Use of protein kinase Czeta in regulating tumor cell chemotactic movement

A protein kinase and tumor cell technology, applied in the field of protein kinase Czeta to regulate tumor cell chemotaxis, can solve the problems of unseen design of specific anticancer drugs and unknown functions

Inactive Publication Date: 2005-08-03
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the function of PKC subtypes in EGFR-induced tumor cell chemotaxis is still unk

Method used

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  • Use of protein kinase Czeta in regulating tumor cell chemotactic movement
  • Use of protein kinase Czeta in regulating tumor cell chemotactic movement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 EGF can induce chemotactic movement of MDA-231 cells

[0032] 1. Experimental method

[0033] Using a chemotaxis chamber to detect the chemotaxis movement of cells is a very mature method, which can be specifically operated using conventional methods in the art. Briefly, different concentrations of the chemokines EGF and SDF-1[alpha] were added to the wells of the bottom plate of the chemotaxis chamber. Separate the upper and lower chambers with a filter membrane, and add MDA-231 cells to the upper chamber. After incubation, the non-specific chemotaxis cells on the upper layer of the membrane were removed, and the membrane was fixed, stained with Diff-Quick, and counted under a microscope (400x field of view). The average number of 3 fields of view in each well was taken as the number of chemoattractant cells in the well. The ratio of the number of chemoattractant cells in the sample well to the number of cells in the control well is the chemotaxis index. ...

Embodiment 2

[0036] Example 2 Inhibition of PKCζ and PI3K Specific Inhibitors on MDA-231 Chemotaxis Movement

[0037] 1. Experimental method: pre-treat the cells with the specific PI3K inhibitor Ly294002 (30μM) and different PKC subtype-specific inhibitors, 100nM G6850, 50nM G6976, 10μM Chelerythrine Chloride at 37°C for 45-60min, then Add it directly to the upper plate hole of the chemotaxis chamber to detect the chemotaxis movement ability of the cells to EGF.

[0038] 2. Experimental results: see figure 1 , PI3K-specific inhibitor Ly294002 can effectively inhibit EGF-induced chemotaxis of MDA-231; similarly, PKCζ-specific inhibitor Chelerythrine Chloride can also completely inhibit the chemotaxis of MDA-231 cells. However, G6850 and G6976, which inhibit cPKC and nPKC, had no significant inhibitory effect on EGF-induced MDA-231 motility.

[0039] 3. Conclusion: Ly294002 and Chelerythrine Chloride can effectively inhibit the chemotaxis of EGF-induced breast cancer cell MDA-231, sugg...

Embodiment 3

[0040] Example 3 Immunofluorescence method to determine whether EGF can specifically induce the activation of PKCζ molecule in MDA-231 cells

[0041] 1. Experimental method

[0042] MDA-231 cells were planted 48 hours before the experiment, and cultured for 3 hours with serum-free FPMI 1640 containing 0.1% BSA. The cells were continued to be treated with serum-free medium (FPMI 1640, 0.1% BSA) containing inhibitor (30 μM Ly294002) or without inhibitor for 60 min. Cells were fixed with 4% PFA after stimulation with medium (FPMI 1640, 0.1% BSA) or EGF (diluted to 50 ng / ml in FPMI 1640, 0.1% BSA) for a specified period of time. After washing twice with PBS, the membrane was permeated with 0.2% Tween 20 at 4°C for 30 min. After washing again, incubate with rabbit anti-human PKC specific polyclonal antibody (diluted 1:100 in PBS) at room temperature for 1 hour, wash thoroughly with PBS for 3 times, and then add FITC-(fluorescein isothiocyanate)-labeled goat anti-rabbit The secon...

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Abstract

The present invention relates to the use of protein kinase C-zeta (PKC-zeta) in regulating tumor cell chemotactic motion. It is found that PKC-zeta is the component essential for the chemotactic motion of tumor cell, so that suppressing the activity of PKC-zeta can suppress EGFR effectively and the chemotactic factor induced chemotactic motion of tumor cell. Therefore, PKC-zeta is the key targeting in designing efficient and specific medicine for resisting the growth and metastasis of cancer cell.

Description

Technical field: [0001] The invention relates to the application of protein kinase Czeta to regulate tumor cell chemotaxis. Background technique: [0002] Chemokines and their receptors play a very important role in the survival, growth and metastasis of tumors at different stages. They not only regulate the growth and metastasis of tumor cells, but also chemoattract different types of white blood cells, forming a tumor cell that is conducive to the survival, growth and differentiation of tumor cells. microenvironment. As Muller and colleagues found, in mouse models, CXCR4 and its ligand SDF-1α control the distribution pattern of breast cancer metastasis, see literature (Muller A, Homey B, Soto H, et al.Involvement of chemokinereceptors in breast cancer metastasis. Nature 2001; 410: 50-56.) Therefore, blocking chemotactic movement can effectively control the metastasis of cancer cells and shrink or eliminate tumors. [0003] It has been found that the chemotactic movement ...

Claims

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Application Information

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IPC IPC(8): A61K38/48A61P35/00
Inventor 张宁孙荣华朱斯特J·奥本海姆马大龙
Owner PEKING UNIV
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