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Method for assessing proliferation inhibiting effect of inhibitor, and method for determining sensitivity of tumor cell to inhibitor

A tumor cell and proliferation inhibition technology, applied in the detection of programmed cell death, biochemical equipment and methods, screening of compounds, etc., can solve problems such as insufficient inhibition of tyrosine kinases

Inactive Publication Date: 2008-04-09
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, when investigating the inhibitory effect of inhibitors on the proliferation of tumor cells and the sensitivity of tumor cells to inhibitors, it is not enough to separately confirm the inhibitory effects on each receptor tyrosine kinase as introduced by N Osherov et al.

Method used

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  • Method for assessing proliferation inhibiting effect of inhibitor, and method for determining sensitivity of tumor cell to inhibitor
  • Method for assessing proliferation inhibiting effect of inhibitor, and method for determining sensitivity of tumor cell to inhibitor
  • Method for assessing proliferation inhibiting effect of inhibitor, and method for determining sensitivity of tumor cell to inhibitor

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0139] (Preparation of reaction samples)

[0140] 50 μl buffer 1 (containing 20mM HEPES pH7.4, 10mM MnCl 2 , 1% NP40, 1mM DTT, 0.2% protease inhibitor (hereinafter referred to as PI), 10% glycerol, 200μM Na 3 VO 4 and 50 mM NaF) and 0.5 pmol of a commercially available receptor tyrosine kinase ICD were mixed, and the resulting mixture was used as a reaction sample for the following enzyme reactions. Here, used as ICD: platelet-derived growth factor-β receptor kinase (hereinafter referred to as PDGFR-β kinase), vascular endothelial cell growth factor receptor 1 kinase (hereinafter referred to as VEGFR1 kinase), vascular endothelial cell growth factor receptor 2 kinase (hereinafter referred to as VEGFR2 kinase), epidermal growth factor receptor 1 kinase (hereinafter referred to as HER1 kinase), epidermal growth factor receptor 2 kinase (hereinafter referred to as HER2 kinase), epidermal growth factor receptor 4 kinase (hereinafter referred to as HER4 kinase), insulin Growth f...

example 1

[0150] Effect of ATP Competitive Tyrosine Kinase Inhibitors

[0151] In this example, a reaction sample containing receptor tyrosine kinase was prepared from cultured cells. Prepared reactions are treated with ATP-competitive tyrosine kinase inhibitors. Furthermore, the activity of receptor tyrosine kinase contained in the treated reaction sample was measured using a GST-Poly (Glu, Tyr) substrate. According to the obtained activity value, the inhibitory effect of the inhibitor on the tyrosine kinase activity of the cultured cells was discussed.

[0152] (sample for reaction)

[0153] Samples for the reaction were prepared from five types of cultured cells (MDA-MB453, MDA-MB468, SKBr3, Hela, and HT29). Specifically, the cultured cells and 1ml cell treatment solution (containing 20mM HEPES pH7.4, 0.2% PI, 10% glycerol, 200μM Na 3 VO 4 and 50mM NaF) were mixed, and the cell membrane of the resulting mixture was destroyed under pressure with a pestle to prepare a cell solutio...

Embodiment 2

[0199] Effect of Combination of Two ATP Competitive Tyrosine Kinase Inhibitors

[0200] In this example, the reaction sample MB468 used in Example 1 was treated by combining two kinds of ATP-competitive tyrosine kinase inhibitors. The activity value of the receptor tyrosine kinase group contained in the treated reaction sample MB468 was measured using a GST-Poly (Glu, Tyr) substrate. According to the obtained activity values, the inhibitory effect of the combination of the two inhibitors on the tyrosine kinase activity of the cultured cells was discussed.

[0201] (sample for reaction)

[0202] Use the reaction sample MB468 that embodiment 1 prepares.

[0203] (ELISA plate)

[0204] The following enzymatic reactions were carried out using the chromatography plate for ELISA prepared in Example 1.

[0205] (ATP Competitive Tyrosine Kinase Inhibitor Treatment)

[0206] In this example, four ATP-competitive tyrosine kinase inhibitors AG1478, 4557W, PDGF receptor tyrosine kina...

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Abstract

A method for assessing the proliferation inhibiting effect of a receptor tyrosine kinase inhibitor is described. In the method, a cytoplasm is separated from a tumor cell to prepare a sample containing various receptor tyrosine kinases. The prepared sample is treated with the inhibitor. The receptor tyrosine kinases in the treated sample, and a substrate for at least two kinds of receptor tyrosine kinases are contacted. The phosphorylated substrate is detected, and the activity value of the receptor tyrosine kinases is measured based on the detection result. The proliferation inhibiting effect is assessed based on the resulting activity value.

Description

Technical field: [0001] The invention relates to a method for evaluating the inhibitory effect of receptor tyrosine kinase inhibitors on tumor cell proliferation according to the activity of receptor tyrosine kinase. The invention also relates to a method for judging the sensitivity of tumor cells to receptor tyrosine kinase inhibitors according to the activity of receptor tyrosine kinase. The invention also relates to a method of screening compounds for receptor tyrosine kinase activity. Background technique: [0002] Transmembrane tyrosine kinases (hereinafter referred to as receptor tyrosine kinases) present on cell membranes play an important role in cell proliferation, cell survival, cell differentiation and angiogenesis. For example, receptor tyrosine kinases are known to have many substances related to cell carcinogenesis, and their abnormal expression and enzyme activity can cause cell carcinogenesis. Specifically, it has been reported that insulin-like growth fact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48
CPCC12Q1/485G01N2500/00G01N2333/9121
Inventor 能登谷伦崇佐藤淳大山朋子吉田智一石原英幹
Owner SYSMEX CORP
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