Application of xanthotoxol in preparation of medicine for treating liver cancer

Abstract

The invention discloses application of xanthotoxol in preparation of a medicine for treating liver cancer, and relates to the technical field of new application of medicines. The invention provides application of xanthotoxol in preparation of a medicine for treating liver cancer, especially hepatocellular carcinoma, xanthotoxol is a natural small molecule compound extracted from plants, and the invention discloses the inhibiting effect of xanthotoxol on liver cancer cells for the first time. The invention not only provides a new drug source and thought for research and development of liver cancer treatment drugs, but also explores new medical value of xanthotoxol.

Description

Technical Field

[0001] This invention relates to the field of novel uses of pharmaceuticals, specifically to the application of xanthotoxin in the preparation of drugs for treating liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. It is closely related to chronic liver diseases (such as hepatitis B, hepatitis C, and cirrhosis), and its early symptoms are often insidious, while late-stage symptoms may include abdominal pain, jaundice, and weight loss. HCC is characterized by high morbidity and mortality. Currently, treatment methods for HCC mainly include surgical resection, liver transplantation, interventional therapy, chemotherapy, and targeted therapy. However, due to the lack of obvious early symptoms, most patients are diagnosed at an advanced stage, missing the optimal window for surgical treatment. Furthermore, existing chemotherapy and targeted therapies have limitations in efficacy, significant side effects, and a tendency to develop drug resistance. Therefore, finding highly effective and low-toxicity anti-HCC drugs has become a current research hotspot.

[0003] Natural products are an important source of drug development, and screening for compounds with anti-tumor activity from them is of great significance. Zanthoxylum bungeanum, a natural small-molecule compound extracted from plants such as Zanthoxylum bungeanum, has anti-asthmatic, expectorant, and anti-allergic effects, and can prevent osteoporosis induced by glucocorticoids. Studies have found that zanthoxylum bungeanum not only inhibits the growth of Gram-positive bacteria such as Staphylococcus aureus, similar to osthol, but also has inhibitory and bactericidal effects on Staphylococcus albus, and also has strong inhibitory and bactericidal effects on Gram-negative bacteria, Salmonella paratyphi B, and even Pseudomonas aeruginosa. Studies have found that zanthoxylum bungeanum can inhibit the progression of lung cancer, but its role in inhibiting hepatocellular carcinoma (HCC) has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of xanthocyanin in the preparation of drugs for treating liver cancer. Based on the known pharmacological effects and previous studies of xanthocyanin, the inventors of this application propose that xanthocyanin can be used to prepare drugs for treating liver cancer and applied to scientific research and clinical treatment.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: the application of xanthotoxin in the preparation of drugs for treating liver cancer.

[0006] In a preferred embodiment of the application described in this invention, the liver cancer is hepatocellular carcinoma.

[0007] As a preferred embodiment of the application described in this invention, the drug for treating liver cancer is a drug that inhibits the proliferation, invasion, and / or metastasis of liver cancer cells.

[0008] Preferably, the final concentration of xanthotoxin in the drug is 40 mM.

[0009] This invention also provides the application of xanthotoxin in the preparation of reagents for inhibiting liver cancer cells.

[0010] In a preferred embodiment of the application described in this invention, the liver cancer cells include human liver cancer Huh7 cells or human liver cancer Hep3B cells.

[0011] The inventors of this application have discovered that treating human liver cancer Huh7 cells or human liver cancer Hep3B cells with low to medium concentrations of xanthotoxin significantly inhibits the growth of liver cancer cells and affects the cell cycle process, indicating that xanthotoxin can inhibit liver cancer cells.

[0012] In a preferred embodiment of the application described in this invention, the inhibition includes inhibiting cell growth and inhibiting the cell cycle.

[0013] This invention also provides the application of xanthotoxin in the preparation of drugs that activate the P53 signaling pathway in liver cancer cells.

[0014] The present invention also provides a drug for treating hepatocellular carcinoma, wherein the drug uses xanthotoxin as a drug prodrug.

[0015] The present invention also provides a pharmaceutical composition for treating hepatocellular carcinoma, the pharmaceutical composition comprising xanthotoxin.

[0016] As a preferred embodiment of the pharmaceutical composition of the present invention, it further includes a pharmaceutically acceptable carrier.

[0017] Beneficial effects of the present invention: The present invention provides the application of xanthotoxin in the preparation of drugs for treating liver cancer, especially hepatocellular carcinoma. Xanthotoxin is a natural small molecule compound extracted from plants. The present invention discloses for the first time the inhibitory effect of xanthotoxin on liver cancer cells. The present invention not only provides a new drug source and idea for the research and development of drugs for the treatment of liver cancer, but also discovers new medicinal value of xanthotoxin. Attached Figure Description

[0018] Figure 1 The molecular structural formula of xanthotoxin is shown.

[0019] Figure 2 Western blot analysis was conducted to detect the effect of xanthotoxin treatment on the p53 signaling pathway in Hep3B cells.

[0020] Figure 3The effects of xanthotoxin on the viability of HCC cell lines were investigated. In this study, A represents the effect of different concentrations of xanthotoxin on the viability of Huh7 and Hep3B cells; B represents the effect of treatment with 40 μM xanthotoxin for different durations on the viability of Huh7 and Hep3B cells; and C represents the effect of xanthotoxin on cell viability after p53 knockdown.

[0021] Figure 4 The effects of xanthotoxin on cell proliferation are shown in Figure A, where xanthotoxin affects the proliferation of Huh7 liver cancer cells; Figure B shows the effect of xanthotoxin affects the proliferation of Hep3B liver cancer cells; and Figure C shows the effect of p53 knockdown on cell proliferation.

[0022] Figure 5 The effects of xanthotoxin on cell migration / invasion are shown in Figure AB, where AB represents the effect of xanthotoxin on cell migration of Huh7 and Hep3B liver cancer cells; CD represents the effect of xanthotoxin on cell invasion of Huh7 and Hep3B liver cancer cells; and EF represents the effect of xanthotoxin treatment on p53 knockdown on cell migration and invasion.

[0023] Figure 6 To explore the potential mechanism by which xanthotoxin inhibits HCC, A represents principal component analysis of RNA-seq data; B represents GESA pathway enrichment studies; and CI represents heatmap analysis showing the expression levels of genes related to tumor activation signaling pathways in xanthotoxin-treated Hep3B cells. Detailed Implementation

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially. Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0025] Example 1 1.1 Experimental Materials: (1) Xanthotoxol (purchased from Shanghai Yuanye Company, item number S31771, specification 5g), its English name is Xanthotoxol, and its molecular formula is C 11 H6O4, the specific molecular structure is as follows: Figure 1 As shown. The concentration of the mother liquor after dissolving xanthotoxin in DMSO was adjusted to 40 mM, and it was stored at -80℃ for long-term storage and -20℃ for short-term storage. (2) Cell culture: Human liver cancer cell lines Huh7 and Hep3B were purchased from Wuhan Pronosei Biotechnology Co., Ltd. Cells were cultured in DMEM high-glucose medium containing 10% FBS in a humid environment at 37°C with 5% CO2.

[0026] 2. Construction of P53 knockdown cell lines (1) Construction of P53 knockdown plasmid: The knockdown primer sequence was obtained from the gene sequence of P53 (Gene ID: 7157) from Yunzhou Biotechnology (https: / / www.vectorbuilder.cn), and the sense and antisense strands with higher scores were selected. Target knockdown sequence: ACCACTGGATGGAGAATATTT. Sh-RNA primers were synthesized by Wuhan Qingke Biotechnology Co., Ltd. The synthesized primer powder was centrifuged at 12000 rpm for 5 minutes, and diluted with buffer at a concentration of 100 μmol / L. The annealing system consisted of Primer-F 4.5ul, Primer-R 4.5ul, and NE buffer 1ul. The PCR reaction was carried out under the following conditions: denaturation at 95℃ for 3 minutes, annealing temperature decreased by 0.5℃ every 30 seconds, and 140 cycles were run.

[0027] (2) Lentiviral packaging: Take a 1.5 mL EP tube, add 200 μL of serum-free culture medium, add the packaging plasmid PMD, PSA, and target plasmid (0.5:0.75:1) and label it, let it stand for five minutes; take another packaging reagent PEI (concentration of 2 mg / mL; the volume required per well is 1.5 times the total amount of plasmid added) and add it to another EP tube and let it stand for five minutes; add the prepared PEI to the plasmid mixing system, mix well and let it stand for 15 minutes; take the prepared 293T cells, add the plasmid to the well plate, shake well and put it in the incubator, change the medium after 6 hours; collect the virus once at 24, 48 and 72 hours, mix the collected virus supernatant, filter it with a 0.45 μm filter membrane (wash the filter membrane with PBS before filtration), and store it at -80℃ for long-term storage.

[0028] (3) Construction of stable cell lines: Take out 1.5 mL of virus solution and shake it in a 37℃ water bath to melt it; take out a 6-well plate of Huh7 / HepG2 cells, wash with 1×PBS, add 1.5 mL of virus suspension, 500 μL of complete culture medium and 2 μL of agglutinin to each well to form an infection solution (the final concentration of agglutinin is 10 μg / mL), shake well and put it in a 37℃ incubator, change the medium after 8 hours; re-inoculate after 24 hours, and use wild-type Huh7 / HepG2 cells as the control group cells for screening stable lines. Add 5 μg / ml puromycin and observe the control group cells after 48 hours. Determine whether to perform secondary screening based on the degree of cell death. Continue until no cells survive in the control group, change the culture medium, and obtain a knockdown P53 cell line (sh-P53) that can stably express the target plasmid.

[0029] 3. Western blotting analysis: Hep3B cells were seeded at a density of 10,000 cells / well in 96-well plates and divided into three groups: DMSO group, xanthocyanin group, and xanthocyanin + sh-P53 group. DMEM medium was mixed with xanthocyanin to prepare a 40 μM solution. An equal volume of DMSO was added to the DMSO group; 40 μM xanthocyanin solution was added to the xanthocyanin group; and the xanthocyanin + sh-P53 group consisted of knocked-down P53 cells followed by the addition of 40 μM xanthocyanin solution. After 24 hours, cells were collected, washed with PBS, and then lysed using NP-40 lysis buffer (P0013F, Beyotime, China) containing protease and phosphatase inhibitors. After lysis, protein samples were quantified using a BCA kit (23227, Thermo Fisher Scientific, USA). The quantified protein samples were then separated by SDS-PAGE and transferred to a PVDF membrane (IPVH00010, Millipore, USA). The membrane was blocked for 1 hour at room temperature with TBST solution (1×TBS, 0.1% Tween 20) containing 5% skim milk powder. The membrane was then incubated overnight with primary antibody at 4°C. The next day, the PVDF membrane was incubated with secondary antibody at room temperature, followed by treatment with the Omni-ECL™ Ultrasensitive Chemiluminescence Detection Kit (Factory Grade) (SQ201, YAG, China) and visualized using an image gel imaging analyzer (Bio-Rad, USA).

[0030] The results are as follows Figure 2As shown, treatment of Hep3B cells with 40 μM xanthocyanin upregulated the expression levels of P53 and P21 proteins, while decreasing the levels of CDK1, CDK4, CDK6, Cyclin D1, and Cyclin E1 proteins. Compared with xanthocyanin treatment alone, knockdown of P53 resulted in decreased expression levels of P53 and P21 proteins, and increased levels of CDK1, CDK4, CDK6, Cyclin D1, and Cyclin E1 proteins. These results indicate that xanthocyanin inhibits HCC by activating P53 to suppress the expression of cell cycle-related proteins, thereby inhibiting the cell cycle.

[0031] 4. CCK-8 cell viability assay (1) Two different liver cancer cells, Huh7 and Hep3B cells, were seeded in 96-well plates at a density of 10,000 cells / well. There was one control group and four experimental groups, with five replicates in each group. After 24 hours, DMEM medium was mixed with xanthotoxin to prepare 5 μM, 10 μM, 20 μM and 40 μM mixtures. An equal amount of DMSO was added to each well in the blank group. After 24 hours, the toxicity of the drug to the cells was detected using the CCK-8 (BMU106, Abbkine, China) endpoint assay kit. After 2 hours, the absorbance at 450 nm was measured using an ELISA reader.

[0032] (2) Two different liver cancer cells, Huh7 and Hep3B cells, were seeded in 96-well plates at a density of 10,000 cells / well. There was one control group and four experimental groups, with five replicates in each group. After 24 hours, DMEM medium was mixed with xanthocyanin to prepare a 40 μM mixture. An equal amount of DMSO was added to each well in the blank group. After 0, 6, 12, 24 and 48 hours, the toxicity of the drug to the cells was detected by the CCK-8 (BMU106, Abbkine, China) endpoint assay kit. After 2 hours, the absorbance at 450 nm was measured by an ELISA reader.

[0033] (3) Hep3B cells were divided into three groups: DMSO group, xanthocyanin group, and xanthocyanin + sh-P53 group. 96-well plates were seeded at a density of 10,000 cells / well, with five replicates per group. After 24 hours, DMEM medium was mixed with xanthocyanin to prepare a 40 μM solution. An equal volume of DMSO was added to the DMSO group; 40 μM xanthocyanin solution was added to the xanthocyanin group; and the xanthocyanin + sh-P53 group was prepared by knocking down P53 cells and adding 40 μM xanthocyanin solution. After 24 hours, the cytotoxic effect of the drug on the cells was detected using a CCK-8 (BMU106, Abbkine, China) endpoint assay kit. Two hours later, the absorbance at 450 nm was measured using a microplate reader.

[0034] The results are as follows Figure 3As shown, xanthotoxin inhibits the activity of hepatocellular carcinoma cells in a concentration-dependent manner. Figure 3 A); Zanthoxylin inhibits the activity of hepatocellular carcinoma cells in a time-dependent manner ( Figure 3 B). In the context of xanthotoxin treatment, knockdown of p53 promotes cell viability ( Figure 3 C).

[0035] 5.5 Plate Cloning Detection: Six-well plates were divided into three groups: DMSO group, xanthocyanin group, and xanthocyanin + sh-P53 group. 2000-3000 Hep3B cells were seeded in each well. After 24 hours, DMEM medium was mixed with xanthocyanin to prepare a 40 μM solution. An equal volume of DMSO was added to the DMSO group; 40 μM xanthocyanin solution was added to the xanthocyanin group; and for the xanthocyanin + sh-P53 group, P53 cells were knocked down and 40 μM xanthocyanin solution was added. After 24 hours of treatment, the medium was replaced with normal medium. Observe the formation of cell clonal clumps and the color change of the culture medium in the 6-well plates daily. When the culture medium turns yellow, replace it with fresh complete culture medium in time. After about 14 days of culture, stop the culture when cell colonies are observed. Discard the culture medium in the 6-well plates, gently wash the cells twice with 1 mL PBS to remove non-adherent cells, add an appropriate amount of 4% paraformaldehyde solution to each well, and fix at room temperature for about 30 min. Add an appropriate amount of crystal violet staining solution and stain at room temperature for 5 min. After staining, wash the cells with PBS. Place the culture plate in a ventilated place to air dry, and take and save the cell colony images under suitable light conditions.

[0036] The results are as follows Figure 4 As shown, xanthotoxin treatment can inhibit the proliferation of hepatocellular carcinoma cells. Knockdown of p53 in the context of xanthotoxin treatment promotes cell proliferation.

[0037] 6. Transwell migration / invasion detection (1) Add approximately 100 μL of serum-free culture medium to the upper chamber of the Transwell plate, allowing the polycarbonate membrane to fully saturate for 15 min, then aspirate the culture medium; add 50,000 Hep3B cells and Huh7 cells to each well, using the five-point hanging drop method to drop the cell suspension into the upper chamber to ensure uniform distribution of the cell suspension, then let it stand for 10 min; in the lower chamber of the 24-well plate, a certain amount of culture medium containing FBS (fetal bovine serum) is usually added, avoiding the generation of air bubbles during addition, and the culture plate is placed in an incubator; divide into two groups: the blank group is given an equal amount of DMSO, and the experimental group is given 40 μM xanthocyanin, and after 24 hours the culture medium is normal culture medium. After culturing for 48 h, carefully aspirate the culture medium from the upper chamber of the Transwell plate, wash with PBS two to three times, and fix the cells with 4% paraformaldehyde for 15 min; add 1 mL of 0.1% crystal violet staining solution to the 24-well plate and stain at room temperature for 5 min. Use a cotton swab dipped in a small amount of PBS to gently wipe away the cells on the membrane. Next, clean with PBS, air dry, and take photos. Thaw the matrix gel at 4°C one day in advance, and dilute the matrix gel with DMEM medium. Place the chambers in a 24-well plate, and spread 100 µL of diluted matrix gel evenly in each chamber; take care to avoid generating air bubbles during the operation; place the chambers with matrix gel in an incubator and let them stand for more than 2 hours to allow the matrix gel to solidify; the rest of the operation is the same as (1).

[0038] (2) Add approximately 100 μL of serum-free medium to the upper chamber of the Transwell plate, allowing the polycarbonate membrane to fully saturate for 15 min, then remove the medium. Add 50,000 Hep3B cells to each well, using the five-point hanging drop method to drop the cell suspension into the upper chamber to ensure uniform distribution of the cell suspension, then let it stand for 10 min. In the lower chamber of the 24-well plate, add a certain amount of medium containing FBS (fetal bovine serum), avoiding the generation of air bubbles during addition, and place the culture plate in an incubator. Divide the plate into three groups: DMSO group, xanthocyanin group, and xanthocyanin + sh-P53 group. After 24 hours, mix DMEM medium with xanthocyanin to prepare a 40 μM mixture. Add an equal amount of DMSO to the blank group; add 40 μM xanthocyanin mixture to the xanthocyanin group; xanthocyanin + sh-P53 group: knock down P53 cells and add 40 μM xanthocyanin mixture, treat for 24 h, then replace with normal medium. After culturing for 48 h, carefully aspirate the culture medium from the upper chamber of the Transwell chamber, wash two to three times with PBS, and fix the cells with 4% paraformaldehyde for 15 min; add 1 mL of 0.1% crystal violet staining solution to the 24-well plate and stain for 5 min at room temperature. Gently wipe away the cells on the membrane with a cotton swab dipped in a small amount of PBS. Then wash with PBS, air dry, and take pictures. Thaw the matrix gel at 4°C one day in advance, and dilute the matrix gel with DMEM culture medium. Place the chamber in the 24-well plate, take 100 µL of diluted matrix gel and spread it evenly in each chamber; be careful to avoid generating air bubbles during the operation; place the chamber with matrix gel in the incubator and let it stand for more than 2 h to allow the matrix gel to solidify; the rest of the operation is the same as (1).

[0039] The results are as follows Figure 5 As shown, xanthocyanin treatment can inhibit the migration / invasion of hepatocellular carcinoma cells. Knockdown of p53 in the context of xanthocyanin treatment promotes cell migration / invasion.

[0040] 6. Transcriptome Analysis: Hep3B cells were seeded into cell culture plates and divided into two groups. After 24 hours, the control group was treated with an equal volume of DMSO, while the experimental group was treated with 40 μM xanthocyanin. After 24 hours, the medium was replaced with normal medium. Total RNA was extracted using the Trizol method and subjected to routine quality control. A cDNA library was constructed using the MGI Easy RNA Library Prep Kit, strictly following the kit's operating instructions. Double-stranded cDNA was synthesized after fragmentation, repaired, and A-tailed, ligated to sequencing terminals, and then amplified by PCR. Fragmentation distribution of the purified products was analyzed using the Agilent DNA 1000 Kit. The PCR products underwent multiple sample pooling, circularization, and digestion to obtain a single-stranded circular DNA library before sequencing. After purification, the PCR products were quality controlled using the Agilent DNA 1000 Kit. Paired-end 150 bp sequencing was performed on the BGISEQ-500 platform, with reference genome comparison. Gene set enrichment analysis was used to interpret differentially expressed gene functional characteristics. Significant pathways with FDR < 0.25 and p < 0.05 were screened.

[0041] The results are as follows Figure 6 As shown, xanthotoxin treatment can inhibit tumor-activated signaling pathways.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of xanthotoxin in the preparation of drugs for treating liver cancer.

2. The application according to claim 1, characterized in that, The liver cancer mentioned is hepatocellular carcinoma.

3. The application according to claim 1, characterized in that, The drugs mentioned for treating liver cancer are those that inhibit the proliferation, invasion, and / or metastasis of liver cancer cells.

4. Application of xanthotoxin in the preparation of reagents that inhibit liver cancer cells.

5. The application according to claim 4, characterized in that, The liver cancer cells include human liver cancer Huh7 cells or human liver cancer Hep3B cells.

6. The application according to claim 4, characterized in that, The inhibition includes inhibiting cell growth and inhibiting the cell cycle.

7. Application of xanthotoxin in the preparation of drugs that activate the P53 signaling pathway in liver cancer cells.

8. A drug for treating hepatocellular carcinoma, characterized in that, The drug uses xanthotoxin as a drug precursor.

9. A pharmaceutical composition for treating hepatocellular carcinoma, characterized in that, The pharmaceutical composition includes xanthotoxin.

10. The pharmaceutical composition according to claim 9, characterized in that, It also includes pharmaceutically acceptable carriers.

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