Development of Tyrosine Kinase Inhibitor Screening Assay for Anti-cancer Agents
Keywords:
Cancer, Tyrosine kinase inhibitor screening assay, Tyrosine kinase inhibitors, Cancer therapeutic targetsAbstract
Tyrosine kinases play a key role in various cellular signaling pathways, linking to proliferation, migration, invasion and angiogenesis of cancer cells. Many of these are therapeutic targets for the treatment of cancers. This study aimed to develop the tyrosine kinase inhibitor assay for compound library screening. The assay development was started by investigating the expression levels of genes associated with these proteins in four cancer cell lines by quantitative real-time PCR, and measuring the tyrosine kinase activity of each cell lysate by ELISA. HeLa cell was chosen for the development. We further optimized the assay conditions, and the following were found: cell lysate, 0.125 mg/mL; peptide substrate,
0.25 μg/well; ATP, 25 nM; and incubation time of 30 minutes was suitable for the kinase reaction. The assay
system was verifi ed by using anti-cancer agents. The results showed that tyrosine kinase inhibitors decreased the assay signal. We also validated this assay by quantitative measurement of signal variability. It was shown that all parameters met the acceptance criteria, indicating that this assay was reproducible and suitable for screening. This developed assay was also used to test 180 medicinal plant extracts, and the active extracts were further tested by MTT and transmembrane assays. The results showed that some extracts exhibited potential anti-cancer activity in cell lines. Thus, the developed assay could be used for screening of tyrosine kinase inhibitors in a large number of samples.
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