Application of non-metabolic function based on CHKalpha as target for cancer treatment, diagnosis and prognostic prediction

A technology for cancer treatment and metabolic function, applied in the field of oncology medicine, can solve problems such as inaccurate specific mechanisms

Pending Publication Date: 2021-09-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the exact mechanism by which choline kinase α (CHKα) enhances tumor progression remains unclear.

Method used

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  • Application of non-metabolic function based on CHKalpha as target for cancer treatment, diagnosis and prognostic prediction
  • Application of non-metabolic function based on CHKalpha as target for cancer treatment, diagnosis and prognostic prediction
  • Application of non-metabolic function based on CHKalpha as target for cancer treatment, diagnosis and prognostic prediction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1. CHKα is a direct sensor of oxidative stress

[0094] 1. The formation of intramolecular disulfide bonds in CHKα is a direct event for cells to respond to oxidative stress

[0095] with 50 μM H 2 o 2 Treatment of LN229 cells. Oxidized proteins were labeled with biotin-maleimide and purified with streptavidin-agarose beads. Such as figure 1 Shown, H 2 o 2 Intramolecular disulfide bonds were formed in CHKα at 2 minutes of treatment.

[0096] 2. The intramolecular disulfide bond of CHKα is formed between C303 and C307

[0097] (1) if figure 2 As shown, the human CHKα structure (PDB: 2CKO) shows the spatial location of C303 and C307. The surrounding areas of C303 and C307 are framed and magnified. An intramolecular disulfide bond is formed between the sulfur atoms of C303 and C307.

[0098] (2) Express HA-tagged wild-type CHKα, CHKαC303A, or CHKαC307A in LN229 cells. Cells were treated with the reducing reagent N-acetylcysteine ​​(NAC) for 30 min, and...

Embodiment 2

[0099] Example 2. Oxidative stress causes CHKα to bind and phosphorylate CBS

[0100] 1.H 2 o 2 CHKα binds to CBS after treatment

[0101] LN229 cells expressing Flag-tagged CHKα and His-tagged CBS were treated with NAC for 10 minutes, and then treated with 50 μM H 2 o 2 Process for 10 minutes. Ni-NTA down experimental results show that H 2 o 2 Treatment resulted in binding of CHKα to CBS, which was blocked by NAC treatment. ( Figure 4 )

[0102] 2. Oxidative stress causes CHKα to phosphorylate the Y484 site of CBS

[0103] Purified wild-type Flag-CHKα was immobilized on magnetic beads, and added H 2 o 2 Treat for 10 minutes, rinse with PBS, and then incubate with purified wild-type His-CBS or His-CBS Y484F in the presence of 32P-ATP for in vitro kinase assay. The result is as Figure 5 , H 2 o 2 Treatment phosphorylates CBS, and the Y484F mutation of CBS blocks this phosphorylation.

Embodiment 3

[0104] Example 3, CHKα-mediated phosphorylation of CBS activates CBS

[0105] Purified wild-type Flag-CHKα or its mutant protein was immobilized on magnetic beads, and 50 μM H 2 o 2 Treat for 10 minutes, 10mM DTT for 30 minutes, rinse with PBS, and then incubate with purified wild-type His-CBS or His-CBSY484F in the presence of ATP for in vitro kinase assay (left) and CBS activity assay (right). The result is as Image 6 , Y484 phosphorylation of CBS enhances CBS activity.

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Abstract

The invention belongs to the technical field of tumor medicine, and relates to application of a non-metabolic function based on CHKalpha as a target for cancer treatment, diagnosis and prognosis prediction. According to the application disclosed by the invention, the tumor generation and development mechanism of choline kinase alpha (CHK alpha) in human cancers is analyzed, and a new action target is provided for cancer treatment, diagnosis and prognosis prediction by utilizing the effects of the non-metabolic function of CHKalpha in phosphatidylcholine synthesis regulation, glycometabolism (glycolysis), glutathione synthesis, lipid droplet lipolysis, beta-oxidation and tumor growth.

Description

technical field [0001] The invention belongs to the technical field of tumor medicine, and relates to the application of the non-metabolic function of CHKα as a target for cancer treatment, diagnosis and prognosis prediction. Background technique [0002] Choline kinase catalyzes the phosphorylation of free choline to phosphorylcholine, which is further converted to cytidine diphosphocholine by phosphorylcholine cytosyltransferase and to phosphatidylcholine by choline phosphotransferase. Choline kinase α promotes the proliferation and survival of tumor cells, is highly expressed in 40%–60% of human tumors, is positively correlated with the poor prognosis of multiple tumors, and plays a role in the occurrence and development of tumors. However, the specific mechanism by which choline kinase α (CHKα) enhances tumor progression remains unclear. [0003] Cellular metabolic reprogramming is a hallmark of tumor cells. The non-metabolic functions of many metabolic enzymes in tumo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/573A61K45/00A61P35/00
CPCG01N33/574G01N33/57488G01N33/573A61K45/00A61P35/00G01N2333/91215G01N2800/52
Inventor 刘锐马清霞邵飞其他发明人请求不公开姓名
Owner ZHEJIANG UNIV
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