Indirect co-culture of lung carcinoma cells with hyperthermia-treated mesenchymal stem cells influences tumor spheroid growth in a collagen-based 3-dimensional microfluidic model.

BACKGROUND: Mesenchymal stem cells (MSCs) have paradoxically been reported to exert either pro- or anti-tumor effects in vitro. Hyperthermia, in combination with chemotherapy, has tumor-inhibiting effects; however, its role, together with MSCs, so far is not well understood. Furthermore, a lot of research is conducted using conventional 2-dimensional in vitro models that do not mimic the actual tumor microenvironment. AIM: In light of this fact, an indirect method of co-culturing human amniotic membrane-derived MSCs (AMMSCs) with collagen-encapsulated human lung carcinoma cells (A549) was performed using a 3-dimensional (3D) tumor-on-chip device. METHODS: The conditioned medium of AMMSCs (AMMSC-CM) or heat-treated AMMSCs (heat-AMMSC-CM) was utilized to create indirect co-culture conditions. Tumor spheroid growth characterization, immunocytochemistry and cytotoxicity assays, and anti-cancer peptide (P1) screening were performed to determine the effects of the conditioned medium. RESULTS: The A549 cells cultured inside the 3D microfluidic chip developed into multicellular tumor spheroids over five days of culture. The AMMSC-CM, contrary to previous reports claiming its tumor-inhibiting potential, led to significant proliferation of tumor spheroids. Heat-AMMSC-CM led to reductions in both spheroid diameter and cell proliferation. The medium containing the P1 peptide was found to be the least cytotoxic to tumor spheroids in co-culture compared with the monoculture and heat-co-culture groups. CONCLUSIONS: Hyperthermia, in combination with the anticancer peptide, exhibited highest cytotoxic effects. This study highlights the growing importance of 3D microfluidic tumor models for testing stem-cell-based and other anti-cancer therapies.

Effects of Rationally Designed Physico-Chemical Variants of the Peptide PuroA on Biocidal Activity towards Bacterial and Mammalian Cells.

Antimicrobial peptides (AMPs) often exhibit wide-spectrum activities and are considered ideal candidates for effectively controlling persistent and multidrug-resistant wound infections. PuroA, a synthetic peptide based on the tryptophan (Trp)-rich domain of the wheat protein puroindoline A, displays strong antimicrobial activities. In this work, a number of peptides were designed based on PuroA, varying in physico-chemical parameters of length, number of Trp residues, net charge, hydrophobicity or amphipathicity, D-versus L-isomers of amino acids, cyclization or dimerization, and were tested for antimicrobial potency and salt and protease tolerance. Selected peptides were assessed for effects on biofilms of methicillin-resistant Staphylococcus aureus (MRSA) and selected mammalian cells. Peptide P1, with the highest amphipathicity, six Trp and a net charge of +7, showed strong antimicrobial activity and salt stability. Peptides W7, W8 and WW (seven to eight residues) were generally more active than PuroA and all diastereomers were protease-resistant. PuroA and certain variants significantly inhibited initial biomass attachment and eradicated preformed biofilms of MRSA. Further, P1 and dimeric PuroA were cytotoxic to HeLa cells. The work has led to peptides with biocidal effects on common human pathogens and/or anticancer potential, also offering great insights into the relationship between physico-chemical parameters and bioactivities, accelerating progress towards rational design of AMPs for therapeutics.

Selective Cytotoxicity of a Novel Trp-Rich Peptide against Lung Tumor Spheroids Encapsulated inside a 3D Microfluidic Device.

There is a globally rising healthcare need to develop new anticancer therapies as well as to test them on biologically relevant in vitro cancer models instead of overly simplistic 2D models. To address both these needs, a 3D lung cancer spheroid model is developed using human A549 cells trapped inside a collagen gel in a compartmentalized microfluidic device and homogenously sized (35-45 µm) multicellular tumor spheroids are obtained in 5 days. The novel tryptophan-rich peptide P1, identified earlier as a potential anticancer peptide (ACP), shows enhanced cytotoxic efficacy against A549 tumor spheroids (>75%) in clinically relevant low concentrations, while it does not affect human amniotic membrane mesenchymal stem cells at the same concentrations (<15%). The peptide also inhibits the formation of tumor spheroids by reducing cell viability as well as lowering the proliferative capacity, which is confirmed by the expression of cell proliferation marker Ki-67. The ACP offers a novel therapeutic strategy against lung cancer cells without affecting healthy cells. The microfluidic device used is likely to be useful in helping develop models for several other cancer types to test new anticancer agents.

Using fluorescence lifetime dequenching to estimate the average quinary stoichiometry of proteins in living cells.

Biological proteins are understood in terms of five structural levels-primary, secondary, tertiary, quaternary and quinary. The quinary structure is defined as the set of macromolecular interactions that are transient in vivo. This includes non-covalent protein-protein interactions occurring within the crowded intracellular environment. For much of twentieth century science, the canonical approach to studying biological proteins involved test tube environments. These uncrowded in vitro studies inadvertently failed to replicate and observe the quinary structures present within the original cells. Consequently, contemporary literature surrounding the fifth level of protein organisation is lacking. In particular, there is a lack of literature on the size of transient clusters within living cells. In an attempt to reconcile this gap in knowledge, we propose a quantitative method for estimating the average quinary stoichiometry in living cells. The method is based on lifetime self-quenching of fluorescently-labelled proteins in living cells. Close approach of two or more proteins in a quinary complex will result in self-quenching of the fluorescence lifetime from the fluorescent labels. Our method utilises the random mixing of proteins during cell division to mix fluorescently labelled with unlabelled proteins. Such mixing reduces the probability of adjacency between labelled proteins and, hence, decreases the probability of fluorescence lifetime quenching from labels. By monitoring fluorescence lifetime dequenching during multiple cell divisions, we can determine the average quinary structure in living proliferating cells. We demonstrate this method with a case study on cultured HeLa cells. The average quinary stoichiometry was found to be between five and six. That is, at any given point in time, there are five or six weakly interacting partners in the immediate neighbourhood of any given protein.

Antimicrobial peptides: biochemical determinants of activity and biophysical techniques of elucidating their functionality.

Antimicrobial peptides (AMPs) have been established over millennia as powerful components of the innate immune system of many organisms. Due to their broad spectrum of activity and the development of host resistance against them being unlikely, AMPs are strong candidates for controlling drug-resistant pathogenic microbial pathogens. AMPs cause cell death through several independent or cooperative mechanisms involving membrane lysis, non-lytic activity, and/or intracellular mechanisms. Biochemical determinants such as peptide length, primary sequence, charge, secondary structure, hydrophobicity, amphipathicity and host cell membrane composition together influence the biological activities of peptides. A number of biophysical techniques have been used in recent years to study the mechanisms of action of AMPs. This work appraises the molecular parameters that determine the biocidal activity of AMPs and overviews their mechanisms of actions and the diverse biochemical, biophysical and microscopy techniques utilised to elucidate these.

Revealing the sequence of interactions of PuroA peptide with Candida albicans cells by live-cell imaging.

To determine the mechanism(s) of action of antimicrobial peptides (AMPs) it is desirable to provide details of their interaction kinetics with cellular, sub-cellular and molecular targets. The synthetic peptide, PuroA, displays potent antimicrobial activities which have been attributed to peptide-induced membrane destabilization, or intracellular mechanisms of action (DNA-binding) or both. We used time-lapse fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM) to directly monitor the localization and interaction kinetics of a FITC- PuroA peptide on single Candida albicans cells in real time. Our results reveal the sequence of events leading to cell death. Within 1 minute, FITC-PuroA was observed to interact with SYTO-labelled nucleic acids, resulting in a noticeable quenching in the fluorescence lifetime of the peptide label at the nucleus of yeast cells, and cell-cycle arrest. A propidium iodide (PI) influx assay confirmed that peptide translocation itself did not disrupt the cell membrane integrity; however, PI entry occurred 25-45 minutes later, which correlated with an increase in fractional fluorescence of pores and an overall loss of cell size. Our results clarify that membrane disruption appears to be the mechanism by which the C. albicans cells are killed and this occurs after FITC-PuroA translocation and binding to intracellular targets.

Anti-biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins.

The broad-spectrum activity of antimicrobial peptides (AMPs) and low probability of development of host resistance make them excellent candidates as novel bio-control agents. A number of AMPs are found to be cationic, and a small proportion of these are tryptophan-rich. The puroindolines (PIN) are small, basic proteins found in wheat grains with proposed roles in biotic defence of seeds and seedlings. Synthetic peptides based on their unique tryptophan-rich domain (TRD) display antimicrobial properties. Bacterial endospores and biofilms are highly resistant cells, with significant implications in both medical and food industries. In this study, the cationic PIN TRD-based peptides PuroA (FPVTWRWWKWWKG-NH2 ) and Pina-M (FSVTWRWWKWWKG-NH2 ) and the related barley hordoindoline (HIN) based Hina (FPVTWRWWTWWKG-NH2 ) were tested for effects on planktonic cells and biofilms of the common human pathogens including Pseudomonas aeruginosa, Listeria monocytogenes and the non-pathogenic Listeria innocua. All peptides showed significant bactericidal activity. Further, PuroA and Pina-M at 2 × MIC prevented initial biomass attachment by 85-90% and inhibited >90% of 6-h preformed biofilms of all three organisms. However Hina, with a substitution of Lys-9 with uncharged Thr, particularly inhibited Listeria biofilms. The PIN based peptides were also tested against vegetative cells and endospores of Bacillus subtilis. The results provided evidence that these tryptophan-rich peptides could kill B. subtilis even in sporulated state, reducing the number of viable spores by 4 log units. The treated spores appeared withered under scanning electron microscopy. The results establish the potential of these tryptophan-rich peptides in controlling persistent pathogens of relevance to food industries and human health. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Archetypal tryptophan-rich antimicrobial peptides: properties and applications.

Drug-resistant microorganisms ('superbugs') present a serious challenge to the success of antimicrobial treatments. Subsequently, there is a crucial need for novel bio-control agents. Many antimicrobial peptides (AMPs) show a broad-spectrum activity against bacteria, fungi or viruses and are strong candidates to complement or substitute current antimicrobial agents. Some AMPs are also effective against protozoa or cancer cells. The tryptophan (Trp)-rich peptides (TRPs) are a subset of AMPs that display potent antimicrobial activity, credited to the unique biochemical properties of tryptophan that allow it to insert into biological membranes. Further, many Trp-rich AMPs cross bacterial membranes without compromising their integrity and act intracellularly, suggesting interactions with nucleic acids and enzymes. In this work, we overview some archetypal TRPs derived from natural sources, i.e., indolicidin, tritrpticin and lactoferricin, summarising their biochemical properties, structures, antimicrobial activities, mechanistic studies and potential applications.