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Cell autophagy-based antitumor drug screening method

An anti-tumor drug and screening method technology, applied in the field of anti-tumor drug composition and autophagy-based anti-tumor drug screening, can solve the problem of no drug combination scheme screening method, etc.

Inactive Publication Date: 2014-11-26
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Another study shows that the combination of anti-tumor drugs often shows a stronger anti-tumor effect than the original drug, and the combination of autophagy inhibitors chloroquine (CQ), hydroxychloroquine (HCQ) and some cytotoxic drugs It has entered clinical trials, and the combination with SAHA has also shown good results in in vitro experiments, but there is no screening method for drug combination regimens

Method used

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  • Cell autophagy-based antitumor drug screening method
  • Cell autophagy-based antitumor drug screening method
  • Cell autophagy-based antitumor drug screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. HDAC inhibitor LBH589 combined with chloroquine to measure antitumor activity

[0022] The breast cancer cell MCF-7 cultured overnight with 10% FBS medium was digested and diluted to a cell suspension containing 20,000 cells / ml, and 200 μl of cell suspension was added to each well, and stored at 37°C and 5% CO 2 After 24 hours of culture under the condition, the medium was sucked off, and the medium containing different concentrations of LBH589 and / or 20 μM chloroquine was added, and the cells containing 5 μM camptothecin and 20 μM chloroquine were used as positive and negative controls, respectively. After 24 hours of co-cultivation, centrifuge at 200g for 10 minutes, and use Roche’s Cell Death Detection ELISA plus The extent of cell death was determined. The results showed that with the increase of the concentration of LBH589, the death of MCF-7 cells was more; 100 nM LBH589 and 20 μM chloroquine combined could improve the ability of LBH589 to kill MCF-7 c...

Embodiment 2

[0024] Example 2. HDAC inhibitor LBH589 enhances the sensitivity of breast cancer cells to chloroquine

[0025] Breast cancer cells MDA-MB-231 cultured overnight in 10% FBS medium were digested and diluted to a cell suspension containing 20,000 cells / ml. Add 200 μl of cell suspension to each well and store at 37°C and 5% CO 2 After culturing for 24 hours under the same conditions, the medium was sucked off, and medium containing 25nM LBH589 and different concentrations of chloroquine was added, and the degree of cell death was detected by the same method as in Example 1 after 24 hours. The results showed that with the increase of chloroquine concentration, the sensitivity of MDA-MB-231 cells to LBH589 was enhanced, and the cell death was increased, while chloroquine in the absence of LBH589 had no effect on the death of MDA-MB-231 cells even if the concentration was as high as 100 μM. Significant effects (such as figure 2 shown).

[0026]

Embodiment 3

[0027] Example 3. Screening of small molecule compounds used in combination with HDAC inhibitor LBH589

[0028] Breast cancer cells MDA-MB-231 cultured overnight in 10% FBS medium were digested and diluted to a cell suspension containing 20,000 cells / ml. Add 200 μl of cell suspension to each well and store at 37°C and 5% CO 2After culturing for 24 hours under the same conditions, suck off the medium, add the medium containing 25nM LBH589 and different concentrations of chloroquine or the medium containing 100μM CQ as a reference, and measure the culture conditions containing 25nM LBH589 and 10μM of each compound, using the same implementation The method of Example 1 detects the degree of cell death of breast cancer cells after 24 hours of culture. Table 1-1 and Table 1-2 show the average of the three experimental results of 62 monomeric compounds combined with 25nM LBH 589, wherein the higher the average OD value, the better the combined effect.

[0029]

[0030] Table 1-1. ...

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Abstract

The invention belongs to the field of biological pharmacy, and relates to a screening method for screening candidate combination molecules through cell death caused by combination of a tumor cell autophagy inducer and a micro-molecular compound, screened compound molecules may not have antitumor activity, and have the antitumor activity after the screened compound molecules are combined with the cell autophagy inducer, such as an HDAC inhibitor, so a drug combination scheme can be determined. In the invention, cancer cells cultured in vitro are respectively processed by the tumor cell autophagy inductor and single compounds as an object, the degree of the cultured cell death caused by the compounds and the cell autophagy inducer is determined, and cell autophagy-based antitumor drugs are screened. A result of cell experiments proves that the method can be used to screen the combination drugs in order to prepare antitumor medicine compositions of the HDAC inhibitor and the micro-molecular compound, and the medicinal compositions are suitable for all dosage forms.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a method for screening antitumor drugs based on cell autophagy; in particular, it relates to a small molecular compound that increases the sensitivity of cancer cells to HDAC inhibitors and a screening method thereof, and the anti-cancer effect of HDAC inhibitors and small molecular compounds. Tumor drug composition. Background technique [0002] With the deepening of lysosome research, Christian de Duve, who focused on the research of vesicles containing cytoplasm and organelles, opened up a new field of autophagy. These lysosome-related vesicles, namely autophagosomes, are in Widely present in organisms, Christian de Duve also won the 1974 Nobel Prize in Physiology and Medicine for his research on lysosomes. In the past decade, studies have found that there are three forms of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). Studies have shown t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02A61K31/4706A61K31/4747A61P35/00A61K31/4045A61K31/167
Inventor 杨永华姜晓筱包勇刘慧娟寇鑫辉
Owner FUDAN UNIV
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