentage of apoptosis cells were analyzed by flow cytometry. Calcusyn software was used to calculate combination index (CI) values. Western blot was used to analysis phosphorylation of Akt and nuclear NF-κB protein expression in SKM-1 cells. Mature miR-3151 level and p53 protein level were detected after HHT or Bortezomib treatment. The cell proliferation and p53 protein level were reassessed in SKM-1 cells infected with lentivirus to overexpress miR-3151.

Results

Simultaneous exposure to HHT and Bortezomib (10.4:1) resulted in a significant reduction of cell proliferation in SKM-1 cells (P < 0.05). Cell cycle arrest at G0/G1 and G2/M phase was observed (P < 0.05). HHT and Bortezomib synergistically induced cell apoptosis by regulating members of caspase 9, caspase 3 and Bcl-2 family (P < 0.01). The mechanisms of the synergy involved Akt and NF-κB signaling pathway inhibition, downregulation of mature miR-3151 and increment of downstream p53 protein level. Overexpression of miR-3151 promoted cell proliferation and inhibited p53 protein expression in SKM-1 (P < 0.01).

Conclusions

HHT and Bortezomib synergistically inhibit SKM-1 cell proliferation and induce apoptosis in vitro. Inhibition of Akt and NF-κB pathway signaling contribute to molecular mechanism of HHT and Bortezomib. miR-3151 abundance is implicated in SKM-1 cell viability, cell proliferation and p53 protein expression.