Application of FASD1 inhibitor in inhibition of proliferation and migration of colorectal cancer

Abstract

The invention provides a small molecule drug for effectively inhibiting proliferation and migration of colorectal cancer tumors, and the proliferation and migration capacities of colorectal cancer cells (HCT116 and SW620) cultured under the condition that the small molecule drug is added are remarkably inhibited. The component of the culture medium is a DMEM culture medium containing 10% of fetal calf serum, and an inhibitor D5D-IN-326 of 2 [mu] M of FADS1 is added according to the proportion 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] This invention relates to the application of a FASD1 inhibitor in inhibiting the occurrence and development of colorectal cancer.

[0002] Colorectal cancer (CRC) is the third most common malignant tumor worldwide, consistently ranking among the top malignant tumors in terms of incidence and mortality. In recent years, the number of new cases globally has exceeded 2.2 million annually, exhibiting significant geographical and population heterogeneity. CRC patients are characterized by both an increasing proportion of right-sided colon cancer and a high rate of late-stage diagnosis. Distant metastasis and excessive growth of CRC cells are also significant causes of death. This presents unique challenges for disease prevention, control, and treatment. Although significant progress has been made in the treatment of colorectal cancer, prevention and control still face a series of challenges, including insufficient screening coverage, a rising proportion of young patients, and difficulties in significantly improving the survival rate of cancer patients. Therefore, further research into the molecular mechanisms of colorectal cancer cell growth and distant metastasis is crucial for the diagnosis and treatment of colorectal cancer. Previous studies have reported a vital link between lipid metabolism and cancer development, progression, and metastasis, and it is highly correlated with many cellular processes. In our preliminary experiments, we screened various small molecules and validated their effects using CCK-8 and Transwell assays. We ultimately found that the addition of D5D-IN-326 to inhibit the activity of fatty acid desaturase 1 (FADS1) significantly suppressed the proliferation and migration of colorectal cancer cells. This aligns with previous reports in other cancer cells that factors affecting polyunsaturated fatty acid metabolism play a key role in cancer cell proliferation, metastasis, and drug resistance. Therefore, inhibiting fatty acid metabolism can serve as a novel approach to studying the molecular mechanisms of colorectal cancer development and progression, while also providing new insights for the diagnosis and treatment of colorectal cancer.

[0003] D5D-IN-326 (FADS1 Inhibitor) is a potent, selective, and orally available fatty acid desaturase 1 (FADS1) inhibitor.

[0004] Adding the small molecule D5D-IN-326 to a culture system of colorectal cancer cells significantly inhibited their proliferation and migration. This, along with other reported effects on polyunsaturated fatty acid metabolism in other cancer cells, suggests that inhibiting fatty acid metabolism can provide a novel approach to understanding the molecular mechanisms of colorectal cancer development and offer new insights for its diagnosis and treatment. Summary of the Invention

[0005] This drug aims to address the challenge of developing a new treatment method for colon cancer, providing a novel drug to inhibit the occurrence and development of colorectal cancer.

[0006] The technical solution adopted by this drug to solve the above-mentioned technical problems involves adding 2 μM D5D-IN-326 to the culture system of colorectal cancer cell lines HCT116 and SW620. We found that the proliferation and migration ability of colorectal cancer cells were significantly inhibited, which shows that it is expected to become a new type of anti-colorectal cancer drug.

[0007] Verification of cell proliferation capacity: (1) Take the P5 human colorectal cancer cell lines HCT116 and SW620 with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium. (2) Add 0.5 mL of 0.05% trypsin and digest the cells for 2-3 min. Observe the cells under a microscope to see if they are round. Use a pipette to blow the cells and aspirate the cell suspension. Transfer the suspension to a 1.5 mL centrifuge tube containing 1 mL of DMEM serum culture medium and blow to mix to stop the digestion. (3) Centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of DMEM culture medium containing 10% FBS to resuspend the cells, count the cells using a cell counting chamber, and calculate the cell density. (4) Add the diluted cell suspension to a 96-well plate, set up two groups, each group has 3-5 replicates, and add 200 μL of culture medium containing cells to each well; (5) Add 1.0 × 10 to each culture well 5 Take 10 cells, shake the cell culture plate horizontally in a cross shape to distribute the cells evenly, and incubate in an incubator for 24 hours until the cells are completely attached to the plate. (6) Place the culture plate in the incubator. After 4 hours, when the cells adhere to the wall, add 2 μM D5D-IN-326 to the second group. Take out the cells from the first plate, which is 0 hours. 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. Detect changes in cell absorbance at 12, 24, 36, 48, 60, and 72 hours using a microplate reader. (8) The changes in cell absorbance were detected by an enzyme-linked immunosorbent assay (ELISA) reader at 12, 24, 36, 48, 60 and 72 h respectively, and the data were processed and analyzed.

[0008] Verification of cell migration ability: (1) Take the P5 human colorectal cancer cell lines HCT116 and SW620 with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium. (2) Add 0.5 mL of 0.05% trypsin and digest the cells for 2-3 min. Observe the cells under a microscope to see if they are round. Use a pipette to blow the cells and aspirate the cell suspension. Transfer the suspension to a 1.5 mL centrifuge tube containing 1 mL of DMEM serum culture medium and blow to mix to stop the digestion. (3) Centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of DMEM culture medium containing 10% FBS to resuspend the cells, count the cells using a cell counting chamber, and calculate the cell density. (4) Add 600 μL of culture medium containing 10% inactivated serum to the well, then gently place the chamber into the lower chamber. Add 8.0 × 10⁻⁶ μL of culture medium to the Transwell chamber. 4 100 cells, total volume of 200 μL of DMEM medium, with 2 μM D5D-IN-326 added; (5) After 12-24 hours, remove the cells and wipe the chamber with one end of a cotton swab. After wiping, place the chamber 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 to prevent wiping the cells.) (6) Take 600 μL of anhydrous methanol into a 24 plate, put the cleaned chamber into it and fix 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, put the chamber into it, and stain for about 15 min. Note that air bubbles should be eliminated during the process. (8) Observe the cell migration phenomenon on the membrane under a microscope and take pictures to record it.

[0009] This drug has the following advantages: (1) After the addition of the small molecule inhibitor D5D-IN-326, the proliferation and migration of colorectal cancer cell lines HCT116 and SW620 were significantly inhibited. (2) Compared with traditional surgical treatment, drug treatment avoids surgical trauma and greatly reduces the patient's pain. (3) The small molecule inhibitor D5D-IN-326 inhibits the activity of fatty acid desaturase 1 (FADS1), inhibits fatty acid metabolism, and thus plays a role in inhibiting the occurrence and development of tumors. It has a higher and more specific targeting. Attached Figure Description

[0010] Figure 1The effect of adding 2 μM D5D-IN-326 to cells cultured for 24 h on the growth of P5 generation colorectal cancer cell lines HCT116 and SW620 is shown. The culture conditions were: ① DMEM containing 10% fetal bovine serum, ② DMEM containing 10% fetal bovine serum + 2 μM D5D-IN-326, ③ DMEM containing 10% fetal bovine serum, and ④ DMEM containing 10% fetal bovine serum + 2 μM D5D-IN-326. It was found that when the culture condition was 10% fetal bovine serum + 2 μM D5D-IN-326, cells cultured under the inhibitor showed death compared to cells cultured under normal conditions.

[0011] Figure 2 The image shows the CCK-8 proliferation assay performed on cells cultured in 100% fetal bovine serum + 2 μM D5D-IN-326. The absorbance at 450 nm was measured using a microplate reader. Compared to the normal control conditions, the addition of D5D-IN-326 significantly inhibited the proliferation of both cell lines.

[0012] Figure 3 The image shows the Transwell assay results of cells cultured under 10% fetal bovine serum + 2 μM D5D-IN-326 conditions. The results show that, compared with the normal control group, the addition of D5D-IN-326 can significantly inhibit the migration ability of colorectal cancer cell lines HCT116 and SW620. Detailed Implementation

[0013] The colorectal cancer cell lines (HCT116 and SW620 cells) used in the examples were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

[0014] Cell state detection (1) Morphological observation: Cells under normal culture and those cultured with D5D-IN-326 were observed using a Leica DMIL inverted microscope. The results showed that more cells died when the small molecule D5D-IN-326 was added to the culture conditions.

[0015] Cell proliferation activity assay (1) Take the P5 human colorectal cancer cell lines HCT116 and SW620 with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (2) Add 0.5 mL of 0.05% trypsin and digest the cells for 2-3 min. Observe the cells under a microscope to see if they are round. Use a pipette to blow the cells and aspirate the cell suspension. Transfer the suspension to a 1.5 mL centrifuge tube containing 1 mL of DMEM serum culture medium and blow to mix to stop the digestion. (3) Centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of DMEM culture medium containing 10% FBS to resuspend the cells, count the cells using a cell counting chamber, and calculate the cell density. (4) Add the diluted cell suspension to a 96-well plate, set up two groups, each group has 3-5 replicates, and add 200 μL of culture medium containing cells to each well; (5) Add 8.0 × 10⁻⁶ ppm to each culture well. 3 Take 10 cells, shake the cell culture plate horizontally in a cross shape to distribute the cells evenly, and incubate in an incubator for 24 hours until the cells are completely attached to the plate. (6) Place the culture plate in the incubator. After 4 hours, when the cells have adhered, add 2 μM D5D-IN-326 to the second group. Remove the cells from the first plate, which is 0 hours. 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. Detect changes in cell absorbance at 12, 24, 36, 48, 60, and 72 hours using a microplate reader. (8) The changes in cell absorbance were detected by an enzyme-linked immunosorbent assay (ELISA) reader at 12, 24, 36, 48, 60 and 72 h respectively, and the data were processed and analyzed.

[0016] Transwell experiment (1) Take the P5 human colorectal cancer cell lines HCT116 and SW620 with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium. (2) Add 0.5 mL of 0.05% trypsin and digest the cells for 2-3 min. Observe the cells under a microscope to see if they are round. Use a pipette to blow the cells and aspirate the cell suspension. Transfer the suspension to a 1.5 mL centrifuge tube containing 1 mL of DMEM serum culture medium and blow to mix to stop the digestion. (3) Centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of DMEM culture medium containing 10% FBS to resuspend the cells, count the cells using a cell counting chamber, and calculate the cell density. (4) Add 600 μL of culture medium containing 10% inactivated serum to the well, then gently place the chamber into the lower chamber. Add 8.0 × 10⁻⁶ μL of culture medium to the Transwell chamber. 4 100 cells, total volume 200 μL of DMEM medium, with 2 μM D5D-IN-326 added (5) After 12–24 hours, remove the cells and wipe the chamber with one end of a cotton swab. After wiping, place the chamber in PBS and wipe again with the other end of the cotton swab. Repeat this process 3 times. (Note: Do not touch or wipe the bottom of the chamber to avoid wiping the cells.) (6) Take 600 μL of anhydrous methanol into a 24 plate, put the cleaned chamber into it and fix 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, put the chamber into it, and stain for about 15 min. Note that air bubbles should be eliminated during the process. (8) Observe the cell migration phenomenon on the membrane under a microscope and take pictures to record it.

Claims

1. A targeted drug for inhibiting the development and progression of colorectal cancer, characterized by the addition of 2 μM D5D-IN-326 small molecules to a culture medium containing 10% fetal bovine serum to inhibit the proliferation and migration of colorectal cancer cells.

2. The method according to claim 1, characterized in that, The aforementioned colorectal cancer cell lines HCT116 and SW620 underwent CCK-8 assays in the presence of the small molecule D5D-IN-326: (1) Take the P5 human colorectal cancer cell lines HCT116 and SW620 with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (2) Add 0.5 mL of 0.05% trypsin and digest the cells for about 2-3 minutes until the cells float to the edge. Use a pipette to blow the cells and aspirate the cell suspension. Transfer the suspension to a 1.5 mL centrifuge tube containing 1 mL of DMEM serum culture medium and continue to blow and mix to stop digestion. (3) Centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of DMEM culture medium containing 10% FBS to resuspend the cells, count them using a cell counting chamber, and calculate the cell density accordingly. (4) Add the diluted cell suspension to a 96-well plate, set up two groups, each group has 3-5 replicates, and add 200 μL of culture medium containing cells to each well; (5) Add 8.0 × 10⁻⁶ ppm to each culture well. 3 One cell, horizontally shaken in a cross shape to distribute the cells evenly, and placed in an incubator for 24 hours to allow the cells to fully adhere to the wall. (6) Place the culture plate in the incubator. After 4 hours, when the cells adhere to the wall, add 2 μM D5D-IN-326 to the experimental group. Take out the first plate of cells, which is 0 hours. 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 an ELISA reader. Measure the changes in cell absorbance at 12, 24, 36, 48, 60, and 72 hours using an ELISA reader.

3. The method according to claim 1, characterized in that, The aforementioned colorectal cancer cell lines HCT116 and SW620 underwent Transwell assays in the presence of the small molecule D5D-IN-326: (1) Take the P5 human colorectal cancer cell lines HCT116 and SW620 with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (2) Add 0.5 mL of 0.05% trypsin and digest the cells for about 2-3 minutes until the cells float to the edge. Use a pipette to blow the cells and aspirate the cell suspension. Transfer the suspension to a 1.5 mL centrifuge tube containing 1 mL of DMEM serum culture medium and continue to blow and mix to stop digestion. (3) Centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of DMEM culture medium containing 10% FBS to resuspend the cells, count them using a cell counting chamber, and calculate the cell density accordingly. (4) Add 600 μL of culture medium containing 10% inactivated serum to the well, then gently place the chamber into the lower chamber. Add 8.0 × 10⁸ μL of culture medium to the Transwell chamber. 4 100 cells, 200 μL of DMEM medium, and 2 μM D5D-IN-326 added; (5) After 12-24 hours, remove the cells and wipe the chamber with one end of a cotton swab. After wiping, put the chamber into 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 to prevent wiping the cells). (6) Take 600 μL of anhydrous methanol into a 24 plate, put the cleaned chamber into it and fix 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, put the chamber into it, and stain for about 15 min. Note that air bubbles should be eliminated during the process. (8) Observe the cell migration phenomenon on the membrane under a microscope and take pictures.

4. The method according to claims 2 and 3, characterized in that, The addition of 2μMD5D-IN-326 significantly inhibited the proliferation and migration of colorectal cancer cells, making it a promising novel anticancer drug.

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