Hyaluronic Acid Synthesis Research Articles

Hyaluronic Acid and Its Derivatives in Coating and Delivery Systems: Applications in Tissue Engineering, Regenerative Medicine and Immunomodulation.

As an Extracellular Matrix (ECM) component, Hyaluronic acid (HA) plays a multi-faceted role in cell migration, proliferation and differentiation at micro level and system level events such as tissue water homeostasis. Among its biological functions, it is known to interact with cytokines and contribute to their retention in ECM microenvironment. In addition to its biological functions, it has advantageous physical properties which result in the industrial endeavors in the synthesis and extraction of HA for variety of applications ranging from medical to cosmetic. Recently, HA and its derivatives have been the focus of active research for applications in biomedical device coatings, drug delivery systems and in the form of scaffolds or cell-laden hydrogels for tissue engineering. A specific reason for the increase in use of HA based structures is their immunomodulatory and regeneration inducing capacities. In this context, this article reviews recent literature on modulation of the implantable biomaterial microenvironment by systems based on HA and its derivatives, particularly hydrogels and microscale coatings that are able to deliver cytokines in order to reduce the adverse immune reactions and promote tissue healing.

CD44 and RHAMM are essential for rapid growth of bladder cancer driven by loss of Glycogen Debranching Enzyme (AGL).

BackgroundLoss of Amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL) drives rapid proliferation of bladder cancer cells by upregulating Hyaluronic acid(HA) Synthase (HAS2) mediated HA synthesis. However the role of HA receptors CD44 and Hyaluronan Mediated Motility Receptor (RHAMM) in regulating the growth of bladder cancer cells driven by loss of AGL has not been studied.MethodsWestern blot analysis and Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was carried out to study cellular apoptosis with HAS2, CD44 and RHAMM loss in bladder cancer cells with and without AGL expression. Proliferation and softagar assays were carried out to study cellular anchorage dependent and independent growth. Clinicopathologic analysis was carried out on bladder cancer patient datasets.ResultsHigher amounts of cleaved Cas3, Cas9 and PARP was observed in AGL low bladder cancer cell with loss of HAS2, CD44 or RHAMM. TUNEL staining showed more apoptotic cells with loss of HAS2, CD44 or RHAMM in AGL low bladder cancer cells. This revealed that bladder cancer cells whose aggressive growth is mediated by loss of AGL are susceptible to apoptosis with loss of HAS2, CD44 or RHAMM. Interestingly loss of either CD44 or RHAMM induces apoptosis in different low AGL expressing bladder cancer cell lines. Growth assays showed that loss of CD44 and RHAMM predominantly inhibit anchorage dependent and independent growth of AGL low bladder cancer cells. Clinicopathologic analysis revealed that high RHAMM mRNA expression is a marker of poor patient outcome in bladder cancer and patients with high RHAMM and low AGL tumor mRNA expression have poor survival.ConclusionOur findings strongly point to the importance of the HAS2-HA-CD44/RHAMM pathway for rapid growth of bladder cancer cells with loss of AGL and provides rational for targeting this pathway at various steps for “personalized” treatment of bladder cancer patients based of their AGL expression status.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2756-5) contains supplementary material, which is available to authorized users.

Multifunctional Hyaluronic Acid and Chondroitin Sulfate Nanoparticles: Impact of Glycosaminoglycan Presentation on Receptor Mediated Cellular Uptake and Immune Activation.

Hyaluronic acid (HA) and chondroitin sulfate (CS) polymers are extensively used for various biomedical applications, such as for tissue engineering, drug delivery, and gene delivery. Although both these biopolymers are known to target cell surface CD44 receptors, their relative cellular targeting properties and immune activation potential have never been evaluated. In this article, we present the synthesis and characterization of novel self-assembled supramolecular HA and CS nanoparticles (NPs). These NPs were developed using fluorescein as a hydrophobic component that induced amphiphilicity in biopolymers and also efficiently stabilized anticancer drug doxorubicin (DOX) promoting a near zero-order drug release. The cellular uptake and cytotoxicity studies of these NPs in different human cancer lines, namely, human colorectal carcinoma cell line HCT116 and human breast cancer cell line MCF-7 demonstrated dose dependent cytotoxicity. Interestingly, both NPs showed CD44 dependent cellular uptake with the CS-DOX NP displaying higher dose-dependent cytotoxicity than the HA-DOX NP in different mammalian cells tested. Immunological evaluation of these nanocarriers in an ex vivo human whole blood model revealed that unlike unmodified polymers, the HA NP and CS NP surprisingly showed platelet aggregation and thrombin-antithrombin complex formation at high concentrations (0.8 mg/mL). We also observed a clear difference in early- and late-stage complement activation (C3a and sC5b-9) with CS and CS NP triggering significant complement activation at high concentrations (0.08-0.8 mg/mL), unlike HA and HA NP. These results offer new insight into designing glycosaminoglycan-based NPs and understanding their hematological responses and targeting ability.

AN OVERVIEW OF VISCOSUPPLEMENTS: THERAPEUTIC MODALITY FOR THE AILMENT OF OSTEOARTHRITIS

Osteoarthritis is a multifarious joint disease and is caused by inflammatory mediators, along with a procedure of wear and tear of cartilage. Osteoarthritis outcome in cartilage degradation, synovial inflammation, subchondral bone eburnation, degeneration of menisci, and capsular hypertrophy, which takes part in the pathogenesis of Osteoarthritis. Pain is the leading symptom of Osteoarthritis. However the precise mechanism causing pain is multifactorial and is still not well known. Viscosupplementation with intraarticular hylauronic acid promotes chondrocyte hyaluronic acid synthesis and prevents the additional cartilage degradation and may possible help to promote the cartilage regeneration. It is also postulated to diminish the production of inflammatory mediators and matrix metalloproteinase involved in Osteoarthritis. Viscosupplementation decline osteoarthritis symptoms in early to judicious Osteoarthritis, and improves functional outcome, if other conservative treatment modalities are ineffective. Hyaluronic acid (HA) is the major constituent of a 1-­‐2μm layer on the surface of articular cartilage; it is also a major constituent of synovial fluid. HA has many properties including exerting an anti-inflammatory effect, it acts as a lubricant when movements in the joint are slow and as a shock absorber when movements are fast effect. Viscosupplementation relieves the symptoms of osteoarthritis of the knee, and this therapeutic effect may last several weeks. However, it is apparently not much greater than that of placebo and, according to some studies, it barely reaches clinical significance. Its pain-relieving effect seems comparable to that of other non-surgical options. The modest clinical benefit of viscosupplementation may never be great enough to outweigh its disadvantages, which include slow onset of action, the need for several weekly injections, the low but present risk of adverse reactions and the relatively high cost of this treatment. It helps in improving the function in early osteoarthritis knee joint and might delay the need for future knee surgery Keywords: Joint disorders, intraarticular hylauronic acid, non-steroidal anti-inflammatory drugs, osteoarthritisÂ

Inhibition of hyaluronic acid formation sensitizes chronic myelogenous leukemia to treatment with doxorubicin.

In the current study we examined the ability of 4-methylumbelliferone (4-MU), which can inhibit hyaluronic acid synthesis, to sensitize K562 chronic myelogenous leukemia (CML) cells to doxorubicin therapy. Exposure of K562 cells to doxorubicin led to increased hyaluronic acid synthase (HAS) gene expression and increased levels of cell surface hyaluronic acid. Furthermore, exposure of K562 cells to exogenous HA caused resistance to doxorubicin-induced cell death. The combination of low dose 4-MU and doxorubicin led to increased apoptosis when compared to higher doses of any agent alone. Additionally, treatment with 4-MU led to a significant reduction in doxorubicin-induced increase in HA cell surface expression. Mechanistically, 4-MU treatment led to an increase in p38 activation and PARP cleavage. The role of p38 in 4-MU/doxorubicin-treated K562 cells was confirmed when p38 inhibitors led to protection from 4-MU/doxorubicin-induced apoptosis. Together, results from this study suggest that treatment with 4-MU increases the sensitivity of CML to chemotherapeutics by decreasing their HA-mediated resistance to apoptosis.

In vitro and in vivo antibiofilm potential of 2,4-Di-tert-butylphenol from seaweed surface associated bacterium Bacillus subtilis against group A streptococcus

Biofilm formation of Group A Streptococcus (GAS) is recognized as an important virulent determinant. The present study reports the antibiofilm potential of seaweed (Gracilaria gracilis) surface associated Bacillus subtilis against GAS. Purification revealed 2,4-Di-tert-butyl-phenol (DTBP) as the active principle. DTBP exhibited a dose dependent antibiofilm activity against GAS (SF370 & six different clinical M serotypes). Microscopic analysis revealed changes in cell surface architecture and reduced thickness upon DTBP treatment. Results of extracellular polymeric substance quantification, microbial adhesion to hydrocarbon assay and fourier transform infrared spectroscopic analysis suggested that DTBP probably interferes with the initial adhesion stage of biofilm formation cascade. Reduction in hyaluronic acid synthesis goes in unison with blood survival assay wherein, increased susceptibility to phagocytosis was observed. In vivo studies using Caenorhabditis elegans manifested the reduction in adherence and virulence, which prompts further investigation of the potential of DTBP for the treatment of GAS infections.

Infiltrative therapy as conservative treatment in hip osteoarthritis: a literature review.

Osteoarthritis (OA) is a very common disease, its prevalence increases with age and is a frequent cause of disability.Osteoarthritis is characterised by joint pain, stiffness and loss of range of motion. Overall, as many as 40% of those aged over 65 years in the community may have symptomatic OA of the knee or hip (1). OA results from a complex interaction of biomechanical and biochemical factors and is characterised by cartilage disruption and hypertrophy of bone. Intraarticular proinflammatory cytokines and proteinases in OA interfere with the synthesis of hyaluronic acid (HA), a complex glycosaminoglycan composed of repeated disaccharide units to form a linear polymer, resulting in an HA with a significantly reduced molecular weight and a reduction in synovial fluid viscoelasticity (2-3). Loss of normal characteristics of HA leads to the degradation of the articular cartilage and the disruption of the mechanical and homeostasis of the joint.Several pharmaceutical approaches, such as analgesics, non steroidal antiinflammatory drugs, COX-2 inhibitors and steroids, have been proposed (4), with the aim of reducing pain and maintaining and/or improving joint function. However, none of these options has shown to delay the progression of osteoarthritis or reverse joint damage.Infiltrative hip therapy involves injecting into the joint the drugs or medicinal substances that are used primarily to control the symptoms of the disease, such as pain and functional limitation.The aim of this review is to analyse existing infiltrative alternatives for hip osteoarthritis, and describe our experience.

387. Transfer of Hyaluronic Acid Synthase-2 Gene into Canine Joints Using Adeno-Associated Viral Vectors

Osteoarthritis (OA) is one of the more common causes of lameness in dogs and is estimated to affect approximately 20% of dogs >1 year old. OA is a progressive and degenerative disease that results in pain, inflammation and reduced joint mobility. Novel, safe and efficacious therapies that improve joint lubrication and reduce the extent of inflammation and pain are potential solutions for the management of canine OA. Here, we evaluated the efficiency of gene transfer to canine joint by intra-articular injections of recombinant adeno-associated virus (rAAV) vectors encoding therapeutic genes. Specifically, we generated rAAV vectors (either AAV2 and AAV5 serotypes vectors) encoding canine codon-optimized hyaluronic acid (HA) synthase-2 (HAS2) to provide local and continuous synthesis of HA in the joint. Twenty-two adult healthy dogs that were seronegative for anti-AAV2 and -AAV5 antibodies were injected intra-articularly with rAAV2/HAS2 (1, 5 and 10×1011 vg/joint), rAAV5/HAS2 (5×1011 vg/joint) or PBS (control). No adverse clinical signs were observed following the 28-day study period. Histopathological analysis showed minimal synovial inflammation in the joints of dogs treated with rAAV5/HAS2 and no significant changes in the rAAV2/HAS2 treated dogs. Vector genomes (VG) were detected in the synovium of all the rAAV-treated joints and in the majority of cartilage samples tested. The rAAV5/HAS2 vector resulted in findings of higher and more consistent detection of VG and transcripts compared to rAAV2/HAS2 in both the synovial and cartilage samples. Preliminary analysis also showed a trend towards increased HA levels in the synovial fluid of the treated joints. In summary, our study demonstrated that rAAV2 and rAAV5-mediated gene transfer to canine joint tissues is feasible and that both vectors present an acceptable safety profile following a single intra-articular injection.

Inhibition of Hyaluronic Acid Synthesis Suppresses Angiogenesis in Developing Endometriotic Lesions.

BackgroundThe development and long-term survival of endometriotic lesions is crucially dependent on an adequate vascularization. Hyaluronic acid (HA) through its receptor CD44 has been described to be involved in the process of angiogenesis.ObjectiveTo study the effect of HA synthesis inhibition using non-toxic doses of 4-methylumbelliferone (4-MU) on endometriosis-related angiogenesis.Materials and MethodsThe cytotoxicity of different in vitro doses of 4-MU on endothelial cells was firstly tested by means of a lactate dehydrogenase assay. The anti-angiogenic action of non-cytotoxic doses of 4-MU was then assessed by a rat aortic ring assay. In addition, endometriotic lesions were induced in dorsal skinfold chambers of female BALB/c mice, which were daily treated with an intraperitoneal injection of 0.9% NaCl (vehicle group; n = 6), 20mg/kg 4-MU (n = 8) or 80mg/kg 4-MU (n = 7) throughout an observation period of 14 days. The effect of 4-MU on their vascularization, survival and growth were studied by intravital fluorescence microscopy, histology and immunohistochemistry.Main ResultsNon-cytotoxic doses of 4-MU effectively inhibited vascular sprout formation in the rat aortic ring assay. Endometriotic lesions in dorsal skinfold chambers of 4-MU-treated mice dose-dependently exhibited a significantly smaller vascularized area and lower functional microvessel density when compared to vehicle-treated controls. Histological analyses revealed a downregulation of HA expression in 4-MU-treated lesions. This was associated with a reduced density of CD31-positive microvessels within the lesions. In contrast, numbers of PCNA-positive proliferating and cleaved caspase-3-positive apoptotic cells did not differ between 4-MU-treated and control lesions.ConclusionsThe present study demonstrates for the first time that targeting the synthesis of HA suppresses angiogenesis in developing endometriotic lesions. Further studies have to clarify now whether in the future this anti-angiogenic effect can be used beneficially for the treatment of endometriosis.

Loss of Glycogen Debranching Enzyme AGL Drives Bladder Tumor Growth via Induction of Hyaluronic Acid Synthesis.

We demonstrated that amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL) is a tumor growth suppressor and prognostic marker in human bladder cancer. Here we determine how AGL loss enhances tumor growth, hoping to find therapeutically tractable targets/pathways that could be used in patients with low AGL-expressing tumors. We transcriptionally profiled bladder cell lines with different AGL expression. By focusing on transcripts overexpressed as a function of low AGL and associated with adverse clinicopathologic variables in human bladder tumors, we sought to increase the chances of discovering novel therapeutic opportunities. One such transcript was hyaluronic acid synthase 2 (HAS2), an enzyme responsible for hyaluronic acid (HA) synthesis. HAS2 expression was inversely proportional to that of AGL in bladder cancer cells and immortalized and normal urothelium. HAS2-driven HA synthesis was enhanced in bladder cancer cells with low AGL, and this drove anchorage-dependent and independent growth. siRNA-mediated depletion of HAS2 or inhibition of HA synthesis by 4-methylumbelliferone (4MU) abrogated in vitro and xenograft growth of bladder cancer cells with low AGL. AGL and HAS2 mRNA expression in human tumors was inversely correlated in patient datasets. Patients with high HAS2 and low AGL tumor mRNA expression had poor survival, lending clinical support to xenograft findings that HAS2 drives growth of tumors with low AGL. Our study establishes HAS2-mediated HA synthesis as a driver of growth of bladder cancer with low AGL and provides preclinical rationale for personalized targeting of HAS2/HA signaling in patients with low AGL-expressing tumors.

Conserved miR-26b enhances ovarian granulosa cell apoptosis through HAS2-HA-CD44-Caspase-3 pathway by targeting HAS2.

The hyaluronan synthase 2 (HAS2)-hyaluronic acid (HA)-CD44-Caspase-3 pathway is involved in ovarian granulosa cell (GC) functions in mammals. HAS2 is a key enzyme required for HA synthesis and is the key factor in this pathway. However, the regulation of HAS2 and the HAS2-mediated pathway by microRNAs in GCs is poorly understood. Here, we report that miR-26b regulates porcine GC (pGC) apoptosis through the HAS2-HA-CD44-Caspase-3 pathway by binding directly to the 3′- untranslated region of HAS2 mRNA. Knockdown of miR-26b reduced pGC apoptosis. Luciferase reporter assays demonstrated that HAS2 is a direct target of miR-26b in pGCs. Knockdown and overexpression of miR-26b increased and decreased, respectively, HA content, and HAS2 and CD44 expression in pGCs. At the same time, inhibition and overexpression of miR-26b decreased and increased the expression of Caspase-3, a downstream factor in the HAS2-HA-CD44 pathway. Moreover, knockdown of HAS2 enhanced pGC apoptosis, reduced the inhibitory effects of a miR-26b inhibitor on pGC apoptosis, repressed HA content and CD44 expression, and promoted Caspase-3 expression. In addition, overexpression of HAS2 has a opposite effect. Collectively, miR-26b positively regulates pGC apoptosis via a novel HAS2-HA-CD44-Caspase-3 pathway by targeting the HAS2 gene.

A novel engineered dermis for in vitro photodamage research.

The realization of biologically relevant human tissue equivalents as an in vitro model to investigate human diseases, as well as to test the efficacy or toxicity of novel compounds, is emerging as a new challenge in tissue engineering. Currently, the in vitro three-dimensional (3D) dermis model mainly involves the use of cells embedded in exogenous non-human matrices. However, such models feature biological and functional disparities with native dermis, therefore limiting their relevance to the in vivo situation. The purpose of this study was to provide a reliable endogenous human dermal equivalent (HDE) able to recapitulate the extracellular matrix (ECM) remodelling of the native dermis occurring after external damage. To this end, UVA irradiation was used to induce photodamage to both the HDE and to a fibroblast-populated collagen matrix. The photodamage was investigated at the cellular and ECM level and the results showed that, although a cellular response was detected in both systems, no ECM reorganization characteristic of the in vivo photo-aged dermis could be detected in the fibroblast-populated collagen matrix. In contrast in the HDE, the neosynthesized ECM recapitulated the characteristic ageing behaviour of the dermis found in vivo, in terms of collagen and hyaluronic acid synthesis as well as collagen organization remodelling. This study therefore demonstrates the role of the endogenous ECM in recapitulating in vitro the functionality of the human dermis and the proposed HDE as a novel tool for photoprotection trials. Copyright © 2016 John Wiley & Sons, Ltd.

Genetic and biochemical characterization of genes involved in hyaluronic acid synthesis in Streptococcus zooepidemicus.

The biosynthetic pathway for hyaluronic acid (HA) has been proposed; however, a thorough genetic and functional analysis is required to further elucidate the roles of genes involved in HA production. Previously, we developed a markerless gene-deletion system for Streptococcus zooepidemicus and confirmed that hasA is essential for HA synthesis. Here, we constructed a comprehensive set of deletion mutants and investigated the roles of ten additional predicted genes in the HA synthetic pathway. Phenotypic assays revealed that all ten genes play a role in cell growth and/or HA synthesis. As expected, the deletion of hasA or hasB abolished HA production with little effect on growth, while the deletion of genes that are also required for peptidoglycan biosynthesis (hasE, glmM, and glmS) significantly reduced cell growth and HA production. Either of the glmU homologues (hasD and gcaD) was sufficient for optimal growth and the mucoid phenotype, while no double mutant could be isolated. Of the two UDP-glucose pyrophosphorylase (UGPase) paralogues, the operon-encoded hasC1 was responsible for 65% of the activity, while hasC2 was responsible for the remaining 35%. The deletion of hasC1 had no effect on cell growth and caused only a moderate decrease in the UDP-glucose level and HA production. The deletion of both hasC1 and hasC2 resulted in a severe growth defect and negligible UDP-glucose accumulation, HA production, and pyrophosphorylase activity. Of the two phosphoglucomutase paralogues, pgm1 and pgm2, the former is responsible for around 10% of activity, while the latter is responsible for 90%. The deletion of pgm1 showed no apparent effect on HA synthesis and growth, while the deletion of pgm2 resulted in the abolishment of HA synthesis and a significantly slower growth. These results should guide the metabolic engineering of S. zooepidemicus to improve HA productivity and quality.

An overview of the beneficial effects of hydrolysed collagen intake on joint and bone health and on skin ageing

Hydrolysate Collagen (HC) consists of small peptides with a molecular weight lower than 5.000 Da. produced from gelatinization and subsequent enzymatic hydrolysis of native collagen which is found in rich collagenic animal tissues. There is much evidence about the HC ingestion positive effect over degenerative joint and bones diseases. The aim of this article is to review the present scientific studies about HC and to evaluate the HC ingestion therapeutical effects on some collagenic tissues as cartilage, bones and skin. Up to date, there are more than 60 scientific studies (in vitro, in vivo, clinics and on bioavailability) about HC ingestion efficacy on reducing collagen damage and loss consequences as joint pain and erosion (osteoarthritis), bone density loss (osteoporosis) and skin ageing. Preclinical studies show that HC stimulates collagenic tissue regeneration by increasing not only collagen synthesis but minor components (glycosaminoglycans and hyaluronic acid) synthesis as well. Clinical studies show that HC continual ingestion helps to reduce and prevent joint pain, bone density loss and skin ageing. These results as well as its high level of tolerance and safety make HC ingestion attractive for a long-term use in bone and joint degenerative diseases and in fight against skin ageing.

Peculiarities of the action of hyaluronidase of different origin to the connective tissue

The lecture is devoted to consideration of mechanism of therapeutic action of the enzyme hyaluronidase in hyperplastic connective tissue. Drugs based on hyaluronidase increase bioavailability of other drugs used in adjuvant therapy; they significantly increase effectiveness of treatment, and also provide targeted synthesis of hyaluronic acid, ths regulating the regeneration process of connective tissue.

Characterization of UDP-glucose dehydrogenase from Pasteurella multocida CVCC 408 and its application in hyaluronic acid biosynthesis

Hyaluronic acid (HA), a vital acid mucopolysaccharide, has immense applied value in foodstuffs, medicaments, and cosmetics among others. UDP-glucose dehydrogenase (UGDH, EC 1.1.1.22) is an essential enzyme for HA synthesis. In this study, a UGDH (PmuHasB, 45.9kDa) from Pasteurella multocida CVCC 408 was expressed in Escherichia coli BL21 (DE3). It was purified by two chromatographic columns with a specific activity of 6.58IU/mg. The optimum pH and temperature were determined to be 10.0 and 37°C, respectively. The activity was stable across the pH range 6–10, and had a half-life of about 3h at 45°C. The estimated apparent Km values for UDP-glucose and NAD+ were 0.11 and 0.069mM, respectively. The results indicated that PmuHasB was an alkaline and mesophilic UGDH. PmuHasB and PmuHasA (HA synthase, HAS) were co-expressed in E. coli BW25113 to obtain a HA high-producing strain pBPAB/BW25113. It produced about 2.39g/L HA in shake flask by using the method of whole-cell catalysis. Investigation of the different UGDHs on HA synthesis revealed that intracellular UGDH activity and HA total yield of pBPAB/BW25113 (0.15IU/mg and 5.4g/L) were higher than from pBPASB/BW25113 (0.013IU/mg and 2.8g/L) and pBPAEB/BW25113 (0.010IU/mg and 2.22g/L). These results indicated that the activity and stability of UGDH plays a significant role in HA production, and should prove useful for further genetic engineering research with a view to construct other glucuronic acid polysaccharide synthesis pathways.

The hyaluronic acid inhibitor 4-methylumbelliferone is an NSMase2 activator—role of Ceramide in MU anti-tumor activity

Increased synthesis of hyaluronic acid (HA) is often associated with increased metastatic potential and invasivity of tumor cells. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis, and has been studied as a potential anti-tumor drug to inhibit the growth of primary tumors and distant metastasis of tumor cells. Although several studies reported that the anticancer effects of MU are mediated by inhibition of HA signaling, the mechanism still needs to be clarified. In a previous study we demonstrated the regulation of HA synthesis by ceramide, and now show how MU activated neutral sphingomyelinase2 (NSMase2) generates ceramides and mediates MU induced inhibition of HA synthesis, cell migration and invasion, and apoptosis of tumor cells. Using a HA enriched mouse oligodendroglioma cell line G26-24 we found that MU elevated the activity of NSMase2 and increased ceramide levels, which in turn increased phosphatase PP2A activity. Further, the activated PP2A reduced phosphorylation of Akt, decreased activities of HA synthase2 (HAS2) and calpains, and inhibited both the synthesis of HA, and the migration and invasion of G26-24 tumor cells. In addition, MU mediated ceramide stimulated activation of p53 and caspase-3, reduced SIRT1 expression and decreased G26-24 viability. The mechanism of the MU anticancer therefore initially involves NSMase2/ceramide/PP2A/AKT/HAS2/caspase-3/p53/SIRT1 and the calpain signaling pathway, suggesting that ceramides play a key role in the ability of a tumor to become aggressively metastatic and grow.

The effect of oral collagen peptide supplementation on skin moisture and the dermal collagen network: evidence from an ex vivo model and randomized, placebo-controlled clinical trials.

Skin dryness and an accelerated fragmentation of the collagen network in the dermis are hallmarks of skin aging. Nutrition is a key factor influencing skin health and consequently its appearance. A wide range of dietary supplements is offered to improve skin health. Collagen peptides are used as a bioactive ingredient in nutricosmetic products and have been shown in preclinical studies to improve skin barrier function, to induce the synthesis of collagen and hyaluronic acid, and to promote fibroblast growth and migration. Our aim was to investigate the effect of oral supplementation with specific collagen peptides on skin hydration and the dermal collagen network in a clinical setting. Two placebo-controlled clinical trials were run to assess the effect of a daily oral supplementation with collagen peptides on skin hydration by corneometry, on collagen density by high-resolution ultrasound and on collagen fragmentation by reflectance confocal microscopy. Human skin explants were used to study extracellular matrix components in the presence of collagen peptides ex vivo. Oral collagen peptide supplementation significantly increased skin hydration after 8 weeks of intake. The collagen density in the dermis significantly increased and the fragmentation of the dermal collagen network significantly decreased already after 4 weeks of supplementation. Both effects persisted after 12 weeks. Ex vivo experiments demonstrated that collagen peptides induce collagen as well as glycosaminoglycan production, offering a mechanistic explanation for the observed clinical effects. The oral supplementation with collagen peptides is efficacious to improve hallmarks of skin aging.

Abstract 4949: HAS2 is a critical effector for AGL mediated regulation of tumor growth

Abstract In bladder cancer, reduced levels of Amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL), an enzyme involved in glycogenolysis and mutated in glycogen storage disease type III, enhances proliferation in vitro and tumor growth in vivo. To identify how reduced levels of AGL promote bladder cancer growth, we gene expression profiled two bladder cancer cell lines with and without siRNA mediated AGL depletion. This identified that hyaluronic acid synthase 2 (HAS2), an enzyme responsible for hyaluronic acid (HA) synthesis, is upregulated with AGL depletion. We validated this finding in several additional bladder cancer cell lines and also found that HA levels were 2-fold higher bladder cancer cells with low AGL compared to control. Interestingly, siRNA induced knockdown of HAS2 preferentially reduced monolayer, anchorage independent and xenograft growth in bladder cancer cells with low AGL. 4-Methylumbelliferone (4-MU), an inhibitor of HA synthesis, had similar effects. Analysis of human bladder cancer tissues showed that AGL and HAS2 mRNA expression are negatively correlated in 5/8 patient datasets (N = 725). Bladder cancer patients with high HAS2 and low AGL expression had worse survival than patients with the reciprocal relationship between these two genes suggesting that HAS2 is a driver of bladder tumor growth with AGL loss establish the HAS2/HA axis as a major driver and target of therapy in bladder tumors with low AGL. Citation Format: Sunny Guin, Yuanbin Ru, Carolyn R. Lew, Neeraj Agarwal, Charles Owens, Dan Theodorescu. HAS2 is a critical effector for AGL mediated regulation of tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4949. doi:10.1158/1538-7445.AM2015-4949

Abstract 2581: Antitumor effect of Trametinib, a selective MEK inhibitor, in combination with 4-methylumbelliferone, a hyaluronic acid synthesis inhibitor, in Malignant pleural mesothelioma cell lines

Abstract Introduction: Malignant pleural mesothelioma (MPM) is a highly aggressive malignancy for which no targeted therapy exists. The mitogen-activated protein kinase (MAPK) pathway plays critical roles in the regulation of tumorigenesis in multiple solid tumors, including MPM. Trametinib, a selective MEK inhibitor, have a survival benefit in patients with V600 BRAF-mutant metastatic melanoma. The effect of trametinib on MPM cells has not been well studied. Hyaluronan (HA) is one of the major components of the extracellular matrix. MPM is in most cases associated with elevated amounts of HA, which has increased the malignant properties of MPM cells. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) has antitumor effects in various malignant tumors, but its effect on MPM cells has not been well studied. Purpose: We evaluated the effects of trametinib, 4-MU and their combination on MPM cells in vitro and in vivo. Experimental Design: The effects of trametinib, 4-MU, and their combination on MPM cells were evaluated using cell viability assay, western blot analysis, and mouse xenograft model. Results: Trametinib exhibited an antiproliferative activity in all four MPM cell lines, NCI-H226, NCI-H2452, NCI-H2052, and MSTO-211H, with IC50 values ranging from 0.15 μM to 12.7 μM. Trametinib blocked the phosphorylation of ERK until 72 hours and decreased the expression of CD44 in a dose-dependent manner. In addition, the expression of CD44 was inhibited (48-72 hours) after the suppression of ERK phosphorylation by trametinib. 4-MU exhibited an antiproliferative activity in MPM cells. 4-MU inhibited ERK phosphorylation but not CD44 expression. In mouse xenograft model, trametinib and 4-MU suppressed tumor growth (trametinib vs. control, P < 0.0001; 4-MU vs. control, P < 0.01), and their combination suppressed more strongly (combination vs. trametinib, P < 0.01; combination vs. 4-MU, P < 0.0001; combination vs. control, P < 0.0001). Conclusions: Trametinib and 4-MU exhibited antitumor activities in MPM cell lines. Furthermore, their combination exhibited more potent antitumor activities. These results suggests that Trametinib and 4-MU are promising therapeutic agents in MPM, and these combination therapies may be effective for the treatment of MPM. Citation Format: Hiroyuki Cho, Seiji Matsumoto, Yoshiko Fujita, Ayumi Kuroda, Masaki Hashimoto, Teruhisa Takuwa, Toshi Menju, Makoto Sonobe, Nobuyuki Kondo, Hiroshi Date, Seiki Hasegawa. Antitumor effect of Trametinib, a selective MEK inhibitor, in combination with 4-methylumbelliferone, a hyaluronic acid synthesis inhibitor, in Malignant pleural mesothelioma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2581. doi:10.1158/1538-7445.AM2015-2581