Application of 2, 3-dihydroxybenzoic acid in preparation of antitumor drugs

Abstract

The invention discloses an application of 2, 3-dihydroxybenzoic acid in preparation of an antitumor drug. Researches find that 2, 3-dihydroxybenzoic acid can inhibit proliferation, migration and invasion of colorectal cancer cells in vitro and promote cell cycle arrest and cell apoptosis, and 2, 3-dihydroxybenzoic acid can inhibit growth of mouse transplanted tumors in vivo and promote cell apoptosis; the 2, 3-dihydroxybenzoic acid has a remarkable treatment effect on mouse colon cancer and can improve the immunotherapy effect; moreover, the 2, 3-dihydroxybenzoic acid small molecule substance, as an inhibitor of fatty acid synthase (FASN), plays an important role in inhibiting colorectum.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of 2,3-dihydroxybenzoic acid in the preparation of antitumor drugs, belonging to the field of biological medicine and pharmaceutical chemistry. BACKGROUND

[0002] Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer death worldwide, posing a serious threat to human health. Despite advances in screening, early diagnosis, and comprehensive treatment, the prognosis of patients with advanced or metastatic CRC remains poor, with a significantly reduced 5-year survival rate.

[0003] Surgery is the main radical means for early and locally advanced colorectal cancer (CRC), but it has limited effect on patients who have developed distant metastasis. Although chemotherapy, targeted therapy and other treatment methods can improve the survival period of some patients with metastatic CRC (mCRC), the response rate is limited, drug resistance is common, and the target only covers part of the patient population. Immune checkpoint inhibitors (ICIs), especially drugs targeting the PD-1 / PD-L1 and CTLA-4 pathways, have achieved revolutionary success in patients with metastatic colorectal cancer with high microsatellite instability or mismatch repair deficiency (MSI-H / dMMR). This subgroup accounts for about 15% of all mCRC. ICIs show a sustained response and significant survival benefit in this subgroup, and some patients even achieve long-term remission or clinical cure. However, the majority of mCRC patients (about 85%) have microsatellite stable or normal mismatch repair function (MSS / pMMR) tumors. These MSS / pMMR CRC tumors usually show primary resistance to single-agent ICIs, with a very low response rate (usually <5%), which constitutes the main bottleneck and unmet clinical need of current CRC immunotherapy.

[0004] Existing strategies to overcome immune resistance in MSS CRC (such as ICI combined with chemotherapy / targeting) have limited efficacy and increased toxicity, and new combination strategies are not mature. Therefore, developing new, efficient, and safe immunotherapy combination strategies, especially those that can effectively convert "cold" MSS CRC tumors into "hot" tumors, is a key scientific problem and major clinical need in the current CRC treatment field. This requires a deep understanding of the unique immune evasion mechanisms of MSS CRC and the design of combination therapies that can specifically reverse these mechanisms and effectively activate anti-tumor immunity.

[0005] Fatty acid synthase (FASN) is a key rate-limiting enzyme that catalyzes the de novo synthesis of long-chain fatty acids, which provides the lipid precursors required for membrane phospholipid synthesis, energy storage, and protein palmitoylation modification in tumor cells. In malignant tumors such as colorectal cancer (CRC), FASN is often highly expressed, and its expression level is significantly negatively correlated with patient prognosis. Meanwhile, targeted inhibition of FASN can synergistically enhance the effects of chemotherapy and immunotherapy. SUMMARY

[0006] The technical problem solved by the present application is to provide the application of 2,3-dihydroxybenzoic acid (2,3-DHBA) in the preparation of an antitumor drug.

[0007] Technical solution: To solve the above technical problems, the present application provides the application of 2,3-dihydroxybenzoic acid in the preparation of an antitumor drug.

[0008] The tumor is colorectal cancer.

[0009] The present application also provides an antitumor drug containing 2,3-dihydroxybenzoic acid.

[0010] The antitumor drug also contains a PD-1 inhibitor or a PD-L1 inhibitor.

[0011] The tumor is colorectal cancer.

[0012] The antitumor drug comprises a pharmaceutically acceptable carrier.

[0013] The dosage form of the antitumor drug is oral or injectable.

[0014] The present application also provides the application of 2,3-dihydroxybenzoic acid in the preparation of a drug for degrading fatty acid synthase.

[0015] The present application also provides a drug for degrading fatty acid synthase, which contains 2,3-dihydroxybenzoic acid.

[0016] The drug comprises a pharmaceutically acceptable carrier.

[0017] Advantages: Compared with the prior art, the present application has the following significant advantages:

[0018] 1. The application range of 2,3-dihydroxybenzoic acid (DHBA) is expanded, and a new antitumor drug is provided: for the first time, the application value of DHBA in antitumor, especially in colorectal cancer (CRC), is disclosed. This lays a solid foundation for the development of new antitumor drugs based on DHBA or its derivatives.

[0019] 2. Directly inhibit the growth of colorectal cancer: DHBA itself exhibits anti-tumor activity against colorectal cancer cells, and can be used as a single drug component or core component for the treatment of CRC, especially for the types with limited effect of current treatment methods.

[0020] 3. Revealing and utilizing a key new mechanism - degrading fatty acid synthase (FASN): The present application first discovers and confirms that DHBA can effectively degrade fatty acid synthase (FASN). FASN is a key rate-limiting enzyme for tumor cell lipid synthesis, which is highly expressed in various malignant tumors (including CRC), and is closely related to tumor proliferation, invasion, metastasis and poor prognosis. By degrading FASN, DHBA can:

[0021] 1) Directly inhibit the growth and survival of tumor cells: disrupt the abnormal vigorous lipid synthesis requirement of tumor cells, and interfere with their energy metabolism and membrane structure synthesis.

[0022] 2) Targeting the metabolic weakness of tumor: taking advantage of the dependence of tumor cells on FASN, it provides a precise intervention strategy for the metabolic pathway of tumor.

[0023] 3. Overcoming the immunotherapy resistance of MSS type colorectal cancer (core advantage): The most prominent beneficial effect of the present application is that the combination of DHBA and PD-1 / PD-L1 inhibitors can effectively overcome the primary drug resistance problem of microsatellite stable (MSS) tumors to immune checkpoint inhibitors (ICI), which accounts for the majority of CRC. The synergistic mechanism may include:

[0024] 1) Reverse the immunosuppressive microenvironment (TME): DHBA degrading FASN can improve TME through multiple pathways; affect the function and activity of immunosuppressive cells (such as M2 type tumor-associated macrophages - TAMs); enhance the presentation or exposure of tumor-associated antigens.

[0025] 2) Promote the transformation of "cold tumor" to "hot tumor": by improving TME, DHBA combined with PD-1 / PD-L1 inhibitors can significantly increase the infiltration and functional activation of effector T cells (especially CD8+ T cells) in tumor tissue, and transform immunologically "cold" MSS CRC tumors into "hot" tumors sensitive to immunotherapy.

[0026] 3) Enhance the efficacy of immune checkpoint blockade: PD-1 / PD-L1 inhibitors aim to relieve the inhibition of T cells, while DHBA provides the necessary foundation and synergistic stimulation for the effective activation and killing of T cells by remodeling the favorable TME of FASN degradation, thereby significantly improving the response rate and efficacy depth of PD-1 / PD-L1 inhibitors.

[0027] 4. Provide efficient and synergistic combination immunotherapy regimen: The "DHBA + PD-1 / PD-L1 inhibitor" combination drug strategy provided by the present application is expected to produce significant synergistic anti-tumor effects compared to single drug (whether DHBA alone or ICI alone in MSS CRC), including tumor growth mitigation, tumor size reduction, etc.

[0028] 5. Targeting the metabolic-immune crosspoint, innovative: This scheme innovatively combines targeting key tumor metabolic enzymes (FASN) with immune checkpoint blockade, overcoming immune resistance by intervening in the "metabolic-immune" key crosspoint, representing a novel and promising strategy for treating MSS CRC.

[0029] 6. Potentially improve the limitations of existing combination therapy: Compared with the current clinical exploration of ICI combined with chemotherapy / targeting (such as anti-angiogenic drugs) regimen, the combination strategy (DHBA + ICI) of the present application may have: different, more optimal synergistic mechanisms; potentially lower toxicity profile (avoiding significant toxicity of cytotoxic chemotherapy drugs or certain targeted drugs); providing new options for patients resistant to chemotherapy or specific targeted therapy.

[0030] 7. Drug development convenience: DHBA, as a known compound, has a relatively solid foundation in terms of its chemical properties, pharmacokinetics, and safety research, and the present application has clearly demonstrated the feasibility of its oral dosage form, which is conducive to accelerating the development process of its drug (especially oral drug) and potential clinical application convenience.

[0031] 8. Provide new treatment hope for MSS CRC patients: The present application directly targets the huge and unmet clinical needs of the MSS CRC population, providing a combination drug regimen based on a new mechanism that has the potential to significantly improve the efficacy of immunotherapy, and has important clinical application value and broad market prospects. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Effect of 2,3-DHBA on inhibition of colorectal cancer cell proliferation, migration, invasion, promotion of cell cycle arrest and apoptosis: (A) CCK8 experiment to detect the effect of different concentrations of 2,3-DHBA on the proliferation of CRC cell lines; (B) colony formation experiment to detect the effect of 400 μM 2,3-DHBA on the proliferation of CRC cells; (C-D) Transwell chamber experiment to detect the effect of 400 μM 2,3-DHBA on the migration (C) and invasion (D) of CRC cell lines; (E) Effect of 400 μM 2,3-DHBA on cell cycle of CRC cell lines; (F) Effect of 400 μM 2,3-DHBA on apoptosis of CRC cell lines; *p<0.05, **p<0.01;

[0033] Figure 2 Figure 2: 2,3-DHBA inhibits mouse transplanted tumor growth and promotes apoptosis: (A) MC38 cells were inoculated subcutaneously into mice, and then treated with solvent control and 2,3-DHBA, respectively. The volume curve of transplanted tumors was shown. (B) The general photo of transplanted tumors after 22 days of inoculation. (C) The weight of transplanted tumors after treatment with control and 2,3-DHBA. (D) The HE staining results of transplanted tumor tissues after treatment with control and 2,3-DHBA. (E) The expression of proliferation index PCNA, apoptosis index Caspase3 and CD8 positive cell marker CD8 in transplanted tumor tissues after treatment with control and 2,3-DHBA were detected by immunohistochemistry. (F) CT26 cells were inoculated subcutaneously into mice, and then treated with solvent control and 2,3-DHBA, respectively. The volume curve of transplanted tumors was shown. (G) The general photo of transplanted tumors after 22 days of inoculation. (H) The weight of transplanted tumors after treatment with control and 2,3-DHBA. (I) The proportion of CD8 positive cells in transplanted tumors after treatment with control and 2,3-DHBA was detected by flow cytometry. (J) The proportion of CD69 positive CD8 T cells in transplanted tumors after treatment with control and 2,3-DHBA was detected by flow cytometry. *p<0.05, **p<0.01, ***p<0.001. + T cells (reflecting CD8 + T cells with killing activity) were shown.

[0034] Figure 3 Figure 3: 2,3-DHBA enhances the effect of anti-PD-1 tumor treatment: (A) CT26 cells (a cell line insensitive to immunotherapy) were inoculated subcutaneously into mice, and then treated with solvent control, 2,3-DHBA, PD-1 antibody and combination, respectively. The volume curve of transplanted tumors was shown. (B) The general photo of transplanted tumors after 24 days of inoculation. (C) The weight of transplanted tumors after treatment with the above groups. *p<0.05, **p<0.01.

[0035] Figure 4Effect diagram of 2,3-DHBA promoting degradation by binding FASN and inhibiting the activity of FASN: (A) SPR experiment detected the affinity of 2,3-DHBA and FASN at different concentrations; (B) Effect of 2,3-DHBA on FASN protein level in CRC cell line; (C) Western blot detected the expression level of FASN protein in CRC cells after 2,3-DHBA or control induced 200 μg / ml CHX treatment for different time; (D) Detection of PA expression level in colorectal cancer cells after 2,3-DHBA and FASN inhibitor C75 stimulation; (E) Detection of TG expression level in colorectal cancer cells after 2,3-DHBA and FASN inhibitor C75 stimulation; (F) Western blot detected the FASN protein level in CRC cells after 2,3-DHBA and FASN inhibitor C75 treatment, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0036] The technical solutions of the present application will be further described below in combination with the drawings.

[0037] Example 1 2,3-DHBA inhibits colorectal cancer cell proliferation, migration, invasion, promotes cell cycle arrest and apoptosis

[0038] Human colorectal cancer cell lines RKO and HCT116 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The above cell lines were analyzed by STR identification and mycoplasma contamination detection, and it was confirmed that there was no mycoplasma contamination.

[0039] RPMI 1640 and DMEM (HyClone, South Logan, UT, USA) complete medium was obtained by adding 10% FBS (Gibco) and 1% three antibiotics (containing penicillin 100 units / ml, streptomycin 100 ug / ml, amphotericin B 0.25 ug / ml) (Xinsaimai Biological) to RPMI 1640 and DMEM basic medium, respectively. HCT116 was cultured in RPMI 1640 complete medium and RKO was cultured in DMEM complete medium. All cell lines were cultured at 37°C in 5% CO2.

[0040] Cell proliferation experiment process: HCT116 or RKO cells were seeded in 96-well plates at a number of 1000 per well, 6-8 hours after adhering, different concentrations (100 μM, 200 μM, 400 μM, 600 μM) of 2,3-DHBA (MCE, HY-Y0202) or control DMSO were added, 10 μl / well of CCK8 reagent (Pronin, Nanjing) was added at the same time, after 2 hours of incubation in the incubator, the absorbance was detected in the microplate reader at 450 nm wavelength. Subsequently, the OD value was detected at 24, 48, 72, 96 hours, respectively.

[0041] Cell colony formation experiment process: HCT116 or RKO cells were seeded in 6-well plates at a number of 1000 per well, and 2,3-DHBA was added at a final concentration of 400 μM, and incubated in the incubator, and the RPMI 1640 or DMEM medium containing 2,3-DHBA at a final concentration of 400 μM was replaced every three days until visible colonies were formed, fixed, generally about 14 days. The 6-well plate was taken out, the original culture medium was removed, and the PBS was gently washed 2-3 times, 4% paraformaldehyde was added for fixation for 10 minutes, and distilled water was washed for 2 minutes, 1 ml of 0.5% crystal violet reagent (Kaiji Biological, Nanjing) was added to each well, and the room temperature was stained for 30 minutes in the dark, and washed with distilled water until clear. After natural air drying, photographing and statistics were performed, and the number of colonies was counted using Image J.

[0042] Transwell cell migration experiment process: HCT116 or RKO cells were digested and centrifuged, resuspended in 1 ml of PBS, and centrifuged to remove the effect of FBS on migration, resuspended the cells in 1 ml of basic medium, counted, and calculated, 60,000 HCT116 cells and 80,000 RKO cells were resuspended in 200 μl of RPMI 1640 or DMEM basic medium and seeded in the upper chamber of the chamber (Corning, NY, USA), and 2,3-DHBA was added at a final concentration of 400 μM, and 600 μl of RPMI 1640 or DMEM complete medium (24-well plate) was added to the lower chamber. After 48 hours, the chamber was taken out and fixed and stained in 10% methanol and 90% 0.1% crystal violet staining solution for 30 minutes, and then the chamber was washed clean, naturally air dried, and photographed (5 fields were selected) using a 100x microscope.

[0043] Transwell cell invasion experiment process: Matrigel matrix gel (Corning, NY, USA) was mixed with serum-free RPMI 1640 or DMEM medium at a ratio of 1:25 on ice, 50 μl of the complex was added to the upper chamber, and the chamber was placed in the incubator for 30 minutes to allow the matrix gel to solidify. Subsequently, the cells were plated, and the specific steps were consistent with the above cell migration.

[0044] The cell cycle experiment procedure is as follows: 3 × 10 5 HCT116 or RKO cells were seeded into 6-well plates. After 8-12 hours of cell attachment, the original culture medium was removed, and the cells were washed 2-3 times with PBS. The culture medium was then replaced with serum-free RPMI 1640 or DMEM for 24 hours, followed by replacement with RPMI 1640 or DMEM complete medium containing 400 μM 2,3-DHBA. Cells were fixed overnight at -20°C with 75% ethanol. Cell staining was performed using the CycleTEST PLUS DNA Reagent Kit (DBiosciences, San Jose, CA), and cell cycle analysis was then performed using an FAC Scan instrument (Becton Dickinson, Heidelberg, Germany) at the Nanjing Medical University Analytical and Testing Center.

[0045] The apoptosis experiment procedure is as follows: 1×10 6 HCT116 or RKO cells were seeded into 6-well plates. After 8-12 hours, once the cells had adhered, the original culture medium was removed, and the cells were washed 2-3 times with PBS. The medium was then replaced with serum-free RPMI 1640 or DMEM and cultured for 24 hours. The culture medium was then replaced with RPMI 1640 or DMEM complete medium containing 400 μM 2,3-DHBA. HCT116 and RKO cells were digested with EDTA-free trypsin. All washings and the old culture medium were centrifuged together at 1800 rpm for 5 minutes. The cells were washed twice with pre-cooled PBS (4°C), resuspended in 100 μl binding buffer, and 5 μl Annexin V / Alexa Fluor 647 and 5 μl propidium iodide solution (Novazia, Nanjing) were added. After mixing, the cells were incubated at room temperature in the dark for 10 minutes. 400 μl binding buffer was added to the reaction tube, and the cells were analyzed by flow cytometry (FACS) at the Nanjing Medical University Analytical and Testing Center. FlowJo 10.8.1 was used for analysis.

[0046] The results are as follows Figure 1 As shown:

[0047] A: When CRC tumor cell lines were stimulated with different concentrations of 2,3-DHBA, the results of the CCK8 experiment showed that, compared with the control group, 2,3-DHBA significantly inhibited the proliferation of HCT116 and RKO cells in a concentration-dependent manner.

[0048] B: A concentration of 400 μM 2,3-DHBA was selected as the subsequent experimental condition. In the colony formation assay, compared with the control group, 2,3-DHBA significantly inhibited the colony formation ability of CRC cells.

[0049] C-D: 2,3-DHBA at a concentration of 400 mM was selected as the subsequent experimental condition. The results of the Transwell chamber experiment showed that 2,3-DHBA significantly inhibited the migration and invasion ability of CRC cells compared with the control group.

[0050] E: The cell cycle was detected by flow cytometry. The results showed that the proportion of G1 phase cells in CRC cells treated with 2,3-DHBA was significantly increased, and the proportion of S phase cells was decreased compared with the control group.

[0051] F: The apoptosis was detected by flow cytometry. The results showed that 2,3-DHBA significantly promoted the apoptosis of CRC cells compared with the control group.

[0052] The above experimental results show that 2,3-DHBA can inhibit the proliferation, migration, and invasion of CRC cells, block the cell cycle, and promote cell apoptosis in vitro.

[0053] Example 2 2,3-DHBA inhibits the growth of mouse transplanted tumors and increases the proportion of CD8-positive T cells

[0054] MC38 was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and CT26 (a colorectal cancer cell line insensitive to immunotherapy) was purchased from Shanghai Fuheng Biological Technology Co., Ltd. The above cell lines were analyzed by STR identification and mycoplasma contamination detection, and it was confirmed that there was no mycoplasma contamination.

[0055] MC38 and CT26 cells were cultured in RPMI 1640 complete medium, respectively. All cell lines were cultured at 37°C in 5% CO2.

[0056] The animal experiment process is as follows: 10 six-week-old male C57BL / 6 and 10 male BALB / C wild-type mice were divided into two groups, namely the control group and the 75 mg / kg group. 4x10 5 MC38 cells or 1x10 6CT26 cells were inoculated into the right scapula of mice at 100 μl / 100 μl, and 2,3-DHBA and its control cosolvent were administered by gavage the next day. The 2,3-DHBA was dissolved in DMSO to prepare a 200 mg / ml mother liquor, and then diluted with cosolvents PEG300 (MCE, HY-Y0873) and Tween80 (MCE, HY-Y1891) (cosolvent ratio = 40% PEG300 + 5% Tween80). The 2,3-DHBA and its control solution were prepared fresh each day. The body weight was measured daily, and 2,3-DHBA was administered by gavage at 75 mg / kg. The volume of gavage solvent was the same for each group. The tumor volume was measured starting on the 7th day after injection, and the measurement was performed daily (subcutaneous tumor volume = length x width x width / 2). After 3 weeks of injection, the mice were anesthetized, and blood samples were collected before the mice were sacrificed. The blood samples were placed at room temperature for 3-4 hours, centrifuged at 3000 rpm for 10 minutes, and the upper serum was collected and stored at -80°C for testing. The subcutaneous tumor was removed and weighed. One part was fixed with 4% paraformaldehyde for 24-48 hours, and then paraffin-embedded and sectioned for subsequent RNA and protein extraction.

[0057] The histological experiment process was as follows: after the tumor tissue was fixed in 4% paraformaldehyde for 24-48 hours, PBS was replaced, and then the tumor tissue of appropriate size was wrapped in a filter paper and placed in an embedding frame for subsequent dehydration and wax immersion. The tumor tissue was sectioned and subjected to routine HE staining and immunohistochemistry. The antibodies and dilution ratios used in immunohistochemistry were as follows: PCNA (Santa, sc-56, 1:200), Caspase3 (Abeam, Ab3251, 1:2000), CD8 (proteintech, 29896-1-AP, 1:1000); the DAB color development times for different antibodies were as follows: PCNA color development for 2 minutes and 30 seconds, Caspase3 color development for 2 minutes, and CD8 color development for 3 minutes.

[0058] Photos were taken with an upright microscope at 200x magnification, and the positive cell proportion and positive area proportion were calculated and analyzed in Image Plus Pro software to compare the proliferation, apoptosis, and immune infiltration levels between samples.

[0059] The flow cytometry experiment process is: the mouse tumor tissue is mechanically chopped with scissors, and is put into DMEM containing 1 mg / mL collagenase I (BioFROX, 1904MG100) and 200 μg / ml DNase I (MCE, HY-108882) for 37°C water bath digestion for 30 minutes. The digested tumor tissue is washed with DMEM containing 10% fetal bovine serum, and filtered through a 70 μm nylon cell filter (BIOFIL, CSS013070). The filtered cell liquid is added with ACK lysis buffer (Gibco, A10492-01), and incubated at room temperature for 5 minutes to remove red blood cells. Then the cells are washed with PBS and counted. In order to evaluate the CD8 + T cell infiltration level and the activation level of infiltrated CD8 + T. First, 1×10 6 cells are incubated with Fixable Viability Stain (Biolegend, 423101) at room temperature for 30 minutes to label living cells, and after incubation, the cells are washed with PBS+2% FBS for 3 times. Then anti-CD45 (BioLegend, 104527), anti-CD3 (BioLegend, 100232), anti-CD8 (BioLegend, 100721), anti-CD69 (BioLegend, 103128) antibodies are added to the cells, and incubated in a refrigerator at 4°C for 30 minutes. After incubation, the cells are washed with PBS+2% FBS, and fixed and permeated with 4% PFA solution in a refrigerator at 4°C for 20 minutes. Finally, the South Medical University Analysis and Testing Center analyzes by flow cytometry (FACS). The analysis is performed using FlowJo 10.8.1 for flow cytometry detection.

[0060] The results are shown as follows: Figure 2

[0061] A-C: MC38 cells are subcutaneously injected into the flank of 6-week-old male C57BL / 6 mice, and the next day, the mice are gavaged with 2,3-DHBA. The results show that after gavage with 2,3-DHBA, the tumor volume growth rate of the mice is significantly slowed down, and the size and weight are also significantly reduced compared with the control group.

[0062] D: The transplanted tumor is fixed, paraffin-embedded, and then tissue sectioned and HE stained.

[0063] E: Immunohistochemistry (IHC) experiments show that the tumor proliferation index PCNA in the 2,3-DHBA treatment group is significantly decreased, and the immune infiltration index CD8 and the apoptosis index Caspase3 are significantly increased.

[0064] ​F-H: CT26 cells were subcutaneously injected into the flank of 6-week-old male BALB / C mice, and the next day, the mice were gavaged with 2,3-DHBA. The results showed that the growth rate of tumor volume in mice after gavage with 2,3-DHBA was significantly slower than that in the control group, and the size and weight were also significantly reduced.

[0065] I-J: Flow cytometry experiments showed that the proportion of CD8-positive T cells in transplanted tumors was significantly increased after 2,3-DHBA treatment and the activity was also increased.

[0066] The above in vivo experiments showed that 2,3-DHBA produced by exercise can play a role in inhibiting CRC and increasing the proportion of CD8-positive T cells.

[0067] Example 3 2,3-DHBA enhances the effect of anti-PD-1 tumor treatment

[0068] CT26 (a colorectal cancer cell line not sensitive to immunotherapy) was purchased from Shanghai Fuheng Biotechnology Co., Ltd. The above cell lines were analyzed by STR identification and mycoplasma contamination detection, and it was confirmed that there was no mycoplasma contamination.

[0069] CT26 cells were cultured in RPMI 1640 complete medium. All cell lines were cultured at 37°C in 5% CO2.

[0070] The animal experiment process was as follows: 20 6-week-old male BALB / C wild-type mice were taken, and 1x10 6 CT26 cells / 100 μl were inoculated into the right scapular part of the mice. After inoculation, the tumor size was measured, and when it grew to 80-100 mm 3 , it was divided into 4 groups, namely Vehicle+IgG group, 2,3-DHBA+IgG group, Vehicle+PD-1 group, and 2,3-DHBA+PD-1 group. 2,3-DHBA and its control cosolvent were gavaged at a dose of 75 mg / kg per day, and PD-1 (HY-P99144, MCE) or IgG (HY-P990679, MCE) treatment was given at the same time (every two days, with PD-1 and IgG injected 100 μl each time, a total of 4 injections). The tumor volume was measured daily during the modeling process (subcutaneous tumor volume = length x width x width / 2), and the mice were sacrificed after 24 days of modeling. After collecting blood samples from the mice under anesthesia, the mice were sacrificed, and the blood samples were collected and centrifuged at 3000 rpm for 10 minutes at room temperature. The serum was collected and stored at -80°C for testing. The subcutaneous tumors were weighed, and a portion was fixed with 4% paraformaldehyde for 24-48 hours, followed by paraffin embedding and tissue sectioning. Another portion was used for subsequent RNA and protein extraction.

[0071] The histological experiment process is as follows: after the subsequent tumor tissue is fixed, embedded, and sectioned, the foot tissue is subjected to a histological experiment, and the conventional HE staining and immunohistochemical experiment are performed, and the specific process is the same as above.

[0072] As shown in Figure 3

[0073] A: Compared with the control group, the growth rate of tumor volume of mice after intraperitoneal injection of anti-PD-1 was significantly slowed down, and compared with 2,3-DHBA treated mice, 2,3-DHBA combined with anti-PD-1 treatment significantly inhibited the growth rate of subcutaneously transplanted tumors.

[0074] B-C: The size and weight of the transplanted tumors of mice during treatment showed that compared with 2,3-DHBA treated mice, the size and weight of the transplanted tumors after 2,3-DHBA combined with anti-PD-1 treatment were significantly decreased.

[0075] The above results show that 2,3-DHBA combined with anti-PD-1 treatment can enhance the effect of immunotherapy and inhibit tumor growth.

[0076] Example 4 2,3-DHBA promotes degradation and inhibits activity by binding FASN.

[0077] The culture conditions of HCT116 and RKO are the same as above.

[0078] The process of surface plasmon resonance (SPR) experiment is as follows: in the experiment, fatty acid synthase (FASN) is fixed on the surface of the sensor chip as the ligand, and 2,3-dihydroxybenzoic acid (2,3-DHBA) is used as the analyte to flow through the chip surface at different concentrations. When the two are combined, the real-time change of SPR signal response value (RU) is caused by the change of chip surface refractive index, and by recording the binding and dissociation curves, the binding rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (KD) can be calculated to quantitatively characterize the interaction affinity.

[0079] ​The process of Western Blot experiment was as follows: after HCT116 or RKO cells were stimulated by 400 μM 2,3-DHBA for 24 hours, and HCT116 cells were stimulated by 20 μM C75 (FASN inhibitor drug, MCE, 218137-86-1) and RKO cells were stimulated by 40 μM C75, total proteins were extracted by using prepared protein lysate (RIPA: PI: PMSF = 100: 1: 1, v / v / v), and after measuring the protein concentration by BCA method, the proteins were quantified and denatured. Then, the same amount of protein samples were subjected to electrophoresis by SDS-PAGE, transferred, blocked, and incubated with FASN (ABclonal, A1905, 1:1000) and β-actin (Santa, sc-58673, 1:200) primary antibodies at 4°C overnight. The next day, the membrane was incubated with diluted HRP-labeled rabbit (Proteintech, SA00001-2, 1:10000) or mouse (Proteintech, SA00001-1, 1:10000) antibodies at room temperature for 1 hour, and then the protein bands were exposed using ECL chemiluminescence solution.

[0080] The process of cycloheximide (CHX) translation inhibition experiment was as follows: after HCT116 or RKO cells were stimulated by 400 μM 2,3-DHBA for 24 hours, the cells were treated with 200 μg / ml cycloheximide (CHX, Cycloheximide, MCE), and then the cell precipitates were collected at different time points of 0, 3, 6, 9, and 12 hours, and the proteins were extracted. Finally, the expression level of FASN was detected by immunoblotting with GAPDH (Proteintech, 60004-1-Ig, 1:100000) as a control, and ImageJ software (Version 1.5.3) was used for quantitative analysis.

[0081] The process of palmitic acid (PA) detection experiment was as follows: after HCT116 and RKO cells were plated in 6-well plates, the cells were stimulated by 400 μM 2,3-DHBA or 20 μM (stimulating HCT116 cells) or 40 μM (stimulating RKO cells) C75 (MCE, 218137-86-1) for 24 hours, and then the cell precipitates were collected, and the content of intracellular palmitic acid was detected by using a human palmitic acid enzyme-linked immunoassay kit (MM-51627H2, Jiangsu Enzyme Immune Industry Co., Ltd.).

[0082] The process of triglyceride (TG) detection experiment is as follows: after HCT116 and / or RKO cells are plated in 6-well plates, the cells are stimulated with 400 mM 2,3-DHBA or 20 mM (for stimulating HCT116 cells) or 40 mM (for stimulating RKO cells) C75 (MCE, 218137-86-1) for 24 hours, and then the cell precipitate is collected, and the content of intracellular triglyceride is detected using a triglyceride detection kit (PTC1238, Promega).

[0083] As shown in Figure 4

[0084] A: Surface plasmon resonance (SPR) technology is used to determine the binding and dissociation constants of 2,3-DHBA and FASN under different conditions by using different concentrations of 2,3-DHBA flowing through the FAS N protein chip. Biacore Insight evaluation software (Cytiva, Marlborough, MA, USA) is used to evaluate the binding of the two, and it is found that 2,3-DHBA can bind to FASN and has a concentration-dependent effect, that is, the higher the concentration, the stronger the binding ability.

[0085] B: After 2,3-DHBA treatment of CRC cells, the expression of FASN protein is reduced.

[0086] C: By treating control and 2,3-DHBA-stimulated CRC cells with actinomycin D (CHX), the degradation rate of FASN protein is detected, and it is found that 2,3-DHBA promotes the degradation of FASN.

[0087] D-E: After 2,3-DHBA and FASN inhibitor C75 treatment of CRC cells, the PA and TG contents of CRC cells are detected, and it is found that 2,3-DHBA stimulation reduces the PA and TG contents in cells, and has a similar effect as C75 treatment.

[0088] F: Compared with C75-stimulated CRC cells, 2,3-DHBA-stimulated CRC cells have a similar effect on reducing the expression of FASN protein.

[0089] The results show that 2,3-DHBA can bind to FASN and promote its degradation.​

Claims

Application of 1,2,3-dihydroxybenzoic acid in the preparation of antitumor drugs.

2. The application according to claim 1, characterized in that, The tumor is colorectal cancer.

3. The application according to claim 1, characterized in that, The anti-tumor drug also contains immunotherapy drugs, including PD-1 inhibitors or PD-L1 inhibitors.

4. An antitumor drug, characterized in that, It contains 2,3-dihydroxybenzoic acid.

5. The antitumor drug according to claim 4, characterized in that, It also contains immunotherapeutic agents, including PD-1 inhibitors or PD-L1 inhibitors.

6. The antitumor drug according to claim 4, characterized in that, The tumor in question is colorectal cancer.

7. The antitumor drug according to any one of claims 4 to 6, characterized in that, The antitumor drug contains a pharmaceutically acceptable carrier.

8. Application of 2,3-dihydroxybenzoic acid in the preparation of drugs that degrade fatty acid synthases.

9. A drug for degrading fatty acid synthase, characterized in that, It contains 2,3-dihydroxybenzoic acid.

10. The drug for degrading fatty acid synthase according to claim 9, characterized in that, The drug contains a pharmaceutically acceptable carrier.

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