Ethidium Bromide Assay Research Articles

Liposomal formulation with thiazolic compounds against bacterial efflux pumps

This study aimed to evaluate the effects of liposome-encapsulated eugenol-based thiazolic derivatives against efflux pump-carrying bacteria. The Minimum Inhibitory Concentration (MIC) was determined to evaluate the antibacterial activity and antibiotic potentiation against Pseudomonas aeruginosa and Staphylococcus aureus, as well as to analyze the inhibition of efflux pumps in S. aureus strains 1199B and K2068 in the ethidium bromide assay. The direct antibacterial activity analysis showed no clinically relevant results since the compounds presented MICs ≥1024 µg/mL. Regarding the analysis of antibiotic potentiation against multidrug-resistant (MDR) strains of S. aureus, compound LF16 reduced norfloxacin MIC from 128 µg/mL to 64 µg/mL. All associated with gentamicin caused a significant antibiotic MIC reduction. None of the compounds could potentate the activity of norfloxacin against P. aeruginosa. However, all of them potentiated the activity of gentamicin against the same strain. Only the LF 26 caused a significant MIC reduction in the ethidium bromide assay, suggesting efflux inhibition in the S. aureus 1199B strain. Similar results were observed with the K2068 strain. Observing antibiotic MIC reduction S. aureus strains carrying the NorA and MepA proteins brought additional evidence of efflux pump inhibition. Our results indicate that while eugenol-based thiazoles didn’t exhibit direct activity, they can potentiate the antibiotics activity against MDR strains of P. aeruginosa and S. aureus. Among them, compound LF 26 potentiated the inhibitory effects of ethidium bromide and antibiotics against S. aureus strains carrying the NorA and MepA proteins, indicating a potential role of this class of compounds as efflux pump inhibitors.

Dellaglioa Algida Cell-Free Supernatant Inhibits Pseudomonas Fluorescence and Pseudomonas Fragi by Destroying Cell Membranes.

The aim of this study was to examine the components of the cell-free supernatant (CFS) derived from a novel strain of psychrophilic Lactobacillus, Dellaglioa algida, and to further elucidate the impact of this CFS on various cellular processes. Specifically, we sought to understand its effects on the cell membrane, protein and DNA release, protease activity, and metabolites of Pseudomonas fluorescens and Pseudomonas fragi, thereby clarifying the antibacterial mechanism involved. The CFS components were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS), the Coomassie Brilliant Blue method, and the phenol-sulfuric acid method. The inhibitory effect of the CFS on Pseudomonas fluorescens and Pseudomonas fragi was assessed using the ethidium bromide (EB) assay, Oxford cup assay, and ultramicroassay. Additionally, we analyzed the metabolites produced by Pseudomonas fluorescens and Pseudomonas fragi when treated with the CFS. The findings reveal that the CFS of Dellaglioa algida contains 94 volatile components, with protein and sugar concentrations of 32.857 ± 0.9705 mg/mL and 98.250 ± 4.210 mg/L, respectively. The CFS induces varying degrees of damage to the cell membranes of both Pseudomonas fluorescens and Pseudomonas fragi, leading to the release of intracellular proteins and DNA. Furthermore, the CFS reduced the protease activity and metabolic capacity of Pseudomonas fluorescens and Pseudomonas fragi. These results enhance our understanding of the mechanism by which psychrophilic Dellaglioa algida inhibits Pseudomonas fluorescens and Pseudomonas fragi, confirming that its inhibitory effect predominantly occurs through damage to the biological cell membranes of Pseudomonas. Dellaglioa algida is a newly identified cold-adapted inhibitor of Pseudomonas, indicating that its CFS is an effective microbial inhibitor in cold environments. This discovery suggests potential applications in inhibiting the growth and reproduction of Pseudomonas fluorescens and Pseudomonas fragi in food, pharmaceuticals, perfumes, and other chemicals, providing a valuable new reference for industrial preservation.

Unravelling the tissue regenerative nature of marine polysaccharide chitosan embedded halloysite reinforced poly (vinyl alcohol) nanocomposite films

The study focuses on developing the poly (vinyl alcohol) (PVA) nanocomposite films reinforced with Halloysite Nano-Tubes (HNT) which were surface functionalised with chitosan forming a Schiff base structure. The work was aimed to traverse its way in tissue engineering. Modification of halloysite with sea polysaccharide chitosan enhanced its ability to bind to the PVA matrix. The modification was assisted by FTIR, XRD, FESEM, TEM, AFM and DSC-TGA techniques. The films could withstand a higher temperature and exhibited high ranges of tensile strength and Young’s modulus. In addition, biocompatible studies such as in-vitro swelling, enzymatic degradation, water contact angle and hemolysis presented extremely well compatibilities proving it to be viable in physiological pH (in phosphate buffered saline). The cell adhesion and proliferation studies conducted on NIH3T3 mouse fibroblasts revealed the cell proliferation and tissue regeneration properties of the films. Both the tests performed for cell growth- Trypan blue dye exclusion and Acridine Orange Ethidium bromide assays showed a doubled rate of cell growth on the films which proves its biomedical nature.Graphical

A unique combination of natural fatty acids from Hermetia illucens fly larvae fat effectively combats virulence factors and biofilms of MDR hypervirulent mucoviscus Klebsiella pneumoniae strains by increasing Lewis acid-base/van der Waals interactions in bacterial wall membranes.

Hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant K. pneumoniae (CR-Kp) are rapidly emerging as opportunistic pathogens that have a global impact leading to a significant increase in mortality rates among clinical patients. Anti-virulence strategies that target bacterial behavior, such as adhesion and biofilm formation, have been proposed as alternatives to biocidal antibiotic treatments to reduce the rapid emergence of bacterial resistance. The main objective of this study was to examine the efficacy of fatty acid-enriched extract (AWME3) derived from the fat of Black Soldier Fly larvae (Hermetia illucens) in fighting against biofilms of multi-drug resistant (MDR) and highly virulent Klebsiella pneumoniae (hvKp) pathogens. Additionally, the study also aimed to investigate the potential mechanisms underlying this effect. Crystal violet (CV) and ethidium bromide (EtBr) assays show how AWME3 affects the formation of mixed and mature biofilms by the KP ATCC BAA-2473, KPi1627, and KPM9 strains. AWME3 has shown exceptional efficacy in combating the hypermucoviscosity (HMV) virulent factors of KPi1627 and KPM9 strains when tested using the string assay. The rudimentary motility of MDR KPM9 and KP ATCC BAA-2473 strains was detected through swimming, swarming, and twitching assays. The cell wall membrane disturbances induced by AWME3 were detected by light and scanning electron microscopy and further validated by an increase in the bacterial cell wall permeability and Lewis acid-base/van der Waals characteristics of K. pneumoniae strains tested by MATS (microbial adhesion to solvents) method. After being exposed to 0.5 MIC (0.125 mg/ml) of AWME3, a significant reduction in the rudimentary motility of MDR KPM9 and KP ATCC BAA-2473 strains, whereas the treated bacterial strains exhibited motility between 4.23 ± 0.25 and 4.47 ± 0.25 mm, while the non-treated control groups showed significantly higher motility ranging from 8.5 ± 0.5 to 10.5 ± 0.5 mm. In conclusion, this study demonstrates the exceptional capability of the natural AWME3 extract enriched with a unique combination of fatty acids to effectively eliminate the biofilms formed by the highly drug-resistant and highly virulent K. pneumoniae (hvKp) pathogens. Our results highlight the opportunity to control and minimize the rapid emergence of bacterial resistance through the treatment using AWME3 of biofilm-associated infections caused by hvKp and CRKp pathogens.

MiR-497-5p Expression and Biological Activity in Gastric Cancer.

Background: This research aims to investigate the expression and biological roles of miR-497-5p in gastric cancer (GC), and its possible mechanisms. Methods: Real Time Quantitative PCR (RT-qPCR) was performed to detect miR-497-5p in GC and normal tissues, as well as GC cell lines versus normal gastric mucosal cells (GES-1). The effects of miR-497-5p overexpression on proliferation were measured by the cell counting kit-8 (CCK8) assay and ethidium bromide (EdU) assay. Flow cytometry was used to assess the cell cycle. The migration and invasion were evaluated by scratch assay and Transwell assay, respectively. Gene targets of miR-497-5p were predicted using "multiMiR" R package combined with mirTarPathway database. And then luciferase reporter experiment was used to evaluate the activity of ERBB2 by miR-497-5p mimics in GC cell line. Besides, functional experiments were performed to verify the impact of miR-497-5p /ERBB2 on phenotypes of GC cells. Results: Compared with the normal tissues and mucosal cells, miR-497-5p was reduced in GC tissues and GC cell lines. miR-497-5p significantly decreased proliferation, migration, and invasion capacity, with an elevated apoptosis ratio of gastric cancer cells. Bioinformatics indicated that ERBB2 might be the potential target of miR-497-5p Dual-luciferase reporter experiments showed it adversely regulated ERBB2 3'UTR luciferase activity. The expression of ERBB2 in GC tissues and cells is significantly higher compared to normal tissues and cells. Over-expression of ERBB2 in gastric cancer cells significantly reduced miR-497-5p's inhibitory effect on the malignant behavior of GC cells. Conclusion: miR-497-5p was significantly down-regulated in GC tissues and cells, which inhibited the malignant features of GC cells by targeting ERBB2.

GATR-3, a Peptide That Eradicates Preformed Biofilms of Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a gram-negative bacterium that causes hospital-acquired and opportunistic infections, resulting in pneumonia, sepsis, and severe wound infections that can be difficult to treat due to antimicrobial resistance and the formation of biofilms. There is an urgent need to develop novel antimicrobials to tackle the rapid increase in antimicrobial resistance, and antimicrobial peptides (AMPs) represent an additional class of potential agents with direct antimicrobial and/or host-defense activating activities. In this study, we present GATR-3, a synthetic, designed AMP that was modified from a cryptic peptide discovered in American alligator, as our lead peptide to target multidrug-resistant (MDR) A. baumannii. Antimicrobial susceptibility testing and antibiofilm assays were performed to assess GATR-3 against a panel of 8 MDR A. baumannii strains, including AB5075 and some clinical strains. The GATR-3 mechanism of action was determined to be via loss of membrane integrity as measured by DiSC3(5) and ethidium bromide assays. GATR-3 exhibited potent antimicrobial activity against all tested multidrug-resistant A. baumannii strains with rapid killing. Biofilms are difficult to treat and eradicate. Excitingly, GATR-3 inhibited biofilm formation and, more importantly, eradicated preformed biofilms of MDR A. baumannii AB5075, as evidenced by MBEC assays and scanning electron micrographs. GATR3 did not induce resistance in MDR A. baumannii, unlike colistin. Additionally, the toxicity of GATR-3 was evaluated using human red blood cells, HepG2 cells, and waxworms using hemolysis and MTT assays. GATR-3 demonstrated little to no cytotoxicity against HepG2 and red blood cells, even at 100 μg/mL. GATR-3 injection showed little toxicity in the waxworm model, resulting in a 90% survival rate. The therapeutic index of GATR-3 was estimated (based on the HC50/MIC against human RBCs) to be 1250. Overall, GATR-3 is a promising candidate to advance to preclinical testing to potentially treat MDR A. baumannii infections.

Modulation of the bacterial virulence and resistance by well-known European medicinal herbs

Ethnopharmacological relevanceSalvia officinalis L., Sambucus nigra L., Matricaria chamomilla L., Agrimonia eupatoria L., Fragaria vesca L. and Malva sylvestris L. are plants that have a long tradition in European folk medicine. To this day, they are part of medicinal teas or creams that help with the healing of skin wounds and the treatment of respiratory or intestinal infections. However, so far these plants have not been investigated more deeply than in their direct antibacterial effect. Aim of the studyOur research is focused on adjuvants that inhibit the mechanism of antibiotic resistance or modulate bacterial virulence. Based on a preliminary screening of 52 European herbs, which commonly appear as part of tea blends or poultice. Six of them were selected for their ability to revert the resistant phenotype of nosocomial bacterial strains. MethodsHerbs selected for this study were obtained from commercially available sources. For the extraction of active compounds ethanol was used. Modulation of virulence was observed as an ability to inhibit bacterial cell-to-cell communication using two mutant sensor strains of Vibrio campbellii. Biofilm formation, and planktonic cell adhesion was measured using a static antibiofilm test. Ethidium bromide assay was used to checked the potential of inhibition bacterial efflux pumps. The antibacterial activities of the herbs were evaluated against resistant bacterial strains using macro dilution methods. ResultsAlcohol extracts had antibacterial properties mainly against Gram-positive bacteria. Of all of them, the highest antimicrobial activity demonstrated Malva sylvestris, killing both antibiotic resistant bacteria; Staphylococcus aureus with MIC of 0.8 g/L and Pseudomonas aeruginosa 0.7 g/L, respectively. Fragaria vesca extract (0.08 g/L) demonstrated strong synergism with colistin (4 mg/L) in modulating the resistant phenotype to colistin of Pseudomonas aeruginosa. Similarly, the extract of S. officinalis (0.21 g/L) reverted resistance to gentamicin (1 mg/L) in S. aureus. However, Sambucus nigra and Matricaria chamomilla seem to be a very promising source of bacterial efflux pump inhibitors. ConclusionThe extract of F. vesca was the most active. It was able to reduce biofilm formation probably due to the ability to decrease bacterial quorum sensing. On the other hand, the activity of S. nigra or M. chamomilla in reducing bacterial virulence may be explained by the ability to inhibit bacterial efflux systems. All these plants have potential as an adjuvant for the antibiotic treatment.

Phenotypic ESBL and non-phenotypic ESBL isolates of Klebsiella pneumoniae exhibit differing responses to induced antimicrobials resistance and subsequent antibiotic cross-resistance.

To investigate the effect of adapting Klebsiella pneumoniae clinical isolates harboring ESBL genes to cetrimide (CT) in terms of subsequent cross-resistance to other biocides and antibiotics, and to investigate changes to virulence markers, such as biofilm formation and efflux activity. The changes between phenotypic extended spectrum β-lactamases (pESBL) expressing isolates and non-phenotypic ESBL (npESBL) isolates was compared. Kl. pneumoniae isolates (14 pESBL and 17 npESBL) were adapted to increasing concentrations of CT, until 4 × MIC was reached. The MIC of the adapted isolates was tested against chloroxylenol (CX) and chlorhexidine. Disk diffusion techniques were used to determine the susceptibility of the isolates to different antibiotics. Biofilm formation was assessed for the isolates using the crystal violet method and efflux pump activity was studied using the ethidium bromide assay. After CT adaptation, 100% of npESBL isolates and 85.7% of pESBL isolates showed increase in CT MIC after CT adaptation. While 41.2% of npESBL and 57.1% of the pESBL isolates showed a cross-resistance with chlorhexidine. CT adaptation resulted in a significant decrease in the susceptibility of npESBL isolates to aztreonam and cefotaxime compared to pESBL isolates, which could be linked to the increase in efflux activity of npESBL compared to pESBL. Biofilm formation was significantly increased after CT adaptation regardless of the type of isolate. The extensive use of biocides in the environment can induce cross-resistance to other biocides and antibiotics, and can increase the ability of bacteria to form biofilms. The response of bacteria to biocide adaptation differs between pESBL and npESBL isolates, although the effect is strain specific.

Monosaccharide Linked Schiff Base Metal Complexes of Cu(II), Zn(II) and Mn(II): Exploring the Antiproliferative Activity and Cell Death Mechanism

AbstractHerein, a new type of Schiff base ligand, HSL2 (where HSL2=2,2,7,7‐tetramethyltetrahydro‐3aH‐bis[1,3]dioxolo[4,5‐b:4′,5′‐d]pyran‐5‐yl) methyl‐4‐(3,5‐dichloro‐2‐hydroxybenzylidene amino)benzoate) anchored with a monosaccharide unit have been synthesized and characterized by FT‐IR, 1H NMR, and ESI‐MS. The ligand are chelated to metal ions Zn(II) (1), Cu(II) (2), and Mn(II) (3) to from square planar/tetrahedral or octahedral complexes. The DFT studies are carried out to optimize the shapes of the compounds. Moreover, Cu(II) and Mn(II) complexes were also characterized by EPR spectroscopy. The cytotoxicity of the complexes was screened towards three different cell lines A549 (lung cancer), HeLa (cervical cancer), and HT29 (colorectal cancer). These complexes exhibited significant cytotoxicity against three aforementioned cell lines and IC50 values are found in the range of 19.77–23 μM on HeLa cells, 9.42–12.25 μM on A549 cells and 8.84–16 μM on HT29 cells. The cell death mechanism was also studied by employing DNA fragmentation, Hoechst 33342 nuclear staining, and Acridine orange/ Ethidium Bromide (AO/EB) assay. In addition, ROS generation was also evaluated with these metal complexes.

Evaluation of isoeugenol in inhibition of Staphylococcus aureus efflux pumps and their toxicity using Drosophila melanogaster model

The Staphylococcus aureus bacteria is a pathogen considered opportunistic and that has been acquiring resistance to several classes of antibiotics, mainly due to the synthesis of efflux pumps, which are proteins that expel these drugs intracellularly, reducing their effectiveness. The objective of this study was to evaluate the ability of isoeugenol to inhibit S. aureus efflux pumps and to determine its toxicity against a eukaryotic model (Drosophila melanogaster). IS-58, K2068 and K4414 S. aureus strains were used in the study. Isoeugenol minimum inhibitory concentration (MIC) and antibiotic modulation were evaluated in efflux pump inhibitory tests as well as in ethidium bromide (EtBr) assays. Toxicity tests against D. melanogaster assessed mortality and negative geotaxis. Isoeugenol obtained a relevant MIC result and a synergism was observed when isoeugenol was associated with the antibiotics, mainly with ciprofloxacin. Isoeugenol was able to affect all three efflux pumps tested, especially in strain K4414. The mortality of D. melanogaster caused by isoeugenol administration started after 12 h of exposure, being volume dependent and having an LC50 of 81.69 μL/L. In the negative geotaxis test, a statistical difference was observed after 24h of exposure compared to the control, demonstrating that damage to the locomotor apparatus had occurred. Based on the results, isoeugenol is a putative efflux pump inhibitor, becoming an alternative in blocking these proteins, and demonstrated acute toxicity against D. melanogaster.

Synthesis, biological evaluation and computational studies of acrylohydrazide derivatives as potential Staphylococcus aureus NorA efflux pump inhibitors

The NorA efflux pump decreases the intracellular concentration of fluoroquinolones (ciprofloxacin, norfloxacin) by effluxing them from Staphylococcus aureus cells. The synthesis of novel acrylohydrazide derivatives was achieved using well-known reactions and were characterized by various spectroscopy techniques. The synthesized 50 compounds were evaluated for the NorA efflux pump inhibition activity against S. aureus SA-1199B (norA++) and K1758 (norA-) strains. The study provided two most active compounds viz. 19 and 52. Compound 19 was found to be most active in potentiating effect of norfloxacin and also it showed enhanced uptake, efflux inhibition in ethidium bromide assay. Further compound 19 also enhanced post antibiotic effect and reduced mutation prevention concentration of norfloxacin. The homology modeling study was performed to elucidate three-dimensional structure of NorA. Docking studies of potent molecules were done to find the binding affinity and interaction with active site residues. Further, all the tested compounds exhibited good ADME and drug-likeness properties in- silico. Based on the in-silico studies and detailed in vitro studies, acrylohydrazides derivatives may be considered as potential NorA EPI candidates.

Poly(2‐[dimethylamino]ethyl methacrylate)‐b‐poly(hydroxypropyl methacrylate)/DNA polyplexes in aqueous solutions

AbstractThis study involves the investigation of the complexation ability of poly(2‐[dimethylamino]ethyl methacrylate)‐b‐poly(hydroxypropyl methacrylate) (PDMAEMA‐b‐PHPMA) amphiphilic pH and thermoresponsive block copolymers, and their quaternized counterparts QPDMAEMA‐b‐PHPMA, toward short DNA in aqueous solutions. The PDMAEMA‐b‐PHPMA amphiphilic block copolymers present various self‐assembly characteristics when inserted into aqueous media, depending on the composition, the solubilization protocol, the acidity and the temperature of the aqueous media. Copolymer aggregates‐DNA interactions and nanostructure formation after complexation are investigated by dynamic light scattering and intensity measurements in aqueous solutions in a fixed temperature range, utilizing two different solubilization protocols for the copolymers. Ethidium bromide assays by fluorescence spectroscopy and ζ‐potential measurements were also utilized to investigate the structure and properties of the DNA/copolymer polyplexes. The interpretation of such physicochemical characterization provides extra comprehension of the novel (Q)PDMAEMA‐b‐PHPMA copolymers self‐assembly characteristics and assesses their ability for DNA complexation, stabilization, and delivery.

GC-MS Profile and Enhancement of Antibiotic Activity by the Essential Oil of Ocotea odorífera and Safrole: Inhibition of Staphylococcus aureus Efflux Pumps.

Considering the evidence that essential oils, as well as safrole, could modulate bacterial growth in different resistant strains, this study aims to characterize the phytochemical profile and evaluate the antibacterial and antibiotic-modulating properties of the essential oil Ocotea odorífera (EOOO) and safrole against efflux pump (EP)-carrying strains. The EOOO was extracted by hydrodistillation, and the phytochemical analysis was performed by gas chromatography coupled to mass spectrometry (GC-MS). The antibacterial and antibiotic-modulating activities of the EOOO and safrole against resistant strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were analyzed through the broth microdilution method. The EP-inhibiting potential of safrole in association with ethidium bromide or antibiotics was evaluated using the S. aureus 1199B and K2068 strains, which carry genes encoding efflux proteins associated with antibiotic resistance to norfloxacin and ciprofloxacin, respectively. A reduction in the MIC of ethidium bromide or antibiotics was used as a parameter of EP inhibition. The phytochemical analysis identified 16 different compounds in the EOOO including safrole as the principal constituent. While the EOOO and safrole exerted clinically relevant antibacterial effects against S. aureus only, they potentiated the antibacterial activity of norfloxacin against all strains evaluated by our study. The ethidium bromide and antibiotic assays using the strains of S. aureus SA1119B and K2068, as well as molecular docking analysis, indicated that safrole inhibits the NorA and MepA efflux pumps in S. aureus. In conclusion, Ocotea odorifera and safrole presented promising antibacterial and antibiotic-enhancing properties, which should be explored in the development of drugs to combat antibacterial resistance, especially in strains bearing genes encoding efflux proteins.

Impact of marbofloxacin administration on the emergence of marbofloxacin-resistant E. coli in faecal flora of goats and elucidation of molecular basis of resistance

ObjectivesThe level of resistance immediately prior to slaughter in food-producing animals is of great public health significance because of likely transmission of resistant bacteria via the food chain. MethodsMarbofloxacin was administered to goats at the dose of 2 mg/kg body weight by intramuscular route for 5 days. Faecal Escherichia coli population was monitored and examined for bacteriological procedures. DNA sequencing of gyrA and parC genes was performed to identify mutations at quinolone-resistance determining region, and interaction between marbofloxacin and GyrA was studied by in silico docking. E. coli isolates were screened for plasmid-mediated quinolone resistance genes qnrA, qnrB, qnrS, aac(6′)Ib-cr, qepA, oqxA and oqxB. Efflux pump-mediated resistance was evaluated by ethidium bromide assay, reduction in minimum inhibitory concentration (MIC) values in the presence of efflux pump inhibitors and relative expression of AcrAB-TolC efflux pump. ResultsDuring the treatment period, emergence of marbofloxacin-resistant E. coli strains was observed in gut flora. Quinolone resistance determining regions (QRDRs) in gyrA identified amino acid codon mutations Ser83Leu and Asp87Asn, and Ser80Ile in parC. Docking analysis implied that marbofloxacin could not form strong complexes with mutated DNA-gyrase. A high prevalnce of PMQR genes, especially qnrS, was observed along with overexpression of AcrAB-TolC efflux pump. ConclusionsThe study highlighted the high prevalence of transferable mechanisms of quinolone resistance and over expression of efflux pumps in marbofloxacin-resistant E. coli isolates apart from classic QRDR mutations. The present study recommends to consider the period of dominance of resistant commensals, being excreted by animals during the antimicrobial treatments, while formulating the withdrawal period for drugs, especially in food-producing animals.

5-Nitro-Thiophene-Thiosemicarbazone Derivatives Present Antitumor Activity Mediated by Apoptosis and DNA Intercalation.

Considering the need for the development of new antitumor drugs, associated with the great antitumor potential of thiophene and thiosemicarbazonic derivatives, in this work we promote molecular hybridization approach to synthesize new compounds with increased anticancer activity. Investigate the antitumor activity and their likely mechanisms of action of a series of N-substituted 2-(5-nitro-thiophene)-thiosemicarbazone derivatives. Methods were performed in vitro (cytotoxicity, cell cycle progression, morphological analysis, mitochondrial membrane potential evaluation and topoisomerase assay), spectroscopic (DNA interaction studies), and in silico studies (docking and molecular modelling). Most of the compounds presented significant inhibitory activity; the NCIH-292 cell line was the most resistant, and the HL-60 cell line was the most sensitive. The most promising compound was LNN-05 with IC50 values ranging from 0.5 to 1.9 µg.mL-1. The in vitro studies revealed that LNN-05 was able to depolarize (dose-dependently) the mitochondrial membrane, induceG1 phase cell cycle arrest noticeably, promote morphological cell changes associated with apoptosis in chronic human myelocytic leukaemia (K-562) cells, and presented no topoisomerase II inhibition. Spectroscopic UV-vis and molecular fluorescence studies showed that LNN compounds interact with ctDNA forming supramolecular complexes. Intercalation between nitrogenous bases was revealed through KI quenching and competitive ethidium bromide assays. Docking and Molecular Dynamics suggested that 5-nitro-thiophene-thiosemicarbazone compounds interact against the larger DNA groove, and corroborating the spectroscopic results, may assume an intercalating interaction mode. Our findings highlight 5-nitro-thiophene-thiosemicarbazone derivatives, especially LNN-05, as a promising new class of compounds for further studies to provide new anticancer therapies.

The rhenium(I)-diselenoether anticancer drug targets ROS, TGF-β1, VEGF-A, and IGF-1 in an in vitro experimental model of triple-negative breast cancers.

The rhenium(I)-diselenoether complex (Re-diSe) is a rhenium tricarbonyl-based drug chelated by a diselenoether ligand. In this work, we compared its inhibitory effects on the hormone-independent MDA-MB231cancer line and other different cancer cell lines after an exposure time of 72h by MTT assays. The sensitivity of MDA-MB231 was in the same range than the hormone-dependent MCF-7 breast cancer, the PC-3 prostate and HT-29 colon cancer cells, while the A549 lung and the HeLa uterine cancer cells were less sensitive. We compared the inhibitory effects of Re-diSe and of its diselenide ligand (di-Se) on MDA-MB231 and a normal HEK-293 human embryonic cell line, after 72h and 120h of exposure. The cytotoxicity was also studied by flow cytometry using ethidium bromide assays, as well as the effects on the ROS production by DFCA-test, while the levels of TGF-β1, VEGF-A, IGF-1 were addressed by ELISA tests. The dose required to inhibit 50% of the proliferation (IC50) of MDA-MB231 breast cancer cells decreased with the time of exposure to 120h, while the free ligand (di-Se) was found poorly active, demonstrating the important role of Re in this Re-diSe combination. The cytotoxic effects of Re-diSe were highly selective for cancer cells, with a significant increase of the number of dead cancer cells at 5μM for an exposure time of 120h, while normal cells were not affected. A remarkable and significant decrease of the production of ROS together with a decrease of VEGF-A, TGF-β1, and IGF-1 by the cancer cells were also observed when cancer cells were exposed to Re-diSe.

Comparative in vitro and in silico characterization of anticancer compounds piceatannol, biochanin-A and resveratrol on breast cancer cells

Background: Biochanin-A and Piceatannol are phytochemical constituents extracted from Sophora interrupta. Although both the compounds were isolated from a single plant, these compounds were not compared against anticancer activity. Objective: A systematic comparative analysis of biochanin-A, piceatannol, and resveratrol was performed to investigate cancer cell viability, motility, metabolic changes in Michigan Cancer Foundation-7 breast cancer cells, and structure compound interaction with the vascular endothelial growth factor (VEGF) receptors were studied. Materials and Methods: Cancer cell viability was studied using 3 (4, 5 dimethyl thiazol 2yl) 2, 5 diphenyltetrazo- lium bromide and acridine orange (AO)/ethidium bromide (EtBr) assay. The wound-healing assay was performed by measuring cell migration from the scratch area. Metabolic changes of the compounds in culture conditions were recorded using Fourier-transform infrared (FT-IR) spectroscopy. Molecular docking and dynamic simulations were performed using Schrodinger software. Results: Our results showed that the half-maximal growth inhibitory concentration for biochanin-A is 150 μM/ml and piceatannol and resveratrol showed 150 μM/ml, which is evident from the uptake of AO and EtBr dyes by live/dead cells. Moreover, drug-treated cells were unable to fill the cleared area from the scratch area, which suggests that all compounds effectively inhibit cell motility. FT-IR fingerprint showed a marked difference in the percentage of transition and dynamic structural changes between untreated and treated samples. Strong hydrogen-bond interaction with VEGF receptor-1 (VEGFR1) and VEGFR2 proteins and their interactions were stable throughout the simulation period. Moreover, these compounds inhibited sprouting of a new blood vessel from the chicken aorta and microvessels formation in the in ovo chorioallantoic membrane assay. Conclusion: Taken together, we conclude that anticancer and anti-angiogenic activity, structure-function relationship of biochanin-A is like well-known anticancer compound resveratrol and its metabolic product piceatannol in breast cancer cells.

Carvacrol activity & morphological changes in Mycobacterium tuberculosis.

Evaluating carvacrol, derivatives and carvacrol plus anti-TB (anti-tuberculous) drug combination activities in Mycobacterium tuberculosis as well as carvacrol cytotoxicity, efflux pump inhibitor activity and morphological changes in M. tuberculosis H37Rv. Carvacrol (CAR) and derivatives' activities were determined by resazurin microtiter assay and drug interaction by resazurin drug combination microtiter. Carvacrol cytotoxicity in VERO cells and efflux pumps inhibitor activity by ethidium bromide assay were determined and scanning electron microscopy performed. Carvacrol MIC ranged from 19 to 156μg/ml and carvacrol plus rifampicin combination showed synergistic effect in clinical isolates. No anti-M. tuberculosis activity improvement was observed with carvacrol derivatives. Carvacrol showed to be selective for M. tuberculosis, to have efflux pumps activity and to induce rough bacillary and agglomerates. Carvacrol shows good anti-M. tuberculosis activity and synergism with rifampicin.

New (arene)ruthenium(II) complexes of 4‑aryl‑4H‑naphthopyrans with anticancer and anti-vascular activities

A series of four 2‑amino‑3‑cyano‑4‑(3/4‑pyridyl)‑4H‑benzo[h]chromenes 2a–d and their dichlorido(p‑cymene)ruthenium(II) complexes 3a–d were tested for antiproliferative, vascular-disruptive, anti-angiogenic and DNA-binding activity. The coordination of the 4‑pyridyl‑4H‑naphthopyrans 2 to ruthenium led to complexes with pleiotropic effects. Unlike the free ligands 2a–d, their ruthenium complexes 3a–d showed a significant affinity for DNA as demonstrated by electrophoretic mobility shift assays (EMSA) and ethidium bromide assays. Binding of 3a–d to calf thymus DNA proceeded about 10-times faster compared with cisplatin. Treatment of HT-29 colon carcinoma, 518A2 melanoma and MCF-7Topo breast cancer cells with 3a and 3b caused an accumulation of cells in the G2/M phase and an increase of the fraction of mitotic cells in the case of HT-29, due to alterations of the microtubule cytoskeleton as shown by immunofluorescence staining. Complexes 3b–c showed a dual effect on the vascular system. They suppressed angiogenesis in zebrafish embryos and they destroyed the vasculature of the chorioallantoic membrane (CAM) in fertilized chicken eggs. They also inhibited the vasculogenic mimicry, typical of U-87 glioblastoma cells in tube formation assays.

Apoptosis, Necrosis and Cytotoxicity of Newly Emerging and Developing Precursor Hepatoblast and Neuroblast Stem Cells After Critical Cell and Nucleoli Core Penetration of Small Size Nano Silver

Background: The present study was conducted to examine apoptotic, cytotoxic and necrotic effects of small size silver nano particles on newly emerging and developing precursor hepatoblast and neuroblast stem cells of vital organs of pregnant Swiss Albino mice and their fresh born fetuses. (10 mother and 10 fetuses from each group including control) Colloidal silver nano were imposed in a stabilizer sodium borohydride environment with disaggregate agent polyvinyl pyrollidone via synthesis and defragmentation of silver nitrate crystal with few drops of 1.5 M sodium chloride (NaCl) solution causes the suspension to turn darker yellow, then gray as the silver nano particles aggregates. Material and method: The size of silver nanoparticles was measured by transmission electron microscopy (TEM), and dynamic light scattering. Zeta potential of silver nanoparticles was determined by laser Doppler micro electrophoresis. After exposing newly emerging and developing precursor hepatoblast and neuroblast stem cells of pregnant Swiss Albino mice (5 to 15 gestational age) and their fresh born fetuses to small size nanosilver for 8 to 24 h in serial 8 micro meter fresh tissue serial cryostat sections cytotoxicity, apoptosis and necrosis was measured by Acridine orange, DAPI, 1:250 dilution Ethidium Bromide and combination assay through con focal microscope view. Result: Cytotoxicity and Apoptosis in newly emerging and developing precursor hepatoblast and neuroblast stem cells of pregnant Swiss Albino mice and their fresh born fetuses were detected by Acridine orange, DAPI immune staining and double staining of both combination with propidium iodide also scattered necrosis detected in same in 1:250 dilution Ethidium Bromide and combination assay through con focal microscope view. In TEM and dynamic light scattering analyses, the sizes of nanosilver found varied from 2.75 nm to 20 nm. AgNps were 71.56 nm in hydrodynamic diameter with a zeta potential of -17.52 mV. The Acridine orange, DAPI, 1:250 dilutions Ethidium Bromide and combination assay resulted in an LC50 value of 30 µg/mL. Small size nanosilver caused cytotoxic, apoptotic and necrotic effects in a dose-dependent manner. (0.5, 1, 10 & 15 mg/kg/b. W.) The apoptotic effect of nanosilver was marked up to a concentration of 30 µg/mL. At higher concentrations, the apoptotic effect depleted while the necrotic effect became upraised. Conclusion: The results indicate that small size nanosilver with a zeta potential of -17.52 mV and hydrodynamic diameter of 71.56 nm with TEM diagnosed diameter 2.75 to 20 nm ranges can be used in vitro at concentrations of up to 30 to40 µg/mL.