Novel Combination of Sorafenib and Celecoxib Provides Synergistic Anti-Proliferative and Pro-Apoptotic Effects in Human Liver Cancer Cells

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Sorafenib, a multikinase inhibitor, recently received FDA approval for the treatment of advanced HCC. However, although sorafenib is well tolerated, concern for its safety has been expressed. Celecoxib (Celebrex®) is a selective cyclooxygenase-2 (COX-2) inhibitor which exhibits antitumor effects in human HCC cells. The present study examined the interaction between celecoxib and sorafenib in two human liver tumor cell lines HepG2 and Huh7. Our data showed that each inhibitor alone reduced cell growth and the combination of celecoxib with sorafenib synergistically inhibited cell growth and increased apoptosis. To better understand the molecular mechanisms underlying the synergistic antitumor activity of the combination, we investigated the expression profile of the combination-treated liver cancer cell lines using microarray analysis. Combination treatment significantly altered expression levels of 1,986 and 2,483 transcripts in HepG2 and Huh7 cells, respectively. Genes functionally involved in cell death, signal transduction and regulation of transcription were predominantly up-regulated, while genes implicated in metabolism, cell-cycle control and DNA replication and repair were mainly down-regulated upon treatment. However, combination-treated HCC cell lines displayed specificity in the expression and activity of crucial factors involved in hepatocarcinogenesis. The altered expression of some of these genes was confirmed by semi-quantitative and quantitative RT-PCR and by Western blotting. Many novel genes emerged from our transcriptomic analyses, and further functional analyses may determine whether these genes can serve as potential molecular targets for more effective anti-HCC strategies

Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures.

Formation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, highlighting the physicochemical features and the possible pathway of formation of the particulate structure. Our findings provide a novel detailed knowledge of such a general and alternative aggregation pathway for proteins, this being crucial for a basic and broader understanding of the aggregation phenomena.This is the author's accepted manuscript and will be under embargo until the 3rd of September 2015. The final version is published by ACS in The Journal of Physical Chemistry Letters here: http://pubs.acs.org/doi/abs/10.1021/jz501614e

Subtle pH variation around pH 4.0 affects aggregation kinetics and aggregate characteristics of recombinant human insulin

Insulin is a biotherapeutic protein, which, depending on environmental conditions such as pH, has been shown to form a large variety of aggregates with different structures and morphologies. This work focuses on the formation and characteristics of insulin particulates, dense spherical aggregates having diameters spanning from nanometre to low-micron size. An in-depth investigation of the system is obtained by applying a broad range of techniques for particle sizing and characterisation. An interesting observation was achieved regarding the formation kinetics and aggregate characteristics of the particulates; a subtle change in the pH from pH 4.1 to pH 4.3 markedly affected the kinetics of the particulate formation and led to different particulate sizes, either nanosized or micronsized particles. Also, a clear difference between the secondary structure of the protein particulates formed at the two pH values was observed, where the nanosized particulates had an increased content of aggregated β-structure compared to the micronsized particles. The remaining characteristics of the particles were identical for the two particulate populations. These observations highlight the importance of carefully studying the formulation design space and of knowing the impact of parameters such as pH on the aggregation to secure a drug product in control. Furthermore, the identification of particles only varying in few parameters, such as size, are considered highly valuable for studying the effect of particle features on the immunogenicity potential.Drug Delivery Technolog

Waterborne Electrospinning of α-Lactalbumin Generates Tunable and Biocompatible Nanofibers for Drug Delivery

This is the author accepted manuscript. The final version is available from the American Chemical Society via the DOI in this recordProtein-based drug carriers are an interesting alternative to traditional polymeric drug delivery systems due to their intrinsic biocompatibility and biodegradability. Electrospinning of neat proteins holds advantages over electrospinning of protein mixtures, e.g., whey isolates, such as better control of the physicochemical and biological function of the resulting nanofiber-based system. In this study, we explore electrospinning of the isolated milk protein α-lactalbumin (ALA), which is a whey protein with important nutritional and pharmacological properties. Via waterborne electrospinning of ALA with a minimum amount of poly(ethylene oxide) (PEO) as a cospininng polymer, nanofibers of high protein content were successfully produced (up to 84% (w/w)). We demonstrate the ability to produce ALA-based nanofibers with a high degree of tunability in terms of size, stability in water, and mechanical properties. The nanofibers displayed excellent biocompatibility in vitro as the viability of cultured TR146 human buccal epithelium and NIH 3T3 murine fibroblast cells was not influenced by exposure to ALA-based nanofibers. ALA-based nanofibers were loaded with up to 6% (w/w) ampicilin, and the nanofibers were capable of maintaining the activity of the antibiotic after electrospinning and cross-linking. Using such a property of the material, we demonstrate that ampicillin-loaded nanofibers successfully inhibit the growth of Gram-negative bacteria in vitro. Importantly, after treatment with ampicillin-loaded nanofibers, no bacterial regrowth was observed, which indicates that this treatment may clear eventual persisters to ampicillin. Finally, the structural properties of the native functional protein were maintained after release of ALA from the nanofibers. This promotes our platform, not only as a sustainable protein-based drug delivery system, but also as an innovative solid form of ALA for food and pharmaceutical applications.Villum FoundationDanish Council for Independent Research; Technology and ProductionRoyal SocietyMedical Research Council (MRC)Wellcome Trus

Synthesis and evaluation of new Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold

Abstract: Ruthenium catalyzed 1,3-cycloaddition (click chemistry) of an azido moiety installed on dihydroxycumene scaffold with differently substituted aryl propiolates, gave a new family of 1,4,5-trisubstitued triazole carboxylic acid derivatives that showed high affinity towards Hsp90 associated with cell proliferation inhibition, both in nanomolar range. The 1,5 arrangement of the resorcinol, the aryl moieties, and the presence of an alkyl (secondary) amide in position 4 of the triazole ring, were essential to get high activity. Docking simulations suggested that the triazoles penetrate the Hsp90 ATP binding site. Some 1,4,5-trisubstitued triazole carboxamides induced dramatic depletion of the examined client proteins and a very strong increase in the expression levels of the chaperone Hsp70. In vitro metabolic stability and in vivo preliminary studies on selected compounds have shown promising results comparable to the potent Hsp90 inhibitor NVP-AUY922. One of them, (compound 18; SST0287CL1) was selected for further investigation as the most promising drug candidate

Tracking the heterogeneous distribution of amyloid spherulites and their population balance with free fibrils

The analysis of amyloidogenic systems reveals the appearance of distinct states of aggregation for amyloid fibrils. For different proteins and under specific experimental conditions, amyloid spherulites are recognized as a significant component occurring in several protein model systems used for in vitro fibrillation studies. In this work we have developed an approach to characterize solutions containing a mixture of amyloid spherulites and individual fibrils. Using bovine insulin as the model system, sedimentation kinetics for the amyloid aggregates were followed using a combination of UV-Vis spectroscopy and cross-polarized optical microscopy. Spherulites were identified as the species undergoing sedimentation. A simple mathematical approach allows the description of the kinetics in terms of decay time/rate distribution. Moreover, based on the sedimentation kinetics, a rough estimate of the balance between amyloid spherulites and individual fibrils can be provided. Fitting the experimental data with the proposed physico-chemical approach shows self-consistent results in reasonable agreement with quantitative imaging analysis previously reported. Our results provide new physical insights into the analysis of amyloidogenic systems, providing a method to characterize the heterogeneous distribution of amyloid spherulites and simultaneously distinguish spherulites and free fibril populations. Importantly, the method can be generally applied to the characterization of polydisperse solutions containing optically traceable spherical particles in the micrometric range

Economic and Financial Comparison between Organic and Conventional Farming in Sicilian Lemon Orchards

Sicily has a long tradition in citrus fruit cultivations that with vineyard and olive tree represent the main Mediterranean tree crops. In this paper we have evaluated the economic and financial sustainability of lemon production, both in organic farming and in conventional farming; the two systems differing just for inputs utilized in production process. Economic analysis has been carried out in a representative case study located in the Sicilian northwestern coast, considering an orchard economic life equal to 50 years. Results, which referred to one hectare area, showed both a higher economic and financial sustainability of organic farming respect to conventional farming. The higher profitability of organic farming was due to minor labor requirement and to greater market appreciation for organic products that granted a premium price respect to conventional prices. Moreover, greater profitability of organic farming and use of environmentally friendly inputs in production process make farms competitive and eco-friendly