Application of FXR agonist as radiation sensitizer in preparation of anti-hepatoma drugs

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of an FXR agonist as a radiation sensitizer in preparation of anti-liver cancer drugs, in particular to application of obeticholic acid (OCA) combined radiation in treatment of liver cancer. When an FXR agonist OCA is used for activating FXR, it is found that activation of FXR can inhibit STAT3 phosphorylation by targeting SOCS3 to inhibit tumor stem cells, and OCA combined radiation can kill tumor stem cells more effectively. OCA can be used as a radiation sensitizer to enhance the killing effect of liver cancer stem cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of an FXR agonist as a radiation sensitizer in the preparation of anti-liver cancer drugs. Background Art

[0002] Primary liver cancer is the sixth most common malignant tumor worldwide and the third leading cause of cancer death. Patients with this type of cancer often have chronic liver disease or cirrhosis, with common causes including chronic hepatitis B or C virus infection, chronic alcohol consumption, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis. Cancer stem cells, a type of cell within tumor tissue capable of continuous self-renewal and differentiation, are a key driver of tumor recurrence and treatment resistance.

[0003] Radiotherapy is a mainstay of cancer treatment, with approximately half to two-thirds of cancer patients receiving radiation therapy during treatment. Different cell types have varying sensitivities to radiation, leading to varying therapeutic outcomes. Radiation resistance is a major challenge in achieving optimal radiation therapy results. Cancer stem cells (CSCs) play a crucial role in this resistance, resisting the damaging effects of radiation through various pathways. STAT3, in particular, plays a crucial role in maintaining the stemness of CSCs.

[0004] Because 70% of cancers exhibit overactivation of STAT3, the therapeutic effects of STAT3 inhibition have attracted research interest. Reports suggest that co-inhibition of STAT3 and FOXM1 may be an effective approach for preventing radioresistance, as they regulate multiple aspects of radioresistance, including cancer stem cells, DNA damage repair, and protection against ROS. While studies have shown that STAT3 inhibition clearly reverses radioresistance, its specific mechanism of action and its relationship to radiation dose and regimen remain poorly understood. Furthermore, due to the complexity of tumorigenesis, no STAT3 inhibitors have reached the market to date. Therefore, the exploration of combined approaches for radiation and STAT3 inhibitors, as well as the development of STAT3 inhibitor drugs, remain pressing challenges in cancer treatment.

[0005] The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily and is highly expressed in multiple tissues, including the liver, intestine, kidney, and adrenal gland. FXR, as a ligand-activated transcription factor, regulates the expression of target genes involved in enterohepatic circulation and metabolic homeostasis. FXR is considered a negative regulator of tumors. FXR deficiency in mice leads to increased colon cell proliferation and spontaneous liver tumors. FXR is also considered a regulator of inflammatory and immune responses in various diseases. Obeticholic acid (OCA), developed by Intercept Pharmaceuticals in the United States, is the first drug developed in 20 years for the treatment of cholestatic liver disease. As a bile acid mimetic, it activates FXR. We have found that FXR inhibits liver cancer stem cells by targeting SOCS3 and inhibiting STAT3 phosphorylation.

[0006] In the present invention, the applicant discovered that radiation cannot effectively kill liver cancer stem cells, but the FXR agonist OCA can effectively inhibit liver cancer stem cells. OCA combined with radiation can effectively kill liver cancer stem cells. OCA can also act as a radiosensitizer to enhance the killing effect of liver cancer stem cells. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides the use of FXR agonists as radiation sensitizers in the preparation of anti-liver cancer drugs.

[0008] The purpose of the present invention is to provide the use of an FXR agonist as a radiation sensitizer in the preparation of an anti-liver cancer drug; the FXR agonist may be obeticholic acid; and the radiation may be SBRT.

[0009] Another object of the present invention is to provide the use of FXR agonists as radiation sensitizers in the preparation of anti-liver cancer drugs, specifically using FXR as a target to obtain radiation sensitizers.

[0010] The radiation sensitizer targeting FXR includes gene drugs that enhance or inhibit FXR expression, and chemical drugs that regulate the expression of FXR and its target genes through FXR ligands.

[0011] The gene drug or chemical drug includes a pharmaceutically acceptable carrier in a clinically acceptable form. The gene drug contains an FXR expression vector or an interference vector; the chemical drug contains a ligand that regulates FXR and a ligand that regulates FXR target genes.

[0012] The ligands of FXR include chenodeoxycholic acid (CDCA), obeticholic acid (OCA), GW4064, cholic acid (CA), and deoxycholic acid (DCA).

[0013] The radiation mentioned above refers to clinically used radiotherapy options, including stereotactic body radiation therapy (SBRT), image-guided radiation therapy (IGRT), volumetric arc modulated radiation therapy (VMAT), conventional radiotherapy, and hypofractionated radiotherapy.

[0014] The radiation mentioned above refers to radiotherapy schemes used in clinical practice, including gamma-ray, X-ray, proton, and heavy ion radiotherapy.

[0015] The present invention has significant technical effects.

[0016] The inventors have discovered that OCA combined with radiation can effectively kill liver cancer stem cells. OCA can be used as a radiation sensitizer to treat liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 OCA enhanced the inhibition rate of tumor spheres by radiation; (a) Effect of OCA on the tumor formation rate of tumor spheres; (b) Effect of OCA combined with radiation on the tumor formation rate of tumor spheres; (c) Effect of OCA combined with radiation on tumor sphere growth; (d) Image of tumor sphere formation; (e) Survival rate of tumor spheres in the OCA combined with radiation group. Compared with the MOCK group, ***P < 0.001, compared with the 15Gy group, # P<0.05.

[0018] Figure 2 OCA enhances the inhibition of tumors by high-dose radiation; (a) Pictures of tumor formation in tumor-bearing mice in different treatment groups; (bc) Statistics of tumor volume and weight in tumor-bearing mice in different treatment groups; (d) Survival curves of tumor-bearing mice in different treatment groups; (e) Tumor growth curves in tumor-bearing mice in different treatment groups; (f) Tumor inhibition rate in tumor-bearing mice in different treatment groups; (g) Liver function test in tumor-bearing mice in different treatment groups. Compared with the MOCK group, **P<0.01, ***P<0.001, compared with the 15Gy group, # P<0.05.

[0019] Figure 3 OCA in tumor spheres enhances the inhibition of high-dose radiation on cancer stem cells; (a) CYP7A1 gene expression; (bc) expression of cancer stem cell markers and stemness genes; (de) protein expression and analysis of cancer stem cell marker CD133. Compared with the MOCK group, *P < 0.05, **P < 0.01, ***P < 0.001, compared with the 15 Gy group, # P<0.05, ## P<0.01, compared with OCA group, & P<0.05, &&& P<0.001.

[0020] Figure 4OCA enhances the inhibition of high-dose radiation on cancer stem cells in tumor-bearing mice; (a) CYP7A1 gene expression; (bc) expression of cancer stem cell markers and stemness genes; (de) protein expression and analysis of the cancer stem cell marker CD133. Compared with the MOCK group, *P < 0.05, **P < 0.01, ***P < 0.001, compared with the 15 Gy group, # P<0.05, ## P < 0.01, ### P<0.001.

[0021] Figure 5 OCA in tumor spheres exerts radiosensitization by inhibiting STAT3 phosphorylation; (a) Expression of genes related to the STAT3 signaling pathway; (bc) Expression and analysis of proteins related to the STAT3 pathway. Compared with the MOCK group, *P < 0.05, **P < 0.01, ***P < 0.001, compared with the 15 Gy group, # P<0.05, ## P<0.01, compared with OCA group, & P<0.05.

[0022] Figure 6 OCA exerts radiosensitization effect in tumor-bearing mice by inhibiting STAT3 phosphorylation; (a) Expression of genes related to the STAT3 signaling pathway; (bc) Expression and analysis of proteins related to the STAT3 pathway. Compared with the MOCK group, *P < 0.05, **P < 0.01, ***P < 0.001, compared with the 15 Gy group, # P<0.05, ## P < 0.01, ### P<0.001. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to specific embodiments.

[0024] Example 1.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] 12-week-old wild-type C57BL / 6N male mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.; methylcellulose was purchased from Taosu, product catalog number 9004-67-5; obeticholic acid (OCA) was purchased from Taosu, product catalog number 459789-99-2; ALB, ALT, and AST detection kits were purchased from Fujifilm, Japan; SOCS3 antibody was purchased from abcam, product catalog number ab280884; p-STAT3 antibody was purchased from abcam am, product catalog number ab32143; CD133 antibody: purchased from Proteintech, product catalog number 18470-1-AP; MYC antibody: purchased from Proteintech, product catalog number 60003-2-IG; CCND1 antibody: purchased from Proteintech, product catalog number 60186-1-IG; BCL2 antibody: purchased from Proteintech, product catalog number 68103-1-IG.

[0027] The present invention relates to an experimental result detection method.

[0028] 1. Tumor-bearing mouse model.

[0029] In each experiment, 4-6 week old C57BL / 6N male mice were selected and 6×10 6 After the Hepa1-6 cells were subcutaneously injected into the groin area of ​​the mice, the mice were maintained in the original environment and the tumor growth of the mice was observed every day. When the tumor grew to 0.8×0.8cm in size, the mice with relatively uniform tumor size were randomly divided into four groups: MOCK group (control group), OCA group (30mg / kg / 2d by oral gavage), 15Gy radiation group, and 15Gy+OCA group (OCA was gavaged 2 days before 15Gy radiation, 30mg / kg / 2d). The tumor size was measured regularly, and when the tumor of the control group mice grew to 1500mm 3 Mice in the experimental group were killed at 8 days after irradiation. Serum was collected and tested for liver function indicators using a biochemical analyzer. Tumors were removed, weighed, and photographed. Tumor tissues were fixed or frozen in a -80°C medical refrigerator for subsequent experiments. In the mouse survival experiment, after the mice in different groups were treated, they continued to be maintained in the original environment. The growth of the tumor in the mice was observed and recorded every day. When the tumor grew to about 1500mm 3 When the size reached 400 μg / cm2, the mice were sacrificed and considered dead.

[0030] 2. Radiotherapy of tumor-bearing mice.

[0031] Mice requiring radiotherapy were anesthetized with tribromoethanol (250 mg / kg). After the mice entered a deep anesthesia state, a phantom design was performed on the mice and a CT scan was performed. The target area was irradiated based on the CT scan image and a radiotherapy plan was prepared. Radiation was performed using the image-guided gamma-ray stereotactic radiotherapy system developed by Xi'an Dayi Group. The mice were placed on the treatment bed for image-guided γ-ray stereotactic radiotherapy.

[0032] 3. Detection of ALB, ALT, AST and TP-D levels in plasma.

[0033] Eyeballs of mice treated with different treatments were removed and blood was collected. The blood was placed in a heparinized centrifuge tube and centrifuged at 6000 rpm at 4°C for 15 minutes. Plasma was then collected and immediately assayed for ALB, ALT, and AST levels using a Fuji Biochemical Analyzer.

[0034] 4. Protein blotting technology.

[0035] (1) Protein preparation of cell / tissue samples

[0036] ① Protein extraction

[0037] Prepare RIPA lysis buffer and protease inhibitors in a 100:1 volume ratio, mix thoroughly, and store on ice. Remove 0.5 g of tissue from a -80°C freezer and place in a centrifuge tube. Add 500 μL of the prepared protein lysis buffer and two 3 mm sterile steel beads. Grind the tissue in a tissue disruptor. After grinding, incubate the tissue homogenate on ice for 30 minutes. Centrifuge at 13,200 rpm at 4°C for 15 minutes. Transfer the supernatant to a fresh centrifuge tube using a pipette and store on ice.

[0038] ②Measure cell / tissue protein concentration

[0039] The protein concentration was determined using a BCA protein quantification kit.

[0040] ③Preparation of protein for Western blot

[0041] Calculate the amount of protein to be loaded as needed, and then add 5× loading buffer (Omni-Easy TM Add the calculated volume of instant protein loading buffer, ddH2O, and protein sample to a 1.5mL centrifuge tube. Vortex to mix thoroughly, then centrifuge briefly. Denature the sample in a dry block at 95°C for 5 minutes. After 5 minutes, allow the sample to cool slightly and to clear the cap. Securely cap the tube and store in a -20°C refrigerator.

[0042] (2) Western blot

[0043] Prepare the necessary gel rack, sandwich frame, sealing gasket, glass plate, and electrophoresis comb. Evenly add the lower layer of gel first, then the upper layer of gel, from the top of the glass plate. Insert the electrophoresis comb and let it sit for 30 minutes to allow the gel to solidify.

[0044] (3) Protein sample electrophoresis

[0045] After the solidified protein electrophoresis gel is fixed, place it in the electrophoresis tank. Add 1× electrophoresis buffer to the tank, pull the electrophoresis comb upward, and use a syringe to rinse any remaining gel from the channels. Add the protein maker and protein sample to the corresponding channels. After loading, adjust the voltage to 150V and terminate the electrophoresis when the maker reaches the target band position and the target band is fully separated.

[0046] (4) Transfer

[0047] Prepare 1x transfer buffer in advance and pre-chill the 1x transfer buffer, sponge pad, filter paper, and transfer cassette in a 4°C refrigerator. After electrophoresis, activate the PVDF membrane in methanol. Remove the glass plate, cut off any excess gel, and place it in the pre-chilled transfer buffer. Remove the transfer cassette and place the following in the correct order: sponge, filter paper, PVDF membrane, PAGE gel, filter paper, and sponge. Secure the transfer cassette and place it in the transfer tank. Pour 1x transfer buffer into the transfer tank to the specified level. Connect the power supply to 100V for 100 minutes.

[0048] (5) Closed

[0049] After the transfer is completed, the PVDF membrane is removed, washed once with 1×TBST, and then incubated on a shaker with protein-free rapid blocking buffer for 25-30 minutes.

[0050] (6) Incubation with primary antibody

[0051] After blocking, rinse three times with 1× TBS (10 min each time with rapid shaking). Prepare the primary antibody as needed. After washing, label the membrane and place it in the primary antibody incubation box on a shaker at 4°C overnight.

[0052] (7) Incubation with secondary antibody

[0053] Prepare the secondary antibody required for the primary antibody in advance and dilute it 2000-fold in 1× TBST. After the primary antibody incubation, rinse three times with 1× TBST for 10 minutes each. Place the membrane in the secondary antibody incubation box and incubate on a shaker at room temperature for 90 minutes.

[0054] (8) Exposure

[0055] After the secondary antibody incubation, rinse three times with 1× TBST for 10 minutes each. Drain the membrane to remove excess liquid, keeping it moist. Use a pipette to evenly drop the luminescent solution onto the membrane, and then place it in a chemiluminescence analyzer to detect the chemiluminescence signal.

[0056] (9) Western blot analysis

[0057] Image J image analysis software was used to quantitatively analyze the results of the target protein.

[0058] 5. Fluorescence quantitative PCR.

[0059] (1) Extract RNA.

[0060] ① Quickly freeze the removed tumor tissue in liquid nitrogen and store in a -80°C freezer until needed. ② Take 0.5g of frozen tissue, add 500μl of TRI-regent, and lyse the cells using a tissue disruptor. ③ Add BCP layered cell / tissue lysis buffer, centrifuge at 12,000g for 15 minutes at 4°C, and transfer the supernatant to a new EP tube. ④ Add 2-protocol (isopropanol) (2-protocol:supernatant = 1:1 volume ratio), mix up and down more than 10 times, and let stand for 10 minutes to allow for full RNA extraction. Centrifuge at 12,000g for 10 minutes at 4°C. ⑤ Discard the supernatant, add 1ml of 75% DEPC ethanol, and wash the RNA once, gently pipetting. Centrifuge at 7,500g for 5 minutes at 4°C. ⑥ Aspirate the supernatant and dry on a laminar flow hood for 10 minutes. Dissolve the RNA in PCR water, place in a 55°C water bath for 10 minutes, and store in a -20°C freezer.

[0061] (2) RNA is reverse transcribed into cDNA.

[0062] ① Add samples in the following order: H2O—RNA—oligdT. Mix thoroughly, centrifuge, and place the PCR tube in a PCR instrument at 65°C for 10 minutes.

[0063] ②Reaction system, see Table 1:

[0064] Table 1 PCR reaction system.

[0065]

[0066] After mixing, add the cDNA to the PCR tube, mix thoroughly, and centrifuge. Place the PCR tube in a PCR instrument and incubate at 42°C for 90 minutes, followed by 95°C for 5 minutes. After the reaction is complete, store the cDNA in a -20°C refrigerator.

[0067] (3)real-time PCR.

[0068] ① Dilute the cDNA sample. ② Serial dilution of the standard curve sample: Take a diluted cDNA sample and serially dilute it to a final concentration of 6 ng / μl, 0.6 ng / μl, 0.06 ng / μl, 0.006 ng / μl, and 0.0006 ng / μl. ③ Prepare the β-actin and CD133 gene mix. ④ Load the sample: Use an 8-well tube set for real-time PCR. Add the following solution: 15 μl / well of the mix → 5 μl / well of the diluted cDNA sample. After loading, vortex the 8-well tube set or 96-well plate on a vortex mixer to mix thoroughly. Centrifuge. Start the 7500 Real-Time PCR System and place the 8-well plate into the system for testing. ⑤ Analysis of results: Divide the value of each well by the corresponding internal reference value (β-actin) before comparing the results of the corresponding experimental groups.

[0069] The specific experimental process of the present invention is as follows.

[0070] 1. OCA enhances the inhibition rate of radiation on tumor nodules

[0071] Hepa1-6 cells were cultured in serum-free tumor sphere medium to form tumor spheres. When the tumor spheres grew to 3-4 cells, they were treated with different concentrations of OCA (5μM, 10μM, 20μM, 50μM, and 80μM). The higher the concentration, the more obvious the inhibitory effect on tumor spheres. When the OCA concentration was 20μM, the tumor sphere formation rate was about 50% ( Figure 1 a). Tumor spheres cultured for 2-3 days in serum-free conditions were treated with different radiation doses (6 Gy, 10 Gy, 15 Gy) and different concentrations (5 μM, 10 μM, 20 μM) of OCA. The results showed that radiation or OCA alone significantly inhibited the formation and growth of tumor spheres, and the combination of the two showed a significant additive effect ( Figure 1 bc). Among them, 15Gy radiation combined with OCA (20μM) showed the best tumor sphere inhibition effect. The formation and generation of tumor spheres were significantly inhibited, accompanied by the production of a large amount of cell debris. In addition, the combined treatment showed a stronger tumor sphere inhibition rate than radiation alone ( Figure 1 The above results indicate that OCA enhances the inhibitory effect of radiation on tumor nodules.

[0072] 2. OCA enhances tumor suppression by high-dose radiation

[0073] 6 × 10 6Hepa1-6 cells were used to establish a tumor-bearing mouse model. When the tumor grew to 6mm×6m, the mice were randomly divided into four groups according to tumor size: MOCK group (control group), OCA group (gavage 30mg / kg / 2d), 15Gy radiation group, and 15Gy+OCA group (gavage OCA 30mg / kg / 2d 2 days before 15Gy radiation). OCA was gavaged every two days, and 15Gy radiation treatment was performed using a domestically produced large-scale stereotactic radiation therapy system. A single dose of 15 Gy was administered. The growth of the tumor was monitored regularly. The mice in the experimental group were killed 8-10 days after radiation depending on the size of the tumor. Mouse survival experiment: The mice in the different treatment groups were kept in their original environment and the growth of the tumor was observed and recorded regularly. 3 When the size of the mouse is less than 1 mm, the mouse is considered dead and is sacrificed.

[0074] The experimental results showed that OCA or 15Gy radiation treatment of tumor-bearing mice could significantly inhibit tumor growth. The combination of the two showed a more significant tumor inhibition effect ( Figure 2 ac). Through mouse survival experiments and growth curve analysis, the 15Gy radiation combined with OCA group had a significantly higher survival rate than the 15Gy radiation group, and the tumor inhibition effect was also more significant ( Figure 2 df). In addition, serum biochemistry results showed that there was no significant change in liver function indicators among mice in different treatment groups, which means that different treatments did not cause side effects in mice ( Figure 2 g). The above results indicate that radiation combined with OCA has a significant additive effect on inhibiting tumor growth in tumor-bearing mice, and OCA enhances the inhibitory effect of radiation on tumor spheres.

[0075] 3. OCA in tumor spheres enhances the inhibition of high-dose radiation on cancer stem cells

[0076] Tumor spheres cultured in serum-free medium with different treatments were collected and RNA and protein were extracted. qRT-PCR was used to detect the expression of CYP7A1, a negative feedback target gene of FXR. It was found that OCA or 15 Gy radiation downregulated CYP7A1 gene expression, and the combination of OCA and 15 Gy radiation further downregulated CYP7A1 gene expression ( Figure 3 a). qRT-PCR detection of cancer stem cell markers and stemness genes revealed that OCA downregulated the gene expression of β-catenin, BMI1, POU5F1, CD133, CD44, and CD24. Radiation downregulated the gene expression of β-catenin, CD326, and CD133, but upregulated the gene expression of BMI1, POU5F1, CD44, and CD24. OCA further downregulated the gene expression of radiation-regulated cancer stem cell markers and stemness genes ( Figure 3Western blot experiments also yielded similar results. OCA or 15Gy radiation downregulated the protein expression of the cancer stem cell marker CD133, while OCA combined with 15Gy radiation further downregulated the expression of CD133 protein ( Figure 3 The above results indicate that OCA inhibits the stemness of cancer stem cells by activating FXR, and the effects of high-dose radiation on cancer stem cells are complex, but OCA combined with high-dose radiation inhibits the stemness of cancer stem cells.

[0077] 4. OCA enhances the inhibition of cancer stem cells by high-dose radiation in tumor-bearing mice

[0078] Tumors from tumor-bearing mice treated with different treatments were isolated, and RNA and protein were extracted. qRT-PCR was used to detect the expression of CYP7A1, a negative feedback target gene of FXR. It was found that OCA or 15Gy radiation downregulated the gene expression of CYP7A1, and the combination of OCA and 15Gy radiation further downregulated the gene expression of CYP7A1 ( Figure 4 a). qRT-PCR detection of cancer stem cell markers and stemness genes revealed that OCA downregulated the gene expression of β-catenin, BMI1, POU5F1, CD326, CD133, CD44, and CD24. Radiation downregulated the gene expression of β-catenin, CD326, and CD133, but upregulated the gene expression of BMI1, POU5F1, CD44, and CD24. OCA further downregulated the gene expression of radiation-regulated cancer stem cell markers and stemness genes ( Figure 4 Western blot experiments also yielded similar results. OCA or 15Gy radiation downregulated the expression of CD133, a cancer stem cell marker, while OCA combined with 15Gy radiation further downregulated CD133 protein expression ( Figure 4 The above results indicate that OCA inhibits the stemness of cancer stem cells by activating FXR, and the effects of high-dose radiation on cancer stem cells are complex, but OCA combined with high-dose radiation inhibits the stemness of cancer stem cells.

[0079] 5. OCA in tumor spheres exerts radiosensitization by inhibiting STAT3 phosphorylation

[0080] Tumor spheres cultured in serum-free medium with different treatments were collected and RNA and protein were extracted. The expression of genes related to the STAT3 signaling pathway was detected by qRT-PCR. The results showed that OCA downregulated the gene expression of STAT3, cMYC, and BCL2, and upregulated the gene expression of SOCS3. The 15Gy radiation group upregulated the gene expression of STAT3, SOCS3, and BCL2, and downregulated the gene expression of cMYC. The 15Gy radiation combined with OCA group downregulated the gene expression of STAT3, cMYC, and BCL2, and upregulated the gene expression of SOCS3 compared with the 15Gy radiation group ( Figure 5 a). Western blot experiments also showed similar results. OCA or 15Gy radiation downregulated the protein expression of p-STAT3, cMYC, BCL2, and CCND1, and upregulated the protein expression of SOCS3. OCA combined with 15Gy radiation further downregulated the protein expression of p-STAT3, cMYC, BCL2, and CCND1, and upregulated the protein expression of SOCS3 ( Figure 5 bc). The above results indicate that OCA upregulates the expression of FXR target gene SOCS3 by activating FXR, thereby inhibiting STAT3 phosphorylation and exerting radiosensitization effect.

[0081] 6. OCA exerts radiosensitization in tumor-bearing mice by inhibiting STAT3 phosphorylation

[0082] Tumors from tumor-bearing mice treated with different methods were isolated, and RNA and protein were extracted. The expression of genes related to the STAT3 signaling pathway was detected by qRT-PCR experiments. The results showed that OCA downregulated the gene expression of STAT3, cMYC, and BCL2, and upregulated the gene expression of SOCS3. The 15Gy radiation group upregulated the gene expression of STAT3, SOCS3, and BCL2, and downregulated the gene expression of cMYC. The 15Gy radiation combined with OCA group downregulated the gene expression of STAT3, cMYC, and BCL2, and upregulated the gene expression of SOCS3 compared with the 15Gy radiation group ( Figure 6 a). Western blot experiments also showed similar results. OCA or 15Gy radiation downregulated the protein expression of p-STAT3, cMYC, BCL2, and CCND1, and upregulated the protein expression of SOCS3. OCA combined with 15Gy radiation further downregulated the protein expression of p-STAT3, cMYC, BCL2, and CCND1, and upregulated the protein expression of SOCS3 ( Figure 6 bc). The above results indicate that OCA upregulates the expression of FXR target gene SOCS3 by activating FXR, thereby inhibiting STAT3 phosphorylation and exerting radiosensitization effect.

Claims

1. Application of FXR agonists as radiosensitizers in the preparation of anti-liver cancer drugs.

2. Use of the FXR agonist according to claim 1 as a radiation sensitizer in the preparation of an anti-liver cancer drug, characterized in that: Specifically, FXR agonists are combined with radiation to obtain drugs for treating liver cancer.

3. Use of the FXR agonist according to claim 2 as a radiation sensitizer in the preparation of an anti-liver cancer drug, characterized in that: Specifically, these are drugs for treating liver cancer that target FXR, including gene drugs that enhance or inhibit FXR expression, or chemical drugs that regulate the expression of FXR or FXR target genes through FXR ligands.

4. Use of the FXR agonist according to claim 2 as a radiation sensitizer in the preparation of an anti-liver cancer drug, wherein the radiation comprises stereotactic radiotherapy, image-guided radiotherapy, volumetric arc-modulated radiotherapy, radiotherapy or hypofractionated radiotherapy.

5. Use of the FXR agonist as claimed in claim 3 as a radiation sensitizer in the preparation of an anti-liver cancer drug, characterized in that: The gene drug or chemical drug includes a pharmaceutically acceptable carrier in a clinically acceptable form; the gene drug contains an FXR expression vector or an interference vector; the chemical drug contains a ligand that regulates FXR or a ligand that regulates FXR target gene.

6. Use of the FXR agonist according to claim 4 as a radiation sensitizer in the preparation of an anti-liver cancer drug, characterized in that: Ligands include chenodeoxycholic acid (CDCA), obeticholic acid (OCA), GW4064, cholic acid (CA), or deoxycholic acid (DCA).

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