Transcriptome Sequencing Reveals the Mechanism behind Chemically Induced Oral Mucositis in a 3D Cell Culture Model.

Oral mucositis is a common side effect of cancer treatment, and in particular of treatment with the mTORC1 inhibitor everolimus. Current treatment methods are not efficient enough and a better understanding of the causes and mechanisms behind oral mucositis is necessary to find potential therapeutic targets. Here, we treated an organotypic 3D oral mucosal tissue model consisting of human keratinocytes grown on top of human fibroblasts with a high or low dose of everolimus for 40 or 60 h and investigated (1) the effect of everolimus on microscopic sections of the 3D cell culture for evidence of morphologic changes and (2) changes in the transcriptome by high throughput RNA-Seq analysis. We show that the most affected pathways are cornification, cytokine expression, glycolysis, and cell proliferation and we provide further details. This study provides a good resource towards a better understanding of the development of oral mucositis. It gives a detailed overview of the different molecular pathways that are involved in mucositis. This in turn provides information about potential therapeutic targets, which is an important step towards preventing or managing this common side effect of cancer treatment.

Citric Acid: A Multifunctional Pharmaceutical Excipient.

Citric acid, a tricarboxylic acid, has found wide application in the chemical and pharmaceutical industry due to its biocompatibility, versatility, and green, environmentally friendly chemistry. This review emphasizes the pharmaceutical uses of citric acid as a strategic ingredient in drug formulation while focusing on the impact of its physicochemical properties. The functionality of citric acid is due to its three carboxylic groups and one hydroxyl group. These allow it to be used in many ways, including its ability to be used as a crosslinker to form biodegradable polymers and as a co-former in co-amorphous and co-crystal applications. This paper also analyzes the effect of citric acid in physiological processes and how this effect can be used to enhance the attributes of pharmaceutical preparations, as well as providing a critical discussion on the issues that may arise out of the presence of citric acid in formulations.

Antimycobacterial Effects of Everolimus in a Human Granuloma Model.

Mycobacterium tuberculosis (M. tb) has been historically and is currently a threat to global public health. First-line antibiotics have been effective but proven to be burdensome as they have many potential adverse side effects. There has been a recent increase in the number of active tuberculosis (TB) cases due to a prevalence of multidrug and extensively drug-resistant strains of M. tb, and an increasing number of highly susceptible people such as those with Type 2 Diabetes (T2DM) and human immunodeficiency virus (HIV) infection. Multidrug-resistant M. tb infection (MDR-TB) is challenging to treat with existing therapeutics, so novel therapeutics and treatment strategies must be developed. Host-Directed Therapy (HDT) has been a potential target mechanism for effective clearance of infection. Host cell autophagy plays an essential role in antibacterial defense. The mammalian target of rapamycin (mTOR) has been negatively correlated with autophagy induction. Everolimus is an mTOR inhibitor that induces autophagy, but with higher water solubility. Therefore, targeting the mTOR pathway has the potential to develop novel and more effective combination drug therapy for TB. This study tested the effect of everolimus, alone and in combination with current first-line antibiotics (isoniazid and pyrazinamide), on the inhibition of M. tb inside in vitro human granulomas. We found that M. tb-infected in vitro granulomas treated with everolimus alone resulted in significantly decreased M. tb burden compared to similar granulomas in the control group. Cells treated with everolimus doses of either 1 nM or 2 nM in conjunction with pyrazinamide (PZA) produced a significant reduction in intracellular M. tb burden. Treatment groups that received everolimus alone in either 1 nM or 2 nM doses experienced a significant reduction in oxidative stress. Additionally, samples treated with 2 nM everolimus alone were observed to have significantly higher levels of autophagy and mTOR inhibition as well. Results from this study indicate that everolimus is efficacious in controlling M. tb infection in the granulomas and has additive effects when combined with the anti-TB drugs, isoniazid and pyrazinamide. This study has shown that everolimus is a promising host-directed therapeutic in the context of in vitro granuloma M. tb infection. Further study is warranted to better characterize these effects.

Molecular Mechanism of Resveratrol's Lipid Membrane Protection.

Resveratrol, a natural compound found in red wine and various vegetables, has drawn increasing interest due to its reported benefit in cardiovascular protection, neurodegenerative disorders, and cancer therapy. The mechanism by which resveratrol exerts such pleiotropic effects remains unclear. It remains as one of the most discussed polyphenol compounds in the debating "French Paradox". In this study, using molecular dynamics simulations of dipalmitoyl phosphatidylcholine (DPPC) bilayer with resveratrol, we generated a free energy map of resveratrol's location and orientation of inside the lipid bilayer. We found that resveratrol increases the surface area per lipid and decreases membrane thickness, which is the opposite effect of the well-studied cholesterol on liquid phase DPPC. Most importantly, based on the simulation observation that resveratrol has a high probability of forming hydrogen bonds with sn-1 and sn-2 ester groups, we discovered a new mechanism using experimental approach, in which resveratrol protects both sn-1 and sn-2 ester bonds of DPPC and distearoyl phosphatidylcholine (DSPC) from phospholipase A1 (PLA1) and phospholipase A2 (PLA2) cleavage. Our study elucidates the new molecular mechanism of potential health benefits of resveratrol and possibly other similar polyphenols and provides a new paradigm for drug design based on resveratrol and its analogs.

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

Genome wide expression after different doses of irradiation of a three-dimensional (3D) model of oral mucosal.

We evaluated a three-dimensional (3D) human oral cell culture that consisted of two types of cells, oral keratinocytes and fibroblasts as a model of oral mucositis which is a debilitating adverse effect of chemotherapy and radiation treatment. The 3D cell culture model was irradiated with 12 or 2 Gy, and total RNA was collected 6 h after irradiation to compare global gene expression profiles via microarray analysis. Here we provide detailed methods and analysis on these microarray data, which have been deposited in Gene Expression Omnibus (GEO): GSE62395.

Transcriptional profiling of radiation damage and preventive treatments in a 3-dimensional (3D) human cell culture model of oral mucositis.

Cancer patients who receive radiation are often afflicted by oral mucositis, a debilitating disease, characterized by mouth sores and difficulty in swallowing. Oftentimes, cancer patients afflicted with mucositis must stop life-saving therapies. Thus it is very important to prevent mucositis before it develops. Using a validated organotypic model of human oral mucosa, a 3-dimensional cell culture model of human oral keratinocytes, it has been shown that a mixture (NAC-QYD) of N-acetyl cysteine (NAC) and a traditional Chinese medicine, Qingre Liyan decoction (QYD), prevented radiation damage (Lambros et al., 2014). Here we provide detailed methods and analysis of microarray data for non-irradiated and irradiated human oral mucosal tissue with and without pretreatment with NAC, QYD and NAC-QYD. The microarray data been deposited in Gene Expression Omnibus (GEO): GSE62397. These data can be used to further elucidate the mechanisms of irradiation damage in oral mucosa and its prevention.

Molecular signatures in the prevention of radiation damage by the synergistic effect of N-acetyl cysteine and qingre liyan decoction, a traditional chinese medicine, using a 3-dimensional cell culture model of oral mucositis.

Qingre Liyan decoction (QYD), a Traditional Chinese medicine, and N-acetyl cysteine (NAC) have been used to prevent radiation induced mucositis. This work evaluates the protective mechanisms of QYD, NAC, and their combination (NAC-QYD) at the cellular and transcriptional level. A validated organotypic model of oral mucosal consisting of a three-dimensional (3D) cell tissue-culture of primary human keratinocytes exposed to X-ray irradiation was used. Six hours after the irradiation, the tissues were evaluated by hematoxylin and eosin (H and E) and a TUNEL assay to assess histopathology and apoptosis, respectively. Total RNA was extracted and used for microarray gene expression profiling. The tissue-cultures treated with NAC-QYD preserved their integrity and showed no apoptosis. Microarray results revealed that the NAC-QYD caused the upregulation of genes encoding metallothioneins, HMOX1, and other components of the Nrf2 pathway, which protects against oxidative stress. DNA repair genes (XCP, GADD45G, RAD9, and XRCC1), protective genes (EGFR and PPARD), and genes of the NFκB pathway were upregulated. Finally, tissue-cultures treated prophylactically with NAC-QYD showed significant downregulation of apoptosis, cytokines and chemokines genes, and constrained damage-associated molecular patterns (DAMPs). NAC-QYD treatment involves the protective effect of Nrf2, NFκB, and DNA repair factors.

The cystine/glutamate antiporter system x(c)(-) in health and disease: from molecular mechanisms to novel therapeutic opportunities.

The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x(c)(-) inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS.

Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis.

Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first step towards the development 3-D tissue as a screening tool.

Suitability of caspofungin for aerosol delivery: physicochemical profiling and nebulizer choice.

BACKGROUND: Aerosolized antifungal therapy is a promising route of drug delivery for pulmonary aspergillosis due to attainment of high localized concentrations. Caspofungin, a new antifungal agent with proven efficacy against invasive aspergillosis, has ideal potential for aerosolization. STUDY OBJECTIVE: To examine in vitro the suitability of caspofungin for aerosol administration by characterizing factors that influence efficacy and airway tolerance of aerosol delivery: physicochemical properties, aerodynamics of drug particles, and efficiency of nebulizing systems. DESIGN: Physicochemical characteristics of caspofungin solutions (10 mg/mL and 30 mg/mL) were analyzed: osmolality, pH, viscosity, and surface tension. A time-of-flight aerosol spectrometer API Aerosizer was used to determine aerosol particle size and distribution. Drug output was quantified by high-performance liquid chromatography assay. Nebulizer efficiency was measured by drug output and respirable fraction (percentage of aerosolized particles with a 1 to 5 mum aerodynamic diameter) and compared among three jet nebulizer/compressor systems: device 1, Micromist (Hudson RCI; Temecula, CA)/Pulmo-Aide (model 5650D; DeVilbiss; Somerset, PA); device 2, Sidestream MS 2400/Envoy model IRC 1192 (Invacare; Elyria, OH); and device 3, Pari LC Star/Proneb Ultra (Pari Respiratory Equipment; Midlothian, VA). MEASUREMENTS AND RESULTS: Caspofungin requires 0.9% NaCl rather than sterile water as the diluent and addition of 0.3N NaOH buffer to adjust acidity of solutions (pH 6.17 to 6.26) in order to achieve optimal physicochemical properties for airway tolerability (osmolality, 150 to 550 milliosmol per kilogram; chloride ion, 31 to 300 mmol/L; and pH 7.4). The drug output rate increased with higher concentrations of drug solution: device 1, 4.0 mg/min vs 12.5 mg/min; device 2, 5.4 mg/min vs 14.7 mg/min; and device 3, 2.3 mg/min vs 12 mg/min, respectively. The percentage of particles within the respirable range varies depending on device and concentration of drug solutions (10 mg/mL vs 30 mg/mL): device 1, 85% vs 38%; device 2, 44% vs 57%; and device 3, 83% vs 93%, respectively. CONCLUSION: Caspofungin solution with adjustments appears to have physicochemical and aerodynamic characteristics suitable for aerosolization when used with either the Pari LC Star/Proneb Ultra or Micromist/Pulmo-Aide devices. Further in vivo testing is warranted.

Effects of cyclosporin A on model lipid membranes.

Cyclosporin A (CSA) is a widely used immunosuppressant drug for transplant therapy, however its limitation is its toxicity. The effect of CSA on model membranes such as dimyristoyl phosphatidylcholine (DMPC) bilayers was studied using small-angle X-ray diffraction and differential scanning calorimetry (DSC). CSA abolishes the pretransition and affects the transition of DMPC model membranes in a concentration-related manner as is shown by DSC. CSA induces a second peak at the high temperature side of the main transition, which is interpreted as a phase separation between areas rich and poor in CSA concentration. Small angle X-ray diffraction shows that the repeat distance of the DMPC bilayers in the lamellar Lalpha state increases as a function of concentration up to 10 mol% and remains constant thereafter. Furthermore, CSA affects the fatty acyl chains of the bilayer, especially the part of the chain proximal to the head group. In conclusion, CSA, as both small-angle X-ray diffraction and DSC show, affects in a concentration-wise manner the DMPC model membranes and perturbs the bilayer, in particular the acyl chain region.