Methyl Tetrazolium Salt Research Articles

Comparative analysis of cytotoxicity effects of two denture hard lining materials on human gingival fibroblasts: an in vitro study

BackgroundThe objective of this study was to compare the cytotoxicity of TDV and Rebase II denture hard liners on human gingival fibroblasts, aiming to address issues associated with incomplete polymerization and free monomers that affect material properties. MethodsSeventy-two specimens (24 each of TDV, Rebase II, and controls) were prepared under aseptic conditions according to factory instructions. Cytotoxicity was determined using the MTT test with methyl tetrazolium salt added to the cell culture medium. A two-way ANOVA and a post-hoc Tukey test was used to evaluate the results of incubation before mitochondrial activity was measured using Multiscan spectrophotometry (570 nm). ResultsThere were significant differences in cell viability between the groups after 24 hours (P < 0.001), with TDV having higher viability than Rebase II. The difference between Rebase II and TDV, however, was not significant at 48 and 96 hours (P > 0.131). At 24 hours, Rebase II exhibited significantly lower viability than TDV liner, with a significant difference between the two groups (P = 0.001). ConclusionDue to the maximum monomer release in the early hours of incubation, the amount of cytotoxicity decreased with increasing incubation time.

Ultraviolet C as a method of disinfecting medical silicone used in facial prostheses: An in vitro study – Part 2

Statement of problemDisinfection is an important factor in preserving facial prostheses and maintaining tissue health. However, whether disinfection with ultraviolet C is an effective disinfection method is unclear. PurposeThe purpose of this in vitro study was to evaluate the effectiveness of irradiation with different exposure durations of an ultraviolet-C light-emitting diode in the disinfection of the silicone (A-588–1; Factor II) used in facial prostheses. Material and methodsA total of 216 specimens were prepared, contaminated by multispecies biofilm, and divided into 9 groups (n=24) for different treatments: chlorhexidine 0.12% (G CHG), ultraviolet-C light-emitting diode for 5 minutes (G UVC5), ultraviolet-C light-emitting diode for 10 minutes (G UVC10), ultraviolet-C light-emitting diode for 20 minutes (G UVC20), their respective untreated controls (Gcontrol CHG, Gcontrol UVC5, Gcontrol UVC10, Gcontrol UVC20), and dimethyl sulfoxide (G DMSO) as the negative control. Cell viability was measured by using the methyl tetrazolium salt (MTT) method. Two statistical analyses were performed. First, a 2×3 ANOVA was carried out to compare the control groups (Gcontrol UVC5, Gcontrol UVC10, and Gcontrol UVC20) and the experimental groups of UV-C LED light with different exposure durations (G UVC5, G UVC10, and G UVC20). The second analysis was performed using generalized linear models to compare the optical density of the groups (G UVC5, G UVC10, G UVC20, G CHG, and G DMSO). ResultsCell viability results demonstrated a microbial reduction after exposure to the ultraviolet-C light-emitting diode for 20 minutes (G UVC20) compared with untreated controls (P<.05). The 5- and 10-minute exposures were statistically similar to their respective control groups (P>.05). The 20 minutes exposure had the lowest average optical density value, being statistically different from the 5-minute exposure (P<.05). A 20-minute exposure to the ultraviolet-C light-emitting diode (G UVC20) was similarly effective when compared with the standard disinfection treatment (G CHG) and dimethyl sulfoxide (G DMSO) (P>.05). ConclusionsIrradiation with an ultraviolet-C light-emitting diode for 20 minutes decreased the in vitro microbial cell viability on the medical silicone used in facial prostheses.

Effect of the surface finish on the mechanical properties and cellular adhesion in (Ce,Y)-TZP/Al2O3 ceramic composites for denture implants

Ceramic composites based on (Ce, Y)-TZP/Al2O3 system have great potential for applications as dental implants due to their unusually great balance between good mechanical properties and resistance to hydrothermal degradation. Surface roughness plays an important role in controlling these properties, but few studies have investigated the relationship between cytocompatibility and surface roughness, at levels considered moderate and low, comparable to titanium implants. In this work, bending strength, hydrothermal degradation and biological evaluation of a ceramic composite based on (Ce,Y)-TZP/Al2O3 system were investigated as a function of surface roughness. Compacted samples were sintered at 1500 °C - 2h and then submitted to different surface treatments: Group 1 composed of samples with smooth surfaces, Group 2 and Group 3 composed of rough surfaces (grinded with 15 μm or 45 μm diamond sandpaper, respectively. Samples were characterized by X-ray diffraction, scanning electron microscopy, contact angle and optical profilometry and then subjected to hydrothermal degradation tests in autoclave (134 °C - 2 bar) using artificial saliva. The Piston-on-three-balls (P–3B) testing was used to determine flexural strength. To assess indirect cytotoxicity, samples were immersed in the culture medium for NIH-3T3 cells for 72 h. Furthermore, cell adhesion and proliferation were investigated using MG63 cells (human osteosarcoma) after 3, 7, 14, and 21 days of culture. Cytotoxicity, adhesion, and cell proliferation were examined by the Methyl Tetrazolium salt (MTS) and Alizarin Red, using a confocal laser microscope. The results indicated that the materials have high resistance to degradation. Furthermore, the (Ce,Y)-TZP/Al2O3 composites are not cytotoxic. The flexural strength of the composites was 913 ± 103 MPa in samples presenting original (smooth) surface, however, a reduction in the order of 17% was observed in samples containing rough surfaces. The rougher samples show the best cellular adhesion and proliferation, leading to the formation of a mineralized matrix after 21 days. These results clearly suggest that the new (Ce,Y)-TZP/Al2O3 brand is strong and highly biocompatible and warrants further study.

Ultraviolet C as a method of disinfecting medical silicone used in facial prostheses: An in vitro study

Statement of problemHygiene and disinfection are important factors for preserving facial prostheses and supporting tissue health. However, a method that does not accelerate degradation or color change is necessary. PurposeThe purpose of this in vitro study was to evaluate the effectiveness of irradiation with ultraviolet C light-emitting diode (UV-C LED) light in the disinfection and initial color stability of the silicone (A-588-1; Factor II) used in facial prostheses. Material and methodsOne hundred and twenty specimens were made, contaminated by multispecies biofilm, and divided into 5 groups (n=24) with different treatments: control, distilled water, 0.12% chlorhexidine, UV-C LED light, and dimethyl sulfoxide (DMSO) as the negative control. Cell viability was measured by the methyl tetrazolium salt method. Statistical analysis was performed by generalized linear models. Additional descriptive analysis was performed for color analysis by using 16 silicone specimens made with light and dark intrinsic coloring in 4 groups (controls and treatments n=4) submitted to UV-C LED light. The ΔE of the specimens was obtained by CIEDE200. ResultsThe results of cell viability demonstrated a statistically significant difference among the groups (P<.001), with a microbial reduction after UVC-LED exposure compared with the control group. Regarding the color, the groups presented an average ΔE (light 0.205 and dark 0.308) compatible with visually imperceptible changes (light <0.7 and dark <1.2). ConclusionsIrradiation with UV-C LED light decreased the in vitro microbial cell viability of the medical silicone used in facial prostheses, demonstrating initial color stability.

Antimicrobial potential of resin matrices loaded with coffee compounds.

This study evaluated the biological behavior of the coffee compounds Trigonelline (T), chlorogenic acid (C), and nicotinic acid (N), correlating with their release from a resin matrix. Minimum inhibitory concentration (MIC) was evaluated against Streptococcus mutans UA159, and cytotoxicity was assessed by methyl tetrazolium salt on OD-21 cells. Resin matrices (bisphenol A-glycidyl-dimethacrylate/triethylene glycol-dimethacrylate 70/30 wt%, camphorquinone/ethyl 4-dimethyl aminobenzoate 0.5/1 wt%) were doped with coffee compounds in different concentrations (10/20/30/40/50 wt%), performing 15 groups (T10-T50, C10-C50, N10-N50), and a control group with no coffee compound. Degree of conversion (DC%) was analyzed by Fourier transform infrared spectroscopy. Antimicrobial properties were evaluated by bioluminescence (Luciferase assay). The release from loaded matrices was analyzed over time (24 hr, 6, 14, 21 and 28 days), using high-performance liquid chromatography (HPLC). Data were submitted to ANOVA/Tukey's test (α = 0.05). MIC for T and C was 6 mg/ml, and 4 mg/ml for N. None of them were cytotoxic. Only T50 and C50 showed lower DC% than control (α < 0.05). Some groups (T30/T40/T50/C40/C50/N50) were strongly antimicrobial, reducing bacterial activity approximately five times compared to control (α < 0.05). For T30, T40, T50, C40, and C50, the HPLC showed a release above or closer to MIC values mainly in 24 hr, but for N50, up to 28 days. In conclusion, the coffee compounds presented antimicrobial activity, depending on their concentration when added in resin matrices, being found a correlation with their release.

In Vitro Analysis of the Proliferation of HaCaT Cells Stimulated by Pigments Used for Maxillofacial Prostheses

Introduction: Although physical and mechanical properties are extremely important for the effectiveness of prosthetic treatment, materials used for facial rehabilitation should be biocompatible, as the maxillofacial prosthesis is in close contact with the patient’s skin. The objective of this study was to evaluate the influence of different eluates formation periods obtained from the methyl tetrazolium salt (MTT) and Alamar Blue assays, analyzed in different silicones and pigments used in the manufacture of maxillofacial prostheses in the proliferation of human keratinocytes (HaCaT cells). Materials and Methods: For the preparation of samples, A2 A-2186 and Silastic MDX4-4210 silicones and three types of pigments (bronze, black, and medium pink) were selected. Two of the pigments were specific for the characterization of the prosthesis and already available in dental market in bronze and black colors, and a new pigment was tested in the medium pink color. Five samples were prepared for each group and distributed according to the type of silicone and pigment added, aiming to prepare the eluates after 24 and 72 h of sample’s immersion in culture medium. In vitro assays of MTT and Alamar Blue were performed for cytotoxic analysis. Data obtained were submitted to analysis of variance and the Tukey test, with a 5% significance level. Results: All groups had cell proliferation percentages higher than 75%, indicating absence of cytotoxicity. Conclusions: It can be concluded that all the materials used are not cytotoxic, including the proposed new pigment, for the preparation of maxillofacial prostheses.

In vitro biocompatibility of new bioactive lithia-silica glass-ceramics

Glass-ceramics based on the Li2O-SiO2 system have been extensively used as restorative dental materials due to their excellent chemical durability, aesthetics, inertness in the buccal environment, and high fracture strength; but they are not bioactive. On the other hand, all known bioactive glasses show ability to bond to bone, teeth and cartilage coupled to osteoconduction and osteoinduction, but their fracture strength and toughness are rather low. The aim of this study is to develop and evaluate the in vitro biocompatibility of a new type of (bioactive and strong) lithia-silica glass-ceramic. For these purposes, two types of glass-ceramics based on a multicomponent lithia-silica system were studied: lithium metasilicate (LM) and lithium disilicate (LD). The in vitro bioactivity study was conducted in a SBF solution, before and after different times of immersion; the new materials were analyzed by XRD, FTIR, and SEM. Some samples were subjected to in vitro biodegradation tests to quantify the release of lithium and the weight loss. Cytotoxicity, adhesion, and cell proliferation on different samples were examined by using the Methyl Tetrazolium salt (MTS) and Alizarin Red. For ~40 vol% crystallinity, lithium metasilicate was detected as the major phase, whereas for ~80 vol% crystallinity, lithium disilicate was the major phase. The LD proved to be strong (3p-bending strength of 233 ± 12 MPa) and bioactive after 14 days of immersion in SBF. In terms of lithium ion release, the LD was outside the toxic range (>8.3 ppm). The LM and LD are not cytotoxic. The LD shows the best cellular adhesion and proliferation, leading to the formation of a mineralized matrix after 21 days. These results clearly suggest that the new LD brand is strong and highly biocompatible and warrants further study.

Raspberry-like poly(γ-glutamic acid) hydrogel particles for pH-dependent cell membrane passage and controlled cytosolic delivery of antitumor drugs.

In this research, we synthesized bioderived poly(amino acid) hydrogel particles that showed pH-dependent membrane-disrupting properties and controlled cytosolic delivery of antitumor drugs. Poly(γ-glutamic acid) (γ-PGA) that has been produced extensively using bacteria, especially those of Bacillus subtilis species, was modified with cholesterol (γ-PGA/Chol), and the γ-PGA/Chol conjugates were used to form polymeric nanoparticles the size of 21.0±1.1 nm in aqueous solution. When the polymeric nanoparticles were mixed with doxorubicin (Dox), raspberry-like hydrogel particles (RBHPs) were formed by the electrostatic interaction between the cationically charged Dox and the anionically charged nanoparticles. The average size and surface charge of the RBHPs in aqueous solution were 444.9±122.5 nm and −56.44 mV, respectively. The loaded amount of Dox was approximately 63.9 μg/mg of RBHPs. The RBHPs showed controlled drug release behavior in both in vitro and ex vivo cell-based experiments. Through fluorescence microscopy and fluorescence-activated cell sorting, the cellular uptake of RBHPs into human cervical cancer cells (HeLa) was analyzed. The cytotoxic effect, evaluated by the methyl tetrazolium salt assay, was dependent on both the concentration of RBHPs and the treatment time. The pH-dependent membrane-disrupting properties of the RBHPs and the subsequent cytosolic delivery of Dox were evaluated using a standard hemolysis assay. Upon an increase in hydrophobicity at the lysosomal acidic pH, RBHPs could easily interact, penetrate cell membranes, and destabilize them. Taken together, the data suggested that RBHPs could be used as drug delivery carriers after loading with other therapeutic drugs, such as proteins or small interfering RNA for cancer therapy.

Polymer nanoparticles for cross-presentation of exogenous antigens and enhanced cytotoxic T-lymphocyte immune response.

Effective induction of an antigen-specific cytotoxic T lymphocyte (CTL) immune response is one of the key goals of cancer immunotherapy. We report the design and fabrication of polyethylenimine (PEI)-coated polymer nanoparticles (NPs) as efficient antigen-delivery carriers that can induce antigen cross-presentation and a strong CTL response. After synthesis of poly(d,l-lactide-co-glycolide) (PLGA) NPs containing ovalbumin (OVA) by the double-emulsion solvent-evaporation method, cationic-charged PLGA NPs were generated by coating them with PEI. In a methyl tetrazolium salt assay, no discernible cytotoxic effect of PEI-coated PLGA (OVA) NPs was observed. The capacity and mechanism of PEI-coated PLGA (OVA) NPs for antigen delivery and cross-presentation on dendritic cells (DCs) were determined by fluorescence microscopy and flow cytometry. PEI-coated PLGA (OVA) NPs were internalized efficiently via phagocytosis or macropinocytosis in DCs and induced efficient cross-presentation of the antigen on MHC class I molecules via both endosome escape and a lysosomal processing mechanism. The DCs treated with PEI-coated PLGA (OVA) NPs induced a release of IL-2 cytokine from OVA-specific CD8-OVA1.3 T cells more efficiently than DCs treated with PLGA (OVA) NPs. Therefore, the PEI-coated PLGA (OVA) NPs can induce antigen cross-presentation and are expected to be used for induction of a strong CTL immune response and for efficient anticancer immunotherapy.

Exposure of Human Endothelial Progenitors to Sevoflurane Improves Their Survival Abilities

Abstract Endothelial progenitor cells (EPCs) have prominent roles in vessel and tissue repair; however, their regenerative efficacy is diminished due to the poor survival in the hostile microenvironment of the injured organs. Recent data suggest a promising potential of volatile anesthetics for improving stem cell biology. Thus, we hypothesized that exposure to sevoflurane could stimulate growth and viability of cultured EPCs. Total mononuclear cells were isolated from human umbilical cord blood by gradient centrifugation. After five days in culture, the cells were exposed for one or two hours to sevoflurane 2% or 4% in air/5% CO2, or only to air/5% CO2 (sham control) in a sealed modular chamber. 24 or 48 hours post-exposure, viability, proliferation and apoptosis were assessed using lactate dehydrogenase (LDH) leakage assay, a methyl tetrazolium salt (MTS) assay and FITC-annexin V/ propidium iodide (PI) staining, respectively. LDH leakage was discretely lowered, whereas the levels of formazan were significantly increased (p &lt; 0.05 for 1 h incubation with 4% sevoflurane at 24 hrs post-exposure, and with 2% sevoflurane at 48 h post-exposure) in the preconditioned cultures, proving no cytotoxic effects and increased proliferation in treated cells versus control samples. Early (p &lt; 0.05) and late apoptosis (p &lt; 0.05 only for 2% sevoflurane) were diminished following the procedure. Thus, the commonly used sevoflurane anesthetic has protective effects on viability and proliferation of human early endothelial progenitor cells in vitro, suggesting a promising potential of anesthetic preconditioning for improving the regeneration of ischemic tissues.

Influence of the addition of β-TCP on the morphology, thermal properties and cell viability of poly (lactic acid) fibers obtained by electrospinning.

Electrospinning is a simple and low-cost way to fabricate fibers. Among the various polymers used in electrospinning process, the poly (lactic acid) (PLA) stands out due to its excellent biodegradability and biocompatibility. Calcium phosphate ceramics has been recognized as an attractive biomaterial because their chemical composition is similar to the mineral component of the hard tissue in the body. Furthermore, they are bioactive and osteoinductive and some are even quite biodegradable. The beta-tricalcium phosphate (β-TCP) particles were synthesized by solid state reaction. Different contents of β-TCP particles were incorporated in polymer matrices to form fibers of PLA/β-TCP composites by electrospinning aiming a possible application as a scaffold for tissue engineering. The fibers were characterized by scanning electron microscopy (SEM), infrared (FTIR), differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA). The average diameter of the fibers varied in the range of 260-519.6 ± 50 nm. The presence of β-TCP particles promoted changes on thermal properties of the fibers. The composite with 8 wt-% of β-TCP showed a low degree of crystallinity and can be used for application in tissue engineering. The cell viability was analyzed by reduction of the methyl tetrazolium salt by the pyruvate dehydrogenase enzymatic complex present in the matrix of mitochondria (MTT test). All PLA fiber groups, with different contents of β-TCP, showed cytocompatibility ability with non-cytotoxicity effect and bioactive properties using SBF assay.

Effects of Ad-p53 plus fulvestrant on induced apoptosis in MCF-7 breast cancer cells

To explore the apoptosis-inducing effect of Ad-p53 plus fulvestrant in MCF-7 breast cancer cells. ER-positive MCF-7 breast cancer cells were used. MCF-7 cells were established with control, fulvestrant alone, Ad-p53 alone and Ad-p53 plus fulvestrant. The morphological changes of MCF-7 after drug combination were observed by inverted microscopy. Methyltetrazolium salt (MTS) assay was used to determine the proliferation of MCF-7 cells. The protein expressions of p53 and ER were examined by Western blot. Flow cytometry was used to detect apoptosis. Significant inhibition of cell growth was observed by inverted microscopy. Flow cytometry revealed that the apoptosis rate of using fulvestrant alone or Ad-p53 alone was (13.3 ± 1.2) % and (14.5 ± 2.9) %. The apoptosis rate of combined group was (38.1 ± 5.9)% and it was higher than Ad-p53 or fulvestrant alone (P = 0.001, P = 0.001). MTS assay indicated that, after treating for 24, 48, 72, 96 h by Ad-p53 alone or fulvestrant alone, the inhibition of cell proliferation rate was (8.21 ± 0.54)%, (28.50 ± 1.42)%, (50.14 ± 0.78)%, (58.25 ± 2.92)% and (9.73 ± 1.68)%, (25.26 ± 0.82)%, (35.25 ± 3.94)% and (46.37 ± 2.56)% respectively. Simultaneously, the inhibition of cell proliferation rate of combined group was (12.42 ± 1.76)%, (35.20 ± 0.58)%, (62.08 ± 2.56)% and (75.43 ± 3.56)%. It further confirmed the increase of cell proliferation inhibition rate after drug combination (P < 0.05). Western blot showed that the p53 protein expression level increased obviously with Ad-p53 in MCF-7cells. Ad-p53 increased the level of ER expression while drug combination reduced the level of ER expression. Ad-p53 plus fulvestrant can significantly inhibit cell growth and induce cell apoptosis. And there is probably some synergistic effects between both.

Differential dendritic cell‐mediated activation and functions of invariant NKT‐cell subsets in oral cancer

Invariant natural killer T (iNKT) cells are unique subset of glycolipid-reactive T lymphocytes with potent antitumour characteristics. This study was planned to understand Th-like cytokine profiles of iNKT-cell subsets and modulation of their functions in response to glycolipid ligand and tumour cell lysate (TL). Cytokine profile of iNKT-cell subsets was evaluated from the peripheral blood of eight oral squamous cell carcinoma (OSCC) patients by flow cytometry and enzyme-linked immunosorbent assay (ELISA), while antitumour activity of iNKT cells was measured by methyl tetrazolium salt assay. CD4(+) (CD4(+) CD8(-)) iNKT subset from OSCC patients showed significant (P < 0.01) expansion and higher IL-4 production following activation with α-GalCer-pulsed DCs, while CD4(-) CD8(-) double negative (DN) and CD8(+) (CD4(-) CD8(+) iNKT subsets produced IFN-γ predominantly. iNKT cells showed significantly (P = 0.02) increased secretion of IFN-γ and enhanced cytotoxicity to KB and SCC-4 tumour cells in response to α-GalCer and TL-pulsed DCs. It appears that mutual balance/ratio of iNKT subsets may be important for their effector functions. Selectively expanded DN and CD8(+) iNKT cells with α-GalCer and TL may be a better candidate vaccine for iNKT-cell-based adoptive cancer immunotherapy.

Uptake mechanism of Furosemide-loaded pegylated nanoparticles by cochlear cell lines

This study tests the hypothesis that pegylated nanoparticles (NPs) could be taken up by the cochlear cells [House Ear Institute-organ of Corti 1 (HEI-OC1) and Stria vascularis K-1 (SVK-1)], through endocytic pathways. Furthermore, the in vitro drug release and the cytotoxicity of Furosemide (FUR)-loaded NPs on these two cochlear cells are investigated. FUR-loaded pegylated NPs are prepared by the emulsion-solvent diffusion method without surfactant. The NPs are characterized for particle mean diameter, polydispersity index (PDI), morphology, percent drug encapsulation efficiency (EE%), and FUR release kinetics. The methyl tetrazolium salt (MTS) and lactate dehydrogenase (LDH) bioassays are used to evaluate in vitro, the cytotoxicity of FUR-loaded NPs and native FUR. The NPs uptake is investigated using confocal microscopy, microplate reader/fluorimetry, and flow cytometry. Spherical NPs with a mean diameter range of 133–210 nm and PDI values varying from 0.037 to 0.41 are produced. The FUR EE% is 86% and the drug is released from the NPs according to the zero-order and Higuchi models. After treatment with blank NPs, the percentage of cell viability and cell death are 95.96% and 8.95%, in HEI-OC1 cells, respectively. The NPs are internalized by HEI-OC1 cells through a clathrin-dependent pathway. In addition, results show that NPs can be taken up via clathrin and cytoskeleton mediated pathways in SVK-1 cells. The internalization of the pegylated NPs can enhance the drug toxicity by necrosis in a dose-dependent and sustained release manner. The formulated NPs provide a promising template for a targeted drug delivery system to the inner ear.

AB0237 Endothelin-1 induces alpha-smooth muscle actin (alpha-SMA) expression in cultured human endothelial cells

BackgroundMyofibroblasts are activated fibroblasts mainly characterized by the expression of α-smooth muscle actin (α-SMA) playing a prominent role in the fibrotic process that characterizes some connective tissue diseases, in particular...

Abstract 1035: Crenolanib, a highly selective platelet-derived growth factor receptor α/β (PDGFRA/B) tyrosine kinase inhibitor, destabilizes ETV1 protein in KIT-mutant gastrointestinal stromal tumor (GIST) cells.

Abstract Background &amp; Aims: Most GIST harbor oncogenic KIT or PDGFRA mutations, which are in most cases heterozygous. However, most GIST co-express both wild-type KIT and PDGFRA with the mutated KIT or PDGFRA. Signaling mediated by heterodimers of the wild-type receptors contribute to GIST growth even after blocking oncogenic signaling by KIT/PDGFRA inhibitors such as imatinib. Previously we found that crenolanib (CP-868596), a PDGFRA/B tyrosine kinase inhibitor with &amp;gt;100-fold selectivity over other tyrosine kinases including KIT and a potent inhibitor of oncogenic PDGFRA signaling in vitro, inhibited the proliferation of KIT+PDGFRA+PDGFRB− GIST-T1 cells with a potency comparable to that of imatinib (IC50: 5.3 nM; imatinib: IC50: 3.5 nM) but had very little effect in the KITlow/−PDGFRAlow/−PDGFRB+ GIST48B cells (maximum inhibition: &amp;lt;50%; IC50&amp;gt;75 nM) and in mouse stem cells for interstitial cells of Cajal (ICC-SC), a presumed cellular source for GIST, which share a phenotype with GIST48B cells (IC50 &amp;gt;600 nM; Hayashi et al., Gastroenterology 2012; 142:S30). Here we investigated whether the antiproliferative effect of crenolanib was dependent on KIT expression, and studied crenolanib's effect on KIT/PDGFRA signaling and on the stability of ETV1, a transcription factor required for GIST growth and survival. Methods: Protein expression and phosphorylation were studied by Western blotting. KITlow/−PDGFRAlow/+PDGFRB+ murine ICC-SC (2xSCS2F10) were retrovirally transduced to express wild-type murine KIT. Cell proliferation was studied by methyl-tetrazolium salt assay. Results: Expression of wild-type KIT in retrovirally transduced ICC-SC dramatically reduced crenolanib's IC50 from 1157 nM to 121 nM. In GIST-T1 cells, crenolanib significantly reduced PDGFRA and KIT protein expression, inhibited PDGFRA phosphorylation (IC50: 5 nM) but had no effect on KIT phosphorylation. Crenolanib applied at 8.3 nM concentration rapidly and profoundly inhibited phosphorylation of ERK1/2 MAP kinases and reduced ETV1 protein levels by &amp;gt;50% in 2 h. The MEK inhibitor PD98059 (50 μM) reproduced crenolanib's effect on ETV1 protein levels with an identical time-course. The inhibitory effects of both crenolanib and PD98059 on ETV1 protein expression could be reversed with the proteasome inhibitor MG132 (10 μM) and accentuated with the protein synthesis inhibitor cycloheximide (10 μg/mL). ETV1 was undetectable in the crenolanib-resistant, KITlow/−PDGFRAlow/−PDGFRB+ GIST48B cells. Conclusions: In KIT mutant, KIT+PDGFRA+PDGFRB− GIST cells, crenolanib induced ETV1 protein degradation by suppressing PDGFRA-dependent MAPK phosphorylation. Crenolanib's antiproliferative effect in these cells may depend on KIT-induced ETV1 expression and reflect the destabilization of ETV1. Grant support: NIH DK58185. Citation Format: Yujiro Hayashi, Michael R. Bardsley, Yoshitaka Toyomasu, Takahiro Taguchi, Brian P. Rubin, Monique Carter, Abhijit Ramachandran, Tamas Ordog. Crenolanib, a highly selective platelet-derived growth factor receptor α/β (PDGFRA/B) tyrosine kinase inhibitor, destabilizes ETV1 protein in KIT-mutant gastrointestinal stromal tumor (GIST) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1035. doi:10.1158/1538-7445.AM2013-1035

In Vitro Study of Thrombin on Tubule Formation and Regulators of Angiogenesis

Angiogenesis occurs within atherosclerotic plaques, and thrombin has been implicated in plaque progression by increasing smooth muscle cell proliferation and upregulating Vascular Endothelial Growth Factor (VEGF) receptor expression. This study investigated the effects of thrombin on key aspects of angiogenesis and expression of pro- and anti-angiogenic regulators: VEGF and Pigment Epithelial Derived Factor. Human umbilical smooth muscle cells (HUASMC) were exposed to vehicle or thrombin at 10 U/ml. To quantify cell proliferation, methyl tetrazolium salt (MTS) solution was added after exposure to thrombin for 24, 48 and 72 hours and the absorbance at 490nm recorded using a plate reader. For Real-time RT-PCR cells were exposed to thrombin for 24hr before analysis of VEGF and PEDF mRNA. A commercial Angiokit was used to construct an in vitro angiogenesis model to measure tubule formation and branching. After 12 days treatment with thrombin 10 U/ml, cells were fixed and the AngioSys 1.0 software was used to analyse tubule morphology. In comparison with controls, thrombin significantly increased HUASMC proliferation (p = 0.002, n = 11) and VEGF mRNA expression by 93% (n = 4, p = 0.024). However in the HUVEC/fibroblast co-cultures it decreased the number of junctions [132(9) vs 196(18), n = 6, p = 0.017] and tubules [537 (17) vs 589 (26), n = 6, p = 0.049], and tubule length [11393 (1601) vs 12195 (1014), n = 6, p = 0.044], indicating an anti-angiogenic effect. Thrombin stimulates vascular smooth muscle cell proliferation and VEGF expression, but attenuates endothelial cell-mediated growth of vascular tubules and branching of new vascular structures.

Macrophage and osteoblast responses to biphasic calcium phosphate microparticles

The aim of this work was to investigate in vitro the biological events leading to ectopic bone formation in contact with microporous biphasic calcium phosphate (BCP) ceramics. After implantation, microparticles may arise from their degradation and induce an inflammatory response involving macrophages. The secretion of pro-inflammatory cytokines may affect the differentiation of osteoblasts. Mouse macrophage-like (J774) and osteoblast-like (MC3T3-E1) cells were cultured in the presence of BCP microparticles of different sizes (<20, 40-80, or 80-200 microm). The smallest microparticles decreased the viability of both cell types as measured with LDH and methyl tetrazolium salt assays, and enhanced the secretion of pro-inflammatory cytokines (IL-6 and TNF-alpha) by macrophages after 24 h, as revealed by ELISA. Osteoblastic cells were then cultured for 96 h in the presence of these pro-inflammatory cytokines and their differentiation studied by RT-PCR. MC3T3-E1 cells cultured with TNF-alpha showed a decrease in osterix, PTH receptor (PTHR1), and osteocalcin gene expression. On the contrary, IL-6 enhanced the expression of osterix, Runx2, alkaline phosphatase, and osteocalcin compared with plastic. In conclusion, this study shows that the inflammatory response initiated by BCP microparticles may have both detrimental and beneficial effects on osteogenesis.

Culture of human mesenchymal stem cells using electrosprayed porous chitosan microbeads

The aim of this study was to fabricate porous chitosan microbeads using an electrospraying method into liquid nitrogen, then thawing and refreezing. The microbeads were then used to evaluate their potential for tissue engineering of human mesenchymal stem cells (hMSCs). Scanning electron microscopy (SEM) and a mercury porosimeter were used to show the morphology of the scaffolds formed and to determine their pore size and porosity. As the chitosan concentration increased (0.5, 1, 1.5, and 2 wt %), the diameter of the porous microbead increased from 350 to 890 microm, and the average pore size and the porosity decreased from 65 to 21 microm and 95 to 38%, respectively. The hMSCs were cultured onto the porous microbeads in a spinner flask. SEM images and methyl tetrazolium salt assays at 3, 7, 14, and 21 days of culture revealed that hMSCs had successfully attached and proliferated inside the porous microbeads. This study demonstrated that electrosprayed porous chitosan microbeads can be used as three-dimensional scaffolds for tissue engineering.

Cisplatin-mediated sensitivity to TRAIL-induced cell death in human granulosa tumor cells

Objectives. The goal of the present study was to determine the efficacy of combinatorial treatment using cisplatin and tumor necrosis factor-related apoptosis including ligand (TRAIL) to promote apoptosis in granulosa cell tumor (GCT) lines, in vitro. Methods. Two human GCT lines (COV434 and KGN) were treated with cisplatin or TRAIL, alone or in combination. The cytotoxic effects of each treatment were evaluated using a methyl tetrazolium salt (MTS) assay. Initiation of TRAIL-induced apoptosis was verified by PARP- and FLIP-cleavage. Overexpression and knockdown studies were conducted to evaluate the role of p53 in TRAIL-induced cell death. Real-time PCR was used for gene expression analysis of the TRAIL receptor dr5 and the pro-apoptotic bax following treatment with cisplatin. Results. Treatment with TRAIL (100–200 ng/ml) led to a slight, but significant, loss of cell viability following an 18-h culture. This effect was enhanced following pre-treatment with cisplatin (25 μM) for 2 or 18 h. Moreover, pre-treatment with cisplatin decreased the maximal effective dose of TRAIL from 100 ng/ml to as low as 3 ng/ml in both cell lines. GCT lines overexpressing or deficient in p53 were used to determine the requirement for p53 on TRAIL-induced apoptosis. While the level of p53 expression enhanced both the death-inducing and TRAIL-sensitizing effects of cisplatin, TRAIL-induced cell death was found to occur independent of p53. Conclusions. These data suggest that the efficacy of cisplatin in GCT cells can be enhanced through combinatorial treatment with TRAIL. This result is due to both p53-dependent (cisplatin) and -independent (TRAIL) mechanisms. Combinatorial treatment of GCTs with cisplatin and TRAIL may provide an efficacious addition to cisplatin-based regimens.