Human Embryonic Kidney Cell Line Research Articles

Synthesis, conformation and cytotoxic activity of short hybrid peptides containing conformationally constrained 1-(aminomethyl)cyclohexanecarboxylic acid and gabapentin

The present work describes the synthesis,conformation and cytotoxic activities of short β/γ hybrid peptides, Boc-β2,2-Ac6c-Gpn-NHMe, BG1; Boc-(β2,2-Ac6c-Gpn)2-OMe, BG2; Boc-(β2,2-Ac6c-Gpn)3-OMe, BG3; H-β2,2-Ac6c-Gpn-NHMe, BG4; H-(β2,2-Ac6c-Gpn)2-OMe, BG5; H-(β2,2-Ac6c-Gpn)3-OMe, BG6, Boc-β2,2-Ac6c-Gpn-OMe, BG7 and H-β2,2-Ac6c-Gpn-OMe, BG8. Mixed C6/C7 conformations were observed for β/γ hybrid peptides. Further, BG1-BG8 were screened against MCF-7 (Breast cancer), A549 (Lung Cancer), PC-3 (Prostate cancer), HCT-116 (Colon cancer), and MDA-MB-231 (Breast cancer) cell lines. Among all, BG6 exhibited potent cytotoxicity against all cancer cell lines with IC50 ranging from 1.6 μM to 6.3 μM with relatively low cytotoxicity against normal epithelial breast cell line fR-2 and human embryonic kidney cell line HEK-293. Minimal hemolytic activity was observed for BG6 against human erythrocytes. Peptide BG6 displayed anti-migratory and anti-invasive potentials showing strong interactions with intrinsic apoptotic markers Bcl-2, Bax, and cleaved-PARP, as well as the induction of the mitochondria maladjustment mediated apoptosis.

Isolation, Characterization, Antioxidant, and Wound Healing Activities of Extracellular Polysaccharide from Endophytic Fungus Talaromyces purpureogenus.

In this study, two extracellular polysaccharides (TEPS1 and TEPS2) were isolatedfrom the endophytic fungus (Talaromyces purpureogenus) and purified by DEAE-Sepharose Fast Flow column using NaCl as gradient eluent. The HPLC analysis displayed that TEPS1 was composed of mannose (38.70%), ribose (25.02%), glucose (19.34%), and galactose (16.94%) while the TEPS2 composed by mannose (100%). The NMR results indicated that TEPS1 exhibited α-glycosidic configurations. The both polysaccharides, TEPS1 and TEPS2 were exhibited a good antioxidant activity interms of DPPH, ABTS, and •OH scavenging. However, TEPS1 showed a higher antioxidant activity than TEPS2. The IC50 of TEPS1 were 32.16, 192.57, and 54.67μg·mL-1, for DPPH, ABTS, and •OH radicalscavenging, respectively. Furthermore, TEPS1 showed the high cellular antioxidant and wound healing activity in the human embryonic kidney (HEK293) cell line. Overall, these two polysaccharides were promising inantioxidant activity.

Bioinspired synthesis of gold nanoparticles from Hemidesmus indicus L. root extract and their antibiofilm efficacy against Pseudomonas aeruginosa

Pseudomonas aeruginosa, is one the major causative agents for nosocomial and biofilm mediated infections. The present study is an attempt to disarm the biofilm forming potential of PAO1 using gold nanoparticles biosynthesized from Hemidesmus indicus L. root extract (HiAuNPs). The HiAuNPs were optimally synthesised using 1 mM HAuCl4 at 70 °C for 20 min with a maximum surface plasmon resonance (SPR) at 531 nm. The average size distribution, elemental composition, crystalline nature and the capping of functional biomolecules of HiAuNPs were ascertained by DLS, SEM-EDAX, XRD, FTIR and HPLC analyses respectively. HRTEM identified that HiAuNPs were predominantly hexagonal with an average particle size of 37 nm. The thermal properties of HiAuNPs were studied using thermal gravimetric analysis. The minimum biofilm inhibitory concentration (300 µg/mL) of HiAuNPs greatly mitigated the biofilm-forming ability as well as the virulence factors such as EPS, hydrophobicity index, pyocyanin, swarming motility, surfactant production, catalase and haemolytic activity of PAO1, without affecting its growth. Moreover, HiAuNPs exhibited significant synergistic antibacterial activity with antibiotics. The cytotoxicity assessment unveiled the non-cytotoxic nature of HiAuNPs against the human embryonic kidney cell lines. Thus, the present study reports HiAuNPs as a nontoxic, potent antibiofilm agent against P. aeruginosa.

An Alternate Approach to Generate Induced Pluripotent Stem Cells with Precise CRISPR/Cas9 Tool.

The induced pluripotent stem cells (iPSCs) are considered powerful tools in pharmacology, biomedicine, toxicology, and cell therapy. Multiple approaches have been used to generate iPSCs with the expression of reprogramming factors. Here, we generated iPSCs by integrating the reprogramming cassette into a genomic safe harbor, CASH-1, with the use of a precise genome editing tool, CRISPR/Cas9. The integration of cassette at CASH-1 into target cells did not alter the pattern of proliferation and interleukin-6 secretion as a response to ligands of multiple signaling pathways involving tumor necrosis factor-α receptor, interleukin-1 receptor, and toll-like receptors. Moreover, doxycycline-inducible expression of OCT4, SOX2, and KLF4 reprogrammed engineered human dermal fibroblasts and human embryonic kidney cell line into iPSCs. The generated iPSCs showed their potential to make embryoid bodies and differentiate into the derivatives of all three germ layers. Collectively, our data emphasize the exploitation of CASH-1 by CRISPR/Cas9 tool for therapeutic and biotechnological applications including but not limited to reprogramming of engineered cells into iPSCs.

In Vitro Evaluation of Cytotoxic Potential of Caladium lindenii Extracts on Human Hepatocarcinoma HepG2 and Normal HEK293T Cell Lines.

Data regarding the therapeutic potential of Caladium lindenii (C. lindenii) are insufficient. It becomes more important to explore plants as an alternative or palliative therapeutics in deadly diseases around the globe. The current study was planned to explore C. lindenii for its anticancer activity of ethanolic and hexane extracts of C. lindenii leaves against hepatic carcinoma (HepG2) and human embryonic kidney (HEK293T) cell lines. HepG2 and HEK293T cells were treated with 10, 50, 100, 200, and 400 μg/mL of ethanolic and hexane extracts of C. lindenii and were incubated for 72 h. Antiproliferative activity was measured by 3-(4,5-dimethylthiazol-2yl)-2,5-biphenyl tetrazolium bromide (MTT) assay, and percentage viability were calculated through crystal violet staining and cellular morphology by Floid Cell Imaging Station. The study showed ethanolic extract exhibiting a significantly higher antiproliferative effect on HepG2 (IC50 = 31μg/mL) in a concentration-dependent manner, while HEK293T (IC50 = 241μg/mL) cells showed no toxicity. Hexane extract exhibited lower cytotoxicity (IC50 = 150μg/mL) on HepG2 cells with no effect on HEK293T (IC50 = 550μg/mL). On the other hand, the percentage viability of HepG2 cells was recorded as 78%, 67%, 50%, 37%, and 28% by ethanolic extracts, and 88%, 80%, 69%, 59%, and 50% by hexane extracts at tested concentrations of both extracts. Toxicity assay showed significantly safer ranges of percentage viabilities in normal cells (HEK293T), i.e., 95%, 90%, 88%, 76%, and 61% with ethanolic extract and 97%, 95%, 88%, 75%, and 62% with hexane extract. The assay validity revealed 100% viability in the control negative (dimethyl sulfoxide treated) and less than 45% in the control positive (cisplatin) on both HepG2 and HEK293T cells. Morphological studies showed alterations in HepG2 cells upon exposure to >50 μg/mL of ethanolic extracts and ≥400 μg/mL of hexane extracts. HEK293T on the other hand did not change its morphology against any of the extracts compared to the aggressive changes on the HepG2 cell line by both extracts and positive control (cisplatin). In conclusion, extracts of C. lindenii are proved to have significant potential for cytotoxicity-induced apoptosis in human cancer HepG2 cells and are less toxic to normal HEK293T cells. Hence C. lindenii extracts are proposed to be used against hepatocellular carcinoma (HCC) after further validations.

The effects of amitriptyline on human embryonic kidney cells growth and proliferation in vitro

Many studies have shown that antidepressants, including amitriptyline, can cause side effects on normal human cells, although the exact effects of amitriptyline on normal human cells are unclear. Accordingly, the present study investigated the cytotoxic effects of amitriptyline on the human embryonic kidney cell line. In this experimental-laboratory study, cells were divided into control group (no drug-treated) and amitriptyline treatment groups (treated with 39.06, 12.78, 156.25, 312.5, 625, 1250 and 2500 μg/ml of amitriptyline). MTT method was used to assay the cytotoxic effects of amitriptyline at 24 and 48 hours. The data were analyzed by one-way analysis of variance. Treatment with 39.06 and 78.12 μg/ml of amitriptyline for 24 hours had no significant effect on the viability of Hek-293 cells, but treatment with 156.25 μg/ml and higher concentrations of amitriptyline significantly reduced the viability of Hek-293 cells. Treatment of cells for 48 hours showed a significant reduction in cell viability at all concentrations. Amitriptyline has cytotoxic effects on normal human embryonic kidney cells and these effects depend on the dose and duration of treatment. The findings of this study can be of significant importance in terms of the side effects of amitriptyline consumption on normal cells of the human body.

The non-glycosylated protein of Toxocara canis MUC-1 interacts with proteins of murine macrophages.

Toxocariasis is a neglected parasitic disease caused predominantly by larvae of Toxocara canis. While this zoonotic disease is of major importance in humans and canids, it can also affect a range of other mammalian hosts. It is known that mucins secreted by larvae play key roles in immune recognition and evasion, but very little is understood about the molecular interactions between host cells and T. canis. Here, using an integrative approach (affinity pull-down, mass spectrometry, co-immunoprecipitation and bioinformatics), we identified 219 proteins expressed by a murine macrophage cell line (RAW264.7) that interact with prokaryotically-expressed recombinant protein (rTc-MUC-1) representing the mucin Tc-MUC-1 present in the surface coat of infective larvae of T. canis. Protein-protein interactions between rTc-MUC-1 and an actin binding protein CFL1 as well as the fatty acid binding protein FABP5 of RAW264.7 macrophages were also demonstrated in a human embryonic kidney cell line (HEK 293T). By combing predicted structural information on the protein-protein interaction and functional knowledge of the related protein association networks, we inferred roles for Tc-MUC-1 protein in the regulation of actin cytoskeletal remodelling, and the migration and phagosome formation of macrophage cells. These molecular interactions now require verification in vivo. The experimental approach taken here should be readily applicable to comparative studies of other ascaridoid nematodes (e.g. T. cati, Anisakis simplex, Ascaris suum and Baylisascaris procyonis) whose larvae undergo tissue migration in accidental hosts, including humans.

Role of Lactiplantibacillus plantarum strain RD1 (Lpb RD1) in mitochondria-mediated apoptosis: an in vitro analysis.

The purpose of this study was to determine the cytotoxicity ofLactiplantibacillus plantarum strain RD1 (Lpb RD1), which was isolated and identified from the curd by 16 S rRNA sequencing. The probiotic properties of the isolated strain were studied by bile and NaCl tolerance and the ethyl acetate extract ofEa-LpRD1, was used to determine the toxicity against human breast cancer (MCF-7) cell lines and human embryonic kidney (HEK-293) cell lines by MTT assay. DNA fragmentation assay was carried out to study apoptosis induction. Flow cytometry analysis was done to determine the % of a cell population using the FTIC-Annexin V staining method. RT-PCR was used to assess gene expression levels in both cell lines. The IC50 concentration of the Ea-LpRD1 in MCF-7 cells was 0.30mg/ml and in HEK-293 was 0.47mg/ml. The expression levels of the BCL-2 gene anti-apoptotic genes in humans were reduced and BAX, caspase-8, caspase-3, and caspase-9 were an increased expression in MCF-7 cell lines.

The Potential Selective Oncolytic Effects of Newcastle Disease Virus (NDV) on Cervical and Colon Cancer Cell Lines

The mesogenic and velogenic strains of the avian Newcastle Disease Viruses (NDV) have been investigated for use in cancer virotherapy. However, despite its promising results, these strains have the potential to cause outbreaks leading to morbidity and mortality in large numbers of avians that can devastate the poultry industry. Therefore, there is a growing interest towards the use of lentogenic strains such as NDV LaSota vaccine strain for virotherapy. This study investigated the oncolytic potential of the NDV LaSota strain on colorectal cancer (HT29) and cervical cancer (HeLa) cell lines. The NDV LaSota stain was propagated in pathogen free embryonated chicken eggs and quantified using a hemagglutination assay. The oncolytic activity was evaluated by comparison of HT29 and HeLa cell lines to the non-cancerous human embryonic kidney (HEK293) cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. The highest NDV concentration of 16 HAU was found to reduce the viability of HT29, HeLa, and HEK293 cell lines to 42±3% (p = 0.0001), 49±12% (p = 0.0009), and 77±5% (p = 0.007), respectively, indicating its potential selective cytotoxicity, as supported by the disturbed cell morphology. The NDV LaSota strain exhibits a potential selective oncolytic activity towards cancer cell lines, thus may serve as an interesting candidate for virotherapy against colorectal and cervical cancer.

Diagnostic kit based on halloysite nanoclay-ionic liquid nanocomposite modified electrode for electrochemical determination of cancer biomarker

Nucleic acid hybridization is occurred between the selective single-stranded nucleic acid sequence and its target sequence, which is one of the essential procedure for electrochemical detection of nucleic acid. microRNA-21 (miRNA-21) is known as a biomarker in various cancers. The determination of miRNA-21 was attained through by hybridization of inosine substituted miRNA-21 specific DNA probe (Pinosine) with its target miRNA-21. In this study, the surface of pencil graphite electrode (PGE) was firstly modified with halloysite nanoclay-ionic liquid (HNT/IL) nanocomposite. The characterization of surface was performed by Scanning Electron Microscope (SEM) images and Energy Dispersive X-Ray Analysis (EDX) analysis, and the differences at surface modifications were also shown by electrochemical methods with electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). For sensitive and selective determination of miRNA-21, Pinosine and target miRNA concentration, immobilization and hybridization time were optimized by using HNT/IL modified PGE in combination with differential pulse voltammetry (DPV). The detection limit was achieved as 0.17 μg/mL (equals to 23.69 nM) in the linear range of 0.25–2 μg/mL miRNA-21. The selectivity of voltammetric method based on HNT/IL-PGE developed for miRNA-21 was examined in the presence of mismatch (MM) and non-complementary (NC) sequences. Because miRNA-21 is over-expressed in cancer cells, it has been tested in total RNA samples isolated from cancer cell line (breast cancer cell line, MCF-7). In the total RNA samples obtained from MCF-7, the detection limit was calculated as 0.28 μg/mL in the linear range of 1–4 μg/mL. Besides, the healthy cell line (human embryonic kidney cell line, HEK-293) was used as a control group and the results obtained by MCF-7 total RNA samples were compared to the results using HEK-293 total RNA samples in terms of miRNA-21 level.

Anticancer and antimicrobial activities of Imine containing compounds

A series of imine derivatives (3a-m) including thio-semicarbazone, semicarbazone, thiazole and oxazole functional moieties were examined for anti-cancer activity in-vitro by MTT assay using glioblastoma cell line (U87). Among all compound 3m was most potent compound with IC50 value of 8.86 ± 0.15 µM. Cytotoxicity of compounds were evaluated using normal human embryonic kidney cell line i.e HEK-293. Moreover, antibacterial activity of synthesized derivatives was also evaluated by disc diffusion method.

Information fields of written texts protect cells from oxidative damage and accelerate repair

ObjectiveWritten words demonstrated specific effects on biochemical measures and cell growth in cultured mammalian cells in previous study. We examined the protection and reparative effects of Chinese texts with positive meaning and the word "Buddha" on oxidative-damaged cells, with the goal of exploring the therapeutic effect of such characters. MethodsThe human embryonic kidney cell line HEK293T was utilized, with the oxidative- damaged cell model produced by hydrogen peroxide (H2O2). Double-blind experiments were set up to ensure all operators involved in the experiments did not know the contents of the texts in envelopes. Petri-dishes (9 plates) containing oxidative-damaged cells were selected randomly for three controls, three treatments by texts with positive meaning, and another three treatment by the word “Buddha”. Cells were sub-sampled at 3 h and 24 h to examine growth and mitochondrial functions. All data are expressed relative to the controls. ResultsCompared with the control group, mitochondrial functions were enhanced as evidenced by increased adenosine triphosphate (ATP) levels, accompanied by the significant improvement in cell growth rate for the groups treated by positive texts and the word “Buddha”. Reactive oxygen species (ROS) levels were decreased significantly in positive texts treatment, which suggested that the antioxidant capacity of cells was also improved. ConclusionsThis study revealed that certain texts can potentially offer protection and accelerate the cellular repair for oxidative-damaged cells.

Synthesis and in vitro anticancer evaluation of 8b-hydroxy-1-(6-substitutedbenzo[d]thiazol-2-yl)-3-(3-substitutedphenyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(3aH)-ones

A series of 1,8b-dihydroindeno[1,2-c]pyrazol-4(3aH)-ones has been efficiently synthesized on reaction of 2-(3-substitutedbenzoyl)-(1H)-indene-1,3(2H)-diones 1 and 2-hydrazinylbenzo[d]thiazole/2-hydrazinyl-6-substitutedbenzo[d]thiazoles 2 in presence of dry ethanol under reflux conditions in good yields. The structures of all the synthesized derivatives were elucidated by using different physical and spectral techniques (FTIR, 1H NMR, 13C NMR, 2D NMR and HRMS). The synthesized hydroxypyrazolones were assayed for their anticancer activity against two human cancer cell lines (i) lung carcinoma (A549) and (ii) breast carcinoma (MCF7) using MTT assay. Among all the tested compounds, 3d and 3k with IC50 values in the range of 30.68 ± 0.57 µM to 34.87 ± 0.76 µM exhibited moderate inhibitory ability against both the tested cancer cell lines in comparison to the standard drug Doxorubicin (0.95 ± 0.10 µM for A549 and 1.09 ± 0.11 µM for MCF7). The moderately active compounds of the series i.e.3d and 3k were further tested against normal human embryonic kidney cell line (HEK 293T) to examine their toxicity against normal cell lines. Selectivity Index (S.I.) of the most active compounds 3d and 3k was calculated for both the cancer cell lines which revealed that these derivatives i.e. 3d (S.I = 2.82 for A549 and 2.90 for MCF7) and 3k (S.I = 2.78 for A549 and 2.95 for MCF7) are safer anticancer agents.

Pan-cancer proteomic map of 949 human cell lines.

SummaryThe proteome provides unique insights into disease biology beyond the genome and transcriptome. A lack of large proteomic datasets has restricted the identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types are analyzed by mass spectrometry. Deploying a workflow to quantify 8,498 proteins, these data capture evidence of cell-type and post-transcriptional modifications. Integrating multi-omics, drug response, and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline reveals thousands of protein biomarkers of cancer vulnerabilities that are not significant at the transcript level. The power of the proteome to predict drug response is very similar to that of the transcriptome. Further, random downsampling to only 1,500 proteins has limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger) is a comprehensive resource available at https://cellmodelpassports.sanger.ac.uk.

Microwave-Assisted Three-Component Synthesis of Novel N-Arylated-Dihydrobenzo[g]quinoline-5,10-Diones and Their Potential Cytotoxic Activity.

A convenient three-component synthetic approach was developed en route to new and significative N-arylated-dihydrobenzo[g]quinoline-5,10-diones using 2-hydroxy-1,4-naphthoquinone, a variety of aromatic aldehydes, and 4-(arylamino)furan-2(5H)-ones. A sequence of steps including Knoevenagel condensation, Michael addition, [1,3]-hydrogen shift, intramolecular cyclization and dehydration led to the formation of products. All the products were structurally characterized by spectroscopic techniques and assessed in terms of their cytotoxicity profile against four cancer cell lines (KB, HepG2, A549, and MCF7), and human embryonic kidney (Hek-293) cell lines.

Synthesis and Biological Evaluation of Arylamide Sulphonate Derivatives as Ectonucleotide Pyrophosphatase/Phosphodiesterase-1 and -3 Inhibitors.

Aberrant level of ectonucleotide pyrophosphatase/phosphodiesterase-1 and -3 is linked with numerous disorders, for instance, diabetes, cancer, osteoarthritis, chondrocalcinosis, and allergic reactions. These disorders may be cured or minimized by blocking the activity of ENPP1 and ENPP3 isozymes. In this study, arylamide sulphonates were synthesized, characterized, and evaluated for their capability to affect the activity of isozymes ENPP1 and ENPP3. Among the selective inhibitors of ENPP1, compounds 4f and 4q exhibited sub-micromolar IC50 values of 0.28 ± 0.08 and 0.37 ± 0.03 μM, respectively, followed by 7a, with IC50 equal to 0.81 ± 0.05 μM, whereas out of the selective inhibitors of isozyme ENPP3, 4t and 7d preferably lessened the activity to half of the maximal inhibitory concentration of 0.15 ± 0.04 and 0.16 ± 0.01 μM alternatively. In addition, many structures including 4c, 4g, 4k, 4l, 4n, 4o, 4r, 4s, 7b, 7c, and 7e inhibited the activity of both isozymes to a significant level. Enzyme kinetic study of compound 4j revealed an uncompetitive mode of inhibition of ENPP1 isozyme, while 7e competitively blocked the activity of ENPP3. Cell viability analysis revealed the compound 4o as a cytotoxic agent against MCF7 (human breast cancer cell line) with a percentage inhibition of 63.2 ± 2.51%, whereas compounds 4c, 4d, 4n, and 7d decreased the HeLa cell viability (human cervical cancer cell line) to more than 50%. The tested compounds were non-cytotoxic against HEK293 (a human embryonic kidney cell line). Molecular docking analysis of selected inhibitors of both isozymes produced optimistic interactions with the influential amino acids, such as Leu290, Lys295, Tyr340, Asp376, His380, and Pro323 of ENPP1, whereas residues Asn226, His329, Leu239, Tyr289, Pro272, Tyr320, and Ala205 of ENPP3 crystallographic structure formed interactions with the potent inhibitors.

Synthesis of water soluble symmetric and asymmetric pillar[5]arene derivatives: Cytotoxicity, apoptosis and molecular docking studies

In this study, two water-soluble pillar[5]arenes, [1-(3-(trimethyl-azane)propoxy)-4-methoxyhydro-chloridepillar[5]arene (asym-P5) and 1,4-bis(2-(trimethyl-azane)ethoxy)hydrobromidepillar[5]arene (sym-P5), were synthesized and characterized using fourier transform infrared spectroscopy (FTIR), 1H and 13C nuclear magnetic resonance (1H-NMR and 13C-NMR). Cytotoxic and apoptotic effects of asym-P5 and sym-P5 on human hepatocellular carcinoma (HepG2), human breast adenocarcinoma (MDA-MB-231), human colon carcinoma (Caco-2), human prostate cancer (LnCap) and human embryonic kidney (HEK293) cell lines were determined by MTT, Annexin V-FITC apoptosis assays and quantitative analysis of mRNA levels of apoptosis related genes (Bax, Bcl-2, caspase 3, 8, and 9), respectively. asym-P5 not only reduced cell viability in all cell lines in a dose-dependent manner but also increased the apoptotic cell percentage significantly. sym-P5 was less effective than asym-P5 in inducing these effects in cancer cell lines. In RT-qPCR studies, it was observed that after asym-P5 treatment, Bax and caspase-3 mRNA levels increased, however, Bcl-2 levels decreased compared to the control group. As a result of the molecular docking, asym-P5 exhibited an energetically more favorable DNA binding affinity (-5.4 kcal/mol) than sym-P5 (-4.5 kcal/mol), where both ligands conformationally snug-fit into the major groove of DNA. These results demonstrated that asym-P5 had cytotoxic and apoptotic effects in cell lines.

Intracellular and Extracellular Antifreeze Protein Significantly Improves Mammalian Cell Cryopreservation.

Cell cryopreservation is an essential part of the biotechnology, food, and health care industries. There is a need to develop more effective, less toxic cryoprotective agents (CPAs) and methods, especially for mammalian cells. We investigated the impact of an insect antifreeze protein from Anatolica polita (ApAFP752) on mammalian cell cryopreservation using the human embryonic kidney cell line HEK 293T. An enhanced green fluorescent protein (EGFP)-tagged antifreeze protein, EGFP–ApAFP752, was transfected into the cells and the GFP was used to determine the efficiency of transfection. AFP was assessed for its cryoprotective effects intra- and extracellularly and both simultaneously at different concentrations with and without dimethyl sulfoxide (DMSO) at different concentrations. Comparisons were made to DMSO or medium alone. Cells were cryopreserved at −196 °C for ≥4 weeks. Upon thawing, cellular viability was determined using trypan blue, cellular damage was assessed by lactate dehydrogenase (LDH) assay, and cellular metabolism was measured using a metabolic activity assay (MTS). The use of this AFP significantly improved cryopreserved cell survival when used with DMSO intracellularly. Extracellular AFP also significantly improved cell survival when included in the DMSO freezing medium. Intra- and extracellular AFP used together demonstrated the most significantly increased cryoprotection compared to DMSO alone. These findings present a potential method to improve the viability of cryopreserved mammalian cells.

Functional Comparison of Human and Murine Equilibrative Nucleobase Transporter 1

BackgroundThiopurines are a key component for the immunosuppressive therapy of various leukemias, inflammatory bowels disease, and other autoimmune disorders. Thiopurine therapy discontinuation/interruption is associated with a higher risk of relapse, but is common due to the prevalence of severe and potentially fatal adverse events, such as hepatotoxicity.Drug transporter expression is a known factor for patient variability in drug response and toxicity. We have recently established that the SLC43A3‐encoded transporter, equilibrative nucleobase transporter 1 (ENBT1), is the primary mechanism by which the thiopurine, 6‐mercaptopurine (6‐MP) enters cells. ENBT1 is known to be highly expressed in human hepatocytes, however, the relationship between ENBT1 and thiopurine‐induced hepatotoxicity has not been explored in the literature.To investigate this paradigm, our lab has proposed developing a novel SLC43A3 knockout mouse model. However, the functional differences between human and murine ENBT1, in mediating 6‐MP transport, has equally not been explored. Evidence that suggests ENBT1 is functionally similar between species, would be an essential foundation to assist bridging the gap between our proposed animal model and clinical studies within the literature.HypothesisWe hypothesize that hSLC43A3‐encoded hENBT1 and mslc43a3‐encoded mENBT1 are functionally similar and will have non‐significantly different 6‐MP transport kinetics and resulting cytotoxicity.MethodsThe ENBT1‐deficient human embryonic kidney 293 (HEK293) cell line was stably transfected with either hSLC43A3 or mslc43a3. [3H]Adenine and [3H]6‐MP were used in an oil‐stop centrifugation assay to assess ENBT1‐mediated transport activity in wildtype and transfected HEK293 cell lines. To determine cytotoxicity, wildtype and transfected HEK293 cell lines were incubated for 48 hours with a range of 6‐MP concentrations (78 nM – 1.28 mM) before being assessed via MTT cell viability assay.ResultsOil stop centrifugation assay of ENBT1‐mediated [3H]adenine and [3H]6‐MP transport revealed that hENBT1 and mENBT1 have similar transport kinetics (Km and Vmax), where resulting Michaelis‐Menten curves were not significantly different (unpaired T‐test: Adenine ‐ t16=0.32, p=0.76, n=5 & 6‐MP ‐ t16=0.64, p=0.53, n=4). Adenine inhibition of ENBT1‐mediated [3H]6‐MP transport and 6‐MP inhibition of ENBT1‐mediated [3H]adenine transport also showed that hENBT1 and mENBT1 have concurring inhibition kinetics (Ki), inhibition curves were not significantly different (unpaired T‐test: Adenine: t17=0.29, p=0.77, n=5 & 6‐MP: t14=0.18, p=0.86, n=4). Subsequent MTT cell viability assay showed that wildtype HEK293 are relatively 6‐MP resistant, while the transfected‐HEK293s are 6‐MP sensitive and have non‐significantly different cell viability curves (unpaired T‐test: t28=0.13, p=0.89, n=5).ConclusionOur results show that hENBT1 and mENBT1 are functionally similar in regards to ENBT1‐mediated adenine and 6‐MP transport in a stably transfected cell line. Additional work is necessary to investigate whether this is the case in human and murine hepatic cell lines/primary hepatocytes that have endogenous SLC43A3/ENBT1 expression and a larger range of xenobiotic metabolizing enzymes that may alter the equilibrium of 6‐MP within cells.

Selective effect of β-catenin on nuclear receptor-dependent hepatitis B virus transcription and replication

β-catenin regulates HBV transcription in cell culture and viral biosynthesis in vivo in the transgenic mouse model of chronic HBV infection. Therefore, it is important to understand which transcription factor activities are coactivated by β-catenin to enhance HBV biosynthesis. The effect of β-catenin expression in the context of nuclear receptor-mediated HBV transcription was evaluated initially in the human embryonic kidney cell line, HEK293T. Reporter gene and viral replication assays revealed that β-catenin can coactivate HBV transcription through some, most predominantly liver receptor homolog 1 (LRH1), but not all nuclear receptors known to activate viral biosynthesis. Similarly, β-catenin activated nuclear receptor-mediated HBV transcription and replication in the human hepatoma cell line, Huh7, primarily through its effect on the farnesoid X receptor α (FXRα). These data indicate that β-catenin can enhance nuclear receptor-mediated HBV biosynthesis, but the relative importance of various transcription factors is dependent upon the precise cellular environment.