Application method of small molecule medicine for inhibiting proliferation and migration of colorectal cancer tumors

Abstract

The invention provides an application method of a small-molecule drug for inhibiting proliferation and migration of colorectal cancer tumors, and the proliferation and migration capabilities of colorectal cancer cells can be obviously inhibited compared with those of a control group under the condition that the small-molecule drug is added. The component of the culture medium is a DMEM culture medium containing 10% of fetal calf serum, and 2 [mu] M of A922500 is added to an experimental group according to a ratio of 1: 1000. The invention discloses a new target for occurrence and development of the colorectal cancer, and provides a new idea for treatment of the colorectal cancer.

Description

Technical Field

[0001] The present invention relates to a novel small molecule drug for inhibiting the occurrence and development of colorectal cancer tumors

[0002] Colorectal cancer (CRC) is the third most common solid tumor and the second most common cause of cancer death worldwide. Excessive growth of cancer cells and distant metastasis are the two main causes of colorectal cancer-related death. Excessive growth of colorectal cancer cells leads to colorectal obstruction and massive bleeding, requiring emergency surgery and may lead to severe complications and high mortality. Colorectal cancer is most likely to metastasize to the liver and lungs, and distant metastasis is the most common cause of colorectal cancer-related death. Despite significant progress in comprehensive treatment, the 5-year overall survival rate of colorectal cancer patients with distant metastasis is still as low as 15%. Therefore, in-depth research on the molecular mechanisms of colorectal cancer growth and distant metastasis is crucial for the diagnosis and treatment of colorectal cancer. Previous studies have reported that lipid accumulation has a profound effect on cancer progression and metastasis. Lipid droplets (LDs) are intracellular forms of lipid deposition, a new marker of cancer cells, and are highly associated with many cellular processes. In the preliminary experiments, we screened various small molecules and performed CCK-8 and Transwell experiments. Finally, it was found that when the activity of diacylglycerol acyltransferase 1 (DGAT1) was inhibited and A922500 was added to inhibit lipid droplet formation, the proliferation and migration ability of colorectal cancer cells were significantly inhibited. As has been reported in other cancer cells, it can play a key role in cancer cell proliferation, metastasis and drug resistance under the condition of reduced lipid droplet formation. Therefore, inhibiting the formation of lipid droplets can open up new ways to study the molecular mechanism of the occurrence and development of colorectal cancer, and at the same time provide new ideas for the treatment of colon cancer.

[0003] A922500 (DGAT-1 Inhibitor 4a) is a potent, selective and orally available diacylglycerol acyltransferase 1 (DGAT-1) inhibitor.

[0004] When the small molecule A922500 was added to the culture system of colorectal cancer cells at a ratio of 1:1000, we found that the proliferation and migration ability of colorectal cancer cells were significantly inhibited. As has been reported in other cancer cells, it can play a key role in cancer cell proliferation, metastasis and drug resistance under the condition of reduced lipid droplet formation. Therefore, inhibiting the formation of lipid droplets can open up new ways to study the molecular mechanism of the occurrence and development of colorectal cancer, and at the same time provide new ideas for the treatment of colon cancer. Summary of the invention

[0005] This drug aims to provide a new method for treating colon cancer and a new drug that can inhibit the occurrence and development of colorectal cancer.

[0006] The technical solution adopted by this drug to solve the above technical problems is to add 2μM A922500 at a ratio of 1:1000 to the culture system of colorectal cancer cell lines HCT116 and SW480. We found that the proliferation and migration ability of colorectal cancer cells were significantly inhibited, and it is expected to become a new anti-colorectal cancer tumor drug.

[0007] Cell proliferation ability verification: (1) P5 human colorectal cancer cell lines HCT116 and SW480 with a growth density of 70-80% were taken, the culture medium was discarded, and the cells were washed once with PBS buffer to remove the residual culture medium; (2) Add 0.3 ml of 0.05% trypsin and digest the cells for about 4 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (3) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (4) Add the diluted cell suspension to a 96-well plate and set up two groups, each with 3-5 replicate wells, and add 200 μL of culture medium containing cells to each well; (5) Add 1.0×10 5 Cells were shaken horizontally in a cross shape to make the cells evenly distributed, and placed in an incubator for 24 hours until the cells were completely attached to the wall; (6) Place the culture plate in an incubator. After 4 hours, when the cells are attached to the wall, add 2 μM A922500 to the second group. Take out the cells from the first plate, which is 0 hours, and dilute the CCK-8 reagent 10 times with culture medium containing inactivated serum; (7) After adding CCK8 reagent to the wells, incubate in an incubator for 1 hour and then measure the absorbance at 450 nm using a microplate reader. At 12, 24, 36, 48, 60, and 72 hours, measure the absorbance changes of the cells using a microplate reader; (8) Measure the absorbance changes of cells with an ELISA reader at 12, 24, 36, 48, 60, and 72 hours, and process and analyze the data;

[0008] Verification of cell migration ability: (1) Take P5 human colorectal cancer cell lines HCT116 and SW480 with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (2) Add 0.3 ml of 0.05% trypsin and digest the cells for about 4 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (3) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (4) Add culture medium (600 μL) containing 10% inactivated serum to the wells, then gently place the chamber into the lower chamber, and add 8.0×10 4 cells, a total volume of 200 μL DMEM medium, and 2 μM A922500 (5) After 12 to 24 hours, take out the cells and wipe the chamber with one end of a cotton swab. After wiping, put it in PBS and wipe it again with the other end of the cotton swab. Repeat 3 times. (Note: Do not touch or wipe the bottom of the chamber during the whole process to prevent the cells from being wiped off.) (6) Take 600 μL of anhydrous methanol in a 24-well plate, place the wiped chamber in it and fix it for 15 min. Then wash the chamber with PBS and gently wipe the upper side of the chamber with a cotton swab. Repeat this three times. (7) Prepare 0.5% crystal violet staining solution with anhydrous methanol, take 600 μL into a 24-well plate, place the chamber in it, and stain for about 15 minutes. Be careful to eliminate bubbles during this process. (8) Prepare 0.5% crystal violet staining solution with anhydrous methanol, take 600 μL into a 24-well plate, place the chamber in it, and stain for about 15 minutes. Be careful to eliminate bubbles during this process. (9) Observe the cell migration phenomenon on the membrane under a microscope and take photos.

[0009] This drug has the following advantages: (1) After adding the small molecule inhibitor A922500, the proliferation and migration abilities of colorectal cancer cell lines HCTl16 and SW480 were significantly inhibited, with obvious effects. (2) Compared with traditional surgical treatment, drug treatment avoids surgical trauma and greatly reduces the patient's pain. (3) The small molecule inhibitor A922500 inhibits the activity of diacylglycerol acyltransferase 1 and the formation of lipid droplets, thereby inhibiting the occurrence and development of tumors, with higher targeting and clearer specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1The figure shows the effect of adding 2μM A922500 for 24 hours on the growth of P5 colorectal cancer cell lines HCT116 and SW480. The culture conditions are ① DMEM containing 10% fetal bovine serum, ② containing 10% fetal bovine serum + 2μM A922500, ③ containing 10% fetal bovine serum DMEM, ④ 10% fetal bovine serum + 2μM A922500; it can be found that when the culture condition is 10% fetal bovine serum + 2μM A922500, the cells all die.

[0011] Figure 2 The figure shows the CCK-8 proliferation test of cells under the culture condition of 10% fetal bovine serum + 2μM A922500, and the absorbance at 450nm was detected by a microplate reader. Compared with the normal control condition, the addition of A922500 significantly inhibited the proliferation ability of the two cell lines.

[0012] Figure 3 The figure shows the results of Transwell experiment on cells under the culture conditions of 10% fetal bovine serum + 2 μM A922500. The results show that compared with the normal control group, the addition of A922500 can significantly inhibit the migration ability of colorectal cancer cell lines HCT116 and SW480. DETAILED DESCRIPTION

[0013] Example The colorectal cancer cell lines (HCT116 and SW480 cells) used in the experiments were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

[0014] Cell state detection (1) Morphological observation: A Leica DMIL inverted microscope was used to observe cells under normal culture conditions and under culture conditions with the addition of A922500. The results showed that more cells died when the small molecule A922500 was added to the culture conditions.

[0015] Cell proliferation activity assay (1) P25 human colorectal cancer cells HCT116 and SW480 with a growth density of 70-80% were taken, the culture medium was discarded, and the cells were washed once with PBS buffer to remove the residual culture medium; (2) Add 0.3 ml of 0.05% trypsin and digest the cells for about 2 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (3) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (4) Add the diluted cell suspension to a 96-well plate. Set up two groups, each with 3-5 replicate wells, and add 200 μL to each well. (5) Add 8.0×10 3 cells, shake them horizontally in a cross shape to make the cells evenly distributed, and place them in an incubator for 24 hours until the cells are completely attached to the wall; (6) Place the culture plate in the incubator. After 4 hours, when the cells are attached to the wall, add 2 μM A922500 to the experimental group. Take out the first plate of cells, which is 0 hours, and dilute the CCK-8 reagent 10 times with culture medium containing inactivated serum; (7) After adding CCK-8 reagent to the wells, incubate in an incubator for 1 hour and then measure the absorbance at 450 nm using a microplate reader. At 12, 24, 36, 48, 60, and 72 hours, measure the absorbance changes of the cells using a microplate reader;

[0016] Transwell experiment (1) P25 human colorectal cancer cells HCT116 and SW480 with a growth density of 70-80% were taken, the culture medium was discarded, and the cells were washed once with PBS buffer to remove the residual culture medium; (2) Add 0.3 ml of 0.05% trypsin and digest the cells for about 2 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (3) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (4) Add culture medium (600 μL) containing 15% or 10% inactivated serum to the wells, then gently place the chamber into the lower chamber. Add 8.0×10 4 cells, with a total volume of 200 μL of DMEM medium, and 2 μM A922500 was added to the two cell experimental groups; (5) After 12 to 24 hours, take out the cells and wipe the chamber with one end of a cotton swab. After wiping, put it in PBS and wipe it again with the other end of the cotton swab. Repeat 3 times. (Note: Do not touch or wipe the bottom of the chamber during the whole process to prevent wiping off the cells). (6) Take 600 μL of anhydrous methanol in a 24-well plate, place the wiped chamber in it and fix it for 15 min, then wash the chamber with PBS, gently wipe the upper side of the chamber with a cotton swab, and repeat 3 times; (7) Prepare 0.5% crystal violet staining solution with anhydrous methanol, take 600 μL into a 24-well plate, place the chamber in it, and stain for about 15 min. Be careful to eliminate bubbles during this process; (8) Prepare 0.5% crystal violet staining solution with anhydrous methanol, take 600 μL into a 24-well plate, place the chamber in it, and stain for about 15 min. Be careful to eliminate bubbles during this process; (9) Observe the cell migration phenomenon on the membrane under a microscope and take photos.

Claims

1. A method for using a small molecule drug to inhibit colorectal cancer tumor proliferation and migration, characterized in that: 2 μM A922500 small molecule was added to the culture medium containing 10% fetal bovine serum to inhibit the proliferation and migration ability of colorectal cancer cells.

2. The method according to claim 1, characterized in that The colorectal cancer cell lines HCT116 and SW480 were subjected to CCK-8 experiments in the presence of the A922500 small molecule: (1) P5 human colorectal cancer cell lines HCT116 and SW480 with a growth density of 70-80% were taken, the culture medium was discarded, and the cells were washed once with PBS buffer to remove the residual culture medium; (2) Add 0.3 ml of 0.05% trypsin and digest the cells for about 4 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (3) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (4) Add the diluted cell suspension to a 96-well plate and set up two groups, each with 3-5 replicate wells, and add 200 μL of culture medium containing cells to each well; (5) Add 8.0×10 3 cells, shake them horizontally in a cross shape to make the cells evenly distributed, and place them in an incubator for 24 hours until the cells are completely attached to the wall; (6) Place the culture plate in the incubator. After 4 hours, when the cells are attached to the wall, add 2 μM A922500 to the experimental group. Take out the first plate of cells, which is 0 hours, and dilute the CCK-8 reagent 10 times with culture medium containing inactivated serum; (7) After adding CCK8 reagent to the wells, incubate in an incubator for 1 hour and measure the absorbance at 450 nm using a microplate reader. At 12, 24, 36, 48, 60, and 72 hours, measure the absorbance changes of the cells using a microplate reader.

3. The method according to claim 1, characterized in that The colorectal cancer cell lines HCT116 and SW480 were subjected to Transwell assay in the presence of small molecules A922500: (1) Take P5 human colorectal cancer cell lines HCT116 and SW480 with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (2) Add 0.3 ml of 0.05% trypsin and digest the cells for about 4 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (3) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (4) Add culture medium (600 μL) containing 10% inactivated serum to the wells, then gently place the chamber into the lower chamber, and add 8.0×10 4 cells, a total volume of 200 μL of DMEM medium, and 2 μM A922500 was added; (5) After 12 to 24 hours, take out the cells and wipe the chamber with one end of a cotton swab. After wiping, put it in PBS and wipe it again with the other end of the cotton swab. Repeat 3 times. (Note: Do not touch or wipe the bottom of the chamber during the whole process to prevent wiping off the cells). (6) Take 600 μL of anhydrous methanol in a 24-well plate, place the wiped chamber in it and fix it for 15 min, then wash the chamber with PBS, gently wipe the upper side of the chamber with a cotton swab, and repeat 3 times; (7) Prepare 0.5% crystal violet staining solution with anhydrous methanol, take 600 μL into a 24-well plate, place the chamber in it, and stain for about 15 min. Be careful to eliminate bubbles during this process; (8) Prepare 0.5% crystal violet staining solution with anhydrous methanol, take 600 μL into a 24-well plate, place the chamber in it, and stain for about 15 minutes. Note that bubbles must be eliminated during this process: (9) Observe the cell migration phenomenon on the membrane under a microscope and take photos.

4. The method according to claim 2 or 3, characterized in that: After adding 2μM A922500, the proliferation and migration ability of colorectal cancer cells were significantly inhibited, and it is expected to become a new anti-cancer drug.

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