Posttranslational processing and targeting of transgenic human defensin in murine granulocyte, macrophage, fibroblast, and pituitary adenoma cell lines

Abstract Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal constituents of the neutrophil's azurophil granules. To determine the tissue specificity of posttranslational processing and subcellular targeting of defensins, the cDNA for a 94 aa human preprodefensin was transduced into murine cell lines (NIH 3T3 embryonic fibroblasts, AtT-20 pituitary adenoma, J774.1 and RAW 264.7 macrophages, and 32D and 32D cl3 granulocytes) using retroviral vectors. All transduced cell types expressed and to a variable extent constitutively secreted a 75 aa prodefensin formed by the removal of the amino terminal signal sequence. In AtT-20 cells, the 75 aa form accumulated intracellularly in granules and was releasable by secretagogues. Proteolytic processing to mature defensins was seen only in myeloid cells (J774.1, RAW 264.7, 32D, and 32D cl3). Newly formed mature defensin was rapidly degraded in J774.1 and RAW 264.7 macrophages, but accumulated stably in multivesicular bodies in 32D cells and in cytoplasmic granules of 32D cl3 cells. Our data suggest that the enzymatic and transport machinery required to process preprodefensin to mature defensin and to store it in cytoplasmic granules is a specialized feature of cells of granulocytic lineage.

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Posttranslational processing and targeting of transgenic human defensin in murine granulocyte, macrophage, fibroblast, and pituitary adenoma cell lines

Blood ◽

10.1182/blood.v82.2.641.bloodjournal822641 ◽

1993 ◽

Vol 82 (2) ◽

pp. 641-650 ◽

Cited By ~ 1

Author(s):

T Ganz ◽

L Liu ◽

EV Valore ◽

A Oren

Keyword(s):

Pituitary Adenoma ◽

Cell Lines ◽

Retroviral Vectors ◽

Proteolytic Processing ◽

Raw 264.7 ◽

Posttranslational Processing ◽

Subcellular Targeting ◽

Raw 264.7 Macrophages ◽

Cytoplasmic Granules ◽

Amino Terminal

Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal constituents of the neutrophil's azurophil granules. To determine the tissue specificity of posttranslational processing and subcellular targeting of defensins, the cDNA for a 94 aa human preprodefensin was transduced into murine cell lines (NIH 3T3 embryonic fibroblasts, AtT-20 pituitary adenoma, J774.1 and RAW 264.7 macrophages, and 32D and 32D cl3 granulocytes) using retroviral vectors. All transduced cell types expressed and to a variable extent constitutively secreted a 75 aa prodefensin formed by the removal of the amino terminal signal sequence. In AtT-20 cells, the 75 aa form accumulated intracellularly in granules and was releasable by secretagogues. Proteolytic processing to mature defensins was seen only in myeloid cells (J774.1, RAW 264.7, 32D, and 32D cl3). Newly formed mature defensin was rapidly degraded in J774.1 and RAW 264.7 macrophages, but accumulated stably in multivesicular bodies in 32D cells and in cytoplasmic granules of 32D cl3 cells. Our data suggest that the enzymatic and transport machinery required to process preprodefensin to mature defensin and to store it in cytoplasmic granules is a specialized feature of cells of granulocytic lineage.

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Secoiridoid Glucosides from Fraxinus Excelsior with Effects on LPS-Induced Nitrite Production in RAW 264.7 Macrophages and Human Cancer Cell Lines

ACS Symposium Series - African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition ◽

10.1021/bk-2013-1127.ch009 ◽

2013 ◽

pp. 115-125

Author(s):

Naisheng Bai ◽

Kan He ◽

Marc Roller ◽

Ching-Shu Lai ◽

Xi Shao ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Human Cancer ◽

Cancer Cell Lines ◽

Fraxinus Excelsior ◽

Raw 264.7 ◽

Human Cancer Cell ◽

Raw 264.7 Macrophages ◽

Human Cancer Cell Lines ◽

Nitrite Production

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The Evaluation of Potential Cytotoxic Effect of Different Proton Pump Inhibitors on Different Human Cancer Cell Lines

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666191029151545 ◽

2020 ◽

Vol 20 (2) ◽

pp. 245-253

Author(s):

Aya Qasem ◽

Violet Kasabri ◽

Eman AbuRish ◽

Yasser Bustanji ◽

Yusuf Al-Hiari ◽

...

Keyword(s):

Cytotoxic Activity ◽

Cell Lines ◽

Cancer Cell ◽

Human Cancer ◽

Cancer Cell Lines ◽

Raw 264.7 ◽

Human Cancer Cell ◽

Raw 264.7 Macrophages ◽

Human Cancer Cell Lines ◽

Ic50 Values

Objective : To assess the differential cytotoxic activity of PPIs on different human cancer cell lines; namely A549 lung cancer, CACO-2 colorectal cancer, MCF-7 breast cancer, and PANC-1 pancreatic cancer, A375 skin melanoma. Methods: In this study, the five human cancer cell lines and human non-cancerous fibroblasts were treated with increasing concentration of PPIs Omeprazole (OMP), Esomeprazole (ESOM), and Lansoprazole (LANSO) (50-300μM), over 24h, 48h, and 72h. Cell viability was determined using 3-(4,5- Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and the IC50 values of PPIs were measured. The most sensitive cell line A375 was used for further investigation. The cytotoxic effects of LANSO on these cells were assessed using Annexin-V Propidium Iodide (AV-PI) flow cytometry. As of action mechanism; anti-inflammatory effects of each PPIs and PPIs-DOXO combination therapy on LPS-stimulated RAW 264.7 mouse macrophages were assessed. Results: Dose and time dependence cytotoxic activity of PPIs on human cancer cell lines was founded. Unlike DOXO; All PPIs had a selective cytotoxic effect in the normal fibroblasts. Unlike the equipotent OMP and ESOM; LANSO was the most potent drug with IC50 values at 72h of 99, 217, 272, 208, 181μM against A375, A549, CACO-2, MCF-7, and PANC-1, respectively. AV-PI flow cytometry revealed dose-dependent apoptotic effects of LANSO alone and substantially enhanced in DOXO-co-treatments. Interestingly unlike ESOM and OMP, LANSO proved more effective than indomethacin in LPS-stimulated RAW 264.7 macrophages. None of the tested compounds, as well as indomethacin, exerted any cytotoxicity against RAW 264.7 macrophages. PPIs-DOXO lacked potential synergistic combination antiinflammation therapies. Conclusion: This study provides the evidence that PPIs induce a direct and differential cytotoxic activity against human cancer cell line by the induction of the apoptosis. Moreover, PPIs increase cancer cell lines sensitivity to doxorubicin via apoptosis augmentation. Nevertheless, PPIs-DOXO lacked potential synergistic combination therapies in either antiproliferation or anti-inflammation.

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Homocysteine elicits an M1 phenotype in murine macrophages through an EMMPRIN-mediated pathway

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2014-0520 ◽

2015 ◽

Vol 93 (7) ◽

pp. 577-584 ◽

Cited By ~ 7

Author(s):

Lee J. Winchester ◽

Sudhakar Veeranki ◽

Srikanth Givvimani ◽

Suresh C. Tyagi

Keyword(s):

Nitric Oxide ◽

Matrix Metalloproteinase ◽

Cell Lines ◽

Inflammatory Diseases ◽

Raw 264.7 ◽

Macrophage Phenotype ◽

Raw 264.7 Macrophages ◽

M1 Macrophage ◽

Endothelial Nitric Oxide ◽

The Impact

Introduction: Hyperhomocysteinemia (HHcy) is associated with inflammatory diseases and is known to increase the production of reactive oxygen species (ROS), matrix metalloproteinase (MMP)-9, and inducible nitric oxide synthase, and to decrease endothelial nitric oxide production. However, the impact of HHcy on macrophage phenotype differentiation is not well-established. It has been documented that macrophages have 2 distinct phenotypes: the “classically activated/destructive” (M1), and the “alternatively activated/constructive” (M2) subtypes. We hypothesize that HHcy increases M1 macrophage differentiation through extracellular matrix metalloproteinase inducer (EMMPRIN), a known inducer of matrix metalloproteinases. Methods: murine J774A.1 and Raw 264.7 macrophages were treated with 100 and 500 μmol/L Hcy, respectively, for 24 h. Samples were analyzed using Western blotting and immunocytochemistry. Results: Homocysteine treatment increased cluster of differentiation 40 (CD40; M1 marker) in J774A.1 and Raw 264.7 macrophages. MMP-9 was induced in both cell lines. EMMPRIN protein expression was also increased in both cell lines. Blocking EMMPRIN function by pre-treating cells with anti-EMMPRIN antibody, with or without Hcy, resulted in significantly lower expression of CD40 in both cell lines by comparison with the controls. A DCFDA assay demonstrated increased ROS production in both cell lines with Hcy treatment when compared with the controls. Conclusion: Our results suggest that HHcy results in an increase of the M1 macrophage phenotype. This effect seems to be at least partially mediated by EMMPRIN induction.

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The pro region of human neutrophil defensin contains a motif that is essential for normal subcellular sorting

Blood ◽

10.1182/blood.v85.4.1095.bloodjournal8541095 ◽

1995 ◽

Vol 85 (4) ◽

pp. 1095-1103 ◽

Cited By ~ 60

Author(s):

L Liu ◽

T Ganz

Keyword(s):

Amino Acid ◽

Cell Lines ◽

Human Neutrophil ◽

Signal Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Signal Peptidase ◽

Cytoplasmic Granules ◽

Amino Terminal ◽

Nih 3T3

Human defensins (human neutrophil peptides) HNP 1–3 are 29–30 amino acid antibiotic and cytotoxic peptides highly abundant in the cytoplasmic granules of polymorphonuclear leukocytes. The peptides are produced from 94 amino acid (aa) prepropeptides by proteolytic cleavage of the signal sequence and stepwise removal of the 44–45 aa anionic propiece. To study the role of the propiece, we constructed five in- frame deletions in preproHNP-1 cDNA between the signal peptidase site and the amino-terminus of the mature defensin region (aa 21–64). The wild type HNP-1 cDNA and the deletion mutants were ligated into the pBabe-Neo retroviral vector, expressed in GP+E86 packaging derivative of NIH 3T3 cells, then transduced into the 32D cl3 granulocytic cell line. For each construction and both cell lines, we measured the accumulation of the various defensin forms in cells and media by 24- hour labeling or pulse-chase with 35S-cysteine- and immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Deletions in the amino-terminal two-fifths of the propiece, delta 21–28 and delta 21–38, had only minor effects on defensin biosynthesis in both cell lines and did not interfere with the accumulation of mature defensin in the granules of 32D cl3 cells. Deletions in the carboxyterminal three-fifths of the propiece (delta 21– 51 and delta 21–64) diminished net defensin synthesis, blocked constitutive secretion of prodefensin in both cell lines, and interfered with defensin accumulation in cytoplasmic granules of 32D cl3 cells. These effects were reproduced by the smaller deletion delta 40–51, which contains highly conserved secondary structure. The propiece segment 40–51 appears to be essential for the subcellular trafficking and sorting of HNP-1 defensin.

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In-vitro Anti-Cancer and Anti-Inflammatory Screening of Dodonaea viscosa

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i39b32194 ◽

2021 ◽

pp. 186-192

Author(s):

R. Ramkumar ◽

S. K. Periyasamy ◽

B. R. Venkatraman ◽

K. G. Sekar

Keyword(s):

Cell Lines ◽

Raw 264.7 ◽

Dodonaea Viscosa ◽

Raw 264.7 Macrophages ◽

Tnf Α ◽

Cervical Disease ◽

Hct 116 ◽

The Impact ◽

Mcf 7

Background: The current investigation was done to assess the in vitro anticancer property of Dodonaea viscosa (D. viscosa) in three malignant growth cell lines and mitigating impact in RAW 264.7 macrophages. Methods: The hydroalcoholic remove D. viscosa was ready and tried against HCT-116 colon malignancy, MCF-7 bosom disease HeLa cervical disease cell lines. The cytotoxicity of concentrate was affirmed by MTT cheeky. The calming movement of concentrate was assessed utilizing LPS invigorated RAW 264.7 macrophages and the degree of incendiary middle people was estimated. Results: The anticancer impact of D. viscosa onHCT-116, MCF-7 and HeLa cell line with the IC50 worth of 60.43 ± 0.76 μg/ml,75.26 ± 0.45 μg/ml and 72.12 ± 0.87μg/ml individually. Further, in LPS stimulatedRAW264.7 macrophage cells, treatment with D. viscosa extract altogether decreased the raised level of NO, TNF-α and PGE2. Conclusion: This examination gave the proof to D. viscosa an anticancer and mitigating specialist. Further bioactive confinement and atomic examinations are needed to prove the impact of plant remove.

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