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Screening method for anti-tumor drugs targeting Smurf1 ubiquitination and degradation of Rhob

An anti-tumor drug and ubiquitination technology, applied in compound screening, biochemical equipment and methods, biological testing, etc., can solve problems such as weakened motor ability, and achieve clear functions, reliable results, and short experimental cycles

Active Publication Date: 2016-01-27
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although RhoB-knockout mice have no obvious physiological defects, the motility of RhoB- / - mouse embryonic fibroblasts (MEFs) on fibronectin is significantly reduced

Method used

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  • Screening method for anti-tumor drugs targeting Smurf1 ubiquitination and degradation of Rhob
  • Screening method for anti-tumor drugs targeting Smurf1 ubiquitination and degradation of Rhob
  • Screening method for anti-tumor drugs targeting Smurf1 ubiquitination and degradation of Rhob

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Western blotting was used to detect the effect of Smurf1 on the stability of RhoB protein.

[0056] The test method is as follows:

[0057] 1) Cell transfection:

[0058] The cells were seeded in a 12-well culture plate, transfected 24 hours later, and the cells were replaced with fresh culture medium before transfection. The transfection steps are: take a 1.5mL centrifuge tube, add 180μL of water, the corresponding plasmid, 2.5MCaCl 2 20 μL, HBS 200 μL, mix gently, let stand at room temperature for 15-30 minutes, then slowly add dropwise to the culture medium, shake the culture plate gently to make the distribution even.

[0059] After 12 hours of transfection, the cell culture medium was replaced with fresh DMEM (Dulbecco's Modified Eagle's Medium), and after continuing to culture for 12 hours, the cells were routinely collected for subsequent experiments.

[0060] Solution recipe:

[0061] HBS: 280mMNaCl, 10mMKCl, 1.5mMNa 2 HPO 4 , 12mM Glucose, 50mM HEPES, pH7...

Embodiment 2

[0077] The combination of Smurf1 and RhoB was detected by GST-Pulldown.

[0078] The test method is as follows:

[0079] 1) Protein expression and purification in vitro

[0080] First, clone the target DNA fragment into a suitable bacterial expression vector, transform E.coliBL-21 or DH-5α cells with the correct plasmid, select transformants on LB / ampicillin plate, and inoculate into appropriate amount of LB / amp medium , 37°C shaking culture overnight. The next day, use this as a seed, inoculate it into a large volume of LB / amp medium at a ratio of 1:100, and grow it to OD with shaking at 37°C 600 It is about 0.6, add IPTG to a final concentration of 1mmol / L to induce the expression of the fusion protein, induce at a low temperature of 20°C to reduce the formation of inclusion bodies, and continue to culture for more than 6h. For different carriers or different proteins, the optimal bacterial concentration before induction, the IPTG concentration required for induction, the...

Embodiment 3

[0087] Intracellular ubiquitination assay was used to detect whether Smurf1 affects the ubiquitination level of RhoB.

[0088] The test method is as follows:

[0089] With 1.5~2×10 5 Cells were seeded at a density of 1 / mL in a 10 cm cell culture dish, and when the cell density reached 40% to 50%, the corresponding combination of plasmids was transfected by the calcium phosphate method. 24 hours after transfection, the medium was changed and drugs were added at the same time. Cells were treated with 10 μM MG-132. The purpose of adding drugs was to inhibit the degradation of substrate proteins that had been ubiquitinated to varying degrees through the lysosomal pathway. Cells were collected 36 hours after transfection and treated with PMSF-containing Lyse with TNTE (0.5%) buffer of NEM. From the obtained 1mL cell lysate, take 45μL as totallysate and add 15μL 4×loadingbuffer for Western blot analysis, and the remaining 955μL is used for the first IP. Add 1 μL Flag primary anti...

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Abstract

The invention provides an antitumor drug screening method using RhoB inhibiting ubiquitination degradation of Smurf1 as a therapeutic target, and relates to the screening of anti-cancer drugs. The screening method comprises the following steps: a candidate compound is in contact with a material substance; the influence of the candidate compound on the detection result of RhoB for inhibiting the E3 ubiquitin ligase Smurf1 ubiquitination degradation substrate is observed, and if the candidate compound enables the detection result in cells to generate negative changes, namely, the ability that the ubiquitination degradation of the Smurf1 for the RhoB is weakened, which indicates that the candidate compound is a potential antitumor drug. After the candidate compound is indicated to be the potential antitumor drug, the compound which can increase the RhoB protein content in cancer cells can be further selected. The specific method comprises the following steps: the candidate compound is in contact with different kinds of tumor cells; the influence of the candidate compound on the RhoB protein content in different kinds of tumor cells is observed. The experiment cycle is short, the detection process is fast, the sensitivity is high, and the application range is wide.

Description

technical field [0001] The invention relates to anticancer drug screening, in particular to an antitumor drug screening method targeting at inhibiting Smurf1 ubiquitination and degrading RhoB. Background technique [0002] In 2010, cancer surpassed cardiovascular and cerebrovascular diseases to become the number one disease that endangers human health. In 2013, the World Health Organization announced that the number of cancer patients in the world has increased significantly every year. So far, more than 14 million new cancer patients have been detected every year. Compared with the statistical result of 12.7 million in 2008, this is a significant increase. During the same period, the number of deaths of cancer patients also increased, from 7.6 million in the past to 8.2 million. An important mark that distinguishes cancer cells from normal cells is the escape from programmed cell death, that is, apoptosis. Apoptosis is an active cell death process triggered by changes in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/539C12Q1/02
CPCG01N33/573G01N33/6863G01N2500/00
Inventor 王洪睿郭磊王梅林曾涛玲林琦
Owner XIAMEN UNIV
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