scholarly journals Intracellular Delivery of DNA and Protein by a Novel Cell-Permeable Peptide Derived from DOT1L

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 217 ◽  
Author(s):  
Jingping Geng ◽  
Xiangli Guo ◽  
Lidan Wang ◽  
Richard Q. Nguyen ◽  
Fengqin Wang ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Green Fluorescence Protein ◽  
Cell Penetrating Peptides ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Targets ◽  
Intracellular Release ◽  
Cargo Delivery ◽  
Future Drug ◽  
Cell Permeable Peptide

Cellular uptake and intracellular release efficiency of biomacromolecules is low because of hurdles in the cell membrane that result in limited access to intra-cellular targets with few functional effects. Cell-penetrating peptides (CPPs) act as cargo delivery vehicles to promote therapeutic molecule translocation. Here, we describe the novel CPP-Dot1l that not only penetrates by itself, but also mediates cargo translocation in cultured cells, as confirmed by fluorescence microscopy and fluorescence spectrophotometry. We conducted cytotoxicity assays and safety evaluations, and determined peptide-membrane interactions to understand the possible pathway for cargo translocation. Additional nucleic acid and covalently conjugated green fluorescence protein (GFP) studies mediated by CPP-Dot1l were conducted to show functional delivery potential. Results indicate that CPP-Dot1l is a novel and effective CPP due to its good penetrating properties in different cell lines and its ability to enter cells in a concentration-dependent manner. Its penetration efficiency can be prompted by DMSO pretreatment. In addition, not only can it mediate plasmid delivery, but CPP-Dot1l can also deliver GFP protein into cytosol. In conclusion, the findings of this study showed CPP-Dot1l is an attractive pharmaceutical and biochemical tool for future drug, regenerative medicine, cell therapy, gene therapy, and gene editing-based therapy development.

scholarly journals Binding of heparin and of the small proteoglycan decorin to the same endocytosis receptor proteins leads to different metabolic consequences.

1991 ◽  
Vol 114 (1) ◽  
pp. 45-52 ◽  
Author(s):  
H Hausser ◽  
H Kresse
Keyword(s):  
Cultured Cells ◽  
Dermatan Sulfate ◽  
Core Protein ◽  
Affinity Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Receptor Proteins ◽  
High Affinity ◽  
Dermatan Sulfate Proteoglycan ◽  
Small Proteoglycan

Decorin, a small interstitial dermatan sulfate proteoglycan, is turned over in cultured cells of mesenchymal origin by receptor-mediated endocytosis followed by intralysosomal degradation. Two endosomal proteins of 51 and 26 kD have been implicated in the endocytotic process because of their interaction with decorin core protein. However, heparin and protein-free dermatan sulfate were able to inhibit endocytosis of decorin in a concentration-dependent manner. After Western blotting of endosomal proteins, there was competition for binding to the 51- and 26-kD proteins between heparin and decorin. In spite of its high-affinity binding, heparin was poorly cleared from the medium of cultured cells and then catabolized in lysosomes. In contrast to decorin, binding of heparin to the 51- and 26-kD proteins was insensitive to acidic pH, thus presumably preventing its dissociation from the receptor in the endosome. Recycling of heparin to the cell surface after internalization could indeed be demonstrated.

Stereoselective Inhibition of Renal Basolateral Human Organic Anion Transporter 3 by Lansoprazole Enantiomers

Pharmacology ◽  
2018 ◽  
Vol 101 (3-4) ◽  
pp. 176-183 ◽  
Author(s):  
Yugo Hamada ◽  
Kenji Ikemura ◽  
Takuya Iwamoto ◽  
Masahiro Okuda
Keyword(s):  
Cultured Cells ◽  
Organic Anion ◽  
Inhibitory Effect ◽  
Organic Anion Transporter ◽  
Racemic Mixture ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Anion Transporter ◽  
Ic50 Value ◽  
Organic Anion Transporter 3

Lansoprazole, a proton pump inhibitor, potently inhibits human organic anion transporter, hOAT3 (SLC22A8). Lansoprazole has an asymmetric atom in its structure and is clinically administered as a racemic mixture of (R)-and (S)-enantiomers. However, little is known about the stereoselective inhibitory potencies of lansoprazole against hOAT3 and its homolog, hOAT1. In the present study, the stereoselective inhibitory effect of lansoprazole was evaluated using hOAT1-and hOAT3-expressing cultured cells. hOAT1 and hOAT3 transported [14C]p-aminohippurate and [3H]estrone-3-sulfate (ES) with Michaelis-Menten constants of 29.8 ± 4.0 and 30.1 ± 9.0 µmol/L respectively. Lansoprazole enantiomers inhibited hOAT1- and hOAT3-mediated transport of each substrate in a concentration-dependent manner. The IC50 value of (S)-lansoprazole against hOAT3-mediated transport of [3H]ES (0.61 ± 0.08 µmol/L) was significantly lower than that of (R)-lansoprazole (1.75 ± 0.31 µmol/L). In contrast, stereoselectivity was not demonstrated for the inhibition of hOAT1. Furthermore, (S)-lansoprazole inhibited hOAT3-mediated transport of pemetrexed and methotrexate (hOAT3 substrates) more strongly than the corresponding (R)-lansoprazole. This study is the first to demonstrate that the stereoselective inhibitory potency of (S)-lansoprazole against hOAT3 is greater than that of (R)-lansoprazole. The present findings provide novel information about the drug interactions associated with lansoprazole.

Cell Penetrating Mechanism of Bax Inhibiting Peptides.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4298-4298
Author(s):  
Jose A. Gomez ◽  
Tomoyuki Yoshida ◽  
Minh Lam ◽  
Clark W. Distelhorst ◽  
Shigemi Matsuyama
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Fluorescent Dyes ◽  
Cultured Cells ◽  
Cell Penetrating Peptides ◽  
Dependent Manner ◽  
Cell Penetration ◽  
New Members ◽  
Cell Penetrating ◽  
High Degree

Abstract Plasma membrane is known to have a high degree of selectivity for molecular trafficking, and it does not allow the penetration of peptides larger than 3 amino acids. Previously known exceptions of large peptides that penetrate the plasma membrane are the Arginine rich peptides such as human immunodeficiency virus (HIV)-tat peptides. However, the mechanism of cell penetration of these peptides is largely unknown. Bax Inhibiting Peptides (BIP) are penta-peptides derived from the Bax binding domain of Ku70. At present, three types of BIP have been developed. Those are: VPMLK, VPTLK, and VPALR. All of these three BIPs directly bind Bax and inhibit Bax-mediated cell death in cultured cells as well as in animal study. Surprisingly, BIPs are cell permeable and autonomously enter the cytoplasm of the cells within 1 hr. Therefore BIPs are recognized as new members of cell penetration peptides. In this study, we investigated the mechanism of cell penetration of BIPs. DAMI cells (a human megakaryocyte cell line) and HeLa cells were used to investigate the detailed mechanism of cell penetration of BIPs. To detect the cell entry of BIPs, fluorescent dyes (fluorescein or tetramethylrodhamine) were conjugated to the N-terminus of BIPs and the cytoplasmic localization of BIPs was confirmed by confocal microscopy. Cell Penetration activities of BIPs were detected at 1 uM concentration in the culture medium. The significant accumulation of BIPs in the cytoplasm were detected within 1 hour of incubation both at 4 °C and 37 °C, suggesting that ATP-independent mechanism of cell penetration of BIP exists. However, cellular uptake of BIPs reaches plateau at 100 uM at 4 °C, whereas it increases in a dose dependent manner up to 1 mM at 37 °C without any sign of cytotoxicity. These results suggest that there are at least two mechanisms contributing to the cell penetration of BIPs that are, “ATP-independent (4 °C)” and “ATP-dependent (37 °C)” mechanisms. In addition to BIPs, we generated a series of mutated BIPs that do not bind Bax but retain cell-penetrating activities. We performed competition assay using fluorescence dye-labeled and non-labeled BIP (and the mutant BIPs), and the preliminary results suggest that there is a specific receptor for each peptide for its delivery into the cells. Our data also indicates that BIPs can deliver a cargo molecule (e.g. fluorescent dye) with at least the same molecular weight. Unlike other cell penetrating peptides, BIP has minimum toxicity due to its nature to inhibit Bax-mediated cell death. Along with the new data showing that BIP protects cells from pathological damages in cell culture and animal model, we will discuss the potential application of BIPs as a new type of drug delivery tool.

scholarly journals Thrombin stimulation of inflammatory breast cancer cells leads to aggressiveness via the EGFR-PAR1-Pak1 pathway

2012 ◽  
Vol 27 (4) ◽  
pp. 305-313 ◽  
Author(s):  
Kazufumi Ohshiro ◽  
Tri M. Bui-Nguyen ◽  
Reddy S. Divijendra Natha ◽  
Arnold M. Schwartz ◽  
Paul Levine ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Inflammatory Breast Cancer ◽  
Growth Factor Receptor ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Targets ◽  
Immunohistochemical Evidence ◽  
P21 Activated Kinase ◽  
Stimulation Of

Inflammatory breast cancer (IBC) accounts for a small fraction but aggressive form of epithelial breast cancer. Although the role of thrombin in cancer is beginning to be unfolded, its impact on the biology of IBC remains unknown. The purpose of this study was to establish the role of thrombin on the invasiveness of IBC cells. The IBC SUM149 cell line was treated with thrombin in the absence or presence of the epidermal growth factor receptor (EGFR) inhibitor erlotinib and protease-activated receptor 1 (PAR1) inhibitor. The effects of pharmacological inhibitors on the ability of thrombin to stimulate the growth rate and invasiveness were examined. We found that the inhibition of putative cellular targets of thrombin action suppresses both the growth and invasiveness of SUM149 cells in a concentration-dependent manner. In addition, thrombin-mediated increased invasion of SUM149 cells was routed through EGFR phosphorylation, and in turn, stimulation of the p21-activated kinase (Pak1) activity in a EGFR-sensitive manner. Interestingly, thrombin-mediated activation of the Pak1 pathway stimulation was blocked by erlotinib and PAR1 inhibitor. For proof-of-principle studies, we found immunohistochemical evidence of Pak1 activation as well as expression of PAR1 in IBC. Thrombin utilizes EGFR to relay signals promoting SUM149 cell growth and invasion via the Pak1 pathway. The study provides the rationale for future therapeutic approaches in mitigating the invasive nature of IBC by targeting Pak1 and/or EGFR.

scholarly journals Comparative Study of Mechanisms of Herpes Simplex Virus Inactivation by Sodium Lauryl Sulfate and n-Lauroylsarcosine

2002 ◽  
Vol 46 (9) ◽  
pp. 2933-2942 ◽  
Author(s):  
Jocelyne Piret ◽  
Sylvie Roy ◽  
Mylène Gagnon ◽  
Sébastien Landry ◽  
André Désormeaux ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Sodium Lauryl Sulfate ◽  
Cultured Cells ◽  
Vero Cells ◽  
Dependent Manner ◽  
Lauryl Sulfate ◽  
Concentration Dependent Manner ◽  
Simplex Virus

ABSTRACT The mechanisms of herpes simplex virus (HSV) inactivation by sodium lauryl sulfate (SLS) and n-lauroylsarcosine (LS), two anionic surfactants with protein denaturant potency, have been evaluated in cultured cells. Results showed that pretreatment of HSV type 1 (HSV-1) strain F and HSV-2 strain 333 with either surfactant inhibited, in a concentration- and time-dependent manner, their infectivities on Vero cells. SLS was a more potent inhibitor of HSV-2 strain 333 infectivity than LS with respect to the concentration (4.8-fold lower) and time (2.4-fold shorter) required to completely inactivate the virus. No inhibition of both herpesvirus strains infectivities was observed when Vero cells were pretreated with either surfactant. LS prevented the binding of HSV-2 strain 333 to cells without affecting the stable attachment and the rate of penetration into cells, whereas SLS exerted the opposite effect. Both SLS and LS inhibited, in a concentration-dependent manner, the HSV-2 strain 333-induced cytopathic effect, probably by affecting newly synthesized virions that come into contact with surfactant molecules present in culture medium. The pretreatment of HSV-2 strain 333 with specific combinations of SLS and LS concentrations inhibited the viral infectivity in a synergistic manner and resulted in only a small increase in their toxicities for exponentially growing Vero cells compared with that caused by each compound alone. Taken together, these results suggest that SLS and LS, alone or combined, could represent potent candidates as microbicides in topical vaginal formulations to prevent the transmission of herpes and possibly other pathogens that cause sexually transmitted diseases, including human immunodeficiency virus type 1.

Effects on the ATP Content of Cultured Cells after Radiographic Contrast Media Exposure

1989 ◽  
Vol 30 (5) ◽  
pp. 541-547 ◽  
Author(s):  
A. Nordby ◽  
K. Thorstensen ◽  
J. Halgunset ◽  
O. A. Haugen ◽  
S. Solberg
Keyword(s):  
Contrast Media ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Established Cell Line ◽  
Dependent Manner ◽  
Atp Content ◽  
Concentration Dependent Manner ◽  
Short Period ◽  
Radiographic Contrast Media ◽  
Established Cell

The ATP content of cultured cells after exposure to meglumine-calcium metrizoate, sodium metrizoate, iohexol, iopamidol and saline was studied. Initially, the ATP content diminished rapidly for a short period and thereafter slowly during the incubation. After incubation with contrast media or saline, the ATP content slowly increased to normal when the cells were reincubated with fresh nutrient medium. Different contrast media and saline with the same final osmolality produced a similar effect on the ATP content of the cultured cells. Cellular association of meglumine-sodium diatrizoate, sodium metrizoate, sodium-iothalamate, iohexol and iopamidol was also examined. The established cell line NHIK 3025 as well as primary cultures of human umbilical endothelium were found to accumulate contrast media in a time-and concentration-dependent manner. When the incubation was carried out at 4°C, the cellular accumulation of contrast medium was less than 35 per cent of that seen at 37°C. It therefore seems that energy-dependent processes are involved to some degree.

scholarly journals A peptide mimetic of human interferon (IFN)-beta

2003 ◽  
Vol 371 (2) ◽  
pp. 603-608 ◽  
Author(s):  
Atsushi SATO ◽  
Saburo SONE
Keyword(s):  
Antiviral Activity ◽  
Cultured Cells ◽  
Type I Interferons ◽  
Human Interferon ◽  
Type I ◽  
Dependent Manner ◽  
Type I Ifn ◽  
Concentration Dependent Manner ◽  
Antitumour Agents ◽  
Potent Agonist

Type I interferons (IFNs) are cytokines that are used clinically as antiviral and antitumour agents. The interaction of IFNs with their heterodimeric type I IFN receptor comprised of IFNAR1 and IFNAR2 is a first step to inducing biological actions. Here, we describe the successful mimicry of IFN-β by a peptide isolated by phage-display screening using a neutralizing anti-IFN-β monoclonal antibody. The 15-mer peptide, designated SYR6, was shown to compete with IFN-β for binding to type I IFN receptor in a concentration-dependent manner, and was shown to elicit antiviral activity on cultured cells. This antiviral activity was not eliminated in the presence of neutralizing monoclonal antibodies to IFN-α, -β and -γ, and a low concentration of soluble type I IFN receptor, suggesting that it was not due to IFN contamination or the induction of endogenous IFNs by SYR6. This peptide might be a potent agonist to provide a mechanism of activating heterodimeric cytokine receptors.

scholarly journals Qigesan reduces the motility of esophageal cancer cells via inhibiting Gas6/Axl and NF-κB expression

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Lingyu Kong ◽  
Zhongbing Wu ◽  
Yang Zhao ◽  
Xin Lu ◽  
Huijuan Shi ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Matrix Metalloproteinase ◽  
Protein Expression ◽  
Cell Lines ◽  
Cultured Cells ◽  
Matrix Metalloproteinase 2 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Protein Activation ◽  
Downstream Signaling

AbstractThe present study is mainly to explore the mechanism that how Qigesan (QGS) affects the movement capacity of esophageal cancer (EC) cell. QGS incubates ECA109 and TE1 cell lines and detecting the motility of tumor cells by different experiments. Growth arrest-specific 6 (Gas6) and Anexelekto (Axl) were co-localized, and then detecting Gas6, Axl signaling pathway, and protein expression after QGS intervention. Similarly, Observing the signal localization and protein expression of P-phosphoinositide3-kinases (PI3K), P-AKT protein kinase B (AKT), P-nuclear factor-kappa B (NF-κB), matrix metalloproteinase-2 (MMP2), and matrix metalloproteinase-9 (MMP9). The results showed that the concentration of QGS was less than 200 ug/ml, and the cultured cells did not exceed 24 h, that no obvious cytotoxicity was observed. QGS significantly inhibited the mobility of ECA109 and TE1 cell lines in the concentration-dependent manner. In addition, QGS can regulate the Gas6/Axl pathway, inhibit the formation and localization of the Gas6/Axl complex, and reduce the protein activation of PI3K/AKT, NF-κB, MMP2, and MMP9. Experimental innovation shows that QGS can significantly slow down the mobility of EC cells by regulating the Gas6/Axl complex and downstream signaling pathways, and provides a theoretical basis for the pharmacological effects of QGS in the therapy of EC.

scholarly journals Evaluation of Resveratrol and Piceatannol Cytotoxicity in Macrophages, T Cells, and Skin Cells

2007 ◽  
Vol 58 (3) ◽  
pp. 293-304 ◽  
Author(s):  
Vijayalaxmi Radkar ◽  
Diane Hardej ◽  
Cesar Lau-Cam ◽  
Blase Billack
Keyword(s):  
T Cells ◽  
Cultured Cells ◽  
Thiobarbituric Acid ◽  
Cell Types ◽  
Structural Analog ◽  
Dependent Manner ◽  
High Concentration ◽  
Concentration Dependent Manner ◽  
Skin Cells ◽  
Diphenyl Tetrazolium Bromide

Evaluation of Resveratrol and Piceatannol Cytotoxicity in Macrophages, T Cells, and Skin CellsThe cytotoxicity of resveratrol and of piceatannol, a structural analog of resveratrol, was examined in cultured cells. Using a MTT-based assay, which measures the conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) to a colored formazan product in living cells, resveratrol was found to inhibit the viability of transformed mouse macrophages, tumor-derived human T cells and human epidermoid carcinoma cells in a concentration-dependent manner, with the effect decreasing in the order: T cells (LC50 ~27 μmol L-1, 24 h; ~9 μmol L-1; 48h) > macrophages (LC50~29 μmol L-1, 24 h; 39 μmol L-1, 48 h) > skin cells (LC50 ~91 μmol L-1, 24 h; ~66 μmol L-1, 48 h). Paradoxically, a high concentration of resveratrol (50 μmol L-1) inhibited the proliferation of all three cell types, and a low concentration (5 μmol L-1) stimulated the proliferation of macrophages. The viability of macrophages was also decreased by piceatannol in a concentration-dependent manner. The stimulation of macrophages with zymosan lowered the cytotoxicity of both resveratrol and piceatannol. Scanning electron microscopy of cells treated with resveratrol revealed changes in cellular morphology that were consistent with toxicity. In macrophages and skin cells, resveratrol (50 μmol L-1) induced a time-dependent increase in reduced glutathione levels but did not alter the background levels of thiobarbituric acid-reactive substances. Taken together, the present data indicate that resveratrol is toxic to cultured macrophages, T cells and skin cells at concentrations ≥25 μmol L-1, and that the cytotoxicity occurs via a mechanism that does not involve oxidative stress. Furthermore, the degree of toxicity of both resveratrol and piceatannol towards macrophages depends on the activation status of these cells, with zymosan-activated cells appearing more resistant than nonstimulated cells.

Comparative analysis of XTT assay and xCELLigence system by measuring cytotoxicity of resveratrol in human cancer cell lines

2016 ◽  
Vol 41 (6) ◽  
Author(s):  
Harika Atmaca ◽  
Emir Bozkurt ◽  
Aslı Kısım ◽  
Rüçhan Uslu
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cultured Cells ◽  
Human Cancer ◽  
Cancer Cell Lines ◽  
Dependent Manner ◽  
Xtt Assay ◽  
Concentration Dependent Manner ◽  
End Point

AbstractObjective:In vitro preliminary oncological and translational studies are mainly based on evaluating the cytotoxic effects of a specific compound on cultured cells. Resveratrol is a commercially available compound which is originally isolated from the roots of white hellebore and later fromMethods:XTT end point assay and real-time cell based xCELLigence system were used to evaluate cytotoxicity. Cytotoxicity results were verified by monitoring cells under phase-contrast microscope which were treated with ICResults:Resveratrol decreased cell viability in a time- and concentration-dependent manner in all cancer cell lines when tested by both the XTT assay and xCELLigence system. Standard deviations of the xCELLigence data were found to be lower than the data from XTT assay.Conclusion:The data from this study strongly imply that xCELLigence system has higher precision, more enlightening and more reproducible than XTT end point assay.

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