Bioactivity Determination of a Therapeutic Recombinant Human Keratinocyte Growth Factor by a Validated Cell-based Bioassay

The therapeutic recombinant human keratinocyte growth factor 1 (rhKGF-1) was approved by the FDA for oral mucositis resulting from hematopoietic stem cell transplantation for hematological malignancies in 2004. However, no recommended bioassay for rhKGF-1 bioactivity has been recorded in the U.S. Pharmacopoeia. In this study, we developed an rhKGF-1-dependent bioassay for determining rhKGF-1 bioactivity based on HEK293 and HaCat cell lines that stably expressed the luciferase reporter driven by the serum response element (SRE) and human fibroblast growth factor receptor (FGFR2) IIIb. A good responsiveness to rhKGF-1 and rhKGF-2 shared by target HEK293/HaCat cell lines was demonstrated. Our stringent validation was completely focused on specificity, linearity, accuracy, precision, and robustness according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, AAPS/FDA Bioanalytical Workshop and the Chinese Pharmacopoeia. We confirmed the reliability of the method in determining rhKGF bioactivity. The validated method is highly timesaving, sensitive, and simple, and is especially valuable for providing information for quality control during the manufacture, research, and development of therapeutic rhKGF.

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Regulation of Human Beta-Defensin 3(hBD-3) in Human Keratinocyte(HaCaT) Cell Lines

Annals of Dermatology ◽

10.5021/ad.2003.15.1.1 ◽

2003 ◽

Vol 15 (1) ◽

pp. 1 ◽

Cited By ~ 1

Author(s):

Yu-Jin Kim ◽

Chang-Kwun Hong ◽

Seong-Jun Seo

Keyword(s):

Cell Lines ◽

Hacat Cell ◽

Human Keratinocyte ◽

Hacat Cell Lines

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Generation and Characterization of Monoclonal Antibodies to Human Keratinocyte Growth Factor Receptor

Hybridoma ◽

10.1089/hyb.2006.25.115 ◽

2006 ◽

Vol 25 (3) ◽

pp. 115-124 ◽

Cited By ~ 2

Author(s):

Ping Wei ◽

Jinghui Zhan ◽

Shuying Liu ◽

David Chang ◽

Raj Haldankar ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Growth Factor ◽

Keratinocyte Growth Factor ◽

Growth Factor Receptor ◽

Human Keratinocyte

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EVALUATION OF ANTIPROLIFERATIVE ACTIVITY OF SIDDHA ANTI-PSORIATIC FORMULATION PANCHAMUGA CHENDHURAM USING CULTURED HUMAN KERATINOCYTE CELL LINES

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2019.v12i6.32594 ◽

2019 ◽

pp. 280-283

Author(s):

RAJALAKSHMI S ◽

RAMYA VT ◽

SAMRAJ K

Keyword(s):

Cell Lines ◽

Antiproliferative Activity ◽

Hacat Cell ◽

Scientific Evidence ◽

Positive Control ◽

Traditional Use ◽

Human Keratinocyte ◽

Ic50 Values ◽

Hacat Cell Lines

Objectives: This study was aimed at scientifically evaluating the in vitro antipsoriatic activity of Siddha drug Panchamuga Chendhuram (PMC) in human keratinocyte (HacaT) cell lines. Methods: The Siddha drug PMC tested for antipsoriatic activity on HacaT cell lines was morphologically examined by phase contrast microscopy, and the cell viability was determined by 3- (4, 5 dimethyl thiazole-2 yl) -2.5-diphenyl tetrazolium bromide assay. About 100 μl of different concentrations (2, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg/ml) of the test samples were prepared in the cell culture medium and incubated for 24 h and 48 h to determine the viable cells. Results: The results revealed that Siddha drug PMC showed hopeful antiproliferative activity. In vitro studies showed that after 24 h and 48 h incubation, the inhibitory concentration 50 (IC50) values of PMC (IC50 20 μg/ml) were 72.08±27.56 μg/ml and 43.91±17.71 μg/ml, respectively, as compared with Asiaticoside as a positive control with an IC50 value of 20.13 μg/ml. Conclusion: Thus, this study provides scientific evidence about the efficacy of the Siddha drug PMC against the HacaT cell lines confirming its traditional use in psoriasis treatment and also emphasizes the need for antipsoriatic evaluation in animal models.

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Epidermal Growth Factor and Adenosine Triphosphate Induce Natrium Iodide Symporter Expression in Breast Cancer Cell Lines

Open Access Macedonian Journal of Medical Sciences ◽

10.3889/oamjms.2019.620 ◽

2019 ◽

Vol 7 (13) ◽

pp. 2088-2092 ◽

Cited By ~ 2

Author(s):

Aisyah Elliyanti ◽

Andani Eka Putra ◽

Yunia Sribudiani ◽

Noormartany Noormartany ◽

Johan S. Masjhur ◽

...

Keyword(s):

Breast Cancer ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Mrna Expression ◽

Cell Line ◽

Cell Lines ◽

Hacat Cell ◽

Breast Cancer Cell Lines ◽

Epidermal Growth ◽

Hacat Cell Lines

AIM: This study aims to investigate the effect of ATP, EGF and combination of those two to the Natrium Iodide Symporter (NIS) expression in MCF7, SKBR3 and HaCaT cell lines. METHODS: MCF7, SKBR3 and HaCaT cell lines were treated with ATP, EGF and combination of those two for 6, 12 and 24 hours. The expression of NIS mRNA was measured through quantitative-reverse transcription-polymerase chain reaction (qRT-PCR). The NIS protein expression was confirmed by immunocytofluorescence. RESULTS: NIS mRNA was expressed in SKBR3 and HaCaT cell lines but not in MCF7. The levels of NIS mRNA expression, after treatment by epidermal growth factor (EGF), adenosine Tri-Phosphate (ATP) or the combination of both for 6 and 12 hours were not significantly different from those of untreated cells. However, the treatment by a combination of ATP and EGF for 24 hours increases the level of NIS mRNA expression by 1.6 fold higher than that of the untreated cells (1.6241 ± 0.3, p < 0.05) and protein NIS expression increase significantly by the treatment than untreated cells (P < 0.05). CONCLUSION: The level of NIS expression varies among the different subtypes of breast cancer cell lines. MCF7 cell line is representing the luminal A subtype of breast cancer does not express NIS. Only SKBR3 cell line express NIS and this subtype might be suitable to receive radioiodine therapy as those cells expressing NIS. A combination treatment of EGF and ATP increases the expression of NIS mRNA and protein at the membrane in SKBR3 cells.

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Computational Evaluation and In Vitro Validation of New Epidermal Growth Factor Receptor Inhibitors

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200603122726 ◽

2020 ◽

Vol 20 (18) ◽

pp. 1628-1639

Author(s):

Sergi Gómez-Ganau ◽

Josefa Castillo ◽

Andrés Cervantes ◽

Jesus Vicente de Julián-Ortiz ◽

Rafael Gozalbes

Keyword(s):

Cell Proliferation ◽

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Binding Affinity ◽

Cell Lines ◽

Growth Factor Receptor ◽

Significant Activity ◽

Epidermal Growth

Background: The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein that acts as a receptor of extracellular protein ligands of the epidermal growth factor (EGF/ErbB) family. It has been shown that EGFR is overexpressed by many tumours and correlates with poor prognosis. Therefore, EGFR can be considered as a very interesting therapeutic target for the treatment of a large variety of cancers such as lung, ovarian, endometrial, gastric, bladder and breast cancers, cervical adenocarcinoma, malignant melanoma and glioblastoma. Methods: We have followed a structure-based virtual screening (SBVS) procedure with a library composed of several commercial collections of chemicals (615,462 compounds in total) and the 3D structure of EGFR obtained from the Protein Data Bank (PDB code: 1M17). The docking results from this campaign were then ranked according to the theoretical binding affinity of these molecules to EGFR, and compared with the binding affinity of erlotinib, a well-known EGFR inhibitor. A total of 23 top-rated commercial compounds displaying potential binding affinities similar or even better than erlotinib were selected for experimental evaluation. In vitro assays in different cell lines were performed. A preliminary test was carried out with a simple and standard quick cell proliferation assay kit, and six compounds showed significant activity when compared to positive control. Then, viability and cell proliferation of these compounds were further tested using a protocol based on propidium iodide (PI) and flow cytometry in HCT116, Caco-2 and H358 cell lines. Results: The whole six compounds displayed good effects when compared with erlotinib at 30 μM. When reducing the concentration to 10μM, the activity of the 6 compounds depends on the cell line used: the six compounds showed inhibitory activity with HCT116, two compounds showed inhibition with Caco-2, and three compounds showed inhibitory effects with H358. At 2 μM, one compound showed inhibiting effects close to those from erlotinib. Conclusion: Therefore, these compounds could be considered as potential primary hits, acting as promising starting points to expand the therapeutic options against a wide range of cancers.

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