Highly fluorescent GFPm2+-based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Study of activity of cloned promoters in slow-growing Mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon-optimized gfpm2+ gene, coding for GFPm2+ of highest fluorescence reported till date, mycobacteriophage L5 attP-int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFPm2+ from M. tuberculosis and M. smegmatis genome. Expression of GFPm2+, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2-promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome-integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long-term in vitro growth or stress conditions, or inside macrophages.

Layer-by-Layer Assembled Thin Film of Albumin Nanoparticles for Delivery of Doxorubicin

Protein nanoparticles (NPs) have found significant applications in drug delivery due to their inherent biocompatibility, which is attributed to their natural origin. In this study, bovine serum abumin (BSA) nanoparticles were introduced in multilayer thin film via layer-by-layer self-assembly for localized delivery of the anticancer drug Doxorubicin (Dox). BSA nanoparticles (similar to 100 nm) show a high negative zeta potential in aqueous medium (-55 mV) and form a stable dispersion in water without agglomeration for a long period. Hence, BSA NPs can be assembled on a substrate via layer-by-layer approach using a positively charged polyelectrolyte (chitosan in acidic medium). The protein nature of these BSA nanoparticles ensures the biocompatibility of the film, whereas the availability of functional groups on this protein allows one to tune the property of the self-assembly to have a pH-dependent drug release profile. The growth of multilayer thin film was monitored by UV-visible spectroscopy, and the films were further characterized by atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM). The drug release kinetics of these BSA nanoparticles and their self-assembled thin film has been compared at a physiological pH of 7.4 and an acidic pH of 6.4.

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3'-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or nmers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3) 8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties.

DNA targeting polyaza macrobicyclic dizinc(II) complexes promoting high in vitro caspase dependent anti-proliferative activity against human carcinoma cancer cells

A series of macrobicyclic dizinc(II) complexes Zn2L1-2B](ClO4)(4) (1-6) have been synthesized and characterized (L1-2 are polyaza macrobicyclic binucleating ligands, and B is the N,N-donor heterocyclic base (viz. 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen)). The DNA and protein binding, DNA hydrolysis and anticancer activity of these complexes were investigated. The interactions of complexes 1-6 with calf thymus DNA were studied by spectroscopic techniques, including absorption, fluorescence and CD spectroscopy. The DNA binding constant values of the complexes were found to range from 2.80 x 10(5) to 5.25 x 10(5) M-1, and the binding affinities are in the following order: 3 > 6 > 2 > 5 > 1 > 4. All the dizinc(II) complexes 1-6 are found to effectively promote the hydrolytic cleavage of plasmid pBR322 DNA under anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 3 and 6 under physiological conditions give observed rate constants (k(obs)) of 5.56 +/- 0.1 and 5.12 +/- 0.2 h(-1), respectively, showing a 10(7)-fold rate acceleration over the uncatalyzed reaction of dsDNA. Remarkably, the macrobicyclic dizinc(II) complexes 1-6 bind and cleave bovine serum albumin (BSA), and effectively promote the caspase-3 and caspase-9 dependent deaths of HeLa and BeWo cancer cells. The cytotoxicity of the complexes was further confirmed by lactate dehydrogenase enzyme levels in cancer cell lysate and content media.

Stabilization and Structural Alteration of the G-Quadruplex DNA Made from the Human Telomeric Repeat Mediated by Troger's Base Based Novel Benzimidazole Derivatives

Ligand-induced stabilization of the G-quadruplex DNA structure derived from the single-stranded 3'-overhang of the telomeric DNA is an attractive strategy for the inhibition of the telomerase activity. The agents that can induce/stabilize a DNA sequence into a G-quadruplex structure are therefore potential anticancer drugs. Herein we present the first report of the interactions of two novel bisbenzimidazoles (TBBz1 and TBBz2) based on Troger's base skeleton with the G-quadruplex DNA (G4DNA). These Troger's base molecules stabilize the G4DNA derived from a human telomeric sequence. Evidence of their strong interaction with the G4DNA has been obtained from CD spectroscopy, thermal denaturation, and UV-vis titration studies. These ligands also possess significantly higher affinity toward the G4DNA over the duplex DNA. The above results obtained are in excellent agreement with the biological activity, measured in vitro using a modified TRAP assay. Furthermore, the ligands are selectively more cytotoxic toward the cancerous cells than the corresponding noncancerous cells. Computational studies suggested that the adaptive scaffold might allow these ligands to occupy not only the G-quartet planes but also the grooves of the G4DNA.

Near-infrared light-responsive graphene oxide composite multilayer capsules: a novel route for remote controlled drug delivery

A novel and simple route for near-infrared (NIR)-light controlled release of drugs has been demonstrated using graphene oxide (GO) composite microcapsules based on the unique optical properties of GO. Upon NIR-laser irradiation, the microcapsules were ruptured in a point-wise fashion due to local heating which in turn triggers the light-controlled release of the encapsulated anticancer drug doxorubicin (Dox) from these capsules.

Extracts of Strawberry Fruits Induce Intrinsic Pathway of Apoptosis in Breast Cancer Cells and Inhibits Tumor Progression in Mice

Background: The consumption of berry fruits, including strawberries, has been suggested to have beneficial effects against oxidative stress mediated diseases. Berries contain multiple phenolic compounds and secondary metabolites that contribute to their biological properties. Methodology/Principal Findings: Current study investigates the anticancer activity of the methanolic extract of strawberry (MESB) fruits in leukaemia (CEM) and breast cancer (T47D) cell lines ex vivo, and its cancer therapeutic and chemopreventive potential in mice models. Results of MTT, trypan blue and LDH assays suggested that MESB can induce cytotoxicity in cancer cells, irrespective of origin, in a concentration-and time-dependent manner. Treatment of mice bearing breast adenocarcinoma with MESB blocked the proliferation of tumor cells in a time-dependent manner and resulted in extended life span. Histological and immunohistochemical studies suggest that MESB treatment affected tumor cell proliferation by activating apoptosis and did not result in any side effects. Finally, we show that MESB can induce intrinsic pathway of apoptosis by activating p73 in breast cancer cells, when tumor suppressor gene p53 is mutated. Conclusions/Significance: The present study reveals that strawberry fruits possess both cancer preventive and therapeutic values and we discuss the mechanism by which it is achieved.

Biocides - resistance, cross-resistance mechanisms and assessment

Importance of the field: Antibiotic resistance in bacterial pathogens has increased worldwide leading to treatment failures. Concerns have been raised about the use of biocides as a contributing factor to the risk of antimicrobial resistance (AMR) development. In vitro studies demonstrating increase in resistance have often been cited as evidence for increased risks. It is therefore important to understand the mechanisms of resistance employed by bacteria toward biocides used in consumer products and their potential to impart cross-resistance to therapeutic antibiotics. Areas covered: In this review, the mechanisms of resistance and cross-resistance reported in the literature toward biocides commonly used in consumer products are summarized. The physiological and molecular techniques used in describing and examining these mechanisms are reviewed and application of these techniques for systematic assessment of biocides for their potential to develop resistance and/or cross-resistance is discussed. Expert opinion: The guidelines in the usage of biocides in household or industrial purpose should be monitored and regulated to avoid the emergence of any MDR strains. The genetic and molecular methods to monitor the resistance development to biocides should be developed and included in preclinical and clinical studies.

Structure of an amidohydrolase, SACOL0085, from methicillin-resistant Staphylococcus aureus COL

Staphylococcus aureus is an opportunistic pathogen that rapidly acquires resistance to frontline antibiotics. The characterization of novel protein targets from this bacterium is thus an important step towards future therapeutic strategies. Here, the crystal structure of an amidohydrolase, SACOL0085, from S. aureus COL is described. SACOL0085 is a member of the M20D family of peptidases. Unlike other M20D peptidases, which are either monomers or dimers, SACOL0085 adopts a butterfly-shaped homotetrameric arrangement with extensive intersubunit interactions. Each subunit of SACOL0085 contains two Mn2+ ions at the active site. A conserved cysteine residue at the active site distinguishes M20D peptidases from other M20 family members. This cysteine, Cys103, serves as bidentate ligand coordinating both Mn2+ ions in SACOL0085.

Cytotoxic and pro-apoptotic effects of Abrus precatorius L. on human metastatic breast cancer cell line, MDA-MB-231

Abrus precatorius is highly regarded as a universal panacea in the herbal medicine with diverse pharmacological activity spectra. This experimental study on the mechanism of the anticancer activity of A. precatorius leaf extracts, may offer new evidence for A. precatorius in the treatment of breast cancer in clinical practice. Cell death was determined by using MTT assay. Further analyses were carried out by doing DNA laddering, PARP cleavage, FACS, semi-quantitative RT-PCR and detection of cellular reactive oxygen species (ROS) by DCFDA assay. A. precatorius showed very striking inhibition on MDA-MB-231 cells. MTT assay showed more than 75 % inhibition of the cells and treated cells indicated visible laddering pattern with thick compact band. PARP cleavage produced 89 kDa cleavage product which was associated with apoptosis. Flow cytometer exhibited a sub-G0/G1 peak as an indicative of apoptosis. mRNA expression level of apoptosis-related genes p21 and p53 was markedly increased in cells treated with the extract as compared to control. The up-regulation of p21 and p53 may be the molecular mechanisms by which A. precatorius extract which induces apoptosis. An increase in the concentration of A. precatorius extract does not generate ROS, instead it reduces ROS formation in MDA-MB-231 cells, as evident from the shift in fluorescence below untreated control. This is the first report showing that A. precatorius leaf extract exhibits a growth inhibitory effect by induction of apoptosis in MDA-MB-231 cells. Our results contribute towards validation of the A. precatorius extract as a potentially effective chemopreventive or therapeutic agent against breast cancer.

Surface modification and paclitaxel drug delivery of folic acid modified polyethylene glycol functionalized hydroxyapatite nanoparticles

Nanoparticles are used for a number of biomedical applications. In this work we report the synthesis of folic acid (FA) modified polyethylene glycol (PEG) functionalized hydroxyapatite (HAp) nanoparticles. The anticancer drug, paclitaxel, is attached to the folic acid modified polyethylene glycol functionalized hydroxyapatite nanoparticles and the in vitro drug release is analyzed. The surface modification and functionalization is confirmed by Fourier transform infrared spectroscopy (FTIR), thermo gravimetric analysis (TGA) and UV spectroscopy. The importance of the paper is the investigation of the release behavior of paclitaxel conjugated folic acid modified polyethylene glycol functionalized hydroxyapatite nanoparticles. The results show an initial rapid release and then a sustained release. (C) 2012 Elsevier B.V. All rights reserved.

Intracellular delivery of doxorubicin encapsulated in novel pH-responsive chitosan/heparin nanocapsules

A novel polyelectrolyte nanocapsule system composed of biopolymers, chitosan and heparin has been fabricated by the layer-by-layer technique on silica nanoparticles followed by dissolution of the silica core. The nanocapsules were of the size range 200 +/- 20 nm and loaded with the positively charged anticancer drug doxorubicin with an efficiency of 89%. The loading of the drug into the capsule happens by virtue of the pH-responsive property of the capsule wall, which is determined by the pKa of the polyelectrolytes. As the pH is varied, about 64% of the drug is released in acidic pH while 77% is released in neutral pH. The biocompatibility, efficiency of drug loading, and enhanced bioavailability of the capsule system was confirmed by MTT assay and in vivo biodistribution studies.

CXI-benzo-84 reversibly binds to tubulin at colchicine site and induces apoptosis in cancer cells

Here, we have discovered CXI-benzo-84 as a potential anticancer agent from a library of benzimidazole derivatives using cell based screening strategy. CXI-benzo-84 inhibited cell cycle progression in metaphase stage of mitosis and accumulated spindle assembly checkpoint proteins Mad2 and BubR1 on kinetochores, which subsequently activated apoptotic cell death in cancer cells. CXI-benzo-84 depolymerized both interphase and mitotic microtubules, perturbed EB1 binding to microtubules and inhibited the assembly and GTPase activity of tubulin in vitro. CXI-benzo-84 bound to tubulin at a single binding site with a dissociation constant of 1.2 +/- 0.2 mu M. Competition experiments and molecular docking suggested that CXI-benzo-84 binds to tubulin at the colchicine-site. Further, computational analysis provided a significant insight on the binding site of CXI-benzo-84 on tubulin. In addition to its potential use in cancer chemotherapy, CXI-benzo-84 may also be useful to screen colchicine-site agents and to understand the colchicine binding site on tubulin. (C) 2013 Elsevier Inc. All rights reserved.

Propyl-2-(8-(3,4-Difluorobenzyl)-2 `,5 `-Dioxo-8-AzaspiroBicyclo3.2.1] Octane-3,4 `-Imidazolidine]-1 `-yl) Acetate Induces Apoptosis in Human Leukemia Cells through Mitochondrial Pathway following Cell Cycle Arrest

Background: Due to the functional defects in apoptosis signaling molecules or deficient activation of apoptosis pathways, leukemia has become an aggressive disease with poor prognosis. Although the majority of leukemia patients initially respond to chemotherapy, relapse is still the leading cause of death. Hence targeting apoptosis pathway would be a promising strategy for the improved treatment of leukemia. Hydantoin derivatives possess a wide range of important biological and pharmacological properties including anticancer properties. Here we investigated the antileukemic activity and mechanism of action of one of the potent azaspiro hydantoin derivative, (ASHD). Materials and Methods: To investigate the antileukemic efficacy of ASHD, we have used MTT assay, cell cycle analysis by FACS, tritiated thymidine incorporation assay, Annexin V staining, JC1 staining and western blot analysis. Results: Results showed that ASHD was approximately 3-fold more potent than the parent compounds in inducing cytotoxicity. Tritiated thymidine assay in conjunction with cell cycle analysis suggests that ASHD inhibited the growth of leukemic cells. The limited effect of ASHD on cell viability of normal cells indicated that it may be specifically directed to cancer cells. Translocation of phosphatidyl serine, activation of caspase 3, caspase 9, PARP, alteration in the ratio of BCL2/BAD protein expression as well as the loss of mitochondrial membrane potential suggests activation of the intrinsic pathway of apoptosis. Conclusion: These results could facilitate the future development of novel hydantoin derivatives as chemotherapeutic agents for leukemia.

DNA and protein targeting 1,2,4-triazole based water soluble dinickel(II) complexes enhances antiproliferation and lactate dehydrogenase inhibition

Water soluble dinickel(II) complexes Ni-2(L)(2)(1-2)](NO3)(4) (1-2), where L1-2 are triazole based dinucleating ligands, were synthesized and characterized. The DNA binding, protein binding, DNA hydrolysis and anticancer properties were investigated. The interactions of complexes 1 and 2 with calf thymus DNA were studied by spectroscopic techniques, including absorption and fluorescence spectroscopy. The DNA binding constant values of the complexes 1 and 2 were found to be 2.36 x 10(5) and 4.87 x 10(5) M-1 and the binding affinities are in the following order: 2 > 1. Both the dinickel(II) complexes 1 and 2, promoted the hydrolytic cleavage of plasmid pBR322 DNA under both anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 1 and 2 under physiological conditions give the observed rate constants (k(obs)) of 5.05 +/- 0.2 and 5.65 +/- 0.1 h(-1), respectively, which shows 10(8)-fold rate acceleration over the uncatalyzed reaction of ds-DNA. Meanwhile, the interactions of the complex with BSA have also been studied by spectroscopy. Both the complexes 1 and 2 display strong binding propensity and the binding constant (K-b), number of binding sites (n) were obtained are 0.71 x 10(6) 1.47] and 5.62 x 10(6) 1.98] M-1, respectively. The complexes 1 and 2 also promoted the apoptosis against human carcinoma (HeLa, and BeWo) cancer cells. Cytotoxicity of the complexes was further confirmed by lactate dehydrogenase enzyme level in cancer cell lysate and content media. (c) 2013 Elsevier Ltd. All rights reserved.