Human Immortalized Cell Line Research Articles

Royal Jelly Extract Accelerates Keratinocyte Proliferation, and Upregulates Laminin <i>α</i>3 and Integrin <i>β</i>1 mRNA Expression, via Akt/mTOR/HIF-1<i>α</i> Pathway

Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract (RJ) on keratinocytes have not been fully elucidated. Objective: The primary objectives of this study were to reveal the effects of RJ on keratinocytes and explore the underlying mechanism. Methods: HaCaT cells, an immortal human epidermis-derived keratinocyte cell line, were used in this study. Laminin α3 (LAMA3), integrin β1 (ITGB1), and hypoxia-inducible factor-1α (HIF-1α) mRNA expression levels were determined using real-time PCR. Cell proliferation rate was measured using a bromodeoxyuridine uptake assay. Results: RJ treatment upregulated LAMA3, ITGB1 and HIF-1α mRNA expression, and accelerated HaCaT cell proliferation. Akt and mTOR inhibitors suppressed the RJ-induced HIF-1α expression and cell proliferation. HIF-1α silencing abrogated RJ-induced LAMA3 and ITGB1 mRNA expression and cell proliferation, whereas LAMA3 silencing and antibody-mediated ITGB1 blockade did not affect the effects of RJ. Conclusion: RJ upregulates LAMA3 and ITGB1 mRNA expression levels by HIF-1α expression enhancement. In addition, RJ accelerates keratinocyte proliferation via Akt/mTOR/HIF-1α/NF-κB signaling pathway. These suggest that RJ is beneficial for anti-aging, as a skin care product ingredient.

MicroRNA global profiling in cystic fibrosis cell lines reveals dysregulated pathways related with inflammation, cancer, growth, glucose and lipid metabolism, and fertility: an exploratory study.

Background and aim:Cystic fibrosis (CF), is due to CF transmembrane conductance regulator (CFTR) loss of function, and is associated with comorbidities. The increasing longevity of CF patients has been associated with increased cancer risk besides the other known comorbidities. The significant heterogeneity among patients, suggests potential epigenetic regulation. Little attention has been given to how CFTR influences microRNA (miRNA) expression and how this may impact on biological processes and pathways.Methods:We assessed the changes in miRNAs and subsequently identified the affected molecular pathways using CFBE41o-, and IB3 human immortalized cell lines since they reflect the most common genetic mutations in CF patients, and 16HBE14o- cells were used as controls.Results:In the CF cell lines, 41 miRNAs showed significant changes (FC (log2) ≥ +2 or FC (log2) ≤ -2 and p-value≤0.05). Gene target analysis evidenced 511 validated miRNA target genes. Gene Ontology analysis evidenced cancer, inflammation, body growth, glucose, and lipid metabolism as the biological processes most impacted by these miRNAs. Protein-protein interaction and pathway analysis highlighted 50 significantly enriched pathways among which RAS, TGF beta, JAK/STAT and insulin signaling.Conclusions:CFTR loss of function is associated with changes in the miRNA network, which regulates genes involved in the major comorbidities that affect CF patients suggesting that further research is warranted. (www.actabiomedica.it)

Immortalizing Cells for Human Consumption.

The need to produce immortal, food-relevant cell lines is one of the most pressing challenges of cellular agriculture, the field which seeks to produce meat and other animal products via tissue engineering and synthetic biology. Immortal cell lines have a long and complicated story, from the first recognized immortal human cell lines taken from Henrietta Lacks, to today, where they are used to assay toxicity and produce therapeutics, to the future, where they could be used to create meat without harming an animal. Although work in immortal cell lines began more than 50 years ago, there are few existing cell lines made of species and cell types appropriate for cultured meat. Cells in cultured meat will be eaten by consumers; therefore, cultured meat cell lines will also require unique attributes not selected for in other cell line applications. Specifically, cultured meat cell lines will need to be approved as safe for consumption as food, proliferate and differentiate efficiently at industrial scales, and have desirable taste, texture, and nutrition characteristics for consumers. This paper defines what cell lines are needed, the existing methods to produce new cell lines and their limitations, and the unique considerations of cell lines used in cultured meat.

Catechol functionalized ink system and thrombin-free fibrin gel for fabricating cellular constructs with mechanical support and inner micro channels

The development of 3D bio printing technology has contributed to protocols for the repair and regeneration of tissues in recent years. However, it is still a great challenge to fabricate structures that mimic the complexity of native tissue, including both the biomechanics and microscale internal structure. In this study, a catechol functionalized ink system was developed to produce tough and elastic scaffolds with built-in micro channels that simulate the vascular structure. And a skin model was designed to evaluate the cytocompatibility of the scaffolds. The mechanical support stemmed from the double network based on catechol-hyaluronic acid (HACA) and alginate, the micro channels were generated using sacrificial gelatin. HACA/alginate and gelatin were firstly printed using a 3D extrusion printer. Thrombin-free fibrinogen were then mixed with human dermal fibroblasts and introduced to the printed scaffolds to induce gelation. An immortal human keratinocyte cell line was introduced on top of the cellular construct to mimic the full thickness skin structure. The printed scaffolds demonstrated high elasticity and supported the formation of a double-layered cell-laden skin like structure. The results suggest the 3D printing platform developed here provides a platform for skin regeneration and could be explored further to engineer functional skin tissue by incorporation of other types of cells.

Transcriptomic Response Dynamics of Human Primary and Immortalized Adrenocortical Cells to Steroidogenic Stimuli.

Adrenal steroid hormone production is a dynamic process stimulated by adrenocorticotropic hormone (ACTH) and angiotensin II (AngII). These ligands initialize a rapid and robust gene expression response required for steroidogenesis. Here, we compare the predominant human immortalized cell line model, H295R cell, with primary cultures of adult adrenocortical cells derived from human kidney donors. We performed temporally resolved RNA-seq on primary cells stimulated with either ACTH or AngII at multiple time points. The magnitude of the expression dynamics elicited by ACTH was greater than AngII in primary cells. This is likely due to the larger population of adrenocortical cells that are responsive to ACTH. The dynamics of stimulus-induced expression in H295R cells are mostly recapitulated in primary cells. However, there are some expression responses in primary cells absent in H295R cells. These data are a resource for the endocrine community and will help researchers determine whether H295R is an appropriate model for the specific aspect of steroidogenesis that they are studying.

Induction of apoptosis by Kola nut extract as a recent and promising treatment strategy for Leukemia

Kola nut extracts have recently been reported to contain chemopreventive compounds providing several pharmacological benefits. This study investigated Kola nut extracts' anti-cancer activity on human immortalized myelogenous leukemia cell line K562 through apoptosis and cell cycle arrest. Fresh Kola nuts were prepared as powder and dissolved in DMSO. Different concentrations (50, 100, 150, 200, and 250 μg/ml) of working solutions were prepared. The K562 cells were treated with the different concentrations of Kola nut extract or vehicle control (10% DMSO) followed by incubation at 37°C for 24, 48, and 72 hours, respectively. Treatment activity was investigated in K562 cells; by Resazurin, and FITC/Propidium Iodide and 7-AAD stained cells to evaluate apoptotic cells and the cell cycle's progression. Inhibition of leukemia cell proliferation was observed. The extract effectively induced cell death, early and late apoptosis by approximately 30% after 24 and 48 hours incubation, and an increase in the rate of dead cells by 50% was observed after 72 hours of incubation. Also, cell growth reduction was seen at high dose concentrations (150 and 200 µg/ml), as evident by cell count once treated with Kola nut extract. The total number of apoptotic cells increased from 5.8% of the control group to 27.4% at 250 µg/ml concentration. Moreover, Kola nut extracts' effects on K562 cells increased gradually in a dose and time-dependent manner. It was observed that Kola nut extracts could arrest the cell cycle in the G2/M phase as an increase in the number of cells by 29.8% and 14.6 % were observed from 9.8% and 5.2% after 24 and 48 hours of incubation, respectively. This increase was detected in a dose and time-dependent manner. Kola nut extracts can be used as a novel anti-cancer agent in Leukemia treatment as it has shown significant therapeutic potential and therefore provides new insights in understanding the mechanisms of its action. Keywords: Kola nut extracts, Leukemia, K562 cell line, Apoptosis, Cancer.

MCPIP1-mediated NFIC alternative splicing inhibits proliferation of triple-negative breast cancer via cyclin D1-Rb-E2F1 axis

Triple-negative breast cancer (TNBC) is the most aggressive subtype with the worst prognosis and the highest metastatic and recurrence potential, which represents 15–20% of all breast cancers in Chinese females, and the 5-year overall survival rate is about 80% in Chinese women. Recently, emerging evidence suggested that aberrant alternative splicing (AS) plays a crucial role in tumorigenesis and progression. AS is generally controlled by AS-associated RNA binding proteins (RBPs). Monocyte chemotactic protein induced protein 1 (MCPIP1), a zinc finger RBP, functions as a tumor suppressor in many cancers. Here, we showed that MCPIP1 was downregulated in 80 TNBC tissues and five TNBC cell lines compared to adjacent paracancerous tissues and one human immortalized breast epithelial cell line, while its high expression levels were associated with increased overall survival in TNBC patients. We demonstrated that MCPIP1 overexpression dramatically suppressed cell cycle progression and proliferation of TNBC cells in vitro and repressed tumor growth in vivo. Mechanistically, MCPIP1 was first demonstrated to act as a splicing factor to regulate AS in TNBC cells. Furthermore, we demonstrated that MCPIP1 modulated NFIC AS to promote CTF5 synthesis, which acted as a negative regulator in TNBC cells. Subsequently, we showed that CTF5 participated in MCPIP1-mediated antiproliferative effect by transcriptionally repressing cyclin D1 expression, as well as downregulating its downstream signaling targets p-Rb and E2F1. Conclusively, our findings provided novel insights into the anti-oncogenic mechanism of MCPIP1, suggesting that MCPIP1 could serve as an alternative treatment target in TNBC.

Investigation of bioactivities of endophytes of Ficus carica L. Fam Moraceae

BackgroundThe medicinal plant Ficus carica L., inhabiting southwest regions of Asia, is known for its wide ethnopharmacological applications. In traditional medicine, it has been applied as a treatment of respiratory, gastrointestinal and cardiovascular disorders, among others. Recent studies confirmed antimicrobial and anticancer activities for the plant extract. To find out whether endophytes of F. carica contribute to its biological activities, endophytes of the aerial parts of the plant were isolated and examined for anticancer and antimicrobial effects.ResultsThe isolated species were identified as Chaetomium globosum, Penicillium oxalicum, Alternaria alternata, Fusarium proliferatum and Aspergillus neoniger. An anticancer assay was carried out on the extracts of fungal strains against human umbilical vein endothelial cells (HUVEC), human immortalized myelogenous leukemia cell line (K-562) and human immortal cervical cancer cell line (HeLa). Results revealed antiproliferative effects for all endophytic strains against HUVEC and K-562 with GI50 values ranging from 4.75 to 13.75 µg mL−1 as well as cytotoxicity against HeLa with CC50 ranging from 8.25 to 18.75 µg mL−1. The antimicrobial activity of the extracts was tested against several test strains in agar diffusion assay, and MIC values were determined by the broth microdilution method. All strain extracts exerted antibacterial and antifungal activities with highest antifungal effect observed for A. neoniger extract (MIC = 3.75 µg mL−1).ConclusionsThese results support the assumption that endophytes of F. carica contribute to the plant’s reported antimicrobial and anticancer activities and its corresponding applications in traditional medicine.

Effects of bio-strength electric field on the motility and CD9 expression of human epidermal cell line HaCaT and mouse epidermal cells

Objective: To investigate the regulatory effect of bio-strength electric field (EF) on the motility and CD9 expression of human epidermal cell line HaCaT and mouse epidermal cells. Methods: The experimental research method was used. Human immortal epidermal cell line HaCaT cells in logarithmic growth phase and primary epidermal cells isolated from 16 BALB/c mice (no matter male or female) aged 1-3 days were used for experiments. HaCaT cells were divided into EF group treated for 3 h at the EF intensity of 200 mV/mm and sham EF group with simulated treatment. The cell migration (direction, displacement velocity, and trajectory velocity, with 46 samples in EF group and 34 samples in sham EF group) and arrangement were observed in the living cell workstation, and the distribution and expression of CD9 protein were detected by immunofluorescence method. Both HaCaT cells and mouse epidermal cells were divided into sham EF group (simulated treatment) and EF groups treated respectively for 3 h at the corresponding EF intensity of 50, 100, 200, and 400 mV/mm. Both HaCaT cells and mouse epidermal cells were divided into blank control group without any treatment, and 1 h group, 3 h group, and 6 h group treated with EF at the intensity of 200 mV/mm for corresponding time respectively. The expression of CD9 protein was detected by Western blotting (n=3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test and least significant difference test. Results: Within 3 hours of treatment, HaCaT cells in EF group tended to move towards the negative electrode obviously, while HaCaT cells in sham EF group moved randomly around the origin; compared with those of sham EF group, the directivity of HaCaT cells in EF group was significantly enhanced, and the displacement velocity and trajectory velocity were significantly increased (Z=-3.975, -6.052, -6.299, P<0.01). After 3 hours of treatment, the long axis of HaCaT cells in EF group was perpendicular to the direction of EF, while HaCaT cells in sham EF group arranged randomly. After 3 hours of treatment, the expression of CD9 protein in HaCaT cells in EF group was significantly down-regulated compared with that of sham EF group (t=4.527, P<0.01), although both expressed on cytomembrane. After 3 hours of treatment, the expression of CD9 protein in HaCaT cells and mouse epidermal cells in sham EF group, 50 mV/mm group, 100 mV/mm group, 200 mV/mm group, and 400 mV/mm group were 0.332±0.021, 0.283±0.032, 0.254±0.020, 0.231±0.041, 0.212±0.031 and 0.565±0.021, 0.453±0.022, 0.389±0.020, 0.338±0.021, 0.233±0.011, respectively. For both types of cells, compared with that of sham EF group, the expression of CD9 protein in cells was significantly decreased in the four groups of EF treatment (P<0.01); compared with that of 50 mV/mm group, the expression of CD9 protein in cells was significantly decreased in the other three groups of EF treatment (P<0.01); compared with that of 100 mV/mm group, the expression of CD9 protein in cells was significantly decreased in 200 mV/mm group and 400 mV/mm group (P<0.01); compared with that of 200 mV/mm group, the expression of CD9 protein in cells was significantly decreased in 400 mV/mm group (P<0.01). The expression levels of CD9 protein in HaCaT cells and mouse epidermal cells in blank control group, 1 h group, 3 h group, and 6 h group were 0.962±0.031, 0.784±0.020, 0.531±0.021, 0.409±0.011 and 0.963±0.031, 0.872±0.031, 0.778±0.040, 0.591±0.041, respectively. For both types of cells, compared with that of blank control group, the expression of CD9 protein in cells was significantly decreased in 1 h group, 3 h group, and 6 h group (P<0.01); compared with that of 1 h group, the expression of CD9 protein in cells was significantly decreased in 3 h group and 6 h group (P<0.05 or P<0.01); compared with that of 3 h group, the expression of CD9 protein in cells was significantly decreased in 6 h group (P<0.01). Conclusions: The bio-strength intensity EF can induce the directional migration and arrangement of HaCaT cells and down-regulate the expression of CD9 in HaCaT cells and mouse epidermal cells in a time-dependent and intensity-dependent manner.

Use of MTT Assay for Proliferation of U937 Cell Line and its Inter-rater Reliability - Best Taken with a Grain of Salt

Background Biomedical research frequently employs immortal human cell lines to study various physiological and pathological topics of interest. An important problem that is often ignored is the question of reliability of observations made while using a particular assay on cell lines, especially MTT Assay. Recent studies have questioned its reliability by highlighting the diverse intracellular environmental factors that affect accuracy of MTT assay results. Objective To assess the inter-rater reliability of MTT assay while observing the differential effect of addition of fetal bovine serum to the culture medium on proliferation of U937 cell line using Intraclass correlation coefficient. Method U937 cell suspension prepared by the tutor to maintain uniformity of immortalized cell line was used. Four trainee investigators who participated in the study underwent rigorous training in pipetting and plating methods for four consecutive days prior to start of the experiment. Each trainee investigator followed the same protocol and all procedures were conducted simultaneously. At the end of the experiment, inter observer reliability was calculated. Result Interrater reliability calculated by using intra class correlation coefficient with two way mixed effect model was found to be poor (p = 0.173). For growth with 10% fetal bovine serum, inter-rater reliability was 0.258 for each item and 0.58 for the average of the triplicates, whereas for 0% fetal bovine serum it was 0.374 for each item and 0.70 for average of the values (p=0.104). Conclusion Though MTT assay is considered the gold standard test for assessment of cell proliferation and viability, the inter-rater reliability of this assay might be poor and needs further investigation.

Fine-mapping and cell-specific enrichment at corneal resistance factor loci prioritize candidate causal regulatory variants

Corneal resistance factor (CRF) is altered during corneal diseases progression. Genome-wide-association studies (GWAS) indicated potential CRF and disease genetics overlap. Here, we characterise 135 CRF loci following GWAS in 76029 UK Biobank participants. Enrichment of extra-cellular matrix gene-sets, genetic correlation with corneal thickness (70% (SE = 5%)), reported keratoconus risk variants at 13 loci, all support relevance to corneal stroma biology. Fine-mapping identifies a subset of 55 highly likely causal variants, 91% of which are non-coding. Genomic features enrichments, using all associated variants, also indicate prominent regulatory causal role. We newly established open chromatin landscapes in two widely-used human cornea immortalised cell lines using ATAC-seq. Variants associated with CRF were significantly enriched in regulatory regions from the corneal stroma-derived cell line and enrichment increases to over 5 fold for variants prioritised by fine-mapping-including at GAS7, SMAD3 and COL6A1 loci. Our analysis generates many hypotheses for future functional validation of aetiological mechanisms.

Betulinic Acid-Azaprostanoid Hybrids: Synthesis and Pharmacological Evaluation as Anti-inflammatory Agents

Background: Prevention and treatment of chronic inflammatory diseases require effective and low-toxic medicines. Molecular hybridization is an effective strategy to enhance the biological activity of new compounds. Triterpenoid scaffolds are in the focus of attention owing to their anti-inflammatory, antiviral, antiproliferative, and immunomodulatory activities. Heteroprostanoids have different pleiotropic effects in acute and chronic inflammatory processes.Objective: The study aimed to develop structurally new and low toxic anti-inflammatory agents via hybridization of betulinic acid with azaprostanoic acids.Methods: A series of betulinic acid-azaprostanoid hybrids was synthesized. The synthetic pathway included the transformation of betulin via Jones' oxidation into betulonic acid, reductive amination of the latter and coupling obtained by 3β-amino-3-deoxybetulinic acid with the 7- or 13-azaprostanoic acids and their homo analogues. The hybrids 1-9 were investigated in vivo on histamine-, formalin- and concanavalin A-induced mouse paw edema models and two models of pain - the acetic acid-induced abdominal writhing and the hot-plate test. The hybrids were in vitro evaluated for cytotoxic activity on cancer (MCF7, U-87 MG) and non-cancer humane cell lines.Results: In the immunogenic inflammation model, the substances showed a pronounced anti-inflammatory effect, which was comparable to that of indomethacin. In the models of the exudative inflammation, none of the compounds displayed a statistically significant effect. The hybrids produced weak or moderate analgesic effects. All the agents revealed low cytotoxicity on human immortalized fibroblasts and cancer cell lines compared with 3β-amino-3-deoxybetulinic acid and doxorubicin.Conclusion: The results indicate that the principal anti-inflammatory effect of hybrids is substantially provided with the triterpenoid scaffold and in some cases with the azaprostanoid scaffold, but the latter makes a significant contribution to reducing the toxicity of hybrids. Hybrid 1 is of interest as a potent low toxic agent against immune-mediated inflammation.

A Solid Ultra Fine Self-Nanoemulsifying Drug Delivery System (S-SNEDDS) of Deferasirox for Improved Solubility: Optimization, Characterization, and In Vitro Cytotoxicity Studies

The research work was designed to develop a solid self-nanoemulsifying drug delivery system (S-SNEDDS) of deferasirox (DFX). According to the solubility studies of DFX in different components, Peceol, Kolliphor EL, and Transcutol were selected as excipients. Pseudo-ternary phase diagrams were constructed, and then SNEDDS formation assessment studies and solubility of DFX in selected SNEDDSs formulations were performed. DFX loaded SNEDDS were prepared and characterized. The optimum DFX-SNEDDS formulations were developed. The relative safety of the optimized SNEDDS formulation was examined in a human immortalized myelogenous leukemia cell line, K562 cells, using the MTT cell viability test. Cytotoxicity studies revealed more cell viability (71.44%) of DFX loaded SNEDDS compared to pure DFX (3.99%) at 40 μM. The selected DFX-SNEDDS formulation was converted into S-SNEDDS by adsorbing into porous carriers, in order to study its dissolution behavior. The in vitro drug release studies indicated that DFX release (Q5%) from S-SNEDDS solidified with Neusilin UFL2 was significantly higher (93.6 ± 0.7% within 5 min) compared with the marketed product (81.65 ± 2.10%). The overall results indicated that the S-SNEDDS formulation of DFX could have the potential to enhance the solubility of DFX, which would in turn have the potential to improve its oral bioavailability as a safe novel delivery system.

Microvesicles from Human Immortalized Cell Lines of Endothelial Progenitor Cells and Mesenchymal Stem/Stromal Cells of Adipose Tissue Origin as Carriers of Bioactive Factors Facilitating Angiogenesis.

Endothelial progenitor cells (EPCs) and mesenchymal stem/stromal cells (MSCs) are associated with maintaining tissue homeostasis and tissue repair. Both types of cells contribute to tissue regeneration through the secretion of trophic factors (alone or in the form of microvesicles). This study investigated the isolation and biological properties of microvesicles (MVs) derived from human immortalized MSC line HATMSC1 of adipose tissue origin and EPC line. The human immortalized cell line derived from the adipose tissue of a patient with venous stasis was established in our laboratory using the hTERT and pSV402 plasmids. The human EPC line originating from cord blood (HEPC-CB.1) was established in our previous studies. Microvesicles were isolated through a sequence of centrifugations. Analysis of the protein content of both populations of microvesicles, using the Membrane-Based Antibody Array and Milliplex ELISA showed that isolated microvesicles transported growth factors and pro- and antiangiogenic factors. Analysis of the miRNA content of isolated microvesicles revealed the presence of proangiogenic miRNA (miR-126, miR-296, miR-378, and miR-210) and low expression of antiangiogenic miRNA (miR-221, miR-222, and miR-92a) using real-time RT-PCR with the TaqMan technique. The isolated microvesicles were assessed for their effect on the proliferation and proangiogenic properties of cells involved in tissue repair. It was shown that both HEPC-CB.1- and HATMSC1-derived microvesicles increased the proliferation of human endothelial cells of dermal origin and that this effect was dose-dependent. In contrast, microvesicles had a limited impact on the proliferation of fibroblasts and keratinocytes. Both types of microvesicles improved the proangiogenic properties of human dermal endothelial cells, and this effect was also dose-dependent, as shown in the Matrigel assay. These results confirm the hypothesis that microvesicles of HEPC-CB.1 and HATMSC1 origin carry proteins and miRNAs that support and facilitate angiogenic processes that are important for cutaneous tissue regeneration.

The potential of Salvia officinalis as a suppressor of cell proliferation in animal feed and human nutrition: an experimental study

The objective of this study was to examine the in vitro cytotoxic activities of Salvia officinalis sage oil on human immortalized keratinocyte HaCaT cell lines by using an [3- 4,5-dimethylthiazol-2-yl -2,5-diphenyltetrazoliumbromide] MTT cytotoxicity assay after the Salvia officinalis oil administration in different doses and time-points. In vitro cytotoxic activities of Salvia officinalis oil on HaCaT cell lines were assessed, and MTT assays were used to determine cell viability. The HaCaT cells 100 µL were cultured in 96- well plates at 2 × 104 cells per well and treated with different concentrations of Salvia officinalis oil 25 µM, 50 µM, 100 µM, 150 µM, and 200 µM for durations of 24, 48, and 72 h. Cell death was determined by collecting and staining with 0.4% Trypan blue for 5 min at room temperature before microscopic examination. At 24 h, there was a significant difference between the 25 and 200 µM doses P = 0.034 . For the 72 h Salvia officinalis oil application, there was a significant difference between the 50 and 200 µM doses only P = 0.002 . On the other hand, for 48 h Salvia officinalis application, there were significant differences between 25 and 150 µM, 25 and 200 µM, 50 and 150 µM, 50 and 200 µM, 100 and 150 µM, and 100 and 200 µM doses P = 0.003, P = 0.000, P = 0.005, P = 0.001, P = 0.028, and P = 0.003, respectively . We concluded that Salvia officinalis oil may help the living organism to have a healthy digestive system by stopping excessive cell proliferation via the apoptotic pathway in the gastrointestinal system and, thus, may be used in both animal feed and human nutrition.

The Desmoglein‐1 Transmembrane Domain Drives Lipid Raft Association and Desmosome Assembly

The epidermis is a barrier to the outside world, but mutations in genes encoding desmosomal proteins can inhibit this function, causing human diseases of skin fragility. Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a severe homozygous recessive disorder of skin fragility caused by mutations in the gene encoding desmoglein 1 (DSG1), a component of desmosomes. Desmosomes are junctional complexes that function in mechanical stress resistance across tissues. In these junctions, desmosomal cadherins such as DSG1 interact across extracellular spaces and with intracellular adaptor proteins to link cytoskeletal elements of adjacent cells. Both assembly and disassembly of desmosomes have been shown to rely on lipid raft membrane microdomains, highly ordered cholesterol and sphingolipid‐enriched regions of the plasma membrane important in signaling and trafficking. Our current model predicts that raft association promotes clustering of desmosomal proteins to drive assembly and mediate adhesion. However, the mechanisms of raft association and desmosome assembly are not fully understood. We created a DSG‐null A431 cell line to examine the relationship between DSG raft association and desmosomal adhesion. A431s are an immortal human epidermal carcinoma cell line commonly used in desmosomal studies that express a single DSG species, DSG2. The absence of DSG2 in these cells was confirmed by western blot and immunofluorescence. Further characterization of these DSG‐null A431 cells revealed mislocalized desmoplakin, a marker of mature desmosomes, and a lack of strong cell‐cell adhesion. Exogenous stable expression of GFP‐tagged wildtype DSG1 (DSG1WT‐GFP) rescued both desmosome formation and function, suggesting that these DSG‐null cells would be suitable for probing the relationship between DSG raft association and desmosome assembly and function. Recently, we discovered a novel, heterozygous mutation in the transmembrane domain (TMD)‐encoding region of DSG1 that causes a glycine to arginine substitution and SAM syndrome. DSG1SAM is a lipid raft targeting mutant suggesting an important role for the TMD in raft association. Stable expression of DSG1SAM‐GFP could not rescue desmosome formation or function in the DSG‐null cells, suggesting that a loss of raft association also impacts desmosome assembly and adhesive function. Intriguingly, a chimeric DSG1 protein containing the TMD from E‐cadherin (DSG1Ecad‐GFP), a non‐raft associating adhesive protein, only partly rescued desmosome formation and function. Furthermore, these results correlated with the predicted lipid raft affinity of each TMD, suggesting that desmosome assembly and function is dependent on raft association. We are currently analyzing a panel of GFP‐tagged DSG1TMD variants with differential raft targeting affinities and assessing their ability to support desmosome assembly and function. In conclusion, the TMD targets desmogleins to lipid rafts and modulates desmoglein adhesive function.Support or Funding InformationNIH R01AR050501, NIH R01AR048266

Silencing of keratin 17 by lentivirus-mediated short hairpin RNA inhibits the proliferation of PANC-1 human pancreatic cancer cells.

Keratin 17 (KRT17) has been demonstrated to be a potential biological marker for the prediction of prognosis in particular types of cancer. The aim of the present study was to investigate the molecular mechanisms underlying the function of KRT17 in the pancreatic cancer (PAC) cell line PANC-1 and the potential of KRT17 as a therapeutic target for PAC. KRT17 expression levels were analyzed using quantitative PCR and compared with histological data using bioinformatics tools in PAC samples and three human PAC cell lines. Cell proliferation was determined using an MTT assay, in addition to cell cycle distribution and apoptosis analysis using flow cytometry, colony formation assay using Giemsa staining and cell motility analysis using a Transwell migration assay. Tumor growth was evaluated in vivo in nude mice. The expression levels of a number of signaling molecules were measured to establish the potential mechanism by which silencing KRT17 expression affected PAC PANC-1 cells. Increased levels of KRT17 expression were observed in human PAC compared with normal tissues, as well as in three human PAC cell lines (MIA PaCa-2, PANC-1 and KP-3 cells) compared with the H6c7 human immortal pancreatic duct epithelial cell line. High expression levels of KRT17 in PAC samples were associated with poor overall survival (P=0.036) and disease-free survival (P=0.017). Lentivirus-mediated KRT17 silencing inhibited cell proliferation, colony formation and migration, but promoted apoptosis and resulted in cell cycle arrest in the G0/G1 phase in PANC-1 cells. In addition, KRT17 knockdown inhibited in vivo tumor growth. KRT17 knockdown induced dysregulation of ERK1/2 and upregulation of the pro-apoptotic Bcl-2 protein Bad. In conclusion, the present study demonstrated that elevated KRT17 levels are positively associated with pancreatic cancer progression; KRT17 knockdown suppressed cell growth, colony formation, migration and tumor growth, and induced apoptosis and cell cycle arrest, affecting ERK1/2/Bad signaling. Therefore, the results of the present study suggested that KRT17 may be a potential target for the treatment of pancreatic cancer.

Differential Effects of Lipid and Vitamin D treatment on Cell Viability and CYP24A1 Expression in Hepatocytes and Hepatic Stellate Cells

AbstractActivated hepatic stellate cells (HSCs) are a key contributor to liver fibrosis and drive the progression to advanced disease for many liver conditions, including non-alcoholic fatty liver disease. Previous studies suggest vitamin D may reduce inflammatory and pro-fibrogenic activity of HSCs in vitro. However, the mechanisms underpinning the effects of vitamin D in HSCs are not fully understood. The overall aim of these experiments was to mimic a lipid loading model on immortalised HSCs to test their responses to 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). Two different human immortalised cell lines: HepG2, hepatocellular carcinoma cells, and LX-2, hepatic stellate cells; were cultured using standard methods. Cell viability in different treatment vehicles (2% DMSO and/or 0.1% ethanol) under serum free conditions was measured by MTT assay after 6 and 24 h. Cells were cultured with increasing concentrations of fatty acids (0–500μM, 1:1 oleic acid: palmitic acid) or vitamin D. Nile red, a neutral lipophilic fluorescent dye, was used to measure total intracellular lipid and quantified relative to vehicle. CYP24A1 mRNA expression was measured by qPCR in response to 1000nM 1α,25(OH)2D3 treatment in both cell lines for 24 h using TaqMan® gene expression assays and normalised to 18S rRNA. Cell viability in response to vehicle was examined at 6 h and 24 h to determine the optimal experimental time points. Whereas, HepG2 cells remained unaffected at 24 h in response to either or both vehicles combined (n = 4; combined vehicles, P = 0.3187), LX-2 cells showed reduced viability even at 6 h (n = 5; combined vehicles, P = 0.0050). Fatty acid treatment led to intracellular lipid accumulation in both cell lines. In response to 500μM fatty acid treatment, intracellular lipid increased by 1.7-fold in LX-2 cells at 6 h (n = 5, P = 0.00174) and 3.9-fold in HepG2 cells after 24 h (n = 4, P = 0.00184). Notably, CYP24A1 mRNA expression was markedly induced by vitamin D treatment in LX-2 cells (136 ± 7.64-fold, n = 3, P = 0.0010) in comparison to HepG2 cells (22 ± 0.78-fold, n = 3, P &lt; 0.0001). In summary, the cell viability data suggested optimal time points for both fatty acid and vitamin D treatments may be 6 h for LX-2 cells, and 24 h for HepG2 cells. While intracellular lipid accumulation differed between the cell lines in response to fatty acid treatment, both cell lines produced a dose-dependent increase in intracellular lipid. Lastly, CYP24A1 mRNA expression confirmed the responsiveness of both cell types to vitamin D treatment. Ongoing experiments are examining microRNA expression in HSCs in response to both vitamin D and lipid loading.

Naturally-occurring bacterial cellulose-hyperbranched cationic polysaccharide derivative/MMP-9 siRNA composite dressing for wound healing enhancement in diabetic rats

The anomalous high expression of matrix metalloproteinase 9 (MMP-9) is one important factor that impedes diabetic wound healing. Therefore, inhibition of MMP-9 expression in a diabetic wound could be a feasible method to promote wound healing. In this study, we studied the possibility of self-therapy using wound dressings that contain bacterial cellulose-hyperbranched cationic polysaccharide (BC-HCP) derivatives that encapsulate siRNA (BC-HCP/siMMP-9) and have controlled release properties. Herein, we used four HCPs (Gly-DMAPA, Gly-D4, Amyp-DMAPA, Amyp-D4) as gene carriers. Our results showed that all HCP derivatives were minimally toxic to cells in vitro, while the cationic properties of HCP could be used as a complexation agent for MMP-9 siRNA (siMMP-9). Upon exposure to bacterial cellulose (BC), the BC slowly released HCP/siMMP-9. The released siMMP-9 effectively reduced the gene expression and protein levels of MMP-9 in a human immortalized epithelial cell line (HaCAT) and in diabetic rat wounds. Inhibition of MMP-9 in the wounds of diabetic rats resulted in a significant enhancement of wound healing, suggesting that the BC-HCP/siMMP-9 composite dressing could be used as a safe and effective dressing to promote wound healing in diabetic rats. STATEMENT OF SIGNIFICANCE: In this work, we evaluated the possibility of using bacterial cellulose-hyperbranched cationic polysaccharide derivatives (BC-HCP) as a self-therapeutic wound dressing with siRNA encapsulated and controlled release properties. Our results showed that the BC-HCP/siMMP-9 composite dressing slowly released HCP/siMMP-9. The released siMMP-9 effectively reduced the gene expression and protein level of MMP-9 in human immortalized epithelial cell line and in the wound of diabetic rats. The BC-HCP/siMMP-9 composite dressing promoted diabetic wound healing by the unique nanostructure of BC and by releasing siMMP-9 for specific MMP-9 inhibition. Therefore, it could be used as a safe and effective dressing to promote wound healing in diabetic rats. This is the first evidence on the study of using BC as a dressing composite by encapsulating HCP/siRNA complexes for efficient RNAi gene silencing for better wound healing in diabetic rats.

General toxicity assessment of the novel aldose reductase inhibitor cemtirestat.

Cemtirestat, 3-mercapto-5H-[1,2,4]-triazino[5,6-b] indole-5-acetic acid was recently designed and patented as a highly selective and efficient aldose reductase inhibitor endowed with antioxidant activity. The aim of the present study was to assess the general toxicity of cemtirestat using in silico predictions, in vitro and in vivo assays. ProTox-II toxicity prediction software gave 17 “Inactive” outputs, a mild hepatotoxicity score (0.52 probability) along with a predicted LD50 of 1000 mg/kg. Five different cell lines were used including the immortalized mouse microglia BV-2, the primary human fibroblasts VH10, the insulinoma pancreatic β-cells INS-1E, the human colon cancer cells HCT116 and the human immortalized epithelial endometrial cell lines HIEEC. In contrast to the clinically used epalrestat, cemtirestat showed remarkably low cytotoxicity in several different cell culture viability tests such as MTT proliferation assay, neutral red uptake, BrdU incorporation, WST-1 proliferation assay and propidium iodide staining followed by flow cytometry. In a yeast spotting assay, the presence of cemtirestat in incubation of Saccaromyces cerevisiae at concentrations as high as 1000 μM did not affect cell growth rate significantly. In the 120-day repeated oral toxicity study in male Wistar rats with daily cemtirestat dose of 6.4 mg/kg, no significant behavioral alterations or toxicological manifestations were observed in clinical and pathological examinations or in hematological parameters. In summary, these results suggest that cemtirestat is a safe drug that can proceed beyond preclinical studies.