Murine Fibroblast Cell Line Research Articles

Immortalized murine fibroblast cell lines are refractory to reprogramming to pluripotent state

To date different cell types of various mammalian species have been reprogrammed to induced pluripotent stem cells (iPSCs) using Yamanaka's cocktail of transcription factors (Oct4, Klf4, Sox2, and cMyc). It has been shown that several primary human cancer cell lines could be reprogrammed to iPSCs. We sought if immortalized mouse fibroblast cell lines could also be reprogrammed to iPSCs. The approach of generating iPSCs from such cells should be valuable in different experimental settings as it allows clonally derive cell lines carrying mutations whose impact on reprogramming could be next evaluated. Therefore, we investigated reprogramming of widely used immortalized cell lines (NIH3T and STO), as well as of de novo immortalized fibroblast line (tKM) with the use of highly effective lentiviral polycistronic OKSM expression system. Our reprogramming experiments have shown that in contrast to mouse embryonic fibroblasts (MEFs), none of the immortalized cell lines can be reprogrammed to pluripotent state. Contrary to colonies derived from MEFs, those derived from the immortalized cells lines (1) developed much later, (2) contained large round cells, not typical for iPSCs, and (3) were negative for trusted markers of matured iPSCs, Nanog and SSEA1. Immortalized cell lines NIH3T and STO are known to be mostly aneuploid, whereas tKM population includes cells with normal karyotype, however, neither cell type can be reprogrammed. Thus our data argue that aneuploidy per se is not a reason for the observed refractoriness of mouse immortalized cells to reprogramming to pluripotent state.

Phenolic Composition, Antioxidant and Cytotoxic Prospective of three Linum species: A Potential Source of Novel Anticancer Pharmacophores

The therapeutic potential of some Linum species has been reported previously. Nonetheless, several species of this genus warrant further scientific consideration. In this study, the phenolic composition, antioxidant activity (DPPH, phosphomolybdenum, iron chelating, hydroxyl radical and lipid peroxidation assays) and cytotoxicity effect (human rhabdomyosarcoma, human cervix carcinoma, and murine fibroblast cell lines) of ethyl acetate, methanol, and water extracts of three Linum species (Linum austriacum subsp. glaucescens, Linum hirsutum subsp. anatolicum, and Linum tenuifolium) were assessed. High performance liquid chromatography with diode-array detection (HPLC-DAD) analysis revealed the presence of ferulic acid, p-hydroxybenzoic acid, synapic acid, rutin, apigenin-glucoside, quercetin, luteolin, and naringenin in all the extracts. It was observed that the extracts (IC50 ranging from 42.07 to 51.66 µg/mL) showed IC50 significantly (p<0.05) lower than ascorbic and gallic acid (> 1000 and 255.43 µg/mL respectively) against lipid peroxidation. According to the American National Cancer Institute guidelines, the extracts showed low cytotoxicity (IC50 < 30 µg/mL). However, the water extracts showed cytotoxicity effects (IC50 17.43±0.13 to 28.12±0.66 µg/mL) against the studied cell lines as compared to the antineoplastic positive control (Cis-DDP). These findings support the use of these plants in the management of ROS mediated disorders and advocates for the need for further scientific evaluations to confirm the observed in vitro bioactivity. The cytotoxic potential coupled with the antioxidant property of the water extracts of these plant species is of particular interest which can open new avenues in the quest for novel anticancer drugs. Keywords: Linum, antioxidant, phenolic, nutraceuticals, anticancer, cytotoxic, Turkey.

Electroconductive hyaluronic acid/gelatin/poly(ethylene oxide) polymeric film reinforced by reduced graphene oxide

AbstractNovel electroconductive polymeric films with enhanced mechanical performance were fabricated by encapsulating reduced graphene oxide (RGO) at different concentrations (0–50 vol %) into a hyaluronic acid/gelatin/poly(ethylene oxide) (HyA/Gel/PEO) polymeric structure. The obtained RGO‐reinforced polymeric films were characterized by Fourier transform infrared and scanning electron microscopy analyses. Mechanical performances were measured with a universal mechanical testing machine. The results verified that the RGO reinforcement significantly enhanced the mechanical performances of the films. To determine the biocompatibility of the polymeric films, L929 (murine fibroblast) cell lines were used. The water uptake capacities were measured using swelling tests. A four‐probe method was used to measure conductivity characteristics. The conductivity results indicated that HyA/Gel/PEO film containing 20 vol % RGO has the highest average electrical conductivity (1.832 × 10−6S/cm). All of the results demonstrated that the obtained electroconductive films could be used in biomedical fields in the future, especially in controlled drug release systems and tissue engineering. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci.2019,136, 46905.

Osteosarcoma-derived extracellular vesicles induce a tumor-like phenotype in normal recipient cells.

Osteosarcoma is the most common primary bone cancer and the most frequent cause of bone cancer-related deaths in children and adolescents. Osteosarcoma cells are able to establish a crosstalk with resident bone cells leading to the formation of a deleterious vicious cycle. We hypothesized that osteosarcoma cells can release, in the bone microenvironment, transforming Extracellular Vesicles (EVs) involved in regulating bone cell proliferation and differentiation, thereby promoting tumor growth. We assessed EV production by three osteosarcoma cell lines with increasing aggressiveness in order to investigate their roles in the communication between osteosarcoma cells and normal recipient cells. Osteosarcoma-derived EVs were used to treat the murine fibroblast cell line NIH3T3 and to study the induction of tumor-like phenotypes. Our results showed that osteosarcoma cell lines are able to produce EVs that fuse to recipient cells, with a very high uptake efficiency. The treatment of recipient NIH3T3 with osteosarcoma-derived EVs induced substantial biological and functional effects, as an enhanced proliferation and survival capability under starved conditions, high levels of activated survival pathways, an increased migration, adhesion, and 3D sphere formation and the acquired capability to grow in an anchorage-independent manner. Moreover, in murine NIH3T3 we found human mRNAs of TNF-α, IL-6, and TGF-β, as well as a de novo expression of murine MMP-9 and TNF-α following the treatment of human osteosarcoma-derived EVs.

Biocompatibility of composites based on chitosan, apatite, and graphene oxide for tissue applications

Novel two-dimensional films and three-dimensional (3D) scaffolds based on chitosan (CHI), apatite (Ap), and graphene oxide (GO) were developed by an in situ synthesis in which self-assembly process was conducted to direct partial reduction of GO by CHI in acidic medium. Physical-chemical characterization was carried out by optical microscopy, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. In vitro biological studies using murine fibroblast (MC3T3) and human neuroblastoma (SH-SY5Y) cell lines were also performed. Cell growth and adherence on composites was also checked using SEM. Live and death staining by confocal microscope and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium of the samples were investigated. The results confirmed the incorporation of both Ap and GO sheets, into CHI polymeric matrix. Furthermore, it was confirmed a physical integration between inorganic Ap and organic CHI and strong chemical interaction between CHI and GO in the obtained composites. SH-SY5Y cell line showed preferential adherence on CHI/GO films surface while MC3T3 cell line displayed a good compatibility for all 3D scaffolds. This study confirms the biocompatibility of materials based on CHI, Ap, and GO for future tissues applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1585-1594, 2018.

Synthesis of new indole derivatives using one- pot multicomponent reaction with antiproliferative towards normal and cancer cell lines

Background and objectives Indoles derivatives are natural products, which are well known for their anticancer activity due to its ability to induce cell death for many cancer cell lines. The aim of this study is to synthesize some new heterocyclic compounds derived from 1H-indole-3-carbaldehyde, malononitrile, and different reagents namely: active methylene derivatives, amine derivatives, and resorcinol. The newly synthesized derivatives were prepared and confirmed by their IR, Mass, and 1NMR spectra; and were also tested for their antiproliferative potency towards human breast cancer (MCF-7) and normal murine fibroblast (BALB/3T3) cell lines. Materials and methods The synthesis of new indole derivatives (3a-e) was achieved by the three-component reactions of 1H-indole-3-carbaldehyde (1), ethyl 3-oxobutanoate, 5,5-dimethyl cyclohexane-1,3-dione (Dimidone), barbituric, thiobarbituric acid, and cyclohexanone (2a-e), respectively; and malononitrile in the presence of triethylamine. Compound (4) was afforded by fusing of compound (3a) with thiourea. Also, compounds (5a, b) were yielded by the grinding of compounds (3c, d) with formic acid. On the other side, one-pot synthesis of 7a-d has been achieved via three-component of 1H-indole-3-carbaldehyde (1), and malononitrile in the presence of amine derivatives (namely, 2,4-dinitroaniline, 3-nitroaniline, 3-bromoaniline, and 4-methoxyaniline) (6a-d), respectively, by condensation. Also, compounds (8, 9) were obtained by refluxing of compounds (7a, b) with formic acid. Compound (10) was afforded by condensing the mixture of 1H-indole-3-carbaldehyde (1) with resorcinol in the presence of malononitrile and triethylamine. The newly synthesized derivatives were tested for their antiproliferative potency towards human breast cancer (MCF-7) and normal murine fibroblast (BALB/3T3) cell lines. Results indicated that they showed significant in-vitro antiproliferative activity. Results and conclusion A novel protocol for the preparation of indole derivatives (3a-e) using the three-component reactions of 1H-indole-3-carbaldehyde (1) with active methylene compounds namely: ethyl 3-oxobutanoate, 5, 5-dimethy cyclohexane-1, 3-dione (Dimidone), barbituric acid, thiobarbituric acid, and cyclohexanone (2a-e); and malononitrile in ethanol and triethylamine as catalyst were proceeded in one step. Also, compounds (3a, 3c, d) were reacted with thiourea/and formic acid, respectively, to give compounds (4 and 5a, b). The 3-indole derivatives (7a-d) were formed via condensation of the amine derivatives (6a-d) with indole aldehydes (1) and malononitrile. Compounds (8 and 9) were afforded by condensation of compounds (7b, c) with formic acid, 2-amino-chromene (10) was produced by reaction of resorcinol, 1H-indole-3-carbaldehyde (1), and malononitrile. The in-vitro study showed that most of the prepared compounds gave similar activity towards breast cancer cell line MCF-7 but did not reveal the cytotoxic potency against normal cell line. This means that most of these compounds kill cancer cells but have no or slight effects on normal cells. The newly synthesized derivatives were tested for their antiproliferative potency towards human breast cancer (MCF-7) and normal murine fibroblast (BALB/3T3) cell lines. The results showed that the in-vitro antiproliferative activity of the prepared compounds was significant and could thus be investigated for further in vivo and pharmacokinetic studies.

Antimicrobial activity of an antimicrobial peptide against amastigote forms of Leishmania major.

Zoonotic cutaneous leishmaniasis caused by Leishmania major is a most common type of vector-borne disease in Iran. The pentavalent antimonial drugs have been used in the treatment of cutaneous leishmaniasis for a long time, but drug resistance and some of serious side effects have been reported. Thus, discovery and development of new therapeutic candidates are needed. The CM11 peptide is one of these peptides that its anti-bacterial activity has been proven. This peptide is a short cecropin–melittin hybrid peptide obtained through a sequence combination approach. The aim of this study was to evaluate in vitro anti-leishmanial activity of CM11 peptide against amastigote forms of Leishmania major. In this study, amastigote forms of Iranian strain of L. major (MRHO/IR/75/ER) were cultured in the presence of different concentrations of meglumine antimoniate (Glucantime®) to find the most appropriate in vitro concentration of Glucantime® against L. major amastigotes. Then, the anti-leishmanial activities of various concentrations of CM11 peptide (8, 16, 32 and 64 µM) were evaluated for 24, 48 and 72 hr by DAPI staining. In addition, MTT assay was used to determine the cytotoxic effects of CM11 peptide on murine fibroblast cell line. The results showed that CM11 peptide has antimicrobial activity against Iranian isolate of L. major in the laboratory conditions. It seems that the CM11 peptide has significant potential to be used as a new anti-leishmanial agent.

New Antiproliferative Polyunsaturated Epoxy-Heneicosane Derivatives Isolated from the Brown Alga Lobophora variegata

Two new polyunsaturated 3,4-epoxy-heneicosane derivatives named as epoxy-lobophorene A and epoxy-lobophorene B were isolated from the brown alga Lobophora variegata, in addition to nine known compounds. The structures of the new compounds were elucidated using a combination of 1D/2D nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). The isolated compounds were submitted to antiproliferative assays against the human colon cancer cell line HCT-116, human metastatic prostate cancer PC-3M, murine metastatic melanoma B16-F10 and murine fibroblast cell line L929 and also tested as antibacterial. Both 3,4-epoxy lobophorene A and 3,4-epoxy lobophorene B depicted moderate antiproliferative effect against cell lines. None of them showed antibacterial activity.

Comparative Evaluation of Cytotoxicity and Genotoxicity of Two Bioceramic Sealers on Fibroblast Cell Line: Anin vitroStudy

The purpose of this study was to evaluate and compare the cytotoxicity and genotoxicity of two bioceramic root canal sealers: EndoSequence BC and iRoot SP with zinc oxide eugenol sealers on fibroblast cell line. The sealers tested were zinc oxide eugenol, EndoSequence BC, and iRoot SP. Each material was mixed according to the manufacturer's instructions and mounted into sterile polyethylene color-coded rings, for cytotoxicity and genotoxicity evaluation. After 48 hours, the set materials were transferred to previously marked wells and cytotoxicity evaluation to L929 murine fibroblast cells was done by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The percentages of viable cells were then calculated and values were statistically analyzed by Kruskal-Wallis test. The evaluation of genotoxicity of the materials to L929 murine fibroblast cells was carried out by Comet assay. To quantify deoxyribonucleic acid (DNA) damage, the following comet parameters were evaluated in the assay using Comet scoring software: tail length, tail moment, and Olive moment. The values were statistically analyzed using Kruskal-Wallis test with a significance value set to p < 0.05. The results of the study showed that both cytotoxicity and genotoxicity evaluation by MTT assay and Comet assay can be done on L929 murine fibroblast cell line. Among the three tested materials, zinc oxide eugenol showed maximum cytotoxicity to the cells (30.64% viable cells), followed by EndoSequence BC (71.33% viable cells) and iRoot SP (75.11% viable cells). The evaluation of DNA damage by genotoxicity assessment showed iRoot SP to be least genotoxic followed closely by EndoSequence BC. Zinc oxide eugenol was genotoxic and induced more DNA damage on the fibroblast cell line studied. The statistical analyses for both the assays were nonsignificant. All the three tested sealers showed varying degrees of cytotoxicity and genotoxicity while using fibro-blast cell line. Zinc oxide eugenol was most toxic in both the assays and iRoot SP showed least toxicity, followed closely by EndoSequence BC.

Synthesis and Evaluation of Biological Activities of Aziridine Derivatives of Urea and Thiourea.

In the present paper, we report the synthesis and evaluation of in vitro antimicrobial activities of aziridine-thiourea derivatives. A series of aziridines in reaction with isocyanates and isothiocyanates to obtain urea and thiourea derivatives were used. The structures of all new products were confirmed based on spectroscopic data (1H-NMR, 13C-NMR, HR-MS). These compounds were screened for their in vitro antimicrobial activity against a panel of Gram-positive and Gram-negative strains of bacteria. Six of the tested compounds appeared to be promising agents against reference strains of Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. Subsequently, compounds exhibiting promising antibacterial activity were tested against twelve clinical isolates of S. aureus from three different sources of infection. The most bactericidal compounds (MIC = 16–32 µg/mL) showed better antibacterial activity against MRSA than ampicillin and streptomycin. The in vitro cytotoxicity analysis on L929 murine fibroblast and HeLa human tumor cell line using the MTT assay allowed us to select the least toxic compounds for future investigation.

Opto-electronic and antibacterial activity investigations of mono-dispersed nanostructure copper oxide prepared by a novel method: reduction of reactive oxygen species (ROS)

A novel approach for synthesis of copper oxide nanoparticles is reported by separation of nucleation and growth. The nano-material was characterized by X-ray diffraction, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and UV–Vis diffuse reflectance spectroscopy, transmission electron microscopy, atomic force microscopy, and Brunauer–Emmett–Teller analyses. Optical analysis of mono-dispersed nanostructure copper oxide by UV–Vis diffused reflectance spectroscopy showed the band gap value of 1.47 eV with a blue-shift in the optical band gap due to quantum confinement effect. The dynamic light scattering and zeta potential results showed fairly narrow size distribution and colloidal stability. The results showed that nano-particles were mono-dispersed spheres of 8 nm with no aggregation. Cell viability of treated murine fibroblast cell line (L-929) treated by different concentrations of nanoparticles showed significant viability up to 96% at concentrations 15 and 30 μg ml−1. The nanoparticles exhibited outstanding and stable antibacterial activity against Staphylococcus aureus ATCC 6538 at 30 µg ml−1. The viability and reactive oxygen species (ROS) generation in the L-929 cell line indicated that the nanoparticles were not toxic at the concentrations which were effective on bacteria. ROS analysis using DCFH-DA probe on L-929 were exposed to 7.5–60 μg ml−1 of copper oxide nanoparticles in 6 h revealed ROS generation was decreased dramatically compare to the untreated cells and positive control.

Cell-Derived Nanoparticles are Endogenous Modulators of Sepsis With Therapeutic Potential.

Cell-derived nanoparticles (CDNPs) containing cytosolic proteins and RNAs/DNAs can be isolated from stressed eukaryotic cells. Previously, CDNPs isolated from cultured cells exerted immunomodulatory activities in different infections. Here, we sought to elucidate the role of CDNPs using a murine model of cecal ligation and puncture (CLP). We hypothesized that CDNPs influence the immune response at the site of infection, where severe cellular stress occurs. We observed early CDNP accumulation in the peritoneum after 4 h and continued CDNP presence 24 h after CLP. To determine whether CDNPs influence the host response to sepsis, we isolated CDNPs from a murine fibroblast cell line stressed by nutrient-deprivation, and injected them into septic mice. CDNP-treated mice demonstrated decreased peritoneal interleukin 6 levels and an approximately 2-log lower bacterial load compared with control mice 24 h after CLP. Additionally, a 20% CFU reduction was observed when incubating CDNPs with Pseudomona aeroginosa, indicating that CDNPs are bactericidal. To identify CDNP-responsive cells, CFSE-labeled CDNPs were injected into mice at the time of CLP. We observed that CDNPs were preferentially ingested by F4/80 macrophages, and to a lesser degree, associated with inflammatory monocytes and neutrophils. Strikingly, CDNP-ingesting cells demonstrated elevated CD11b and MHCII expression compared with control cells. Altogether, our data indicate that CDNPs enhance the immune response at the site of infection and promote bacterial clearance, by direct bacterial killing and increasing phagocyte activation. Thus, CDNPs represent a novel, unexplored endogenous sepsis modulator with therapeutic potential.

Structure and intracellular antioxidant activity of pectic polysaccharide from acerola (Malpighia emarginata)

Malpighia emarginata is a tropical fruit plant, found naturally in the Caribbean islands and South America that produces an edible fruit known as acerola or Barbados Cherry. Its polysaccharides were obtained by aqueous extraction, subjected to a freezing and thawing process and ultrafiltration. A homogeneous fraction (ACWS-01E) was analyzed by sugar composition, HPSEC, methylation and NMR spectroscopy analyses. The results showed an arabinan-rich pectic polysaccharide, with 6.1×104g/mol and formed mainly by a high methyl esterified (DM=86%) homogalacturonan and branched arabinan. This latter is anchored in type I rhamnogalacturonan regions. The main chain of arabinan consisted of (1→5)-linked α-Araf, branched only at O-3. The potential ACWS-01E intracellular antioxidant activity against H2O2-induced oxidative stress in murine fibroblast cell line (3T3) was determined by DCFH-DA assay. The treatment with ACWS-01E significantly reduced H2O2-induced cytotoxic effect and the levels of reactive oxygen species (ROS). These findings suggested that ACWS-01E protected and improved NIH 3T3 cell viability from H2O2-induced toxicity by decreasing intracellular levels of ROS.

Multimodal Lentiviral Vectors for Pharmacologically Controlled Switching Between Constitutive Single Gene Expression and Tetracycline-Regulated Multiple Gene Collaboration.

Multimodal lentiviral vectors (LVs) allow switching between constitutive and tetracycline-regulated gene co-expressions in genetically modified cells. Transduction of murine primary hematopoietic progenitor cells (HPCs) with multimodal LVs in the absence of doxycycline ensures the constitutive expression of gene of interest 1 (GOI1) only. In the presence of doxycycline, induced tetracycline-regulated expression of a second GOI (GOI2) allows evaluation of the collaboration between two genes. Drug removal retains constitutive expression, which allows the contribution of an individual gene into created networks to be studied. Doxycycline-dependent switching can be tracked via fluorescent markers coupled to constitutive and tetracycline-regulated GOIs. This article describes transduction of murine primary HPCs with different doses of multimodal LVs, distinct cytokine conditions, and their influence on the number and viability of cells co-expressing both collaborating GOIs upon doxycycline induction. A 2-week protocol is provided for multimodal LV production, titer determination, and evaluation of tetracycline responsive promoter background activity in a murine fibroblast cell line. The power of this model to assess the dose/time/order-controlled contribution of single and multiple genes into hematopoietic networks opens new routes in reprogramming, stem cell, and leukemia biology.

Synergy of Iron Chelators and Therapeutic Peptide Sequences Delivered via a Magnetic Nanocarrier.

Here, we report the synthesis, characterization, and efficacy study of Fe/Fe3O4-nanoparticles that were co-labeled with a tumor-homing and membrane-disrupting oligopeptide and the iron-chelator Dp44mT, which belongs to the group of the thiosemicarbazones. Dp44mT and the peptide sequence PLFAERL(D[KLAKLAKKLAKLAK])CGKRK were tethered to the surface of Fe/Fe3O4 core/shell nanoparticles by utilizing dopamine-anchors. The 26-mer contains two important sequences, which are the tumor targeting peptide CGKRK, and D[KLAKLAK]2, known to disrupt the mitochondrial cell walls and to initiate programmed cell death (apoptosis). It is noteworthy that Fe/Fe3O4 nanoparticles can also be used for MRI imaging purposes in live mammals. In a first step of this endeavor, the efficacy of this nanoplatform has been tested on the highly metastatic 4T1 breast cancer cell line. At the optimal ratio of PLFAERD[KLAKLAK]2CGKRK to Dp44mT of 1 to 3.2 at the surface of the dopamine-coated Fe/Fe3O4-nanocarrier, the IC50 value after 24 h of incubation was found to be 2.2 times lower for murine breast cancer cells (4T1) than for a murine fibroblast cell line used as control. Based on these encouraging results, the reported approach has the potential of leading to a new generation of nanoplatforms for cancer treatment with considerably enhanced selectivity towards tumor cells.

Combination of biochemical and mechanical cues for tendon tissue engineering.

Tendinopathies negatively affect the life quality of millions of people in occupational and athletic settings, as well as the general population. Tendon healing is a slow process, often with insufficient results to restore complete endurance and functionality of the tissue. Tissue engineering, using tendon progenitors, artificial matrices and bioreactors for mechanical stimulation, could be an important approach for treating rips, fraying and tissue rupture. In our work, C3H10T1/2 murine fibroblast cell line was exposed to a combination of stimuli: a biochemical stimulus provided by Transforming Growth Factor Beta (TGF‐β) and Ascorbic Acid (AA); a three‐dimensional environment represented by PEGylated‐Fibrinogen (PEG‐Fibrinogen) biomimetic matrix; and a mechanical induction exploiting a custom bioreactor applying uniaxial stretching. In vitro analyses by immunofluorescence and mechanical testing revealed that the proposed combined approach favours the organization of a three‐dimensional tissue‐like structure promoting a remarkable arrangement of the cells and the neo‐extracellular matrix, reflecting into enhanced mechanical strength. The proposed method represents a novel approach for tendon tissue engineering, demonstrating how the combined effect of biochemical and mechanical stimuli ameliorates biological and mechanical properties of the artificial tissue compared to those obtained with single inducement.

Biocompatibility study on Ni-free Ti-based and Zr-based bulk metallic glasses

Safety and reliability are crucial issues for medical instruments and implants. In the past few decays, bulk metallic glasses (BMGs) have drawn attentions due to their superior mechanical properties, good corrosion resistance, antibacterial and good biocompatibility. However, most Zr-based and Ti-based BMGs contain Ni as an important element which is prone to human allergy problem. In this study, the Ni-free Ti-based and Zr-based BMGs, Ti40Zr10Cu36Pd14, and Zr48Cu36Al8Ag8, were selected for systematical evaluation of their biocompatibility. Several biocompatibility tests, co-cultural with L929 murine fibroblast cell line, were carried out on these two BMGs, as well as the comparison samples of Ti6Al4V and pure Cu. The results in terms of cellular adhesion, cytotoxicity, and metallic ion release affection reveal that the Ti40Zr10Cu36Pd14 BMG and Ti6Al4V exhibit the optimum biocompatibility; cells still being attached on the petri dish with good adhesion and exhibiting the spindle shape after direct contact test. Furthermore, the Ti40Zr10Cu36Pd14 BMG showed very low Cu ion release level, in agreement with the MTT results. Based on the current findings, it is believed that Ni-free Ti-based BMG can act as an ideal candidate for medical implant.

Lung cells support osteosarcoma cell migration and survival

BackgroundOsteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined.MethodsThree human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment.ResultsLung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining.ConclusionsLung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.

On the mechanism of genotoxicity of ethephon on embryonic fibroblast cells

Ethephon is one of the most widely used plant growth regulator in agriculture that its application has been increased in recent years. Many reports have raised concern over the safety of this organophosphorus compound. The aim of the current study was to assess the potential genotoxic effect of ethephon on murine embryonic fibroblast (MEF) cell line, using two genotoxicity endpoints: γH2AX expression and comet assay. γH2AX served as an early and sensitive biomarker of genotoxic damage. Oxidative stress biomarkers, including reactive oxygen species (ROS), lipid peroxidation (LPO) and total antioxidant capacity were also examined. The results showed a significant increase in cell proliferation, 24 h post-treatment with 10, 40,160 μg/ml ethephon, while at the higher concentrations cytotoxic effect was observed. The γH2AX expression and γH2AX foci count per cell were significantly increased at non-cytotoxic concentrations of ethephon, accompanied with increased DNA damage as illustrated by comet assay. LPO and ROS levels were elevated only at 160 μg/ml and higher doses. The results interestingly showed that low non-cytotoxic doses of ethephon promoted DNA damage inducing cell proliferation, raising the possibility of ethephon mutagenicity. The genotoxic effect of ethephon at low doses might not relate to oxidative damage and that increased in the level of ROS and LPO generation at higher doses could account for the cytotoxic effect of ethephon. Taken together, our study provides strong in vitro evidence on potential genotoxicity of ethephon at low doses. More precise studies are needed to clarify the mutagenic effect of chronic exposure to ethephon.

Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines.

Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”.