A rapid and specific method to simultaneously quantify eukaryotic elongation factor 1A1 and A2 protein levels in cancer cells.
Drug resistance
Immunohistochemistry
In Cell Western
LoVo DX
LoVo109
PC-3
PZHPV-7
eEF1A1
eEF1A2
siRNA
Journal
Journal of pharmaceutical and biomedical analysis
ISSN: 1873-264X
Titre abrégé: J Pharm Biomed Anal
Pays: England
ID NLM: 8309336
Informations de publication
Date de publication:
30 Nov 2019
30 Nov 2019
Historique:
received:
24
03
2019
revised:
12
08
2019
accepted:
14
08
2019
pubmed:
27
8
2019
medline:
11
3
2020
entrez:
27
8
2019
Statut:
ppublish
Résumé
The two isoforms of the eukaryotic Elongation Factor 1A (eEF1A1 and eEF1A2), sustain the progression/aggressiveness of cancer cells. Thus, they are considered promising therapeutic targets and prognostic markers. It follows that their precise quantification is of utmost relevance in research and development. The simultaneous quantification of A1 and A2 proteins in the cells helps the comprehension of cancer biology mechanisms and response to drug treatments. However, the high homology at the amino-acidic level (92%) can cause antibodies cross-reaction. Moreover, the commonly employed western blotting just gives semi-quantitative data and does not allow the detection of both protein targets within the same cell. Thus, we developed an in cell western (ICW) technique to bypass the above limitations. Firstly, relevant antibodies cross-reaction was excluded by immunohistochemistry on normal pancreatic tissue; then eEF1A1-A2 protein levels were quantitated by ICW in prostate and colorectal cancer cell lines in 96 well plates under different conditions, which include: 1) drug treatment, 2) siRNA silencing, 3) cell seeding density. We show that: 1) eEF1A1-A2 levels vary depending on the cell type following drug treatment, 2) ICW can accurately detect eEF1A1-A2 protein levels following siRNA silencing, 3) cell seeding density influences eEF1A1-A2 levels, depending on cell type. ICW is a valuable tool to specifically determine the intracellular level of eEF1A1-A2 proteins thus contributing to better define their role as potential therapeutic targets and prognostic markers in human tumors as well as for drug effects screening.
Sections du résumé
BACKGROUND
BACKGROUND
The two isoforms of the eukaryotic Elongation Factor 1A (eEF1A1 and eEF1A2), sustain the progression/aggressiveness of cancer cells. Thus, they are considered promising therapeutic targets and prognostic markers. It follows that their precise quantification is of utmost relevance in research and development. The simultaneous quantification of A1 and A2 proteins in the cells helps the comprehension of cancer biology mechanisms and response to drug treatments. However, the high homology at the amino-acidic level (92%) can cause antibodies cross-reaction. Moreover, the commonly employed western blotting just gives semi-quantitative data and does not allow the detection of both protein targets within the same cell. Thus, we developed an in cell western (ICW) technique to bypass the above limitations.
METHODS
METHODS
Firstly, relevant antibodies cross-reaction was excluded by immunohistochemistry on normal pancreatic tissue; then eEF1A1-A2 protein levels were quantitated by ICW in prostate and colorectal cancer cell lines in 96 well plates under different conditions, which include: 1) drug treatment, 2) siRNA silencing, 3) cell seeding density.
RESULTS
RESULTS
We show that: 1) eEF1A1-A2 levels vary depending on the cell type following drug treatment, 2) ICW can accurately detect eEF1A1-A2 protein levels following siRNA silencing, 3) cell seeding density influences eEF1A1-A2 levels, depending on cell type.
CONCLUSIONS
CONCLUSIONS
ICW is a valuable tool to specifically determine the intracellular level of eEF1A1-A2 proteins thus contributing to better define their role as potential therapeutic targets and prognostic markers in human tumors as well as for drug effects screening.
Identifiants
pubmed: 31450069
pii: S0731-7085(19)30728-9
doi: 10.1016/j.jpba.2019.112814
pii:
doi:
Substances chimiques
Antineoplastic Agents
0
Biomarkers, Tumor
0
EEF1A1 protein, human
0
EEF1A2 protein, human
0
Peptide Elongation Factor 1
0
Types de publication
Evaluation Study
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
112814Informations de copyright
Copyright © 2019 Elsevier B.V. All rights reserved.