Murine Embryonic Fibroblast Cells Research Articles

The non-vesicle cell-free DNA (cfDNA) induces cell transformation associated with horizontal DNA transfer.

Cell-free DNA (cfDNA) is a source for liquid biopsy used for cancer diagnosis, therapy selection, and disease monitoring due to its non-invasive nature and ease of extraction. However, cfDNA also participates in cancer development and progression by horizontal transfer. In humans, cfDNA circulates complexed with extracellular vesicles (EV) and macromolecular complexes such as nucleosomes, lipids, and serum proteins. The present study aimed to demonstrate whether cfDNA not associated with EV induces cell transformation and tumorigenesis. Supernatant of the SW480 human colon cancer cell line was processed by ultracentrifugation to obtain a soluble fraction (SF) and a fraction associated with EV (EVF). Primary murine embryonic fibroblast cells (NIH3T3) underwent passive transfection with these fractions, and cell proliferation, cell cycle, apoptosis, cell transformation, and tumorigenic assays were performed. Next, cfDNA was analyzed by electronic microscopy, and horizontal transfer was assessed by human mutantKRASin recipient cells via PCR and recipient cell internalization via fluorescence microscopy. The results showed that the SF but not the EVF of cfDNA induced proliferative and antiapoptotic effects, cell transformation, and tumorigenesis in nude mice, which were reduced by digestion with DNAse I and proteinase K. These effects were associated with horizontal DNA transfer and cfDNA internalization into recipient cells. The results suggest pro-tumorigenic effects of cfDNA in the SF that can be offset by enzyme treatment. Further exploration of the horizontal tumor progression phenomenon mediated by cfDNA is needed to determine whether its manipulation may play a role in cancer therapy.

AS1411aptamer conjugated liposomes for targeted delivery of arsenic trioxide in mouse xenograft model of melanoma cancer

Development of AS1411aptamer-conjugated liposomes for targeted delivery of arsenic trioxide is the primary goal of this study. AS1411aptamer was used as ligand to target nucleolin, which is highly expressed on the surface of melanoma cancer cells. The targeted liposomes were constructed by the thin film method, and arsenic trioxide was loaded as cobalt (II) hydrogen arsenite (CHA) to increase the loading efficiency and stability of the liposomes. The liposomal structure was characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and field emission scanning electron microscopy (FESEM). In addition, particle sizes and zeta potential of the CHA-loaded liposomes (CHAL) and aptamer-functionalized CHA-loaded liposomes (AP-CHAL) were determined. In vitro cytotoxicity of CHAL and AP-CHAL were evaluated using MTT assay in murine melanoma (B16) and mouse embryonic fibroblast (MEF) cell lines. The encapsulation efficiency of CHAL and AP-CHAL was reported as 60.2 ± 6.5% and 58.7 ± 4.2%, respectively. In vivo antitumor activity of CHAL and AP-CHAL in the B16 tumor-xenograft mouse model was dramatically observed. All mice of both groups survived until the end of treatment and showed body weight gain. The tumor protrusion completely disappeared in 50% of the mice in these groups. Furthermore, histopathology studies demonstrated that CHAL and AP-CHAL did not induce significant toxicity in healthy mice tissues. However, unlike the CHAL group, which showed an initial increase in tumor volume, a specific antitumor effect was observed in the AP-CHAL group from the beginning of treatment. The results showed that AP-CHAL can be used as an effective drug delivery system with high potential in the treatment of solid tumors.

Bionanomining of copper-based nanoparticles using pre-processed mine tailings as the precursor

The bacterial synthesis of copper nanoparticles emerges as an eco-friendly alternative to conventional techniques since it comprises a single-step and bottom-up approach, which leads to stable metal nanoparticles. In this paper, we studied the biosynthesis of Cu-based nanoparticles by Rhodococcus erythropolis ATCC4277 using a pre-processed mining tailing as a precursor. The influence of pulp density and stirring rate on particle size was evaluated using a factor-at-time experimental design. The experiments were carried out in a stirred tank bioreactor for 24 h at 25 °C, wherein 5% (v/v) of bacterial inoculum was employed. The O2 flow rate was maintained at 1.0 L min−1 and the pH at 7.0. Copper nanoparticles (CuNPs), with an average hydrodynamic diameter of 21 ± 1 nm, were synthesized using 25 g.L−1 of mining tailing and a stirring rate of 250 rpm. Aiming to visualize some possible biomedical applications of the as-synthesized CuNPs, their antibacterial activity was evaluated against Escherichia coli and their cytotoxicity was evaluated against Murine Embryonic Fibroblast (MEF) cells. The 7-day extract of CuNPs at 0.1 mg mL−1 resulted in 75% of MEF cell viability. In the direct method, the suspension of CuNPs at 0.1 mg mL−1 resulted in 70% of MEF cell viability. Moreover, the CuNPs at 0.1 mg mL−1 inhibited 60% of E. coli growth. Furthermore, the NPs were evaluated regarding their photocatalytic activity by monitoring the oxidation of methylene blue (MB) dye. The CuNPs synthesized showed rapid oxidation of MB dye, with the degradation of approximately 65% of dye content in 4 h. These results show that the biosynthesis of CuNPs by R. erythropolis using pre-processed mine tailing can be a suitable method to obtain CuNPs from environmental and economical perspectives, resulting in NPs useful for biomedical and photocatalytic applications.

Drug-eluting biodegradable metals and metal-ceramic composites

Biodegradable metals emerged as promising temporary bone implants. The integration of local drug delivery (LDD) can prevent biomaterial-centered infections that are difficult to treat. LDD is achieved by drug-eluting coatings or porous implants where the drug is impregnated after implant fabrication because the high temperatures used during conventional production processes would result in their thermal decomposition. This work reports on the production and characterization of vancomycin hydrochloride (VH)-loaded Fe and Fe-iron oxide (Fe2O3) composites (Fe-Fe2O3) by a cold sintering technique. We use focused ion beam milling for the first time to investigate the drug-metal interface. We investigate the mechanical and degradation properties of VH-free and VH-loaded Fe and Fe-Fe2O3. Results show very high mechanical strength of drug-eluting Fe and Fe-Fe2O3 composites (up to than 780 MPa under compression, exceeding the maximum strength of cancellous bone more than three times) accompanied by a delayed drug release. Then, we confirm the good antimicrobial activity against Staphylococcus aureus and cell compatibility with the murine embryonic fibroblast cell line NIH/3T3 in vitro. Overall results confirm the promise of drug-eluting metals and metal-ceramic composites for LDD in bone.

CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII

On-target integration of large cassettes via homology-directed repair (HDR) has several applications. However, the HDR-mediated targeted knock-in suffered from low efficiency. In this study, we made several large plasmids (12.1–13.4 kb) which included the CRISPR/Cas9 system along with a puromycin transgene as part of the large DNA donor (5.3–7.1 kb insertion cassettes) and used them to evaluate their targeted integration efficiency into a transgenic murine embryonic fibroblast (MEF) cell line carrying a single copy of a Venus transgene. We established a detection assay by which HDR events could be discriminated from the error-prone non-homologous end-joining (NHEJ) events. Improving the plasmid quality could considerably leverage the cell toxicity impediment of large plasmids. The use of the TILD (targeted integration with linearized dsDNA) cassettes did not improve the HDR rate compared to the circular plasmids. However, the direct inclusion of nocodazole into the electroporation solution significantly improved the HDR rate. Also, simultaneous delivery of RNase HII and the donor plasmids into the electroporated cells considerably improved the HDR events. In conclusion, the results of this study showed that using cell synchronization reagents in the electroporation medium can efficiently induce HDR rate in the mammalian genome.

New amides derived from sclareolide as anticholinesterase agents

A series of 25 amides (15 new) derived from (3aR)-(+)-sclareolide were prepared and subjected to Ellman’s assay to determine their efficacies as inhibitors for AChE or BuChE. Five amides (9, 13, 14, 15 and 17) caused inhibition of one of the enzymes greater than 60%; thereby those that inhibited BuChE were more active than positive control galantamine, and they showed better Ki values (1.07 to 8.49). In general, it was found that molecules holding a meta-substituted phenyl group showed a higher percentage of enzymatic inhibition. Molecular modelling calculations indicated the putative interactions of compounds with the amino acids residues of both enzymes AChE and BuChE. The cytotoxicity of compounds 9, 13, 14, 15 and 17 was evaluated against a non-malignant murine embryonic fibroblast cell line (NIH 3T3). Of special note is compound 15, as it presented the second-best Ki value for BuChE (1.71), was not cytotoxic (EC50 > 30 μM). Compound 15 also does not violate Lipinski rules, and showed permeability in the blood brain barrier, indicating that it can be considered a lead for the development of new drugs to treat Alzheimer’s disease.

TBK1 inhibitors enhance transfection efficiency by suppressing p62/SQSTM1 phosphorylation.

DNA transfection is an essential technique in the life sciences. Non-viral transfection reagents are widely used for transfection in basic science. However, low transfection efficiency is a problem in some cell types. This low efficiency can be primarily attributed to the intracellular degradation of transfected DNA by p62-dependent selective autophagy, specifically by p62 phosphorylated at the S403 residue (p62-S403-P). To achieve efficient DNA transfection, we focused on a phosphorylation process that generates p62-S403-P and investigated whether inhibition of this process affects transfection efficiency. One of the kinases that phosphorylate p62 is TBK1. The TBK1 gene depletion in murine embryonic fibroblast cells by genome editing caused a significant reduction or loss of p62-S405-P (equivalent to human S403-P) and enhanced transfection efficiency, suggesting that TBK1 is a major kinase that phosphorylates p62 at S403. Therefore, TBK1 is a viable target for drug treatment to increase transfection efficiency. Transfection efficiency was enhanced when cells were treated with one of the following TBK1 inhibitors BX795, MRT67307, or amlexanox. This effect was synergistically improved when the two inhibitors were used in combination. Our results indicate that TBK1 inhibitors enhanced transfection efficiency by suppressing p62 phosphorylation.

AS1411aptamer conjugated liposomes for targeted delivery of arsenic trioxide in mouse xenograft model of melanoma cancer

Development and characterizing of AS1411aptamer conjugated liposomes for targeted delivery of arsenic trioxide is the primary goal of this study to increase the drug safety and efficacy in the treatment of solid tumors. The targeted liposomes were constructed by the thin film method, and arsenic trioxide was loaded as cobalt (II) hydrogen arsenite (CHA). The liposomal structure was examined by Fourier Transform Infra-red Spectroscopy and field emission scanning electron microscopy. In addition, particle sizes and zeta potential of the CHA-loaded liposomes (CHAL) and aptamer-functionalized CHA-loaded liposomes (AP-CHAL) were determined. An atomic absorption spectrometer was used to quantify the encapsulation efficiency and in vitro drug release. In vitro cytotoxicity of CHAL and AP-CHAL were evaluated using MTT assay in Murine melanoma (B16) and Mouse Embryonic Fibroblast cell lines. The encapsulation efficiency of CHAL and AP-CHAL was reported as 60.2 ± 6.5 % and 58.7 ± 4.2 %, respectively. The enhanced cellular uptake of aptamer-conjugated liposomes was displayed by Fluorescence imaging. In vivo antitumor activity of CHAL and AP-CHAL in the B16 tumor-xenograft mouse model was dramatically observed. All mice in the CHAL group survived to the end of treatment and showed body weight gain. However, the distinct antitumor effect was observed in the AP-CHAL-treated group, and the tumor protrusion completely disappeared in 50 % of the mice. The inhibition rate of tumor growth of CHAL and AP-CHAL was 92.73 % and 97.67 %, respectively. Furthermore, histopathology studies demonstrated that CHAL and AP-CHAL did not induce significant toxicity in mice tissues. AP-CHAL can be used as an effective drug delivery system with high potential in treating solid tumors.

Hydrophilic platinum(II) complexes containing 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine: Spectroscopic and in vitro cytotoxic characterization

Mononuclear platinum(II) complexes with 5,7-methyl-1,2,4-triazolo[1,5-a]pyrimidine (dmtp) of the general formula cis-[Pt(X)2(dmtp)2], where X - acetate (1), trichloroacetate (2), trifluoroacetate (3), nitrate (4) have been synthesized and characterized by multinuclear magnetic resonance (1H, 13C, 15N, 195Pt) and infrared spectroscopy methods. Spectroscopic parameters indicated the presence of the PtO2N2 chromophore system with two monodentate N(3)-bonded 5,7-methyl-1,2,4-triazolo[1,5-a]pyrimidines, and two monodentate O-donor ligands (carboxylate or nitrate).A cytotoxicity assay of hydrophilic platinum(II) complexes (1–4) against three human tumour cell lines A549 (non-small cell lung carcinoma), T47D (breast cancer), HT-29 (colon adenocarcinoma), and normal murine embryonic fibroblast cells (BALB/3T3) was performed.Considering the cytotoxic parameters of the studied complexes, the best in vitro results against the human breast tumour cells (T47D) were found for cis-[Pt(OOCCCl3)2(dmtp)2] although all the tested complexes (except complex (4)) exhibited moderate in vitro activity.

Two plus One: Combination Therapy Tri-systems Involving Two Membrane-Disrupting Antimicrobial Macromolecules and Antibiotics.

The escalating issue of multidrug-resistant (MDR) bacteria indicates the urgent need for new and effective strategies to combat this global health challenge. Here, we describe a new combinatorial approach that can be put forward for experimental therapy application against MDR bacteria. Specifically, we have developed a tri-system that includes the coadministration of two different membrane-disrupting-type antimicrobial agents─a synthetic antimicrobial polymer P and an antimicrobial peptide (AMP) colistin methanesulfonate (Col)─in conjunction with an antibiotic [doxycycline (Dox), rifampicin (Rif), or azithromycin (Azi)]. Traditionally, the administration of membrane-disrupting antimicrobial agents causes toxicity, but, in comparison, we demonstrated synergy and biocompatibility using this combinatorial approach. Checkerboard assays showed the occurrence of synergistic interactions in Col-Dox-P, Col-Rif-P, and Col-Azi-P tri-systems against wild-type and MDR Pseudomonas aeruginosa, with the Col-Dox-P system being the most effective. The ability to synergize thus enables the use of a lower dosage in combinations compared to the standalone agents. The tri-systems not only demonstrated bacteriostatic activity but were also bactericidal. For example, the Col-Dox-P system (at 8, 4, and 8 μg mL-1, respectively) and the Col-Rif-P system (at 4, 8, and 16 μg mL-1, respectively) were able to kill >99.999% of planktonic P. aeruginosa cells within 3 h of treatment. More importantly, an improvement of the therapeutic/selectivity index was achieved via combination therapy. Taking the Col-Dox-P system as an example, its biocompatibility with murine embryonic fibroblast cells was found to be comparable to that of polymer P alone despite the synergistic enhancement in antimicrobial activity of the combination. This resulted in a significant increase in selectivity by 16-fold for the Col-Dox-P combination system compared to P alone. Furthermore, the broad applicability of this tri-system strategy was demonstrated via the successful application of the AMP melittin in place of Col or P. Overall, this study sheds new insights on the application of membrane-disrupting antimicrobial agents in combination therapy and their potential for safer clinical use. Additionally, the information gathered in this study could inform the development of future combination therapy systems involving the simultaneous employment of multiple AMPs with antibiotics.

Species-specific formation of paraspeckles in intestinal epithelium revealed by characterization of NEAT1 in naked mole-rat.

Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.

Diamond Nanoparticles-Porphyrin mTHPP Conjugate as Photosensitizing Platform: Cytotoxicity and Antibacterial Activity.

Conjugation of photosensitizers (PS) with nanoparticles has been largely used as a strategy to stabilize PS in the biological medium resulting in photosensitizing nanoparticles of enhanced photoactivity. Herein, (Meso-5, 10, 15, 20-tetrakis (3-hydroxyphenyl) phorphyryn (mTHPP) was conjugated with diamond nanoparticles (ND) by covalent bond. Nanoconjugate ND-mTHPP showed suitable stability in aqueous suspension with 58 nm of hydrodynamic diameter and Zeta potential of −23 mV. The antibacterial activity of ND-mTHPP was evaluated against Escherichia coli for different incubation times (0–24 h). The optimal activity was observed after 2 h of incubation and irradiation (660 nm; 51 J/cm2) performed right after the addition of ND-mTHPP (100 μg/mL) to the bacterial suspension. The inhibitory activity was 56% whereas ampicillin at the same conditions provided only 14% of bacterial growth inhibition. SEM images showed agglomerate of ND-mTHPP adsorbed on the bacterial cell wall, suggesting that the antimicrobial activity of ND-mTHPP was afforded by inducing membrane damage. Cytotoxicity against murine embryonic fibroblast cells (MEF) was also evaluated and ND-mTHPP was shown to be noncytotoxic since viability of cells cultured for 24 h in the presence of the nanoconjugate (100 μg/mL) was 78%. Considering the enhanced antibacterial activity and the absence of cytotoxic effect, it is possible to consider the ND-mTHPP nanoconjugate as promising platform for application in antimicrobial photodynamic therapy (aPDT).

BUBR1 deficiency results in abnormal megakaryopoiesis

AbstractThe physiologic function of BUBR1, a key component of the spindle checkpoint, was examined by generating BUBR1-mutant mice. BUBR1–/– embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1+/– blastocysts grew relatively normally in vitro, BUBR1–/– blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1+/– mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes, which was correlated with an increase in megakaryocytic, but a decrease in erythroid, progenitors in bone marrow cells. RNA interference–mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However, enhanced megakaryopoiesis in BUBR1+/– mice was not correlated with a significant increase in platelets in peripheral blood, which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together, these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis.

Platinum(II) Complexes with Bulky Disubstitute Triazolopyrimidines as Promising Materials for Anticancer Agents.

Herein, we present dicarboxylate platinum(II) complexes of the general formula [Pt(mal)(DMSO)(L)] and [Pt(CBDC)(DMSO)(L)], where L is dbtp 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine) or ibmtp (7-isobutyl-5-methyl-1,2,4- triazolo[1,5-a]pyrimidine), as prospective prodrugs. The platinum(II) complexes were synthesized in a one-pot reaction between cis-[PtCl2(DMSO)2], silver malonate or silver cyclobutane-1,1-dicarboxylate and triazolopyrimidines. All platinum(II) compounds were characterized by FT-IR, and 1H, 13C, 15N and 195Pt NMR; and their square planar geometries with one monodentate N(3)-bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine, one S-bonded molecule of dimethyl sulfoxide and one O,O-chelating malonato (1, 2) or O,O-chelating cyclobutane-1,1-dicarboxylato (3, 4) was determined. Additionally, [Pt(CBDC)(dbtp)(DMSO)] (3) exhibited (i) substantial in vitro cytotoxicity against the lung adenocarcinoma epithelial cell line (A549) (IC50 = 5.00 µM) and the cisplatin-resistant human ductal breast epithelial tumor cell line (T47D) (IC50 = 6.60 µM); and (ii) definitely exhibited low toxicity against normal murine embryonic fibroblast cells (BALB/3T3).

Large-scale expansion of feeder-free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation.

Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/ml). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (109 cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output mESCs were analyzed for pluripotency marker expression (SSEA-1, SOX-2, and Nanog) through flow cytometry, and spontaneous differentiation and teratoma analysis was used to demonstrate functional maintenance of pluripotency.

A simple procedure to obtain nanodiamonds from leftover of HFCVD system for biological application

Nanodiamonds (NDs) are amongst the most investigated carbon-based nanostructures due to their chemical stability and favorable mechanical properties. Despite the number of works on methods for NDs production, one of the main challenges is to achieve their colloidal stability in aqueous suspension. Additionally, NDs are normally obtained by expensive, complex and time-consuming process. Herein, it was presented a facile method to obtain NDs in aqueous suspension by using columnar structure diamond from Hot-Filament Chemical Vapour Deposition reactor (HFCVD). CVD diamond leftover thick film from CVDVale Company was used. Therefore, this method has the advantage of being not only practical but also cost-effective since it brings a profitable use of CVD diamond leftover. The Diamond thick film was submitted to ultrasonic cavitation in the presence and absence of ZrO2 microbeads in aqueous medium. The NDs hydrodynamic diameter and the stability in aqueous suspension were monitored by light scattering, size and morphology were analyzed by transmission electronic microscopy. Considering the wide application of NDs in biomedical devices, cytotoxicity of aqueous suspensions of NDs was evaluated against murine embryonic fibroblast cells. Furthermore, NDs were functionalized with hydrogen and carboxyl groups. NDs aqueous suspension of straight size distribution was obtained even in the absence of ZrO2 beads, indicating that they may be dispensable in order to decrease NDs size. NDs of average hydrodynamic diameter of 22 nm and − 35 mV of Zeta-potential were obtained after ultrasonic cavitation followed by 2 h of centrifugation, not demonstrating cytotoxicity to cells at very low (0.05–0.5 μg/mL) nor at higher concentrations (116 μg/mL). Nevertheless, NDs showed a moderate cytotoxicity at intermediary concentration range (0.5–2.2 μg/mL). From our knowledge, this is the first work that reports on a facile method for providing NDs aqueous suspension with high colloidal stability from HFCVD diamond leftover.

Silanization improves biocompatibility of graphene oxide.

Evaluation of the biological properties of silanized graphene oxide is important in the context of biomedical applications of the material. In this study, we have evaluated the toxicity, immunogenicity and other biological properties like osteogenicity of silanized graphene oxide (SiGO). Graphene oxide (GO) was silanized using a common silanizing agent namely (3-aminopropyl) triethoxysilane (APTES). Silanization was confirmed through infrared spectroscopy and elemental mapping. Post-silanization, we did not observe any significant changes in the morphology of GO. Silanization leads to an increase in the interlayer distance and disorder in the lattice. Study of in vitro toxicity of SiGO on three different cell lines namely primary human dermal fibroblast, murine embryonic fibroblast and human osteosarcoma cell lines revealed that toxicity of SiGO was significantly less than GO. We further showed that in vitro immune activation of macrophage was less in the case of SiGO in comparison to GO. Profiling of osteogenic differentiation of human mesenchymal stem cell revealed that SiGO is less osteogenic than GO. Study of acute toxicity in the murine model indicated that GO was hepatotoxic at experimental concentration whereas SiGO did not show any significant toxicity. This study implied that SiGO is a better biocompatible material than GO.

Control of doxorubicin-induced, reactive oxygen-related apoptosis by glutathione peroxidase 1 in cardiac fibroblasts

Reactive oxygen formation plays a mechanistic role in the cardiotoxicity of doxorubicin, a chemotherapeutic agent that remains an important component of treatment programs for breast cancer and hematopoietic malignancies. To examine the role of doxorubicin-induced reactive oxygen species (ROS) in drug-related cardiac apoptosis, murine embryonic fibroblast cell lines were derived from the hearts of glutathione peroxidase 1 (Gpx-1) knockout mice. Cells from homozygous Gpx-1 knockout mice and parental animals were propagated with (Se+) and without (Se-) 100 nM sodium selenite. Activity levels of the peroxide detoxifying selenoprotein glutathione peroxidase (GSHPx) were marginally detectable (<1.6 nmol/min/mg) in fibroblasts from homozygous knockout animals whether or not cells were supplemented with selenium. GSHPx activity in Se- cells from parental murine fibroblasts was also <1.6 nmol/min/mg, whereas GSHPx levels in Se+ parental murine fibroblasts were 12.9 ± 2.7 nmol/min/mg (mean ± SE; P < 0.05). Catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glucose 6-phosphate dehydrogenase, and reduced glutathione activities did not differ amongst the four cell lines. Reactive oxygen production increased from 908 ± 122 (arbitrary units) for untreated control cells to 1668 ± 54 following exposure to 1 μM doxorubicin for 24 h in parental fibroblasts not supplemented with selenium (P < 0.03); reactive oxygen formation in doxorubicin-treated parental fibroblasts propagated in selenium was 996 ± 69 (P = not significant compared to untreated control cells). Reactive oxygen levels in homozygous Gpx-1 knockout fibroblasts, irrespective of selenium supplementation status, were increased and equivalent to that in selenium deficient wild type fibroblasts. When cardiac fibroblasts were exposed to doxorubicin (0.05 μM) for 96 h and examined for cell cycle alterations by flow cytometry, and apoptosis by TUNEL assay, marked G2 arrest and TUNEL positivity were observed in knockout fibroblasts in the presence or absence of supplemental selenium, and in parental fibroblasts propagated without selenium. Parental fibroblasts propagated with selenium and exposed to the same concentration of doxorubicin demonstrated modest TUNEL positivity and substantially diminished amounts of low molecular weight DNA. These results were replicated in cardiac fibroblasts exposed to doxorubicin (1–2 μM) for 2 h (to mimic clinical drug dosing schedules) and examined 96 h following initiation of drug exposure. Doxorubicin uptake in cardiac fibroblasts was similar irrespective of the mRNA expression level or activity of GSHPx. These experiments suggest that the intracellular levels of doxorubicin-induced reactive oxygen species (ROS) are modulated by GSHPx and play an important role in doxorubicin-related apoptosis and altered cell cycle progression in murine cardiac fibroblasts.

Anticancer Activity of a Highly Potent Small Molecule Tubulin Polymerization Inhibitor, AB8939

We have identified the small chemical molecule AB8939 as being a structurally novel, synthesized tubulin inhibitor that can circumvent resistance mechanisms known to limit the effectiveness of existing tubulin inhibitors; e.g., P-glycoprotein (Pgp) and myeloperoxidase (MPO) mediated resistance. A series of in vitro preclinical studies provide proof-of-concept that AB8939 has broad applicability as a potent anticancer drug, particularly in tumors of hematopoietic and lymphoid tissues, including acute myeloid leukemia (AML). Regarding mechanism of action, x-ray crystallography demonstrated that AB8939 binds to the colchicine-binding site on the beta-subunit of tubulin. Cell cycle arrest in the G2/M phase was evaluated using HCT116 cells (a human colorectal tumor), treated at various concentrations of AB8939 for 24 hours. It was seen that AB8939 produced a strong mitotic arrest at the sub-micromolar concentration range (90% of cells in G2/M phase at 10 nM), which was of comparable strength to that of established microtubule targeting agents, each at a concentration of 100 nM. Additional assays using cytarabine (Ara-C) resistant MOLM14 AML cells confirmed this activity, also demonstrating dose dependent (2 to 20 nM) G2/M phase cell cycle arrest in patient-derived AML blasts and that G2/M cell cycle arrest lead to cellular death by apoptosis at nanomolar concentrations. The effect of AB8939 (100 nM) on the integrity of the microtubule and actin networks was tested in 3T3NIH cells (murine embryonic fibroblast cell line). AB8939 induced a rapid (within 1 hour) and radical destabilization of the microtubule network but did not affect the actin network. Similarly, destabilization of the microtubule network was observed in human primary cardiomyocytes and primary human lung fibroblast cells treated for 24 hours at 10 to 1000 nM AB8939. Further in vitro analysis showed that AB8939 produces a direct and potent, dose-dependent depolymerization effect (50% inhibition of in vitro microtubule polymerization at around 1 µM, with 100% inhibition at &gt;5 µM). The potential of AB8939 to overcome resistance to chemotherapeutic agents in Pgp-dependent multidrug-resistant cell lines was assessed using the drug-sensitive human sarcoma cell line MES-SA (parental) and its multidrug-resistant counterparts MES-SA/MX2 and MES-SA/Dx5 in a 6-day proliferation/survival assay. AB8939 efficiently inhibited each of these cells with an IC50 ≤10 nM. By comparison, the MES-SA/MX2 and MES-SA/Dx5 cell lines were highly resistant to the chemotherapeutic agents of doxorubicin and vincristine, as compared with the effect on parental cells (IC50 &lt;1.5 - 2.0 µM versus 20 nM, respectively). Additional tests showed that AB8939 is a very poor substrate of Pgp efflux pump, comparable with combretastatin-4, and therefore has the potential to overcome multidrug resistance in cancer patients. The anti-proliferative activity of AB8939 in various hematopoietic tumors and solid tumors was evaluated using a colorimetric cell proliferation and viability assay. AB8939 produced good anti-tumor activity after 72 hours (IC50 of ≤50 nM) in 19 hematopoietic tumor cell lines tested, including AML (3 cell lines), B cell lymphoma (8 cell lines), T cell lymphoma (6 cell lines), and multiple myeloma (2 cell lines). AB8939 also showed good anti-tumor activity after 6 days (IC50 of ≤10 nM) in several solid tumor cell lines, including breast, colon, glioblastoma, head and neck, lung, kidney, melanoma neuroblastoma, ovary, pancreas and prostate cell lines. The therapeutic potential of AB8939 in refractory/resistant AML was investigated further on doxorubicin-resistant AML cell lines (HL60 and U937), doxorubicin being a commonly used AML induction drug and Pgp substrate. AB8939 produced a strong anti-proliferative effect in both cell lines whereas both were resistant to doxorubicin, thus demonstrating AB8939's potential to overcome refractory/resistant AML. Notably, HL60 and U937 are respectively MPO-positive and MPO-negative, indicating that unlike vinca alkaloids (e.g. vincristine or vinblastine) AB8939 it is not deactivated by this myeloid enzyme. These data show that AB8939 is a prolific and highly potent (nanomolar concentrations) Pgp-independent, next-generation microtubule-destabilizer drug for cancer therapy; in particular, difficult to treat hematopoietic tumors such as relapsed/refractory AML. Disclosures Humbert: AB Science: Employment. Goubard:AB Science: Employment. Mansfield:AB Science: Employment, Patents &amp; Royalties. Hermine:AB Science: Membership on an entity's Board of Directors or advisory committees. Dubreuil:AB Science: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding. AB8939 Study Group:AB Science: Consultancy, Employment.

Cytocompatibility of Potential Bioactive Cerium-Doped Glasses based on 45S5.

The cytocompatibility of potential bioactive cerium-containing (Ce3+/Ce4+) glasses is here investigated by preparing three different glasses with increasing amount of doping CeO2 (1.2, 3.6 and 5.3 mol% of CeO2, called BG_1.2, BG_3.6 and BG_5.3, respectively) based on 45S5 Bioglass® (called BG). These materials were characterized by Environmental Scanning Electron Microscopy (ESEM) and infrared spectroscopy (FTIR) after performing bioactivity tests in Dulbecco’s Modified Eagle Medium (DMEM) solution, and the ions released in solution were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Optical Emission Spectrometry (ICP-OES). The data obtained clearly show that the glass surfaces of BG, BG_1.2 and BG_3.6 were covered by hydroxyapatite (HA), while BG_5.3 favored the formation of a cerium phosphate crystal phase. The cytotoxicity tests were performed using both murine long bone osteocyte-like (MLO-Y4) and mouse embryonic fibroblast (NIH/3T3) cell lines. The cerium-containing bioactive glasses show an increment in cell viability with respect to BG, and at long times, no cell aggregation and deformation were observed. The proliferation of NIH/3T3 cells increased with the cerium content in the glasses; in particular, BG_3.6 and BG_5.3 showed a higher proliferation of cells than the negative control. These results highlight and enforce the proposal of cerium-doped bioactive glasses as a new class of biomaterials for hard-tissue applications.