628,409 research outputs found

    Novel 5-oxo-hexahydroquinoline derivatives: design, synthesis, in vitro P-glycoprotein-mediated multidrug resistance reversal profile and molecular dynamics simulation study

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    Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-hexahydroquinoline derivatives containing different nitrophenyl moieties at C-4 and various carboxamide substituents at C-3 were designed, synthesized and evaluated for their ability to inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds with highest MDR reversal activities was further evaluated by measuring the alterations of MES-SA/Dx5 cells' sensitivity to doxorubicin (DXR) using MTT assay. The results of both biological assays indicated that compounds bearing 2-nitrophenyl at C-4 position and compounds with 4-chlorophenyl carboxamide at C-3 demonstrated the highest activities in resistant cells, while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active derivatives, 5c, significantly increased intracellular Rh123 at 100 mu M, and it also significantly reduced the IC50 of DXR by 70.1% and 88.7% at 10 and 25 mu M, respectively, in MES-SA/Dx5 cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular dynamics simulation study was also performed to investigate the possible binding site of 5c in complex with human P-gp, which showed that this compound formed 11 average H-bonds with Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found between the results of the computational and experimental studies. The findings of this study show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for the discovery of new agents for P-gp-mediated MDR reversal

    Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing.

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    Chronic wounds are a major disease burden worldwide. The breach of the epithelial barrier facilitates transition of skin commensals to invasive facultative pathogens. Therefore, we investigated the potential effects of Staphylococcus aureus (SA) on dermal fibroblasts as key cells for tissue repair. In co-culture systems combining live or heat-killed SA with dermal fibroblasts derived from the BJ-5ta cell line, healthy individuals, and patients with systemic sclerosis, we assessed tissue repair including pro-inflammatory cytokines, matrix metalloproteases (MMPs), myofibroblast functions, and host defense responses. Only live SA induced the upregulation of IL-1β/-6/-8 and MMP1/3 as co-factors of tissue degradation. Additionally, the increased cell death reduced collagen production, proliferation, migration, and contractility, prerequisite mechanisms for wound closure. Intracellular SA triggered inflammatory and type I IFN responses via intracellular dsDNA sensor molecules and MyD88 and STING signaling pathways. In conclusion, live SA affected various key tissue repair functions of dermal fibroblasts from different sources to a similar extent. Thus, SA infection of dermal fibroblasts should be taken into account for future wound management strategies

    Impact of copper and iron binding properties on the anticancer activity of 8-hydroxyquinoline derived Mannich bases.

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    The anticancer activity of 8-hydroxyquinolines relies on complex formation with redox active copper and iron ions. Here we employ UV-visible spectrophotometry and EPR spectroscopy to compare proton dissociation and complex formation processes of the reference compound 8-hydroxyquinoline (Q-1) and three related Mannich bases to reveal possible correlations with biological activity. The studied derivatives harbor a CH2-N moiety at position 7 linked to morpholine (Q-2), piperidine (Q-3), and chlorine and fluorobenzylamino (Q-4) substituents. Solid phase structures of Q-3, Q-4·HCl·H2O, [(Cu(HQ-2)2)2]·(CH3OH)2·Cl4·(H2O)2, [Cu(Q-3)2]·Cl2 and [Cu(HQ-4)2(CH3OH)]·ZnCl4·CH3OH were characterized by single-crystal X-ray diffraction analysis. In addition, the redox properties of the copper and iron complexes were studied by cyclic voltammetry, and the direct reaction with physiologically relevant reductants (glutathione and ascorbic acid) was monitored. In vitro cytotoxicity studies conducted with the human uterine sarcoma MES-SA/Dx5 cell line reveal the significant cytotoxicity of Q-2, Q-3, and Q-4 in the sub- to low micromolar range (IC50 values 0.2-3.3 μM). Correlation analysis of the anticancer activity and the metal binding properties of the compound series indicates that, at physiological pH, weaker copper(ii) and iron(iii) binding results in elevated toxicity (e.g.Q4: pCu = 13.0, pFe = 6.8, IC50 = 0.2 μM vs.Q1: pCu = 15.1, pFe = 13.0 IC50 = 2.5 μM). Although the studied 8-hydroxyquinolines preferentially bind copper(ii) over iron(iii), the cyclic voltammetry data revealed that the more cytotoxic ligands preferentially stabilize the lower oxidation state of the metal ions. A linear relationship between the pKa (OH) and IC50 values of the studied 8-hydroxyquinolines was found. In summary, we identify Q-4 as a potent and selective anticancer candidate with significant toxicity in drug resistant cells

    Effect of Aspirin and Salicylic Acid on LPA Induced Differentiation of P19 Stem Cells into Cardiomyocytes

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    The use of stem cell-based therapy in conjunction with existing medical interventions, to target complications caused by coronary artery disease (CAD) is not fully examined. In parallel, the role of lysophosphatidic acid (LPA); an important endogenous bioactive phospholipid, has shown cardioprotective characteristics at low physiological concentrations, providing a potential for future treatment plans. In addition, studies have indicated the promise of aspirin (ASA)/ salicylic acid (SA) or LPA to induce and promote cardiac differentiation of SCs in various models. Therefore, in this project, we investigated the effects of ASA/SA in the presence or absence of LPA to induce the differentiation of the murine P19 teratocarcinoma stem cell line into cardiomyocytes. Routine cell culture was undertaken using P19 stem cells cultured in complete α-minimal essential medium (α-MEM). In the first instance, the protocol was optimised to ensure that efficient and reproducible differentiation was achieved. Embryoid bodies (EB) were formed by seeding cells and left to aggregate over a period of 2 days in ultra-low attachment 96-well plates, to establish differentiation. P19 stem cells were pre-incubated for 1 hour with ASA and SA at varying concentrations (0.1mM, 0.3mM, 1mM and 3mM) and selective NFκB inhibitor (0.1nM CAY10470) were pre-incubated 1 hour prior to adding LPA (5µM). Control cells were cultured in complete α-MEM alone. 6-8 EBs were isolated and seeded into 12-well tissue culture plates and cultured for 6 days. Western blotting was used to confirm differentiation, examining for the expression of ventricular myosin light chain (MLC-1v), relative to β-actin. To determine the potential mechanism through which differentiation may be induced, changes in phosphorylation of activated NFκB and IκB were determined. Optimisation of the differentiation protocol revealed that 1 x 104 cells grown for 2 days, produced consistent EBs sizes which ranged between 350-450µm in diameter. These EBs efficiently differentiated into cardiomyocytes. Differentiation was consistently achieved using LPA (5µM) and at selected concentrations of ASA (0.3 -1mM, at day 3) and SA (1mM, at day 3). Maximal expression of MLC-1v in ASA/SA conditions was seen at 1mM. However, LPA induced differentiation was inhibited by both in combination treatment with ASA and SA, despite both inducing differentiation independently. Analysis of phosphorylated and native proteins associated with the NFκB complex was successfully detected. These initial studies indicated substantial expression of phospho NFκB in LPA, SA and ASA treated cells and increases were seen at the 6-9-hour time points. The expression of phospho IκB in LPA treated cells peaked at 10-15 mins, while ASA/SA treated cells showed phospho IκB peaking at a later time point (3 hours). In conclusion, the experiments conducted in this thesis have shown that both ASA/SA and LPA induced cardiomyocyte differentiation. However, when ASA or SA are used in combination with LPA, an antagonistic effect is seen, preventing LPA to induce differentiation

    Diacetylenic lipids in the design of stable lipopolymers able to complex and protect plasmid DNA

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    Different viral and non-viral vectors have been designed to allow the delivery of nucleic acids in gene therapy. In general, non-viral vectors have been associated with increased safety for in vivo use; however, issues regarding their efficacy, toxicity and stability continue to drive further research. Thus, the aim of this study was to evaluate the potential use of the polymerizable diacetylenic lipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) as a strategy to formulate stable cationic lipopolymers in the delivery and protection of plasmid DNA. Cationic lipopolymers were prepared following two different methodologies by using DC8,9PC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the cationic lipids (CL) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), stearylamine (SA), and myristoylcholine chloride (MCL), in a molar ratio of 1:1:0.2 (DMPC:DC8,9PC:CL). The copolymerization methodology allowed obtaining cationic lipopolymers which were smaller in size than those obtained by the cationic addition methodology although both techniques presented high size stability over a 166-day incubation period at 4C. Cationic lipopolymers containing DOTAP or MCL were more efficient in complexing DNA than those containing SA. Moreover, lipopolymers containing DOTAP were found to form highly stable complexes with DNA, able to resist serum DNAses degradation. Furthermore, neither of the cationic lipopolymers (with or without DNA) induced red blood cell hemolysis, although metabolic activity determined on the L-929 and Vero cell lines was found to be dependent on the cell line, the formulation and the presence of DNA. The high stability and DNA protection capacity as well as the reduced toxicity determined for the cationic lipopolymer containing DOTAP highlight the potential advantage of using lipopolymers when designing novel nonviral carrier systems for use in in vivo gene therapy. Thus, this work represents the first steps toward developing a cationic lipopolymer-based gene delivery system using polymerizable and cationic lipids.Instituto Multidisciplinario de Biología Celula

    Diacetylenic lipids in the design of stable lipopolymers able to complex and protect plasmid DNA

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    Different viral and non-viral vectors have been designed to allow the delivery of nucleic acids in gene therapy. In general, non-viral vectors have been associated with increased safety for in vivo use; however, issues regarding their efficacy, toxicity and stability continue to drive further research. Thus, the aim of this study was to evaluate the potential use of the polymerizable diacetylenic lipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) as a strategy to formulate stable cationic lipopolymers in the delivery and protection of plasmid DNA. Cationic lipopolymers were prepared following two different methodologies by using DC8,9PC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the cationic lipids (CL) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), stearylamine (SA), and myristoylcholine chloride (MCL), in a molar ratio of 1:1:0.2 (DMPC:DC8,9PC:CL). The copolymerization methodology allowed obtaining cationic lipopolymers which were smaller in size than those obtained by the cationic addition methodology although both techniques presented high size stability over a 166-day incubation period at 4C. Cationic lipopolymers containing DOTAP or MCL were more efficient in complexing DNA than those containing SA. Moreover, lipopolymers containing DOTAP were found to form highly stable complexes with DNA, able to resist serum DNAses degradation. Furthermore, neither of the cationic lipopolymers (with or without DNA) induced red blood cell hemolysis, although metabolic activity determined on the L-929 and Vero cell lines was found to be dependent on the cell line, the formulation and the presence of DNA. The high stability and DNA protection capacity as well as the reduced toxicity determined for the cationic lipopolymer containing DOTAP highlight the potential advantage of using lipopolymers when designing novel nonviral carrier systems for use in in vivo gene therapy. Thus, this work represents the first steps toward developing a cationic lipopolymer-based gene delivery system using polymerizable and cationic lipids.Instituto Multidisciplinario de Biología Celula

    The acridonecarboxamide GF120918 potently reverses P-glycoprotein-mediated resistance in human sarcoma MES-Dx5 cells

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    The doxorubicin-selected, P-glycoprotein (P-gp)-expressing human sarcoma cell line MES-Dx5 showed the following levels of resistance relative to the non-P-gp-expressing parental MES-SA cells in a 72 h exposure to cytotoxic drugs: etoposide twofold, doxorubicin ninefold, vinblastine tenfold, taxotere 19-fold and taxol 94-fold. GF120918 potently reversed resistance completely for all drugs. The EC50s of GF120918 to reverse resistance of MES-Dx5 cells were: etoposide 7 ± 2 nM, vinblastine 19 ± 3 nM, doxorubicin 21 ± 6 nM, taxotere 57 ± 14 nM and taxol 91 ± 23 nM. MES-Dx5 cells exhibited an accumulation deficit relative to the parental MES-SA cells of 35% for [3H]-vinblastine, 20% for [3H]-taxol and [14C]-doxorubicin. The EC50 of GF120918, to reverse the accumulation deficit in MES-Dx5 cells, ranged from 37 to 64 nM for all three radiolabelled cytotoxics. [3H]-vinblastine bound saturably to membranes from MES-Dx5 cells with a KD of 7.8 ± 1.4 nM and a Bmax of 5.2 ± 1.6 pmol mg–1 protein. Binding of [3H]-vinblastine to P-gp in MES-Dx5 membranes was inhibited by GF120918 (Ki = 5 ± 1 nM), verapamil (Ki = 660 ± 350 nM) and doxorubicin (Ki = 6940 ± 2100 nM). Taxol, an allosteric inhibitor of [3H]-vinblastine binding to P-gp, could only displace 40% of [3H]-vinblastine (Ki = 400 ± 140 nM). The novel acridonecarboxamide derivative GF120918 potently overcomes P-gp-mediated multidrug resistance in the human sarcoma cell line MES-Dx5. Detailed analysis revealed that five times higher GF120918 concentrations were needed to reverse drug resistance to taxol in the cytotoxicity assay compared to doxorubicin, vinblastine and etoposide. An explanation for this phenomenon had not been found. © 1999 Cancer Research Campaig

    Mechanisms into the development of fatty liver disease: role of free fatty acids and alcohol

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    Background: Alcohol and Free fatty acids such as palmitate are known to promote liver injury. However less mechanistic information is available regarding omega fatty acids ratios with/out alcohol. In healthy populations omega 6/3 ratios are between 1:1 to 4:1, whereas high ratios (>15:1) are thought to correlate with the pathogenesis of fatty liver disease. This study aimed to investigate liver lipotoxicity and mitochondrial dysfunction due to imbalanced omega 6/3 ratios alone or in the presence of alcohol. Method: Human hepatoma cell line, VL-17A cells were treated with individual fatty acids (Palmitic (PA), Stearic (SA), Linoleic (LA), alpha-Linolenic (ALA), Arachidonic (AA) and Docosahexaenoic (DHA) acids) with various concentrations ranging between 0.5 μM to 300 μM and omega 6/3 ratios (1:1, 4:1, 15:1 and 25:1) with/out alcohol (100 mM) for 24, 48 and 72 h after which lipid accumulation and cell toxicity was assessed. Subsequent studies at 24 hr examined oxidative stress, mitochondrial function and lipogenic proteins. Results: PA treatment showed a detrimental effect on cell viability and lipid accumulation than SA after 48 h and 72 h (P0.05), sterol regulatory element-binding protein 1 (SREBP1) did not show any change. AA/DHA ratios also showed a significant decrease in ATP production (P<0.01), basal respiration, maximal respiration and spare mitochondrial capacity and this effect was greater with high ratios (P<0.001). Reactive oxygen species (ROS) production increased significantly, particularly with high AA/DHA ratios (15:1 and 25:1) (P<0.001) alone and in the presence of alcohol (P<0.01). Conclusion: The data suggests that lipid accumulation and toxicity occur with saturated and unsaturated fatty acids and high omega 6/3 ratios. The latter possibly due to the pro-inflammatory products of AA. This study confirms that high AA/DHA ratios with/out alcohol increase ROS production and high AA/DHA alone induce mitochondrial dysfunction and increase lipogenesis pathways by activating lipogenic factors causing steatosis and consequently promoting the development of fatty liver disease. Further work aims to elucidate the effect of fatty acid/alcohol on lipid synthetic and endocannabinoid pathways, which will further our understanding of fatty liver disease development

    Efficacy and safety of crizotinib in the treatment of advanced non-small cell Lung cancer with ROS1 gene fusion: A systematic literature review and Meta-Analysis of real-world evidence

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    Background: Crizotinib was approved to treat patients with advanced non -small cell lung cancer (aNSCLC) with ROS proto-oncogene 1 (ROS1) gene fusion in 2016. We conducted a systematic literature review to identify realworld evidence (RWE) studies and estimated the efficacy and safety of crizotinib using meta-analyses (MA) for objective response rate (ORR), real -world progression -free survival (PFS), and overall survival (OS). Methods: We searched MEDLINE (R), Embase, and Cochrane CENTRAL from January 2016 to March 2023 using Ovid (R) for published single -arm or comparative RWE studies evaluating patients (N >= 20) receiving crizotinib monotherapy for aNSCLC with ROS1 gene fusion. Pooled estimates for ORR and grade 3/4 adverse events (AEs) were derived using the metafor package in R while pooled estimates for median real -world PFS (rwPFS) and OS were derived using reconstructed individual patient data from published Kaplan -Meier curves. The primary analysis included all studies regardless of crizotinib line of therapy; a subgroup analysis (SA) was conducted using studies evaluating patients receiving first -line crizotinib. Results: Fourteen studies met the eligibility criteria and were considered feasible for MA. For the primary analysis, the pooled ORR (N = 9 studies) was 70.6 % (95 % confidence interval [CI]: 57.0, 81.3), median rwPFS was 14.5 months (N = 11 studies), and OS was 40.2 months (N = 9 studies). In the SA, the pooled ORR (N = 4 studies) was 81.1 % (95 % CI: 76.1, 85.2) and the median rwPFS (N = 4 studies) and OS (N = 2 studies) were 18.1 and 60 months, respectively. All MAs were associated with significant heterogeneity (I2 > 25 %). Grade 3/4 AEs occurred in 18.7 % of patients (pooled estimate). Conclusion: The results from this study are consistent with clinical trial data and, taken collectively, supports crizotinib as a safe and effective treatment across different lines of therapy in patients with ROS1 aNSCLC in the real -world setting

    Tracking down gene intefrity within fragile sites: Do they play a role in oesoplageal cancer?

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    Faculty of Science School of Pathology 9900713m Cell #: 083 718 9093Oesophageal cancer (OC) is the third most common malignancy in South Africa (SA), affecting 1 in 20 and 1 in 76 black males and females respectively. Squamous cell carcinoma (SSC) is an aggressive disease showing a poor prognosis due to late diagnosis. Identification of genetic changes associated with these tumours may shed light on its pathophysiology and aetiology in SA. The chromosomal status of five OC cell lines, established in SA, was assessed to identify possible common chromosomal alterations by M-FISH (multicolour fluorescence in situ hybridisation) and specifically the fragile site loci, FRA3B and FRA16D by FISH (Fluorescence in situ hybridisation). The genes at these loci, FHIT (Fragile Histidine Triad) and WWOX (WW domain containing oxidoreductase) respectively, were analysed by RT-PCR (Reverse transcriptase polymerase chain reaction). FHIT was aberrantly expressed in four of the five cell lines while WWOX expression was normal. The EGFR (epidermal growth factor receptor) locus is frequently amplified and this gene is also over-expressed in OC. Increased EGFR expression was previously found in three of the cell lines, for this reason, particular attention was paid to markers involving the EGFR locus on 7p. An interesting marker chromosome seven was identified in one of the cell lines and further analysis, using a specific EGFR probe, revealed an amplification unit involving EGFR in this cell line. Common translocations involving chromosomes 3 and 1 as well as 3 and 22 were identified in two cell lines; these may involve a locus involved in OC and warrants further investigation
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