Application of triterpenoid saponin extract in preparation of medicine for enhancing radiation therapy effect

Abstract

The invention discloses application of a triterpenoid saponin extract in preparation of a medicine for enhancing the effect of radiotherapy, and belongs to the technical field of biomedicine.The Chinese pulsatilla saponin D (SB365) in the formula (I) is a novel triterpenoid saponin extract extracted and identified from Chinese pulsatilla roots, is small in toxic and side effect, can induce apoptosis of liver cancer cells, and can be used for preparing a medicine for enhancing the effect of radiotherapy. A good proliferation inhibition effect is achieved on liver cancer cells; on the other hand, the SB365 can inhibit an HR pathway by inhibiting an AKT / ATM / DDR signal transduction axis and performing targeted combination with RAD51 protein, so that the sensitivity of ionizing radiation to hepatocellular carcinoma is improved.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and specifically relates to the application of triterpenoid saponin extracts in the preparation of drugs for enhancing the effect of radiotherapy. Background Art

[0002] Pulsatilla chinensis is a traditional Chinese herbal medicine widely used in clinical practice of traditional Chinese medicine, with the effects of "cooling blood", detoxifying, anti-infective, anti-viral and enhancing immunity. It was used to treat diseases such as intestinal amoeba bacterial infection, malaria and cancer in ancient China.

[0003] Pulsatilla saponin D (SB365) is a triterpenoid saponin extract of Pulsatilla chinensis, with the chemical formula C 47 H 76 O 17 , and has a broad-spectrum anti-tumor effect, including inhibiting the growth and proliferation of tumor cells, inhibiting migration, invasion and metastasis, and blocking the tumor cell cycle.

[0004] Hepatocellular carcinoma cells are prone to resist ionizing radiation therapy, often leading to the failure of hepatocellular carcinoma treatment or the generation of drug resistance. Therefore, finding a new drug that can enhance the sensitivity of tumor cells to ionizing radiation is of great significance for the clinical treatment of hepatocellular carcinoma. Summary of the Invention

[0005] This application aims to solve one of the above technical problems in the prior art and provides a drug that can enhance the sensitivity of ionizing radiation therapy.

[0006] The technical solution adopted by the present invention is:

[0007] In the first aspect of this application, there is provided the use of triterpenoid saponin extracts in the preparation of drugs for enhancing the effect of radiotherapy, wherein the triterpenoid saponin extracts are compounds represented by formula (I), pharmaceutically acceptable salts thereof, stereoisomers thereof or prodrug molecules thereof:

[0008]

[0009]

[0010] More specifically, in the above technical solution, the radiotherapy is ionizing radiation.

[0011] More specifically, in the above technical solution, the triterpenoid saponin extracts increase DNA damage and apoptosis by inhibiting the activation of AKT, ATM, p-ATM and DNA-PKcs caused by ionizing radiation.

[0012] More specifically, in the above technical solution, the dosage form is selected from: injection, tablet, capsule, kit or patch. Those skilled in the art can prepare the above drug composition into various dosage forms according to specific circumstances, and the preparation method is a well-known technology in the art, so it will not be elaborated in this application.

[0013] More specifically, in the above technical solution, the drug is used for treating liver cancer.

[0014] More specifically, in the above technical solution, the cancer cells of the liver cancer are Huh7 and SMMC-7721.

[0015] In a second aspect, the present application provides an application of a triterpenoid saponin extract in the preparation of a radiosensitizer for radiotherapy, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

[0016]

[0017] In a third aspect, the present application provides an application of a triterpenoid saponin extract in a drug for increasing cancer cell apoptosis, wherein the drug is used in radiotherapy, and the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

[0018]

[0019] In a fourth aspect, the present application provides an application of a triterpenoid saponin extract in the preparation of a drug for inhibiting cancer cell proliferation, wherein the drug is used in radiotherapy, and the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

[0020]

[0021] The "pharmaceutically acceptable salts" are conventional non-toxic salts formed by the reaction of the compound of general formula (I) with inorganic acids or organic acids. For example, the conventional non-toxic salts can be prepared by reacting the compound of general formula (I) with inorganic acids or organic acids. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid, etc., and the organic acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid, etc.; or the sodium salts, potassium salts, calcium salts, aluminum salts or ammonium salts formed by reacting the compound of general formula (I) with inorganic bases after forming esters with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid; or the methylamine salts, ethylamine salts or ethanolamine salts formed by reacting the compound of general formula (I) with organic bases; or the corresponding inorganic acid salts formed by reacting the compound of general formula (I) with lysine, arginine, ornithine after forming esters and then reacting with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid or the corresponding organic acid salts formed by reacting with formic acid, acetic acid, picric acid, methanesulfonic acid and ethanesulfonic acid.

[0022] Stereoisomers refer to different stereoisomers obtained by changing the configurations of multiple chiral centers in the SB365 molecule. Stereoisomers may have different pharmacokinetic properties (such as absorption, distribution, metabolism and excretion) in vivo, but as long as their core structures are similar to SB365, they can still retain their anti-tumor activity. Therefore, stereoisomers are also regarded as having the same pharmacological effects as SB365.

[0023] Prodrug molecules refer to compounds that can release SB365 after metabolism or chemical reactions in vivo. The design of prodrugs is usually to improve the stability, targeting or reduce the toxic and side effects of drugs. After prodrugs are converted into the active ingredient SB365 in vivo, they can still exert their anti-tumor effects.

[0024] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0025] 1. The inventors found that SB365 significantly inhibits the contents of proteins related to the AKT signaling pathway and key proteins of the DDR signaling pathway in Huh7 and SMMC-7721 liver cancer cells, and increases the contents of the DNA damage marker γ-H2AX and the apoptosis marker Cleaved-PARP. And SB365 can target and bind to the RAD51 protein to inhibit the HR pathway.

[0026] 2. SB365 significantly inhibited the cell viability of Huh7 and SMMC-7721 cells, with the highest inhibition on SMMC-7721 cells, with an IC of 50 Only 1.108μM.

[0027] 3. SB365 can inhibit the AKT / ATM / DDR pathway, aggravate DNA damage, sensitize ionizing radiation, significantly inhibit the proliferation of liver cancer cells and increase the apoptosis of liver cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 Schematic diagram of SB365 inhibiting AKT and DDR pathways and targeting RAD51 protein in the HR pathway;

[0030] Figure 2 Schematic diagram of SB365 increasing the sensitivity of liver cancer cells to ionizing radiation;

[0031] Figure 3 Schematic diagram showing that SB365 improves the efficacy of ionizing radiation on liver cancer in animal experiments. DETAILED DESCRIPTION

[0032] In order to more clearly understand the technical content of the present invention, the following examples are specifically described in detail in conjunction with the accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0033] SB365 is a triterpenoid saponin extract from the traditional Chinese herbal medicine Pulsatilla chinensis, with the chemical formula C 47 H 76 O 17 10 mM concentrated SB365 solution was purchased from MCE (purity: 98.60%) and stored at -80°C. When used in the experiment, part of the mother solution was taken and diluted with DMSO to a working concentration of 1 mM.

[0034]

[0035] 1. Experimental Materials and Methods

[0036] Cell lines: The cell lines used in the experiment were human liver cancer Huh7 cells and SMMC-7721 cells, both adherent-growing cells, provided by the Biochemical Laboratory of the Basic Medical College of Jinan University and stored at ultra-low temperature.

[0037] Table 1 Antibodies required for the experiment and their suppliers:

[0038]

[0039]

[0040] Methods: Western blotting experiments were used to detect the protein content in Huh7 cells and SMMC-7721 cells after treatment with SB365 and SB365 combined with ionizing radiation respectively; MTT experiments were used to detect the effects of SB365 and SB365 combined with ionizing radiation on the survival rate of liver cancer cells; cell colony formation experiments were used to detect the effects of SB365 and SB365 combined with ionizing radiation on the proliferation ability of Huh-7 cells; cell thermal migration experiments were used to clarify the action target of SB365. Annexin V-FITC / PI staining and flow cytometry experiments were used to detect the apoptosis rate of Huh-7 cells after treatment with SB365 combined with ionizing radiation. Animal experiments were used to detect the effects of SB365 combined with ionizing radiation on the growth volume and weight of nude mouse xenografts of Huh7 liver cancer cells. It can be understood that ionizing radiation is a known conventional method, and its specific operation method is not described in this application. Those skilled in the art can operate according to actual needs, and the drug and ionizing radiation can be used sequentially or simultaneously.

[0041] Western blotting experiment:

[0042] (1) Protein extraction. Seed the cells in the logarithmic growth phase in a 6-well plate, set the groups, and after overnight adherent culture, perform radiotherapy irradiation treatment. After 24 - 48 h, aspirate the supernatant culture medium, wash away the excess culture medium with PBS, collect the cells and add RIPA strong lysis buffer containing 1% protease inhibitor and 1% protein phosphatase inhibitor for lysis, and lyse on ice for 30 min. Then transfer the lysed cells to a centrifuge tube, centrifuge at a pre-cooled high-speed centrifuge (4°C, 12000 rpm) for 15 min to obtain the protein supernatant, measure the protein concentration of the protein supernatant using a BCA micro protein concentration assay kit, then add 1 / 4 volume of 5×SDS-PAGE protein loading buffer, mix well and heat at 100°C in a metal bath for 5 min to denature the protein, and store the obtained protein sample at -80°C.

[0043] (2) Gel electrophoresis and membrane transfer. Assemble the 4-20% precast protein gel with the electrophoresis apparatus, add 1× electrophoresis buffer to both the inner and outer chambers, then load the Rainbow Protein Maker and the protein samples onto the gel and run the electrophoresis (120V) for about 30 min until the indicator band is close to the bottom of the precast gel. Stop the electrophoresis, take out the gel, cut the gel containing the target protein according to the Maker bands, place it flat on a 0.45 or 0.22 μm PVDF membrane (select different thickness membranes according to the molecular weight of the target protein, and the membrane is pre-soaked and activated with methanol). After confirming that the membrane and the gel are completely adhered, insert them into the membrane transfer clip, place it in the membrane transfer tank, fill it with 1× transfer buffer, and transfer the membrane at a constant current of 250 mA for 90 min. Ice cubes should be filled around the membrane transfer tank to cool it down.

[0044] (3) Blocking and antibody incubation. After the membrane transfer is completed, soak the membrane in 5% blocking milk and incubate it on a shaker at room temperature for 1 h. Then wash off the excess milk with 1× TBST buffer, soak the membrane in the corresponding primary antibody dilution and incubate it overnight (4℃). After the primary antibody incubation, wash the membrane 3 times with 1× TBST buffer. Then put the membrane into the corresponding secondary antibody dilution and incubate it on a shaker at room temperature for 1.5 h. After that, wash the membrane 3 times with 1× TBST buffer again.

[0045] (4) Protein band luminescence and development. After washing the membrane, apply the hypersensitive ECL chemiluminescence solution on the membrane, expose it through a fully automatic chemiluminescence image analysis system, collect the image and analyze the result bands.

[0046] MTT assay:

[0047] (1) Experimental grouping: Design experimental groups, control groups and zero calibration groups. In the control group, add cells and the volume of DMSO in the maximum drug concentration group. In the zero calibration group, only add the culture medium.

[0048] (2) Seed 3000 cells in logarithmic growth phase per well evenly in a 96-well plate. Set 5 replicates for each group, keep the liquid volume in each well consistent, fill the outer circle of the well plate with 200 μL of 1× PBS to reduce the evaporation of the culture medium, and place it in the incubator overnight for cell attachment. Then treat the cells according to the experimental requirements (radiotherapy irradiation), and then culture them in the incubator for 48 hours.

[0049] (3) After culturing for 48 hours, add 20 μL of MTT solution to each well and continue to place it in a 37℃ incubator for 4 h. Discard the supernatant, and add 150 μL of DMSO solution to each well. After the formazan purple crystals are fully dissolved, use a multifunctional microplate reader to detect the absorbance at 570 nm. Subtract the absorbance value of the zero calibration group from the absorbance value of each well to get the effective value. Calculate the average absorbance value of each group, calculate the survival rate of the cells in each group, and calculate the IC using GraphPad Prism 9.1 50And perform statistical analysis, with the mean ± standard deviation (Mean ± SD) representing the values of each group.

[0050] Cell colony formation assay:

[0051] (1) Set up a control group and an experimental group. Take cells in the logarithmic growth phase and seed about 500 - 1000 cells per well evenly into a six-well plate. Shake the plate gently to spread the cells evenly, and then place it in a cell culture incubator overnight to allow the cells to adhere to the plate.

[0052] (2) After the cells adhere to the plate, treat the cells according to the experimental requirements (radiotherapy irradiation). Keep the volume of the culture medium in each group consistent and continue to culture for 7 - 14 days until the clone spots in the control group reach 0.3 - 1.0 mm in size.

[0053] (3) Remove the culture medium, wash the cells 2 - 3 times with PBS. Add an appropriate amount of paraformaldehyde solution to each well and fix at room temperature for 30 min. Then remove the fixative and wash with PBS. Add an appropriate amount of 0.1% crystal violet solution to each well and stain for 30 min. Remove the staining solution, and then gently rinse the plate with running water. After drying, take pictures or scan to save the images. Add 30% glacial acetic acid to dissolve the crystal violet completely. Then transfer the solution in each well to three replicate wells of a 96-well plate respectively. Read the absorbance OD value at a wavelength of 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader to represent the clone value, and use GraphPad Prism 9.1.1 to plot the graph. The mean ± standard deviation (Mean ± SD) represents the values of each group.

[0054] Annexin V-FITC / PI staining and flow cytometry assay:

[0055] (1) Set up a control group and an experimental group. Take cells in the logarithmic growth phase and seed about 200,000 cells per well evenly into a six-well plate. Place it in a cell culture incubator overnight to allow the cells to adhere to the plate. After the cells adhere to the plate, treat the cells according to the experimental requirements (radiotherapy irradiation).

[0056] (2) After 24 h or 48 h, take out the six-well plate and collect the culture medium in the wells into a centrifuge tube. Wash each well 2 times with 1 mL of 1×PBS, and collect the aspirated PBS into the centrifuge tube as well. Add an appropriate amount of trypsin to each well to digest the cells. Transfer the cells into the centrifuge tube and centrifuge at a speed of 1000 rpm for 5 min. Note that the operation should be gentle to avoid physical damage to the cells.

[0057] (3) Discard the supernatant, add 1 mL of PBS to resuspend the cells, repeat centrifugation and discard the supernatant again, retaining the cell pellet. Use the Annexin V / PI double-staining cell apoptosis kit, and sequentially add Binding Buffer, Annexin V, and PI staining solution at a volume ratio of 100:1:1 to suspend and mix the cells evenly. Keep the process in the dark and incubate at room temperature for 10 min. Then, use an intelligent analytical flow cytometer for detection, analyze the apoptosis of the cells, and draw a graph.

[0058] Cell thermal migration experiment:

[0059] (1) Take tumor cells in the logarithmic growth phase, divide them into an experimental group and a control group. Digest the cells and centrifuge, discard the supernatant, and then use the culture medium to resuspend the cell pellet. The number of cells in both groups is kept the same. Add an appropriate concentration of SB365 drug to the cell suspension of the experimental group for treatment, and add an equal amount of DMSO to the cell suspension of the control group. Incubate in an incubator for 2 h.

[0060] (2) Centrifuge the two groups of cells, discard the supernatant, resuspend the cells with 1 ml of PBS, divide the two groups of cells evenly into 9 parts, and heat them gradiently in a thermal cycler (the temperature range of gradient heating can be designed by yourself, such as setting 9 temperature ranges between 40°C and 80°C). Heat each range for 3 min, incubate at room temperature for 3 min, and then quickly freeze.

[0061] (3) Freeze-thaw the cells 3 times repeatedly between liquid nitrogen and room temperature, 3 min each time, to lyse the cells. Then centrifuge the lysate at 12,000 rpm at 4°C for 10 min, take the supernatant, add 1 / 4 volume of 5× SDS-PAGE protein loading buffer, denature the protein in a metal bath at 100°C for 5 min, and then perform Western blotting to detect the changes in the protein contents of AKT, RAD51, β-actin, etc. with temperature. Finally, use ImageJ software to calculate the gray value of the protein band, and use GraphPad Prism software to draw the CETSA curve and perform thermal stability analysis.

[0062] Animal experiment:

[0063] (1) Establish a nude mouse xenograft tumor model by subcutaneous injection of tumor cells. Use BALB / c-nu nude mice, and subcutaneously inoculate 400×10 4 Huh7 cells into each mouse; when the tumor grows to about 100 mm 3 , perform grouping.

[0064] (2) Animal experiments: The groups were divided into a control group, an SB365 (5 mg / kg) group, a radiotherapy (6 Gy) group, and an SB365 combined with radiotherapy group. In the drug treatment groups, the drug (SB365) was intraperitoneally injected once every other day, with a dosage of 5 mg / kg each time, and a total of 12 injections were given. In the radiotherapy group, important organs of the mice were avoided, and radiotherapy with a dose of 6 Gy was performed on the 7th day after grouping. During the experiment, the tumor growth was observed, and the tumor volume was measured every 2 days. The longest diameter a and the shortest diameter b of the tumor were measured, and the tumor volume V was calculated according to the formula a*b 2 / 2. The tumor growth curve was plotted. On the 24th day, the mice were euthanized (anesthetized and decapitated by dislocation of the posterior neck) according to the ethics of experimental animals, the tumor tissues were dissected and weighed; parts of the hearts, livers, kidneys, spleens and other organs of the mice in each group were taken for HE staining and photographed for observation.

[0065] Data processing and statistical analysis:

[0066] The experimental data were statistically analyzed and processed using software SPSS 21.0 and GraphPad Prism 9.1. A representative experiment from three independent experiments was presented, and the data were expressed as mean ± standard deviation (Mean ± SD). The differences between two groups of samples were tested by Student's t-test, and the differences between more than two groups of samples were compared by One Way ANOVA. A P value < 0.05 was considered to have statistically significant differences; image processing was performed using software Adobe Illustrator 2021; schematic diagrams were drawn using FigDraw software.

[0067] Example 1

[0068] To clarify the effect of SB365 on liver cancer, the inventors detected the protein content in Huh7 and SMMC-7721 liver cancer cells after treatment with different concentrations of SB365 for 24 h by Western blotting experiment. As Figure 1 shown in A, SB365 significantly inhibited the contents of AKT signaling pathway-related proteins (AKT, p-AKT S473, p-mTOR) and DDR key proteins (p-ATM, ATM, DNA-PKcs, RAD51, etc.) in Huh7 and SMMC-7721 liver cancer cells, and increased the contents of DNA damage marker γ-H2AX and apoptosis marker Cleaved-PARP. This indicates that SB365 can inhibit the AKT and DDR pathways in liver cancer cells, exacerbate DNA damage and induce apoptosis.

[0069] Next, the MTT experiment was used to detect the effect of SB365 on cell viability. As Figure 1 shown in B, the 48 h IC of SB365 on Huh7 and SMMC-7721 cells 50They were 2.529 μM and 1.108 μM respectively. Further, the inhibitory effect of SB365 on the proliferation of Huh7 cells was detected by colony formation assay. As Figure 1 shown in D, low-concentration SB365 could significantly inhibit the proliferation of Huh7 cells and had a good anti-hepatocellular carcinoma effect.

[0070] To identify the target of SB365 action, the inventors conducted a Cellular Thermal Shift Assay (CETSA). The principle of the CETSA experiment is that when a drug molecule binds to a target protein, it usually becomes stable. As the temperature increases, the protein bound to the drug molecule shows less protein thermal degradation compared to the protein not bound to the drug molecule, that is, the content of undegraded protein is higher, and the thermal degradation curve of this protein shifts to the right. Through the CETSA experiment, it was found that compared with the control group, there was no obvious shift in the temperature degradation curve of AKT protein in Huh7 hepatocellular carcinoma cells in the SB365 treatment group, while the temperature degradation curve of RAD51 protein shifted significantly to the right, as Figure 1 shown in C. This indicates that SB365 binds to the key molecule RAD51 protein of the target HR rather than AKT protein. In addition to inhibiting the AKT / mTOR pathway, SB365 can also target and bind to RAD51 protein to inhibit the HR pathway.

[0071] Example 2

[0072] To investigate whether SB365 can be used as a sensitizer for DNA damage therapy, the inventors treated hepatocellular carcinoma cells with SB365 combined with ionizing radiation. The MTT assay showed that SB365 (1 μM) combined with 2 Gy of ionizing radiation significantly reduced the survival rate of Huh7 and SMMC-7721 cells, as Figure 2 shown in B. Then, through the colony formation assay, the effects of different doses of ionizing radiation on Huh7 cells were detected. As Figure 2 shown in C, ionizing radiation (0, 2, 4, 8 Gy) could inhibit the clonal proliferation of Huh7 cells, which verified the reliability and dose accuracy of the ionizing radiation experiment. Further, SB365 (0.5 μM) combined with ionizing radiation (4 Gy) could synergistically inhibit the clonal proliferation of Huh7 and SMMC-7721 cells, as Figure 2 shown in D. The above results indicate that SB365 can increase the inhibitory effect of ionizing radiation on the proliferation of hepatocellular carcinoma cells.

[0073] The apoptosis rate of Huh-7 cells after treatment with SB365 (1 μM) combined with ionizing radiation (4 Gy) for 24 h was detected by Annexin V-FITC / PI staining and flow cytometry. As Figure 2As shown in E, SB365 combined with ionizing radiation significantly increased the apoptosis rate of Huh7 cells, indicating that SB365 and ionizing radiation have a synergistic effect on inducing apoptosis in liver cancer cells.

[0074] To further confirm whether SB365 exerts its effect by inhibiting the AKT / ATM / DDR pathway and aggravating DNA damage, the inventors detected the protein levels of the AKT pathway, DDR pathway, γ-H2AX, and Cleaved-PARP in Huh7 cells after treatment with SB365 (1 μM) combined with ionizing radiation (4 Gy) through Western blotting experiments. The results are as Figure 2 shown in A. SB365 can inhibit the activation of AKT, ATM, p-ATM, and DNA-PKcs caused by ionizing radiation, and increase the content of the DNA damage marker γ-H2AX and the apoptosis index. This indicates that SB365 sensitizes ionizing radiation by inhibiting the AKT / ATM / DDR pathway and aggravating DNA damage, and SB365 and ionizing radiation have a good synergistic anti-hepatic cancer effect.

[0075] Example 3

[0076] To further confirm the effect of SB365 in sensitizing ionizing radiation against liver cancer, the inventors conducted an experiment on nude mice bearing transplanted tumors of Huh7 liver cancer cells. As Figure 3 shown in B - D, treatment of nude mice with SB365 (5 mg / kg / time, intraperitoneal injection) combined with radiotherapy (6 Gy) significantly inhibited the tumor growth volume and tumor weight of subcutaneous transplanted tumors of Huh7 liver cancer cells in nude mice. The tumor volume and tumor weight of the combined treatment group were significantly lower than those of the other three groups, indicating that SB365 combined with ionizing radiation has a good synergistic anti-hepatic cancer effect, and SB365 is a good radiotherapy sensitizer. The body weights of mice in each group did not show a significant decrease during the experiment. As Figure 3 shown in E, it is suggested that SB365 has low toxicity and has little impact on the mouse body.

[0077] In this application, the inventors first found through Western blotting experiments that SB365 can significantly inhibit the proteins of AKT, p-AKT S473, p-mTOR in Huh7 and SMMC-7721 liver cancer cells, as well as key proteins in the DDR pathway such as ATM, DNA-PKcs, RAD51, etc., inhibit the damage repair of double-stranded DNA, and aggravate DNA damage. MTT experiments found that SB365 significantly inhibited the cell viability of liver cancer cell lines, among which the inhibitory effect on the cell viability of SMMC-7721 cells was the highest, and the IC 50It is only 1.108 μM. The colony formation assay further demonstrated that SB365 could significantly inhibit the proliferation of Huh-7 cells. In addition, the inventors used the CETSA assay to study the target of SB365 and found that the target of SB365 is the RAD51 protein. All these indicate that SB365 has the potential to become a new anti-hepatocellular carcinoma drug.

[0078] The inventors further found through the MTT assay that the combination of SB365 and ionizing radiation could significantly reduce the survival rate of Huh7 hepatocellular carcinoma cells. The colony formation assay showed that SB365 and ionizing radiation could synergistically inhibit the colony proliferation of Huh7 and SMMC-7721 cells. The Annexin V-FITC / PI staining and flow cytometry assay found that the combination of SB365 and ionizing radiation significantly increased the apoptosis rate of Huh7 cells. The Western blotting assay showed that SB365 could inhibit the activation of AKT, ATM, p-ATM, and DNA-PKcs caused by ionizing radiation, and increase the levels of the DNA damage marker γ-H2AX and the apoptosis index Cleaved PARP. All these evidences indicate that SB365 and ionizing radiation have a good synergistic anti-hepatocellular carcinoma effect. The nude mouse xenograft tumor assay of Huh7 hepatocellular carcinoma cells showed that the tumor volume and weight of the group treated with the combination of SB365 and ionizing radiation were significantly lower than those of the control group, the group treated with SB365 alone, or the group treated with ionizing radiation alone, and SB365 did not cause a significant decrease in the body weight of mice. This further confirmed the sensitizing effect of SB365 on ionizing radiation in the treatment of hepatocellular carcinoma.

[0079] In summary, the inventors found that SB365 not only has a good anti-tumor effect on hepatocellular carcinoma, but also can improve the curative effect of radiotherapy on hepatocellular carcinoma by inhibiting the AKT pathway and the DNA damage response, and there will be no obvious increase in toxic side effects. This study clarified the anti-tumor effect and its molecular mechanism of the triterpenoid saponin extract SB365 extracted from Pulsatilla chinensis in hepatocellular carcinoma cells, as well as the mechanism of enhancing the curative effect of radiotherapy on hepatocellular carcinoma cells, providing an experimental basis for the future clinical application of SB365 in the treatment of hepatocellular carcinoma.

[0080] The above-described embodiments only represent several embodiments of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. Use of a triterpenoid saponin extract in the preparation of a medicament for enhancing the effect of radiotherapy, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

2. The application according to claim 1, characterized in that, The radiotherapy is ionizing radiation.

3. The application according to claim 2, wherein The triterpenoid saponin extract increases DNA damage and apoptosis by inhibiting the activation of AKT, ATM, p-ATM, and DNA-PKcs caused by ionizing radiation.

4. The application according to any one of claims 1-3, characterized in that, Its dosage form is selected from: injection, tablet, capsule, kit or patch.

5. The application according to any one of claims 1 to 3, characterized in that The medicament is used for treating liver cancer.

6. The application according to claim 5, wherein The cancer cells of the liver cancer are Huh7 and SMMC-7721.

7. Use of a triterpenoid saponin extract in the preparation of a radiosensitizer for radiotherapy, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

8. Use of a triterpenoid saponin extract in a medicament for increasing cancer cell apoptosis, the medicament being used in radiotherapy, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

9. Use of a triterpenoid saponin extract in the preparation of a medicament for inhibiting cancer cell proliferation, the medicament being used in radiotherapy, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

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