Application of trifluoromethyl tubercle bacillus in preparation of antitumor drugs
By using trifluoromethyl tuberculin (TFMT) to inhibit the proliferation of liver cancer cells and promote anti-tumor immune response, the problem of limited efficacy of existing liver cancer targeted drugs has been solved, and significant anti-tumor effects and extended survival time have been achieved.
Patent Information
- Application Number
- CN202510085555.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-05-13
AI Technical Summary
The current targeted liver cancer drugs have limited efficacy and drug resistance problems, so more effective treatment options are needed.
Trifluoromethyl tuberculin (TFMT) is used as an anti-tumor drug to inhibit the proliferation of liver cancer cells by inhibiting the 2-position oxygen methyltransferase CMTR1 of RNA, and promote the infiltration of follicular helper T cells in the tumor, enhancing the anti-tumor immune response.
TFMT significantly inhibits the proliferation of liver cancer cells, reduces tumor volume, prolongs the survival time of mice, has significant anti-tumor effects, and enhances anti-tumor immune response.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of trifluoromethyl-tubercidin (TFMT) in the preparation of anti-tumor drugs. Background Art
[0002] Liver cancer is one of the most common malignant tumors. Since HCC has hidden early symptoms and progresses rapidly, about 70% of liver cancer patients are already in the middle or late stage when first diagnosed, missing the best time for surgical treatment. According to the diagnosis and treatment guidelines for primary liver cancer, targeted drugs represented by lenvatinib and sorafenib are the first choice for the treatment of middle and late stage liver cancer. Although this type of drug has achieved good results in clinical practice, there are still problems such as limited efficacy and drug resistance, so there is an urgent need to find more effective treatment options.
[0003] Epigenetics refers to the heritable variation of genes without changing the nucleotide sequence, mainly including chromatin remodeling, histone modification, DNA modification and RNA modification. Epigenetic changes are an important feature of the occurrence and development of tumors. In recent years, the role of small molecule targeted drugs targeting epigenetics in tumor treatment has become increasingly prominent. For example, inhibitors targeting DNA methyltransferase (such as 5-azacytidine, etc.) have shown certain advantages in clinical and preclinical studies of tumor treatment including leukemia and liver cancer.
[0004] Trifluoromethyl-tubercidin (TFMT) is a small molecule compound that can inhibit the RNA 2-oxygen methyltransferase CMTR1. The compound can inhibit CMTR1 by interacting in the S-adenosyl-l-methionine binding pocket of CMTR1, thereby limiting the replication of influenza virus. Our previous studies have found that silencing the expression of CMTR1 in liver cancer cells can significantly inhibit the growth of liver cancer. However, the clinical application of trifluoromethyl-tubercidin as an anticancer drug remains unknown. Summary of the invention
[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide an application of TFMT in the preparation of a drug for treating liver cancer, so as to overcome the limitation of the limited efficacy of current clinical liver cancer targeted drugs.
[0006] The objective of the present invention is achieved through the following technical solutions:
[0007] In a first aspect, the present invention provides a use of TFMT in the preparation of a drug for treating liver cancer.
[0008] The present invention has found through research that TFMT can inhibit the proliferation of liver cancer cells, reduce the tumor volume of mice with subcutaneous liver cancer tumor models, prolong the survival time of mice, and has a significant anti-tumor effect.
[0009] In a second aspect, the present invention provides the use of TFMT in enhancing anti-tumor immune response.
[0010] The present invention has found through research that TFMT can enhance the anti-tumor immune response by promoting the infiltration of follicular helper T cells in tumors, thereby achieving an anti-tumor effect.
[0011] As a preferred embodiment of the application of the present invention, the effective dose of TFMT is 1 mg-10 mg / kg / day, and the most preferred effective dose is 5 mg / kg / day.
[0012] Compared with the prior art, the present invention has the following beneficial effects:
[0013] 1. The present invention found that trifluoromethyltuberculin (TFMT) can inhibit the proliferation of liver cancer cells and promote the infiltration of follicular helper T cells into the tumor site.
[0014] 2. The present invention found that trifluoromethyltuberculin (TFMT) has a significant anti-tumor effect and prolongs the survival time of tumor-bearing mice.
[0015] The present invention discovers a new use of trifluoromethyltuberculin (TFMT), expands the application range of trifluoromethyltuberculin (TFMT), and provides a new choice for clinical therapeutic drugs for liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The results of TFMT inhibiting the proliferation of liver cancer cells in Example 1;
[0017] Figure 2 The results of TFMT inhibiting the ability of liver cancer cells to form cell colonies in Example 1, A is the human liver cancer cell line MHCC97-H, B is the human liver cancer cell line SNU449, C is the human liver cancer cell line SMMC7721, and D is the mouse liver cancer cell line Hepa1-6;
[0018] Figure 3 The effect of TFMT on tumor growth and survival of liver cancer-bearing mice in Example 2;
[0019] Figure 4 The effects of TFMT on liver function, body weight and important organs of liver cancer-bearing mice in Example 2;
[0020] Figure 5 This is the effect of TFMT on immune cells in the tumor microenvironment of liver cancer-bearing mice in Example 3. DETAILED DESCRIPTION
[0021] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0022] In the following examples, the use of trifluoromethyl tuberculin (TFMT) in the preparation of a drug for treating liver cancer is provided. The CAS number of trifluoromethyl tuberculin (TFMT) is 1854086-05-7, and the chemical formula is C 12 H 13 F3N4O4, the structural formula is as follows:
[0023]
[0024] The drug for treating liver cancer can be administered orally, or can be administered via other administration routes acceptable to clinical treatment, such as local administration, intraperitoneal injection, intravenous injection, etc.
[0025] In the following examples, the liver cancer cells used were human liver cancer cell lines MHCC97-H, SMMC7721, SNU449 and mouse liver cancer cell line Hepa1-6; trifluoromethyl tuberculin (TFMT) used was purchased from Taoshu Biological. Other reagents, instruments and equipment used were all conventional experimental materials.
[0026] Example 1
[0027] This example demonstrates the effect of TFMT on the proliferation ability of liver cancer cells through in vitro cell experiments. The specific method is as follows:
[0028] 1. Cell proliferation assay:
[0029] Human liver cancer cell lines MHCC97-H, SMMC7721, SNU449 and mouse liver cancer cell line Hepa1-6 were selected as subjects for liver cancer cell proliferation experiments, and the liver cancer cells were cultured using appropriate culture medium and protease. The liver cancer cells in the logarithmic growth phase were inoculated into 96-well plates, with 1000 cells per well.
[0030] The next day, different concentrations of trifluoromethyl tuberculin (TFMT) were added to intervene in the above liver cancer cells. The concentration range of trifluoromethyl tuberculin was 0.02-0.24 μM, and the corresponding concentration of dimethyl sulfoxide (DMSO) was used as a negative control group. The 96-well plate inoculated with cells was placed in a cell culture incubator and cultured at 37°C and 5% CO2 for 1-3 weeks. After that, the proliferation of cells was detected every 24 hours using the CCK8 kit for a total of 4 days.
[0031] The experimental results are as follows Figure 1As shown, compared with the negative control group, for the human liver cancer cell lines MHCC97-H, SMMC7721, SNU449 and the mouse liver cancer cell line Hepa1-6 intervened with trifluoromethyltuberculin (TFMT), the concentrations of TFMT shown in the figure can significantly inhibit the proliferation of liver cancer cells.
[0032] 2. Cell colony assay:
[0033] The liver cancer cells in the logarithmic growth phase in step 1 above were seeded into a six-well plate, with 1000 cells per well.
[0034] The next day, 0.06 μM trifluoromethyl tuberculin (TFMT) was added to intervene in the above liver cancer cells, and the corresponding concentration of dimethyl sulfoxide (DMSO) was used as a negative control group. The 6-well plate inoculated with cells was placed in a cell culture incubator and cultured at 37°C and 5% CO2. After 14 days of culture, when clones (colonies) visible to the naked eye appeared in the culture dish, the culture was terminated to end the experiment, the cells were fixed, crystal violet stained, the number of colonies was counted and photographed.
[0035] Cell colony photos and corresponding colony number statistics, such as Figure 2 As shown in the figure, for human liver cancer cell lines MHCC97-H, SMMC7721, SNU449 and mouse liver cancer cell line Hepa1-6, the number of cell colonies decreased significantly after TFMT intervention compared with the negative control cells, indicating that TFMT can significantly inhibit the proliferation of liver cancer cells.
[0036] Example 2
[0037] This example uses the Hepa1-6 mouse liver cancer subcutaneous tumor model to demonstrate the inhibitory effect of trifluoromethyl tuberculin (TFMT) on liver cancer at the animal level. The specific method is as follows:
[0038] (1) Constructing a Hepa1-6 subcutaneous tumor model, digesting mouse liver cancer cells Hepa1-6 cells in the logarithmic growth phase, counting and resuspending in PBS to 3×10 6 / ml of cell suspension, selected 30 6-8 week old C57BL / 6 mice with the same growth condition and good condition, removed the armpit hair of C57BL / 6 mice, extracted Hepa1-6 cell suspension with a 1ml syringe, and injected 100ul subcutaneously in the armpit of each mouse. After pulling out the needle tip, quickly press the puncture point with a cotton swab for 2 minutes.
[0039] (2) After tumor inoculation, subcutaneous tumors of approximately 100 mm were observed. 3The 20 mice were randomly divided into 2 groups, and 10 mice in each group were injected. The tumor-bearing mice were injected once every 3 days (on the 0th day, the 3rd day, and the 6th day). The experimental group was injected with TFMT at a dosage of 5 mg / kg / mouse, and the control group was injected with DMSO at the same concentration.
[0040] (3) The weight changes and tumor growth of mice were recorded every 2 days. After 2 weeks, the mice were killed, the tumors were removed, and the tumor size was measured.
[0041] (4) The mouse serum was isolated and its liver function was measured. The main organs of the mice including the heart, liver, spleen, lung and kidney were isolated, fixed with formalin and sliced for histopathological analysis.
[0042] The results are as follows, Figure 3 A in the figure shows the growth of subcutaneous liver cancer tumors in mice after TFMT intervention. Figure 3 Panel B shows a picture of the subcutaneous tumor after TFMT intervention. Figure 3 C in Figure 2 shows the statistical results of mouse survival time. Figure 3 The results showed that compared with the control group, TFMT intervention could significantly inhibit the growth of subcutaneous tumors in mice and prolong the survival of tumor-bearing mice.
[0043] Figure 4 A in the figure shows that all the detection indicators of mice after TFMT intervention were within the normal range and had no significant differences compared with the control group. Figure 4 Panel B shows that the body weight of mice after TFMT intervention was not significantly different from that of the control group. Figure 4 C in the figure shows that the structures of organs in all experimental groups are intact, the nucleus and cytoplasm are clearly distinguished, and no obvious changes in tissue morphology were observed, indicating that TFMT intervention will not cause obvious damage to normal organs.
[0044] Example 3
[0045] This example uses a mouse liver cancer subcutaneous tumor model to demonstrate that TFMT has the ability to regulate the tumor immune microenvironment. The specific method is as follows:
[0046] (1) Constructing a Hepa1-6 subcutaneous tumor model, digesting mouse liver cancer cells Hepa1-6 cells in the logarithmic growth phase, counting and resuspending in PBS to 3×10 6 / ml of cell suspension, selected 30 6-8 week old C57BL / 6 mice with the same growth condition and good condition, removed the armpit hair of C57BL / 6 mice, extracted Hepa1-6 cell suspension with a 1ml syringe, and injected 100ul subcutaneously in the armpit of each mouse. After pulling out the needle tip, quickly press the puncture point with a cotton swab for 2 minutes.
[0047] (2) After tumor inoculation, a subcutaneous tumor of approximately 100 mm was observed. 3 The 20 mice were randomly divided into 2 groups, and 10 mice in each group were injected. The tumor-bearing mice were injected once every 3 days (on the 0th day, the 3rd day, and the 6th day). The experimental group was injected with TFMT at a dosage of 5 mg / kg / mouse, and the control group was injected with DMSO at the same concentration.
[0048] (3) After 28 days of intervention, the mice were killed, the tumors were removed, and transcriptome sequencing was performed on the tumor tissues. Based on the transcriptome sequencing results, the abundance of follicular helper T cells (Tfh) in the tumor tissues was analyzed using CIBERSORT software. Figure 5 As shown in Figure A, TFMT intervention promotes the abundance of follicular helper T cells in tumor tissues. After digesting the tumor tissue into a single cell suspension, flow cytometry was used to detect the abundance of CD3+CD4+CXCR5+PD1+T cells to reflect the level of tumor-infiltrating follicular helper T cells. The results are shown in Figure 5 As shown in Figure 2B, TFMT intervention promoted the infiltration of follicular helper T cells in the tumor site.
[0049] Since follicular helper T cells directly affect the differentiation and maturation of B cells by regulating germinal centers (GC), thereby promoting anti-tumor immune response, the above experiments collectively show that TFMT can inhibit liver cancer by promoting the infiltration of follicular helper T cells in the tumor site and enhancing the anti-tumor immune response.
Claims
1. A use of trifluoromethyl tuberculin in the preparation of a drug for treating cancer.
2. The use according to claim 1, characterized in that: The cancer includes hepatocellular carcinoma.
3. The use according to claim 2, characterized in that: The drug for treating hepatocellular carcinoma can inhibit the proliferation of liver cancer cells.
4. The use according to claim 3, characterized in that: The drug for treating hepatocellular carcinoma can promote the infiltration of follicular helper T cells in liver cancer sites.
5. The use according to any one of claims 2 to 4, characterized in that: The medicine for treating hepatocellular carcinoma comprises trifluoromethyl tuberculin and pharmaceutically acceptable excipients.
6. The use according to claim 5, characterized in that: The effective dose of trifluoromethyl tuberculin is 1 mg-10 mg / kg / day.