Effects of apolipoprotein C-II (apoC-II) on the lipolysis of very low density lipoproteins from apoC-II deficient patients

Lipoprotein lipase requires apolipoprotein C-II (apoC-II) from plasma very low density lipoproteins (VLDL) and high density lipoproteins (HDL) for maximal activity. To understand the mechanism by which apoC-II enhances the activity of the enzyme, the kinetic parameters for the hydrolysis of VLDL-triglycerides and phospholipids by purified bovine milk lipoprotein lipase have been determined in two patients with apoC-II deficiency. The absence of apoC-II in these patients was demonstrated by a specific radioimmunoassay for apoC-II (<0.05 mg apoC-II/dl plasma; normals ?5.0 mg/dl) and by isoelectric focusing of the isolated apoVLDL. The plasma levels of apoC-III, another apoprotein of VLDL, in the two subjects were 18.8 and 22.0 mg/dl (normals 11.1 ± 0.9 mg/dl). The kinetics of lipolysis of VLDL in either the presence or absence of apoC-II were monitored by using the patients' VLDL which were labeled in vitro with tri[1-¹⁴C]oleoyl glycerol and dansyl phosphatidylethanolamine (DPE). The release of [¹⁴C]oleic acid and the rate and extent of the DPE fluorescence increase were dependent on lipoprotein lipase and apoC-II concentration. Maximal hydrolysis of VLDL-triglycerides by lipoprotein lipase occurred at 2.5 μg apoC-II/mg triglyceride. The value of the Michaelis-Menten constant (K_m) of lipoprotein lipase for apoC-II deficient VLDL-triglycerides decreased from 7.8 mM in the absence of apoC-II to 1.0 mM at 2.5 μg of apoC-II; there was only a slight change in V_max. When normal HDL were used as the source of apoC-II, the rate of lipolysis of apoC-II deficient VLDL also increased and the value of K_m decreased to 1.0 mM. These results suggest that the effects of apoC-II on the rate of lipolysis of VLDL result from an apoC-II induced decrease in the apparent K_m of the enzyme for the substrate. One possible explanation for this decrease in K_m is that apoC-II enhances the interaction between lipoprotein lipase and triglyceride within the surface monolayer of the lipoprotein particle.     

Extracellular RNA facilitates hypoxia‐induced leukocyte adhesion and infiltration in the lung through TLR3‐IFN‐γ‐STAT1 signaling pathway

Endogenous ligands released from dying cells, including extracellular RNA (eRNA), cause TLR activation, which is associated with inflammation and vascular diseases. However, the importance of this response in acute hypoxia (AH) remains unexplored. Here, we observed eRNA‐mediated TLR3 activation during exposure of mice to AH in the absence of exogenous viral stimuli. RNaseA treatment diminished AH‐induced expression of IFN and cell adhesion molecules (CAMs) and myeloid cell infiltration in the lung, and TLR3 gene silencing or neutralization with antibodies markedly attenuated AH‐ or poly I:C‐induced IFN and CAM expression and leukocyte adhesion (LA) and myeloid cell infiltration in the lung. However, RNaseA treatment or TLR3 gene silencing failed to alter AH‐induced cell death and proliferation in lung vasculature. Furthermore, IFN‐γ—but not IFN‐α—regulated AH‐induced CAM expression and LA. Treatment with RNaseA, TLR3 siRNA, neutralizing antibodies, or a STAT1 inhibitor substantially decreased AH‐ and poly I:C‐induced STAT1 phosphorylation, CAM expression, and myeloid cell infiltration, suggesting a central role for STAT1 phosphorylation in AH‐induced LA and infiltration. We conclude that eRNA activates TLR3 and facilitates, through in vivo IFN‐γ?STAT1 signaling, AH‐induced leukocyte infiltration in the lung. Thus, RNaseA might provide a therapeutic alternative for patients with lung diseases.     

Enhanced expression of DNA polymerase eta contributes to cisplatin resistance of ovarian cancer stem cells

Cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance are believed to be responsible for treatment failure and tumor relapse in ovarian cancer patients. However, it is still unclear how CSCs survive DNA-damaging agent treatment. Here, we report an elevated expression of DNA polymerase η (Pol η) in ovarian CSCs isolated from both ovarian cancer cell lines and primary tumors, indicating that CSCs may have intrinsically enhanced translesion DNA synthesis (TLS). Down-regulation of Pol η blocked cisplatin-induced CSC enrichment both in vitro and in vivo through the enhancement of cisplatin-induced apoptosis in CSCs, indicating that Pol η-mediated TLS contributes to the survival of CSCs upon cisplatin treatment. Furthermore, our data demonstrated a depletion of miR-93 in ovarian CSCs. Enforced expression of miR-93 in ovarian CSCs reduced Pol η expression and increased their sensitivity to cisplatin. Taken together, our data suggest that ovarian CSCs have intrinsically enhanced Pol η-mediated TLS, allowing CSCs to survive cisplatin treatment, leading to tumor relapse. Targeting Pol η, probably through enhancement of miR-93 expression, might be exploited as a strategy to increase the efficacy of cisplatin treatment.Ovarian cancer is the most lethal malignancy of the female reproductive tract with a 5-y survival rate of only 27% in advanced stages (1). The American Cancer Society estimates that in 2014, about 21,980 new cases of ovarian cancer will be diagnosed and 14,270 women will die of ovarian cancer in the United States (1). The mainline treatment of ovarian cancer is cytoreductive surgery followed by platinum (Pt)-based chemotherapy (2). Chemotherapy with Pt is initially effective for most patients. However, the majority eventually becomes refractory to Pt treatment, and around 70% of patients have tumor relapses (3). Poor understanding of the underlying mechanisms of this acquired drug resistance and tumor relapse poses a critical cancer research challenge.cis-diamminedichloroplatinum(II) (cisplatin), the first member of Pt-based chemotherapeutic agents, has been widely used to treat various malignant tumors, including ovarian cancer (4). Mechanistically, cisplatin reacts with DNA bases to cross-link adjacent purines. These cross-links block DNA replication and invoke apoptosis in rapidly dividing cells (5). Thus, the preferential activation of the DNA damage responses, especially the efficient removal of these DNA lesions, or prompt rescue of the replication, will prevent replication fork collapse and promote survival of the cells upon cisplatin treatment, eventually leading to cisplatin resistance. The cisplatin-induced DNA cross-links are primarily removed by the nucleotide excision repair (NER) pathway (6) or bypassed during replication through translesion DNA synthesis (TLS) (7–10). TLS is mediated by specialized DNA polymerases (Pols), which are characterized by low fidelity and an ability to replicate across certain types of damaged sites in template DNA with the assistance of monoubiquitylated proliferating cell nuclear antigen (ub-PCNA) (11). TLS rescues cells from the collapse of the replication fork and thus is believed to contribute to the development of cisplatin resistance (8, 12–17).It has been increasingly evident that heterogeneous ovarian cancers contain a subpopulation of cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance. These CSCs are believed to be responsible for treatment failure and tumor relapse. Ovarian CSCs have been successfully isolated, based on the expression of distinctive cell surface markers CD44 and CD117 (18, 19), their ability to efflux the Hoechst 33342 fluorescent dye (Side population, SP) (20), the activity of ALDH (21), and their ability to grow as floating spheres in serum-free medium (19). The CD44⁺CD117⁺ cells, SP cells, ALDH⁺ cells, and spheroid cells isolated from both ovarian cancer cell lines and primary human ovarian tumors fulfill all currently accepted criteria for the existence of a subpopulation of tumor-initiating cells (19, 22, 23). Most importantly, these CSCs also demonstrate increased cisplatin resistance. However, it is still unclear how CSCs survive cisplatin treatment. In this study, we demonstrated that the expression level of TLS Pol η is higher in ovarian CSCs isolated from both cancer cell lines and primary tumors than the bulk cancer cells. Down-regulation of Pol η expression blocked cisplatin-induced enrichment of the CSC population, through facilitating the killing of CSCs by cisplatin. Mechanistic investigation demonstrated that decreased expression of miR-93 in ovarian CSCs contributes, at least partially, to the enhanced expression of Pol η. Taken together, our study suggests that Pol η-mediated TLS could be a target to facilitate the eradication of ovarian CSCs by cisplatin.     

Coincidental loss of DOCK8 function in NLRP10-deficient and C3H/HeJ mice results in defective dendritic cell migration

Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.Dendritic cells (DCs) are crucial for the initiation of an adaptive immune response. Upon acquiring antigens in the periphery, DCs undergo a maturation process that includes antigen processing, cytokine production, and up-regulation of costimulatory molecules. A mature DC must then migrate from peripheral tissues to draining lymph nodes (LNs) to fulfill its role as an antigen-presenting cell that primes naïve T cells (1). Although the signals that induce this maturation process are now well-established (1), relatively little is understood about DC migration aside from the primary chemotactic cue provided by CCR7 that guides DCs to the LN (2, 3).We recently described a genetically modified NLRP10 (NLR family, pyrin domain containing 10) knockout strain in which this migration step was disrupted while leaving the remainder of the DC maturation program, including CCR7 expression, intact (4). NLRP10 is the only NOD-like receptor (NLR) without a leucine-rich repeat domain, the putative pathogen-associated molecular pattern (PAMP)–binding domain. It has been proposed to both positively and negatively regulate other NLRs, such as NOD1 and NLRP3, respectively (5, 6). Although we found that NLRP3 inflammasome activation was unaltered in the absence of NLRP10, we discovered that Nlrp10^−/− mice could not mount a productive T- or B-cell immune response due to a DC-intrinsic failure to emigrate out of inflamed tissues (4, 7).To understand the mechanism by which NLRP10 governs DC migration, we used an expression proteomic approach to identify molecules with altered expression in DCs generated from the Nlrp10^−/− strain and discovered a profound reduction in DOCK8 (dedicator of cytokinesis 8). DOCK8 is a guanine nucleotide exchange factor (GEF) that has two functional domains, DOCK homology region (DHR) 1 and DHR2 (8). In murine DCs, the DHR2 domain has been implicated in regulating the Rho GTPase CDC42 (cell division control protein 42 homolog), which in turn maintains cell polarity of mature DCs during migration (9, 10). Furthermore, mice harboring inactivating mutations in Dock8 lack marginal zone B-cell development, long-term antibody production following immunization, and memory CD8⁺ T-cell responses to viral infections (11, 12). In humans, inactivating mutations in Dock8 were recently identified as the primary genetic cause underlying autosomal recessive hyper-IgE syndrome (13). This syndrome presents with eczema, recurrent infections of the skin and respiratory tract, increased serum IgE, eosinophilia, recurrent fungal and viral infections, extensive food and environmental allergies, and, in certain patients, squamous cell dysplasia and carcinomas (14).Given that DOCK8 regulates a wide array of immunologic processes in mouse and human, we sought to understand how NLRP10 regulates DOCK8. To our surprise, we discovered that loss of DOCK8 in the Nlrp10^−/− strain was secondary to a point mutation within the Dock8 gene itself. In this study, we demonstrate that restoring DOCK8 function in the Nlrp10^−/− strain leads to normal DC migration in vivo. We further show that deletion of Dock8, as well as spontaneous mutation of Dock8 in another inbred strain of mice, results in defective DC migration and, depending on the degree of impaired migration, also abrogates CD4⁺ T-cell activation.     

Effect of oral anticoagulant during pregnancy with prosthetic heart valve

This retrospective study aimed to evaluate the risks and outcome of oral anticoagulant use during pregnancy in women with prosthetic heart valves. Between December 1989 and November 1998, 192 females of childbearing age underwent heart valve replacement with a mechanical prosthesis. There were 37 pregnancies in 30 patients during follow-up. Pregnancy was terminated on medical grounds in 5 cases, there were 2 (6%) spontaneous abortions, and 1 (3%) premature birth of a normal baby who died 24 hours later due to asphyxia. The other 29 pregnancies (91%) went to full term and the mothers continued taking oral anticoagulants until a week before the expected date of delivery, then switched to heparin. There was no thromboembolism, valve thrombosis, or maternal mortality. Three babies (10%) had a skeletal deformity: nasal hypoplasia in all 3, with cleft pinna in 1. Continuation of oral anticoagulants during pregnancy provided adequate protection against thromboembolism and valve thrombosis, but the risks of fetal abnormalities and premature delivery should be explained to women of childbearing age with a mechanical valve prosthesis.     

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar[5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(±0.3) × 10⁻¹⁴ cm³/s or 3.5(±1.0) × 10⁸ water molecules per s, which is in the range of AQPs (3.4∼40.3 × 10⁸ water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10⁸ water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼10⁷ water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼2.6 × 10⁵ pores per μm²) is two orders of magnitude higher than that of CNT membranes (0.1∼2.5 × 10³ pores per μm²). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.The discovery of the high water and gas permeability of aquaporins (AQPs) and the development of artificial analogs, carbon nanotubes (CNTs), have led to an explosion in studies aimed at incorporating such channels into materials and devices for applications that use their unique transport properties (1–9). Areas of application include liquid and gas separations (10–13), drug delivery and screening (14), DNA recognition (15), and sensors (16). CNTs are promising channels because they conduct water and gas three to four orders of magnitude faster than predicted by conventional Hagen–Poiseuille flow theory (11). However, their use in large-scale applications has been hampered by difficulties in producing CNTs with subnanometer pore diameters and fabricating membranes in which the CNTs are vertically aligned (4). AQPs also efficiently conduct water across membranes (∼3 billion molecules per second) (17) and are therefore being studied intensively for their use in biomimetic membranes for water purification and other applications (1, 2, 18). The large-scale applications of AQPs is complicated by the high cost of membrane protein production, their low stability, and challenges in membrane fabrication (1).Artificial water channels, bioinspired analogs of AQPs created using synthetic chemistry (19), ideally have a structure that forms a water-permeable channel in the center and an outer surface that is compatible with a lipid membrane environment (1). Interest in artificial water channels has grown in recent years, following decades of research and focus on synthetic ion channels (19). However, two fundamental questions remain: (i) Can artificial channels approach the permeability and selectivity of AQP water channels? (ii) How can such artificial channels be packaged into materials with morphologies suitable for engineering applications?Because of the challenges in accurately replicating the functional elements of channel proteins, the water permeability and selectivity of first-generation artificial water channels were far below those of AQPs (SI Appendix, Table S1) (20–25). In some cases, the conduction rate for water was much lower than that of AQPs as a result of excess hydrogen bonds being formed between the water molecules and oxygen atoms lining the channel (20). The low water permeability that was measured for first-generation water channels also highlights the experimental challenge of accurately characterizing water flow through low-permeability water channels. Traditionally, a liposome-based technique has been used to measure water conduction, in which the response to an osmotic gradient is followed by measuring changes in light scattering (26, 27) or fluorescence (28). The measured rates are then converted to permeability values. These measurements suffer from a high background signal due to water diffusion through the lipid bilayer, which, in some cases, can be higher than water conduction through the inserted channels, making it challenging to resolve the permeability contributed by the channels (29). Thus, there is a critical need for a method to accurately measure single-channel permeability of artificial water channels to allow for accurate comparison with those of biological water channels. Furthermore, first-generation artificial water channels were designed with a focus on demonstrating water conduction and one-dimensional assembly into tubular structures (20–24), but how the channels could be assembled into materials suitable for use in engineering applications was not explored. To derive the most advantage from their fast and selective transport properties, artificial water channels are ideally vertically aligned and densely packed in a flat membrane. These features have been long desired but remain a challenge for CNT-based systems (4).Here we introduce peptide-appended pillar[5]arene (PAP; Fig. 1) (30) as an excellent architecture for artificial water channels, and we present data for their single-channel permeability and self-assembly properties. Nonpeptide pillar[5]arene derivatives were among first-generation artificial water channels (1, 23). Pillar[5]arene derivatives, including the one used in this study, have a pore of ∼5 Å in diameter and are excellent templates for functionalization into tubular structures (31–34). However, the permeability of hydrazide-appended pillar[5]arene channels was low (∼6 orders of magnitude lower than that of AQPs; SI Appendix, Table S1). We addressed the challenges of accurately measuring single-channel water permeability and improving the water conduction rate over first-generation artificial water channels by using both experimental and simulation approaches. The presented PAP channel contains more hydrophobic regions (30) compared with its predecessor channel (23), which improves both its water permeability and its ability to insert into membranes. To determine single-channel permeability of PAPs, we combined stopped-flow light-scattering measurements of lipid vesicles containing PAPs with fluorescence correlation spectroscopy (FCS) (35, 36). Stopped-flow experiments allow the kinetics of vesicle swelling or shrinking to be followed with millisecond resolution and water permeability to be calculated, whereas FCS makes it possible to count the number of channels per vesicle (36, 37). The combination of the two techniques allows molecular characterization of channel properties with high resolution and demonstrates that PAP channels have a water permeability close to those of AQPs and CNTs. The experimental results were corroborated by molecular dynamics (MD) simulations, which also provided additional insights into orientation and aggregation of the channels in lipid membranes. Finally, as a first step toward engineering applications such as liquid and gas separations, we were able to assemble PAP channels into highly packed planar membranes, and we experimentally confirmed that the channels form 2D arrays in these membranes.Open in a separate windowFig. 1.Structure of the peptide-appended pillar[5]arene (PAP) channel. (A) The PAP channel (C₃₂₅H₃₂₀N₃₀O₆₀) forms a pentameric tubular structure through intramolecular hydrogen bonding between adjacent alternating d-l-d phenylalanine chains (d-Phe-l-Phe-d-Phe-COOH). (B) Molecular modeling (Gaussian09, semiempirical, PM6) of the PAP channel shows that the benzyl rings of the phenylalanine side chains extend outward from the channel walls (C, purple; H, white; O, red; N, blue). (C and D) MD simulation of the PAP channel in a POPC bilayer revealed its interactions with the surrounding lipids. The five chain-like units of the channel are colored purple, blue, ochre, green, and violet, with hydrogen atoms omitted. In C, the POPC lipids are represented by thin tan lines; in D, water is shown as red (oxygen) and white (hydrogen) van der Waals spheres.     

Inhibition of β-Catenin signaling suppresses pancreatic tumor growth by disrupting nuclear β-Catenin/TCF-1 complex: Critical role of STAT-3

Aberrant activation of β-catenin/TCF signaling is related to the invasiveness of pancreatic cancer. In the present study, we evaluated the effect of capsaicin on β-catenin/TCF signaling. In a concentration and time-dependent study, we observed that capsaicin treatment inhibits the activation of dishevelled (Dsh) protein DvI-1 in L3.6PL, PanC-1 and MiaPaCa-2 pancreatic cancer cells. Capsaicin treatment induced GSK-3β by inhibiting its phosphorylation and further activated APC and Axin multicomplex, leading to the proteasomal degradation of β-catenin. Expression of TCF-1 and β-catenin-responsive proteins, c-Myc and cyclin D1 also decreased in response to capsaicin treatment. Pre-treatment of cells with MG-132 blocked capsaicin-mediated proteasomal degradation of β-catenin. To establish the involvement of β-catenin in capsaicin-induced apoptosis, cells were treated with LiCl or SB415286, inhibitors of GSK-3β. Our results reveal that capsaicin treatment suppressed LiCl or SB415286-mediated activation of β-catenin signaling. Our results further showed that capsaicin blocked nuclear translocation of β-catenin, TCF-1 and p-STAT-3 (Tyr705). The immunoprecipitation results indicated that capsaicin treatment reduced the interaction of β-catenin and TCF-1 in the nucleus. Moreover, capsaicin treatment significantly decreased the phosphorylation of STAT-3 at Tyr705. Interestingly, STAT-3 over expression or STAT-3 activation by IL-6, significantly increased the levels of β-catenin and attenuated the effects of capsaicin in inhibiting β-catenin signaling. Finally, capsaicin mediated inhibition of orthotopic tumor growth was associated with inhibition of β-catenin/TCF-1 signaling. Taken together, our results suggest that capsaicin-induced apoptosis in pancreatic cancer cells was associated with inhibition of β-catenin signaling due to the dissociation of β-catenin/TCF-1 complex and the process was orchestrated by STAT-3.     

Apigenin blocks IKKα activation and suppresses prostate cancer progression

IKKα has been implicated as a key regulator of oncogenesis and driver of the metastatic process; therefore is regarded as a promising therapeutic target in anticancer drug development. In spite of the progress made in the development of IKK inhibitors, no potent IKKα inhibitor(s) have been identified. Our multistep approach of molecular modeling and direct binding has led to the identification of plant flavone apigenin as a specific IKKα inhibitor. Here we report apigenin, in micro molar range, inhibits IKKα kinase activity, demonstrates anti-proliferative and anti-invasive activities in functional cell based assays and exhibits anticancer efficacy in experimental tumor model. We found that apigenin directly binds with IKKα, attenuates IKKα kinase activity and suppresses NF-ĸB/p65 activation in human prostate cancer PC-3 and 22Rv1 cells much more effectively than IKK inhibitor, PS1145. We also showed that apigenin caused cell cycle arrest similar to knockdown of IKKα in prostate cancer cells. Studies in xenograft mouse model indicate that apigenin feeding suppresses tumor growth, lowers proliferation and enhances apoptosis. These effects correlated with inhibition of p-IKKα, NF-ĸB/p65, proliferating cell nuclear antigen and increase in cleaved caspase 3 expression in a dose-dependent manner. Overall, our results suggest that inhibition of cell proliferation, invasiveness and decrease in tumor growth by apigenin are mediated by its ability to suppress IKKα and downstream targets affecting NF-ĸB signaling pathways.