Chondroitin Sulfate-Based Nanocapsules as Nanocarriers for Drugs and Nutraceutical Supplements.

Oil-core nanocapsules (NCs, also known as nanoemulsions) are of great interest due to their application as efficient carriers of various lipophilic bioactives, such as drugs. Here, we reported for the first time the preparation and characterization of NCs consisting of chondroitin sulfate (CS)-based shells and liquid oil cores. For this purpose, two amphiphilic CS derivatives (AmCSs) were obtained by grafting the polysaccharide chain with octadecyl or oleyl groups. AmCS-based NCs were prepared by an ultrasound-assisted emulsification of an oil phase consisting of a mixture of triglyceride oil and vitamin E in a dispersion of AmCSs. Dynamic light scattering and cryo-transmission electron microscopy showed that the as-prepared core-shell NCs have typical diameters in the range of 30-250 nm and spherical morphology. Since CS is a strong polyanion, these particles have a very low surface potential, which promotes their stabilization. The cytotoxicity of the CS derivatives and CS-based NCs and their impact on cell proliferation were analyzed using human keratinocytes (HaCaTs) and primary human skin fibroblasts (HSFs). In vitro studies showed that AmCSs dispersed in an aqueous medium, exhibiting mild cytotoxicity against HaCaTs, while for HSFs, the harmful effect was observed only for the CS derivative with octadecyl side groups. However, the nanocapsules coated with AmCSs, especially those filled with vitamin E, show high biocompatibility with human skin cells. Due to their stability under physiological conditions, the high encapsulation efficiency of their hydrophobic compounds, and biocompatibility, AmCS-based NCs are promising carriers for the topical delivery of lipophilic bioactive compounds.

Bronchial Fibroblasts from Asthmatic Patients Display Impaired Responsiveness to Direct Current Electric Fields (dcEFs).

Accumulating evidence suggests that an important role is played by electric signals in modifying cell behaviour during developmental, regenerative and pathological processes. However, their role in asthma has not yet been addressed. Bronchial fibroblasts have recently been identified having important roles in asthma development. Therefore, we adapted an experimental approach based on the lineages of human bronchial fibroblasts (HBF) derived from non-asthmatic (NA) donors and asthmatic (AS) patients to elucidate whether their reactivity to direct current electric fields (dcEF) could participate in the asthmatic process. The efficient responsiveness of NA HBF to an electric field in the range of 2-4 V/cm was illustrated based on the perpendicular orientation of long axes of the cells to the field lines and their directional movement towards the anode. These responses were related to the activity of TGF-ß signalling, as the electrotaxis and re-orientation of NA HBF polarity was impaired by the inhibitors of canonical and non-canonical TGF-ß-dependent pathways. A similar tendency towards perpendicular cell-dcEF orientation was observed for AS HBF. However, their motility remained insensitive to the electric field applied at 2-4 V/cm. Collectively, these observations demonstrate the sensitivity of NA HBF to dcEF, as well as the inter-relations between this parameter and the canonical and non-canonical TGF-ß pathways, and the differences between the electrotactic responses of NA and AS HBF point to the possible role of their dcEFs in desensitisation in the asthmatic process. This process may impair the physiologic behaviour of AS HBF functions, including cell motility, ECM deposition, and contractility, thus promoting bronchial wall remodelling, which is a characteristic of bronchial asthma.

Saponin Fraction CIL1 from Lysimachia ciliata L. Enhances the Effect of a Targeted Toxin on Cancer Cells.

Saponins are plant metabolites that possess multidirectional biological activities, among these is antitumor potential. The mechanisms of anticancer activity of saponins are very complex and depend on various factors, including the chemical structure of saponins and the type of cell they target. The ability of saponins to enhance the efficacy of various chemotherapeutics has opened new perspectives for using them in combined anticancer chemotherapy. Co-administration of saponins with targeted toxins makes it possible to reduce the dose of the toxin and thus limit the side effects of overall therapy by mediating endosomal escape. Our study indicates that the saponin fraction CIL1 of Lysimachia ciliata L. can improve the efficacy of the EGFR-targeted toxin dianthin (DE). We investigated the effect of cotreatment with CIL1 + DE on cell viability in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, on proliferation in a crystal violet assay (CV) and on pro-apoptotic activity using Annexin V/7 Actinomycin D (7-AAD) staining and luminescence detection of caspase levels. Cotreatment with CIL1 + DE enhanced the target cell-specific cytotoxicity, as well as the antiproliferative and proapoptotic properties. We found a 2200-fold increase in both the cytotoxic and antiproliferative efficacy of CIL1 + DE against HER14-targeted cells, while the effect on control NIH3T3 off-target cells was less profound (6.9- or 5.4-fold, respectively). Furthermore, we demonstrated that the CIL1 saponin fraction has a satisfactory in vitro safety profile with a lack of cytotoxic and mutagenic potential.

PPARδ Agonist GW501516 Suppresses the TGF-ß-Induced Profibrotic Response of Human Bronchial Fibroblasts from Asthmatic Patients.

The airway wall remodeling observed in asthma is associated with subepithelial fibrosis and enhanced activation of human bronchial fibroblasts (HBFs) in the fibroblast to myofibroblast transition (FMT), induced mainly by transforming growth factor-ß (TGF-ß). The relationships between asthma severity, obesity, and hyperlipidemia suggest the involvement of peroxisome proliferator-activated receptors (PPARs) in the remodeling of asthmatic bronchi. In this study, we investigated the effect of PPARδ ligands (GW501516 as an agonist, and GSK0660 as an antagonist) on the FMT potential of HBFs derived from asthmatic patients cultured in vitro. This report shows, for the first time, the inhibitory effect of a PPARδ agonist on the number of myofibroblasts and the expression of myofibroblast-related markers-α-smooth muscle actin, collagen 1, tenascin C, and connexin 43-in asthma-related TGF-ß-treated HBF populations. We suggest that actin cytoskeleton reorganization and Smad2 transcriptional activity altered by GW501516 lead to the attenuation of the FMT in HBF populations derived from asthmatics. In conclusion, our data demonstrate that a PPARδ agonist stimulates antifibrotic effects in an in vitro model of bronchial subepithelial fibrosis. This suggests its potential role in the development of a possible novel therapeutic approach for the treatment of subepithelial fibrosis during asthma.

SB203580-A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma.

Subepithelial fibrosis is a component of the remodeling observed in the bronchial wall of patients diagnosed with asthma. In this process, human bronchial fibroblasts (HBFs) drive the fibroblast-to-myofibroblast transition (FMT) in response to transforming growth factor-ß1 (TGF-ß1), which activates the canonical Smad-dependent signaling. However, the pleiotropic properties of TGF-ß1 also promote the activation of non-canonical signaling pathways which can affect the FMT. In this study we investigated the effect of p38 mitogen-activated protein kinase (MAPK) inhibition by SB203580 on the FMT potential of HBFs derived from asthmatic patients using immunocytofluorescence, real-time PCR and Western blotting methods. Our results demonstrate for the first time the strong effect of p38 MAPK inhibition on the TGF-ß1-induced FMT potential throughout the strong attenuation of myofibroblast-related markers: α-smooth muscle actin (α-SMA), collagen I, fibronectin and connexin 43 in HBFs. We suggest the pleiotropic mechanism of SB203580 on FMT impairment in HBF populations by the diminishing of TGF-ß/Smad signaling activation and disturbances in the actin cytoskeleton architecture along with the maturation of focal adhesion sites. These observations justify future research on the role of p38 kinase in FMT efficiency and bronchial wall remodeling in asthma.

Encapsulation of Curcumin in Polystyrene-Based Nanoparticles-Drug Loading Capacity and Cytotoxicity.

Nanoparticles made of amphiphilic block copolymers are commonly used in the preparation of nano-sized drug delivery systems. Poly(styrene)-block -poly(acrylic acid) (PS-PAA) copolymers have been proposed for drug delivery purposes; however, the drug loading capacity and cytotoxicity of PS-PAA nanoparticles are still not fully recognized. Herein, we investigated the accumulation of a model hydrophobic drug, curcumin, and its spatial distribution inside the PS-PAA nanoparticles. Experimental methods and atomistic molecular dynamics simulations were used to understand the molecular structure of the PS core and how curcumin molecules interact and organize within the PS matrix. The hydrophobic core of the PS-PAA nanoparticles consists of adhering individually coiled polymeric chains and is compact enough to prevent post-incorporation of curcumin. However, the drug has a good affinity for the PS matrix and can be efficiently enclosed in the PS-PAA nanoparticles at the formation stage. At low concentrations, curcumin is evenly distributed in the PS core, while its aggregates were observed above ca. 2 wt %. The nanoparticles were found to have relatively low cytotoxicity to human skin fibroblasts, and the presence of curcumin further increased their biocompatibility. Our work provides a detailed description of the interactions between a hydrophobic drug and PS-PAA nanoparticles and information on the biocompatibility of these anionic nanostructures which may be relevant to the development of amphiphilic copolymer-based drug delivery systems.

Responsiveness of human bronchial fibroblasts and epithelial cells from asthmatic and non-asthmatic donors to the transforming growth factor-ß1 in epithelial-mesenchymal trophic unit model.

BACKGROUND: The asthma-related airway wall remodeling is associated i.a. with a damage of bronchial epithelium and subepithelial fibrosis. Functional interactions between human bronchial epithelial cells and human bronchial fibroblasts are known as the epithelial-mesenchymal trophic unit (EMTU) and are necessary for a proper functioning of lung tissue. However, a high concentration of the transforming growth factor-ß1 (TGF-ß1) in the asthmatic bronchi drives the structural disintegrity of epithelium with the epithelial-to-mesenchymal transition (EMT) of the bronchial epithelial cells, and of subepithelial fibrosis with the fibroblast-to-myofibroblast transition (FMT) of the bronchial fibroblasts. Since previous reports indicate different intrinsic properties of the human bronchial epithelial cells and human bronchial fibroblasts which affect their EMT/FMT potential beetween cells derived from asthmatic and non-asthmatic patients, cultured separatelly in vitro, we were interested to see whether corresponding effects could be obtained in a co-culture of the bronchial epithelial cells and bronchial fibroblasts. In this study, we investigate the effects of the TGF-ß1 on the EMT markers of the bronchial epithelial cells cultured in the air-liquid-interface and effectiveness of FMT in the bronchial fibroblast populations in the EMTU models. RESULTS: Our results show that the asthmatic co-cultures are more sensitive to the TGF-ß1 than the non-asthmatic ones, which is associated with a higher potential of the asthmatic bronchial cells for a profibrotic response, analogously to be observed in '2D' cultures. They also indicate a noticeable impact of human bronchial epithelial cells on the TGF-ß1-induced FMT, stronger in the asthmatic bronchial fibroblast populations in comparison to the non-asthmatic ones. Moreover, our results suggest the protective effects of fibroblasts on the structure of the TGF-ß1-exposed mucociliary differentiated bronchial epithelial cells and their EMT potential. CONCLUSIONS: Our data are the first to demonstrate a protective effect of the human bronchial fibroblasts on the properties of the human bronchial epithelial cells, which suggests that intrinsic properties of not only epithelium but also subepithelial fibroblasts affect a proper condition and function of the EMTU in both normal and asthmatic individuals.

Asthma-derived fibroblast to myofibroblast transition is enhanced in comparison to fibroblasts derived from non-asthmatic patients in 3D in vitro culture due to Smad2/3 signalling.

The basic hallmarks of bronchial asthma, one of the most common chronic diseases occurring in the world, are chronic inflammation, remodelling of the bronchial wall and its hyperresponsiveness to environmental stimuli. It was found out that the fibroblast to myofibroblast transition (FMT), a key phenomenon in subepithelial fibrosis of the bronchial wall, was crucial for the development of asthma. Our previous studies showed that HBFs derived from asthmatic patients cultured in vitro display some inherent features which facilitate their TGF-b-induced FMT. Although usefulness of standard '2D' cultures is invaluable, they have many limitations. As HBFs interact with extracellular matrix proteins in the connective tissue, which can affect the FMT potential, we have decided to expand our '2D' model to in vitro cell cultures in 3D using collagen gels. Our results showed that 1.5 mg/ml concentration of collagen is suitable for HBFs growth, motility, and phenotypic shifts. Moreover, we demonstrated that in the TGF-ß1-activated HBF populations derived from asthmatics, the expression of fibrosis-related genes (ACTA2, TAGLN, SERPINE1, COL1A1, FN1 and CCN2) was significantly increased in comparison to the non-asthmatic ones. We also confirmed that it is related to the TGF-ß/Smad2/3 profibrotic pathway intensification. In summary, the results of our study undoubtedly demonstrate that HBFs from asthmatics have unique intrinsic features which predispose them, regardless the culture conditions, to the increased FMT under the influence of TGF-ß1.

Enhanced asthma-related fibroblast to myofibroblast transition is the result of profibrotic TGF-ß/Smad2/3 pathway intensification and antifibrotic TGF-ß/Smad1/5/(8)9 pathway impairment.

Airway remodelling with subepithelial fibrosis, which abolishes the physiological functions of the bronchial wall, is a major issue in bronchial asthma. Human bronchial fibroblasts (HBFs) derived from patients diagnosed with asthma display in vitro predestination towards TGF-ß1-induced fibroblast-to-myofibroblast transition (FMT), a key event in subepithelial fibrosis. As commonly used anti-asthmatic drugs do not reverse the structural changes of the airways, and the molecular mechanism of enhanced asthma-related TGF-ß1-induced FMT is poorly understood, we investigated the balance between the profibrotic TGF-ß/Smad2/3 and the antifibrotic TGF-ß/Smad1/5/9 signalling pathways and its role in the myofibroblast formation of HBF populations derived from asthmatic and non-asthmatic donors. Our findings showed for the first time that TGF-ß-induced activation of the profibrotic Smad2/3 signalling pathway was enhanced, but the activation of the antifibrotic Smad1/5/(8)9 pathway by TGF-ß1 was significantly diminished in fibroblasts from asthmatic donors compared to those from their healthy counterparts. The impairment of the antifibrotic TGF-ß/Smad1/5/(8)9 pathway in HBFs derived from asthmatic donors was correlated with enhanced FMT. Furthermore, we showed that Smad1 silencing in HBFs from non-asthmatic donors increased the FMT potential in these cells. Additionally, we demonstrated that activation of antifibrotic Smad signalling via BMP7 or isoliquiritigenin [a small-molecule activator of the TGF-ß/Smad1/5/(8)9 pathway] administration prevents FMT in HBFs from asthmatic donors through downregulation of profibrotic genes, e.g., α-SMA and fibronectin. Our data suggest that influencing the balance between the antifibrotic and profibrotic TGF-ß/Smad signalling pathways using BMP7-mimetic compounds presents an unprecedented opportunity to inhibit subepithelial fibrosis during airway remodelling in asthma.

Cinnamic acid derivatives as chemosensitising agents against DOX-treated lung cancer cells - Involvement of carbonyl reductase 1.

Doxorubicin (DOX) therapy is limited by both cancer cells resistance and cardiotoxicity. DOX biotransformation to doxorubicinol (DOXol) by reductases enzymes (mainly by CBR1; carbonyl reductase 1) is a key process responsible for DOX adverse effects development. Thus, inhibition of CBR1 can increase the therapeutic effect of DOX. In the present study, we used a group of new synthetized cinnamic acid (CA) derivatives to improve the effectiveness and safety profile of DOX therapy against cancer cells in vitro. The possible mechanism of CBR1 inhibition was simulated by molecular modelling studies. The kinetics of DOX reduction in the presence of active CA derivatives were measured in cytosols. The chemosensitising activity of CA derivatives including proapoptotic, anti-invasiveness activity were investigated in A549 lung cancer cell line. In our research 7 from 16 tested CA derivatives binded to the active site of CBR1 enzyme and improved DOX stability by inhibition of DOXol formation. Co-treatment of A549 cells with active CA derivatives and DOX induced cells apoptosis by activation of caspase cascade. At the same time we observed decrease of invasive properties (cell migration and transmigration assays) and the rearangments of F-actin cytoskeleton in CA derivatves + DOX treated cells. Meanwhile, control, human lung fibroblasts stay realtivelly unvulnerable and viable. New synthetized CA derivatives may inhibit the activity of CBR1 leading to the stabilization of DOX therapeutic levels in cancer cells and to protect the myocardium against DOXol cytotoxic effect. Favourable physicochemical properties supported by a safety profile and multidirectional chemosensitising activity render CA derivatives a promising group for the development of agent useful in combined therapy.

Saponins as chemosensitizing substances that improve effectiveness and selectivity of anticancer drug-Minireview of in vitro studies.

Triterpene saponins (saponosides) are found in higher plants and display a wide range of biological and pharmacological activities. The antitumor effects of saponins have been proved by their cytotoxic, cytostatic, proapoptotic, and anti-invasive effects in many cellular models. Saponins hold great potential for being developed into chemopreventive and chemotherapeutic drugs. A promising way of reducing the adverse effects of chemotherapy without attenuating its efficiency is provided by the combined application of chemotherapeutic agents and saponosides in subtoxic concentrations. Until recently, saponosides were primarily used as adjuvants that enhance the effect of vaccines. In cancer therapy, saponins are applied in combination with immunotoxins because they increase the selectivity of given immunotoxins against cancer cells and therefore inure normal cells to the cytotoxic effects of immunotoxins. Significantly, certain saponins have been identified that drastically enhance the efficacy of many chemotherapeutic agents, including cisplatin, paclitaxel, doxorubicin, docetaxel, mitoxantrone, and cyclophosphamide. Moreover, saponins used in combination therapy enhance the sensitivity of chemoresistant tumor cells to clinically used chemotherapeutic agents. This review sheds light on the molecular mechanisms underlying cancer co-treatment with saponins and chemotherapy, with a particular focus on modulation of the cell signaling pathways associated with the promotion and progression of cancer cell proliferation, apoptosis, and metastasis.

Increased elasticity of melanoma cells after low-LET proton beam due to actin cytoskeleton rearrangements.

Cellular response to non-lethal radiation stress include perturbations in DNA repair, angiogenesis, migration, and adhesion, among others. Low-LET proton beam radiation has been shown to induce somewhat different biological response than photon radiation. For example, we have shown that non-lethal doses of proton beam radiation inhibited migration of cells and that this effect persisted long-term. Here, we have examined cellular elasticity and actin cytoskeleton organization in BLM cutaneous melanoma and Mel270 uveal melanoma cells. Proton beam radiation increased cellular elasticity to a greater extent than X-rays and both types of radiation induced changes in actin cytoskeleton organization. Vimentin level increased in BLM cells after both types of radiation. Our data show that cell elasticity increased substantially after low-LET proton beam and persisted long after radiation. This may have significant consequences for the migratory properties of melanoma cells, as well as for the cell susceptibility to therapy.

Synergistic anticancer activity of doxorubicin and piperlongumine on DU-145 prostate cancer cells - The involvement of carbonyl reductase 1 inhibition.

One of the causes of therapeutic failure of chemotherapy is cancer cell resistance. In the case of anthracyclines, many resistance mechanisms have been described. One of them assumes the role of carbonyl reductase 1 (CBR1), a cytosolic enzyme that is responsible for the biotransformation process of anthracyclines to less active, undesirable metabolites. Therefore, CBR1 inhibitors are considered for use as a chemosensitizing agents. In the present study, piperlongumine (PL), a Piper longum L. alkaloid that has previously been described as a CBR1 inhibitor, was investigated for its chemosensitizing properties in co-treatment with doxorubicin (DOX). The biotransformation process of DOX in the presence of PL was tracked using human cytosol fraction and LC-MS, then a molecular modeling study was conducted to predict the interaction of PL with the active site of the CBR1. The biological interaction between DOX and PL was investigated using DU-145 prostate cancer cells. Cytotoxic and antiproliferative properties of DOX and PL were examined, and the type and potency of interaction was quantified by Combination Index. The mechanism of the cell death induced by the agents was investigated by flow cytometry and the anti-invasive properties of the drugs were determined by monitoring the movement of individual cells. PL showed dose-dependent inhibition of DOX metabolism in cytosol, which resulted in less doxorubicinol (DOXol) metabolite being formed. The possible mechanism of CBR1 inhibition was explained through molecular modeling studies by prediction of PL's binding mode in the active site of the enzyme's crystal structure-based model. DOX and PL showed a synergistic antiproliferative and proapoptotic effect on cancer cells. Significant anti-invasive properties of the combination of DOX and PL were found, but when the drugs were used separately they did not alter the cancer cells' motility. Cell motility inhibition was accompanied by significant changes in cytoskeleton architecture. DOX and PL used in co-treatment showed significant synergistic anticancer properties. Inhibition of DOX metabolism by PL was found to be a mechanism that was likely to be responsible for the observed interaction.

Major terpenoids from Telekia speciosa flowers and their cytotoxic activity in vitro.

In addition to known constituents of Telekia speciosa, an acetone extract from ray florets of the plant yielded: 5,5'-dibutoxy-2,2'-bifuran (1), 5,5'-diisobutoxy-2,2'-bifuran (2), α-tocopherol (3), ß-tocopherol (4), loliolide palmitate (5), a mixture of calenduladiol esters - 16ß-hydroxylupeol-3-O-palmitate (7) and 16ß-hydroxylupeol-3-O-myristate (8), 1-epiinuviscolide (12), inuviscolide (13), 3-epiisotelekin (16), 4α-hydroxy-9ß,10ß-epoxy-1ß(H)-11(13)-guaien-8α,12-olide (17), 4α-hydroxy-1ß(H)-9(10),11(13)-guaiadien-8α,12-olide (18), loliolide (19) and 4ß,10ß-dihydroxy-1α(H),5α(H)-11(13)-guaien-8α,12-olide (20). Calenduladiol esters and asperilin (14) were the major constituents of the extract. Their cytotoxic effect on human normal prostate epithelial cells (PNT-2), human prostate carcinoma cell lines, human skin fibroblasts (HSF) and human melanoma cell lines was examined in vitro. Triterpene esters showed no cytotoxicity against nearly all cell lines tested, except for Du145 prostate carcinoma cells (IC50 - 62.0 µΜ). Asperilin displayed activity against the cell lines under study, especially against three tested lines of melanomas (A375, IC50 - 17.6 µΜ, WM793, IC50 - 28.2 µΜ and Hs 294T, IC50 - 29.5 µΜ).

Correction to: Fibroblast-to-myofibroblast transition in bronchial asthma.

In the original publication, funding information was inadvertently omitted.

Invasive bronchial fibroblasts derived from asthmatic patients activate lung cancer A549 cells in vitro.

Epidemiological data suggests that there are functional links between bronchial asthma and lung carcinogenesis. Bronchial fibroblasts serve a prominent role in the asthmatic process; however, their involvement in lung cancer progression remains unaddressed. To estimate the effect of the asthmatic microenvironment on the invasiveness of lung cancer cells, the present study compared the behavior of human non-small cell lung cancer A549 cells exposed to the signals from human bronchial fibroblasts (HBFs) derived from non-asthmatic donors (NA HBFs) and from asthmatic patients (AS HBFs). NA HBFs did not significantly affect A549 motility, whereas AS HBFs and the media conditioned with AS HBF/A549 co-cultures increased Snail-1/connexin43 expression and motility of A549 cells. In contrast to NA HBFs, which formed A549-impenetrable lateral barriers, α-SMA+ AS HBFs actively infiltrated A549 monolayers and secreted chemotactic factors that arrested A549 cells within AS HBF/A549 contact zone. However, small sub-populations of A549 cells could release from this arrest and colonize distant regions of AS HBF monolayers. These data indicated that the interactions between lung cancer cells and HBFs in asthmatic bronchi may facilitate the colonization of lung tumors by fibroblasts. It further stabilizes the tumor microenvironment and potentially facilitates collective colonization of novel bronchial loci by cancer cells. Potential mechanistic links between the asthmatic process and lung cancer progression suggest that bronchial asthma should be included in the list of potential prognostic markers for lung cancer therapy.

Fibroblast-to-myofibroblast transition in bronchial asthma.

Bronchial asthma is a chronic inflammatory disease in which bronchial wall remodelling plays a significant role. This phenomenon is related to enhanced proliferation of airway smooth muscle cells, elevated extracellular matrix protein secretion and an increased number of myofibroblasts. Phenotypic fibroblast-to-myofibroblast transition represents one of the primary mechanisms by which myofibroblasts arise in fibrotic lung tissue. Fibroblast-to-myofibroblast transition requires a combination of several types of factors, the most important of which are divided into humoural and mechanical factors, as well as certain extracellular matrix proteins. Despite intensive research on the nature of this process, its underlying mechanisms during bronchial airway wall remodelling in asthma are not yet fully clarified. This review focuses on what is known about the nature of fibroblast-to-myofibroblast transition in asthma. We aim to consider possible mechanisms and conditions that may play an important role in fibroblast-to-myofibroblast transition but have not yet been discussed in this context. Recent studies have shown that some inherent and previously undescribed features of fibroblasts can also play a significant role in fibroblast-to-myofibroblast transition. Differences observed between asthmatic and non-asthmatic bronchial fibroblasts (e.g., response to transforming growth factor ß, cell shape, elasticity, and protein expression profile) may have a crucial influence on this phenomenon. An accurate understanding and recognition of all factors affecting fibroblast-to-myofibroblast transition might provide an opportunity to discover efficient methods of counteracting this phenomenon.

Fenofibrate Reduces the Asthma-Related Fibroblast-To-Myofibroblast Transition by TGF-Β/Smad2/3 Signaling Attenuation and Connexin 43-Dependent Phenotype Destabilization.

The activation of human bronchial fibroblasts by transforming growth factor-ß1 (TGF-ß1) leads to the formation of highly contractile myofibroblasts in the process of the fibroblast⁻myofibroblast transition (FMT). This process is crucial for subepithelial fibrosis and bronchial wall remodeling in asthma. However, this process evades current therapeutic asthma treatment strategies. Since our previous studies showed the attenuation of the TGF-ß1-induced FMT in response to lipid-lowering agents (e.g., statins), we were interested to see whether a corresponding effect could be obtained upon administration of hypolipidemic agents. In this study, we investigated the effect of fenofibrate on FMT efficiency in populations of bronchial fibroblasts derived from asthmatic patients. Fenofibrate exerted a dose-dependent inhibitory effect on the FMT, even though it did not efficiently affect the expression of α-smooth muscle actin (α-SMA; marker of myofibroblasts); however, it considerably reduced its incorporation into stress fibers through connexin 43 regulation. This effect was accompanied by disturbances in the actin cytoskeleton architecture, impairments in the maturation of focal adhesions, and the fenofibrate-induced deactivation of TGF-ß1/Smad2/3 signaling. These data suggest that fenofibrate interferes with myofibroblastic differentiation during asthma-related subepithelial fibrosis. The data indicate the potential application of fenofibrate in the therapy and prevention of bronchial remodeling during the asthmatic process.

Connective tissue growth factor regulates transition of primary bronchial fibroblasts to myofibroblasts in asthmatic subjects.

Fibroblast to myofibroblast transition (FMT) contributes to bronchial wall remodelling in persistent asthma. Among other numerous factors involved, transforming growth factor type ß (TGF-ß) plays a pivotal role. Recently it has been demonstrated that connective tissue growth factor (CTGF), a matricellular protein, combines with TGF-ß in the pathomechanism of many fibrotic disorders. However, it is not clear whether this interaction takes place in asthma as well. Primary cultures of human bronchial fibroblasts from asthmatic and non-asthmatic subjects were used to investigate the impact of CTGF and TGF-ß1 on the fibroblast to myofibroblast transition. The combined activity of TGF-ß1 and CTGF resulted in an average of 90% of FMT accomplished in cell lines derived from asthmatics. In this group FMT was highly dependent on the presence of CTGF produced by the cells, as shown by gene silencing experiments with the specific siRNA. Results support the important role of CTGF biosynthesis in the asthmatic bronchi amplifying FMT. This is evidenced by inhibition of TGF-ß1-induced FMT following CTGF silencing in asthmatic bronchial fibroblasts. CTGF is produced by fibroblasts and contributes to the FMT phenomenon in positive loop-back, inducing and boosting TGF-ß1 triggered FMT. Thus, CTGF is a promising target for pharmacological intervention in secondary prevention of bronchial remodelling in asthma.

Proton beam irradiation inhibits the migration of melanoma cells.

PURPOSE: In recent years experimental data have indicated that low-energy proton beam radiation might induce a difference in cellular migration in comparison to photons. We therefore set out to compare the effect of proton beam irradiation and X-rays on the survival and long-term migratory properties of two cell lines: uveal melanoma Mel270 and skin melanoma BLM. MATERIALS AND METHODS: Cells treated with either proton beam or X-rays were analyzed for their survival using clonogenic assay and MTT test. Long-term migratory properties were assessed with time-lapse monitoring of individual cell movements, wound test and transpore migration, while the expression of the related proteins was measured with western blot. RESULTS: Exposure to proton beam and X-rays led to similar survival but the quality of the cell colonies was markedly different. More paraclones with a low proliferative activity and fewer highly-proliferative holoclones were found after proton beam irradiation in comparison to X-rays. At 20 or 40 days post-irradiation, migratory capacity was decreased more by proton beam than by X-rays. The beta-1-integrin level was decreased in Mel270 cells after both types of radiation, while vimentin, a marker of EMT, was increased in BLM cells only. CONCLUSIONS: We conclude that proton beam irradiation induced long-term inhibition of cellular motility, as well as changes in the level of beta-1 integrin and vimentin. If confirmed, the change in the quality, but not in the number of colonies after proton beam irradiation might favor tumor growth inhibition after fractionated proton therapy.