CN112402417B - Medicine for inhibiting migration and invasion of liver cancer stem cells - Google Patents

Abstract

The invention provides a medicine for inhibiting migration and invasion of liver cancer stem cells. The invention proves the new application of the small molecular inhibitor YYA-021 in reducing the survival of liver cancer cells HepG2 and liver cancer stem cell transfer, so that the small molecular inhibitor YYA-021 can be used for preparing the medicine for treating liver cancer.

Description

Medicine for inhibiting migration and invasion of liver cancer stem cells

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a medicine for inhibiting migration and invasion of liver cancer stem cells.

Background

Hepatocellular carcinoma is a common malignancy of the digestive system, and the mortality rate of patients with hepatocellular carcinoma has long been in the front of patients with global tumor deaths. Hepatocellular carcinoma is a malignant tumor with a very complex pathological mechanism, and the occurrence and development of the hepatocellular carcinoma are a polygenic and multistage process and are related to various factors. At present, the most common treatment mode of stem cell cancer is surgical resection, and auxiliary means such as chemotherapy or radiotherapy and the like are adopted. However, the existing treatment mode is difficult to completely eradicate hepatocellular carcinoma, and the recurrence rate of patients after 5 years of operation reaches 70 percent.

Liver cancer metastasis is the leading cause of death in patients with liver cancer, while the leading cause of tumor metastasis is the presence of liver cancer stem cells in patients. Liver cancer stem cells are a small population of stem cell-like cells in tumor tissue, and have self-renewal and multipotential differentiation potential. Numerous studies have demonstrated that liver cancer stem cells are closely associated with resistance, recurrence and metastasis of hepatocellular carcinoma chemotherapy and radiotherapy. Therefore, the selection of effective drugs for inhibiting liver cancer stem cell metastasis can provide new possibility for treating liver cancer.

YYA-021 is a small molecule CD4 mimic, has the characteristics of high-efficiency HIV resistance and low cytotoxicity, and has no report on YYA-021 in liver cancer stem cells at present.

Disclosure of Invention

The invention aims to provide a new application of YYA-021 in inhibiting liver cancer stem cell metastasis.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides application of a small molecular inhibitor YYA-021 in preparing a medicament for treating liver cancer.

Preferably, the molecular formula of the small molecule inhibitor YYA-021 is C₁₈H₂₇N₃O₂。

Preferably, the application comprises the application of a small molecule inhibitor YYA-021 in promoting liver cancer cell death.

Preferably, the application comprises the application of a small molecule inhibitor YYA-021 in inhibiting the migration of liver cancer stem cells.

In addition, the invention provides a liver cancer medicament, which comprises an effective dose of YYA-021.

Preferably, YYA-021 is the only effective component or one of the effective components of the liver cancer medicament.

Preferably, the liver cancer drug promotes liver cancer cell death and the liver cancer drug inhibits liver cancer stem cell metastasis.

In addition, the invention provides application of a small molecule inhibitor YYA-021 in preparing a medicine for inhibiting liver cancer stem cell metastasis.

Preferably, the molecular formula of the small molecule inhibitor YYA-021 is C₁₈H₂₇N₃O₂。

In addition, the invention provides a medicament for inhibiting the metastasis of the liver cancer stem cells, which comprises an effective dose of YYA-021.

The invention has the beneficial effects that:

the invention proves the new application of a small molecular inhibitor YYA-021 in inhibiting the survival of liver cancer cells and the migration and invasion of liver cancer stem cells.

Drawings

FIG. 1 shows the inhibition effect of small molecule inhibitor YYA-021 on liver cancer cell HepG 2.

FIG. 2 expression differences of stem cell-associated proteins in HepG2 cells and liver cancer stem cells.

FIG. 3 regulation of EMT-associated proteins in liver cancer stem cells by YYA-021.

FIG. 4 regulation of YYA-021 on liver cancer stem cell migration and invasion.

Detailed Description

In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.

Example 1

YYA-021 regulation and control of liver cancer cell HepG2

(1) Inoculating the liver cancer cells into a cell culture plate, and carrying out experimental treatment after overnight culture;

(2) DMSO was added to the control group, and 10. mu.M YYA-021 and 20. mu.M YYA-021 were used for the treatment, and after 24 hours, photographing was performed for recording.

The experimental result of example 1 is shown in fig. 1, and it can be seen that the addition of 20 μ M YYA-021 significantly causes the death of liver cancer HepG2 cells, indicating that YYA-021 can inhibit liver cancer by promoting the death of liver cancer cells.

Example 2

Screening and detection of liver cancer stem cells

1. Liver cancer stem cell screening

(1) Cleaning HepG2 cells with good growth state by using PBS buffer solution, adding 0.25% trypsin to digest the cells into uniform single cell suspension, and centrifuging for 10min at 300 g;

(2) removing supernatant, dispersing the cells into single cell suspension again by using PBS, and counting the cells;

(3) get 10⁷100 mu l of the mixture is distributed in a sterile flow tube, a negative control tube and a plurality of groups of experimental sorting tubes are arranged, 20 mu 1FcR blocker is added into each tube to block nonspecific binding for 5 min;

(4) adding Mouse IgG1-PE antibody 10 mu 1 into a negative control tube, adding CD133/1-PE antibody 20 mu 1 into an experimental sorting tube, and incubating for 20min at 4 ℃ in a dark place;

(5) after the incubation is finished, adding 4ml of PBS buffer solution for washing, and centrifuging for 10min at 300 g;

(6) discarding the supernatant, sieving with 400 mesh cell sieve, resuspending 1ml of sorting solution into single cell suspension, sorting with flow cytometer, and receiving HepG2 CD133 in 15ml centrifuge tube containing stem cell culture solution⁺And CD133^-A cell.

2. Liver cancer stem cell detection

Detection of CD133 Using Western Blot⁺Expression of tumor stem cell associated proteins C-MYC and OCT4

(1) Taking HepG2 cells with good growth state and sorting CD133⁺Adding RIPA protein lysate into the cells, and transferring cell fragments into a 1.5ml EP tube;

(2) standing on ice for 30min, and disrupting the cells with an ultrasonic disruptor;

(3) after sufficient lysis, centrifugation was carried out at 12000rpm for 10min at 4 ℃ and the supernatant was aspirated into a fresh precooled EP tube;

(4) quantifying the protein concentration of the sample according to a BCA method, and adjusting each group of proteins to be detected by using 5 Xloading buffer solution to ensure that each group is 25 mu g/mu l finally;

(5) preparing 10% separating gel and 5% concentrating gel, rapidly inserting into comb, and coagulating for 10 min;

(6) pouring the freshly prepared electrophoresis solution into an electrophoresis tank, placing a sample loading plate, and sequentially adding the Mark protein, the HepG2 cell protein and the CD133⁺Switching on a cell protein, running the concentrated gel at a constant voltage of 90V, and adjusting the constant voltage to be 120V when the bromophenol blue enters the separation gel until the bromophenol blue runs to the bottom;

(7) cutting a PVDF membrane, soaking in methanol, placing in an electric transfer liquid, installing electric transfer clamps according to the sequence of black clamp-sponge-filter paper-glue-PVDF membrane-filter paper-sponge-white clamp, and carrying out constant-current 250mA ice-bath wet transfer for 90 min;

(8) after the electrotransformation is finished, putting the PVDF membrane into 5% skimmed milk powder, and sealing for 1h at room temperature;

(9) incubating CD133, OCT, C-MYC and GAPDH primary antibodies, and shaking-culturing overnight at 4 ℃;

(10) after the primary antibody incubation is finished, washing the membrane for 3 times by using TBST, wherein each time is 10 min;

(11) incubating the secondary antibody at room temperature for 90min, and washing the membrane for 3 times by using TBST after the incubation is finished, wherein each time is 10 min;

(12) adding luminous liquid, and developing and exposing.

The experimental results of example 2 are shown in FIG. 2, from which CD133 can be seen⁺The expression levels of CD133, OCT and C-MYC in the HepG2 cells are obviously higher than those of the ordinary HepG2 cells, which indicates that the HepG2 cells with CD133+ are successfully obtained.

Example 3

Detecting the inhibition effect of YYA-021 on liver cancer stem cells

(1) HepG2 cells of CD133+ were inoculated in a cell culture plate, DMSO was added to a control group, and 5. mu.M YYA-021 and 10. mu.M YYA-021 were used for the experimental group;

(2) after 48h of treatment, RIPA protein lysate was added and cell debris was transferred to a 1.5ml EP tube;

(3) standing on ice for 30min, and disrupting the cells with an ultrasonic disruptor;

(4) after sufficient lysis, centrifugation was carried out at 12000rpm for 10min at 4 ℃ and the supernatant was aspirated into a fresh precooled EP tube;

(5) quantifying the protein concentration of the sample according to a BCA method, and adjusting each group of proteins to be detected by using 5 Xloading buffer solution to ensure that each group is 25 mu g/mu l finally;

(6) preparing 10% separating gel and 5% concentrating gel, rapidly inserting into comb, and coagulating for 10 min;

(7) pouring the freshly prepared electrophoresis solution into an electrophoresis tank, placing a sample loading plate, sequentially adding Mark protein, HepG2 cell protein and CD133+ cell protein, switching on a power supply, running the concentrated gel at a constant voltage of 90V, and adjusting the constant voltage to be 120V when the bromophenol blue enters the separation gel until the bromophenol blue runs to the bottom;

(8) cutting a PVDF membrane, soaking in methanol, placing in an electric transfer liquid, installing electric transfer clamps according to the sequence of black clamp-sponge-filter paper-glue-PVDF membrane-filter paper-sponge-white clamp, and carrying out constant-current 250mA ice-bath wet transfer for 90 min;

(9) after the electrotransformation is finished, putting the PVDF membrane into 5% skimmed milk powder, and sealing for 1h at room temperature;

(10) incubating N-cadherin, E-cadherin, Vimentin and GAPDH primary antibody, and incubating overnight in a shaking table at 4 ℃;

(11) after the primary antibody incubation is finished, washing the membrane for 3 times by using TBST, wherein each time is 10 min;

(12) incubating the secondary antibody at room temperature for 90min, and washing the membrane for 3 times by using TBST after the incubation is finished, wherein each time is 10 min;

(13) adding luminous liquid, and developing and exposing.

The experimental results obtained in example 3 are shown in FIG. 3, from which it can be seen that the addition of YYA-021 can suppress the expression of the mobilization proteins N-cadherin and Vimentin, and can promote the expression of the mobilization protein E-cadherin.

Example 4

1. Cell migration

(1) Cultured CD133⁺The HepG2 cells were digested to prepare a single cell suspension, and washed 1 time with PBS;

(2) control group was resuspended to 1X 10 using DMEM medium supplemented with DMSO⁶Experiment group cells were resuspended to 1X 10 using 10. mu.M of DMEM medium for YYA-02⁶；

(3) Adding 600 mu l of DMEM medium containing 10% FBS into the lower chamber of the Transwell chamber, and adding 100ul of cell resuspension of the control group and the experimental group into the upper chamber respectively;

(4) placing the Transwell chamber in a constant-temperature cell culture box for culturing for 24 h;

(5) after the culture is finished, taking out the Transwell chamber, and scrubbing the upper chamber by using a cotton swab dipped with PBS;

(6) placing the small chamber into methanol, and fixing for 20 min;

(7) staining with 5% crystal violet for 20min, and washing with PBS;

(8) observation and photographing with an inverted microscope

2. Cell invasion

(1) Placing BD Matrigel gel frozen in a refrigerator at-80 ℃ overnight at 4 ℃, and unfreezing to liquid state;

(2) serum-free medium was used as follows 1: 8, diluting to 50 mu g/ml Matrigel, fully mixing uniformly, and adding 60 mu l of Matrigel glue into an upper chamber of a Transwell chamber;

(3) the cultured HepG2 cells of the CD133+ are digested to prepare single cell suspension, and the single cell suspension is washed 1 time by PBS;

(4) control group was resuspended to 1X 10 using DMEM medium supplemented with DMSO⁶Experiment group cells were resuspended to 1X 10 using 10. mu.M of DMEM medium for YYA-02⁶；

(5) Adding 600 mu l of DMEM medium containing 10% FBS into the lower chamber of the Transwell chamber, and adding 100ul of cell resuspension of the control group and the experimental group into the upper chamber respectively;

(6) placing the Transwell chamber in a constant-temperature cell culture box for culturing for 24 h;

(7) after the culture is finished, taking out the Transwell chamber, scrubbing the upper chamber by using a cotton swab dipped with PBS, and removing Matrigel glue;

(8) placing the small chamber into methanol, and fixing for 20 min;

(9) staining with 5% crystal violet for 20min, and washing with PBS;

(10) observation and photographing were performed using an inverted microscope.

The results obtained in example 4 are shown in fig. 4, and it can be seen from the figure that the addition of YYA-02 significantly suppresses the migration and invasion of CD133+ liver cancer stem cells.

The combination of examples 1-4 shows that the small molecule inhibitor YYA-02 can effectively kill liver cancer cells and effectively inhibit the migration and invasion of liver cancer stem cells.

The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.

Claims (3)

1. The application of the small molecular inhibitor YYA-021 in preparing the medicine for treating liver cancer is characterized in that the molecular formula of the small molecular inhibitor YYA-021 is C₁₈H₂₇N₃O₂。

2. The use of claim 1, wherein the use comprises use of a small molecule inhibitor YYA-021 in the preparation of a medicament for promoting hepatoma cell death.

3. The use according to claim 1, wherein the use comprises use of a small molecule inhibitor YYA-021 in preparation of a medicament for inhibiting migration of liver cancer stem cells.

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