Application of lasiokaurin and cis-platinum combined drug in preparation of liver cancer treatment drug

Abstract

The invention relates to the technical field of medicines, in particular to application of lasiokaurin and cis-platinum combined medicine in preparation of a medicine for treating liver cancer. As a diterpenoid compound, lasiokaurin shows significant potential in the anti-cancer field. However, in medical research and clinical application at present, related reports about combined use of lasiokaurin and a classical anti-cancer drug cis-platinum for treating liver cancer do not exist. Based on the current research situation, the invention creatively provides the application of the lasiokaurin and cis-platinum combined medicine in the aspect of preparing the liver cancer treatment medicine. In-depth study finds that lasiokaurin and cis-platinum can generate a synergistic effect and jointly play a strong anti-tumor role, and a brand new way is opened up for treatment of liver cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically the application of the combined use of tannin and cisplatin in the preparation of drugs for the treatment of liver cancer. Background Technology

[0002] Primary liver cancer (PLC) mainly includes hepatocellular carcinoma, intrahepatic cholangiocarcinoma, mixed hepatocellular carcinoma, and cholangiocarcinoma, with hepatocellular carcinoma (HCC) being the most common, accounting for 75%-85% of all PLCs worldwide. However, early symptoms of liver cancer are often insidious, and the disease progresses rapidly, with most patients diagnosed at an advanced stage. It is characterized by high malignancy, high recurrence rate, poor treatment efficacy, and unfavorable prognosis. Surgery and radiotherapy / chemotherapy are common treatments for liver cancer. However, surgery is only suitable for early-stage, non-metastatic patients; most patients have missed the optimal window for surgery by the time of diagnosis, and postoperative recurrence rates are high with poor survival rates. Therefore, chemotherapy is more commonly used. Cisplatin is a commonly used chemotherapy drug; however, most liver cancer patients develop resistance to it, leading to reduced efficacy and increased recurrence risk. Therefore, there is an urgent need to find drugs that can reduce cisplatin resistance to overcome the challenges in liver cancer treatment.

[0003] *Heliotropium indicum* is a precious traditional Chinese medicine with properties of clearing heat and detoxifying, promoting blood circulation and reducing swelling. It can be used to treat hepatitis, traumatic injuries, and snake and insect bites. A diterpenoid compound, tannin (LAS), has been isolated from *Heliotropium indicum*. Studies have found that LAS has significant anticancer activity, significantly inhibiting the growth of various tumor cells, including gastric cancer, glioblastoma, nasopharyngeal carcinoma, lung adenocarcinoma, promyelocytic leukemia, Ehrlich ascites carcinoma, and histiocytic lymphoma. In vitro and in vivo experiments have confirmed that tannin possesses good antitumor activity, with a tumor inhibition rate exceeding 40% in animal models. However, there are currently no reports on the combined use of tannin and cisplatin for the treatment of liver cancer.

[0004] To overcome the shortcomings of the prior art, the present invention provides the application of tannin combined with cisplatin in the preparation of a drug for treating liver cancer. Summary of the Invention

[0005] The purpose of this invention is to provide the application of tannin combined with cisplatin in the preparation of drugs for the treatment of liver cancer, so as to solve the problems raised in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The application of tannin combined with cisplatin in the preparation of a drug for treating liver cancer, wherein tannin and cisplatin are used in combination to prepare a drug that inhibits the proliferation of liver cancer cells.

[0007] In a more optimized manner, tannin and cisplatin are combined to prepare a drug that inhibits the migration and invasion of liver cancer cells.

[0008] Ideally, the liver cancer pathological type used in the liver cancer treatment is hepatocellular carcinoma.

[0009] An optimal ratio of tannin to cisplatin is (2.0-2.3):50.

[0010] The more optimized application of tannin in combination with cisplatin in the preparation of drugs that reduce the toxic side effects of cisplatin monotherapy for liver cancer.

[0011] More optimized application of tannin in combination with cisplatin in the preparation of drugs that reduce cisplatin resistance in liver cancer.

[0012] In a more optimized manner, in vitro experiments demonstrated that the combined use of tannin and cisplatin inhibited the survival, proliferation, and migration of Huh7 / CDDP cells; Huh7 / CDDP cells were cells with cisplatin resistance obtained after cisplatin treatment.

[0013] In a more optimized manner, the in vitro experiments include CCK8, clonogenesis, scratch assay, and Transwell assay.

[0014] In a more optimized manner, the cultured Huh7 / CDDP cells were subcutaneously seeded, and in vivo experiments were conducted to demonstrate the inhibitory effect of the combined use of tannin and cisplatin on the growth of liver cancer tumors in vivo.

[0015] A liver cancer treatment drug comprising tannin and cisplatin.

[0016] The beneficial effects of this invention are: Tannin, a diterpenoid compound, has shown significant potential in the field of anticancer treatment. Extensive experimental data strongly confirms that tannin exhibits a tumor inhibition rate exceeding 40% in animal models, demonstrating its substantial anticancer activity. However, currently, there are no reports in medical research or clinical applications regarding the combined use of tannin and the classic anticancer drug cisplatin for the treatment of liver cancer. Based on this research status, this invention innovatively proposes the application of combined tannin and cisplatin in the preparation of liver cancer therapeutic drugs. In-depth research has revealed that tannin can produce a synergistic effect with cisplatin, jointly exerting a powerful antitumor effect, opening up a completely new avenue for the treatment of liver cancer.

[0017] The combined use of tannin and cisplatin in the preparation of drugs for treating liver cancer has shown several valuable applications. In inhibiting the proliferation of liver cancer cells, the combined use of these two drugs can precisely target the growth cycle of liver cancer cells, effectively blocking their division and proliferation, thereby significantly reducing the number of cancer cells. This combination therapy can not only be used directly to inhibit the proliferation of liver cancer cells, but also to prepare products specifically designed to inhibit the proliferation of liver cancer cells, providing more targeted treatment options for liver cancer patients.

[0018] In inhibiting the migration and invasion of liver cancer cells, the combined use of tannin and cisplatin can interfere with the movement mechanism of cancer cells, disrupt their adhesion to surrounding tissues, and prevent cancer cells from spreading and metastasizing to surrounding tissues and distant organs. Whether directly applied clinically to inhibit the migration and invasion of liver cancer cells or used in the preparation of related products, it will help reduce the risk of liver cancer metastasis and improve patients' survival rate and quality of life.

[0019] In inhibiting liver cancer survival, the synergistic effect of tannin and cisplatin can influence the survival environment and metabolic pathways of liver cancer cells at multiple levels, inducing apoptosis and inhibiting their survival. This combination therapy not only allows for direct inhibition of liver cancer survival but also enables the development of products specifically targeting liver cancer survival, providing a more effective strategy for liver cancer treatment.

[0020] In conclusion, the combined use of tannin and cisplatin has broad application prospects and important clinical value in the preparation of drugs for the treatment of liver cancer, and is expected to bring new hope to liver cancer patients. Attached Figure Description

[0021] Figure 1 The effect of combined administration of tannin and cisplatin on the survival rate of Huh7 / CDDP cells; Figure 2 The effect of combined use of tannin and cisplatin on the invasive ability of Huh7 / CDDP cells; Figure 3 The effect of combined use of tannin and cisplatin on the metastatic ability of Huh7 / CDDP cells; Figure 4 The effect of combined use of tannin and cisplatin on clone formation in Huh7 / CDDP cells; Figure 5 This study investigated the effect of combined use of tannin and cisplatin on the growth of liver cancer tumors in vivo. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Testing and experimentation: I. Effect of CCK-8 assay on the survival rate of Huh7 / CDDP cells treated with a combination of tannin and cisplatin: (1) Cell seeding and culture: Huh7 / CDDP cells were seeded in 96-well plates at a seeding density of 1×10⁶ cells / well. 4 Cells / wells were cultured in an incubator (at 5% CO2 and 37°C) for 24 hours. After 24 hours of culture, the cells adhered to the bottom of the wells and continued to grow and multiply. When the cell density reached approximately 70%, the cells were in a suitable growth state and could be used for subsequent drug treatment experiments.

[0024] (2) Drug treatment: The cultured cells were divided into different treatment groups. One group was treated with cisplatin alone (cisplatin 50 μM), and the other group was treated with tannin and cisplatin in combination (cisplatin 50 μM + tannin 2 μM). The treatment time was 48 h.

[0025] (3) Preparation and addition of CCK-8 working solution: CCK-8 is a reagent used to detect cell viability, and DMEM is a commonly used cell culture medium. CCK-8 reagent and DMEM were mixed at a volume ratio of 1:9 to prepare the CCK-8 working solution. The original culture medium containing the drug was then discarded to avoid interference with the CCK-8 detection results. The prepared CCK-8 working solution was added to each well, and the cells were then incubated in the incubator for 2 hours.

[0026] (4) Detection and calculation: An ELISA reader is an instrument used to measure the absorbance of a solution. After incubation, the absorbance (OD value) of the CCK-8 working solution in each well was read at a wavelength of 450 nm using an ELISA reader, and the cell viability was calculated, where RGR% = (mean OD value of experimental group - mean OD value of blank control group) / (mean OD value of control group - mean OD value of blank control group) × 100%.

[0027] II. Transwell assay to detect the effect of combined administration of tannin and cisplatin on the migration of Huh7 / CDDP cells: (1) Cell seeding: 100 μL of Huh7 / CDDP cell suspension was seeded into a Transwell chamber at a seeding density of 1 × 10⁻⁶ cells / mL. 5 Cells / wells. After seeding cells into the upper layer of the Transwell chamber, the drug was added to the cell-containing upper culture medium. The experiment was divided into a control group, a cisplatin group (50 μM), and a combination of cisplatin and tannin (50 μM cisplatin + 2 μM tannin). For the cisplatin group, cisplatin was added; for the combination therapy group, the combination drug was added; and for the control group, an equal volume of cell culture medium was added.

[0028] (2) Culture medium addition: DMEM medium without fetal bovine serum (FBS) was added to the upper layer of the Transwell chamber, and medium containing 10% FBS was added to the lower layer. By using FBS-free medium in the upper layer, the cells in the upper chamber were in a relatively nutrient-deficient environment; while the medium containing 10% FBS in the lower layer created a nutrient-rich environment, which generated a chemotactic gradient, inducing the cells in the upper chamber to migrate to the nutrient-rich lower chamber.

[0029] (3) Culture and fixation: After culturing for 24 hours, fix the cells with 4% paraformaldehyde to maintain the structure and composition of the cells in their current state and prevent the cells from changing or being damaged in subsequent operations.

[0030] (4) Cell removal and staining: Gently remove the cells in the upper chamber with a cotton swab, and then stain the cells in the lower chamber with 0.1% methanol crystal violet staining solution.

[0031] (5) Observation and counting: Observe and photograph under a microscope, and then count the cells that have migrated to the lower membrane.

[0032] III. Scratch assay to detect the effect of combined administration of tannin and cisplatin on Huh7 / CDDP cell invasion: (1) Cell seeding: Huh7 / CDDP cells were seeded in 6-well plates at a seeding density of 4 × 10⁶ cells / well. 5 Cells / pores.

[0033] (2) Overnight incubation: Place the cells in an incubator overnight to allow them to adhere to the walls and grow in the wells.

[0034] (3) Scratch creation and initial photography: A scratch was created in each well using the tip of a 200 μL sterile pipette to simulate the condition of cells after damage in vivo. Initial photographs (0h) were taken under a microscope to record the state of the scratch immediately after its formation, serving as a baseline for subsequent comparison.

[0035] (4) The experiment was divided into a control group, a cisplatin group (50 μM), and a combination of tannin and cisplatin (50 μM cisplatin + 2 μM tannin). Cells were treated according to the experimental requirements of each group. For the control group, cell culture medium was added; for the cisplatin group, cisplatin was added; for the combination of tannin and cisplatin, both tannin and cisplatin were added. The treated cells were cultured for 24 h and then photographed. By comparing the photographs taken at 0 h and 24 h, the effects of different treatment groups on the migration ability of Huh7 / CDDP cells were evaluated.

[0036] IV. Clonogenesis assay to detect the effect of combined administration of tannin and cisplatin on the proliferation of Huh7 / CDDP cells: (1) Cell seeding and initial culture: Huh7 / CDDP cells were seeded in 6-well plates at a seeding density of 1000 cells / well and then cultured in DMEM medium containing 10% fetal bovine serum for 24 h.

[0037] (2) Drug treatment: The experiment was divided into a control group, a cisplatin group (50 μM), and a combination of tannin and cisplatin (50 μM cisplatin + 2 μM tannin). Cells were treated according to the experimental requirements of different groups. For the control group, cell culture medium was added to the culture medium; for the cisplatin group, cisplatin was added to the culture medium; for the combination of tannin and cisplatin, tannin and cisplatin were added to the culture medium.

[0038] (3) Change the culture medium and continue culturing: After 48 hours of drug treatment, discard the drug-containing culture medium and add DMEM containing 10% fetal bovine serum to continue culturing for 4-5 days (under 5% CO2 and 37℃ conditions) to form cell clones.

[0039] (4) Fixation and staining of cell clones: The cells were washed with PBS buffer, then fixed with 4% paraformaldehyde at 25°C for 15 min and stained with crystal violet for 30 min to make the cell clones more clearly visible and facilitate subsequent observation and counting.

[0040] (5) Observation and counting: The cell clones were observed and photographed under a microscope and then counted. By comparing the number of clones in different treatment groups, the effects of different treatment factors on cell proliferation can be evaluated.

[0041] V. Effect of Huh7 / CDDP cell xenograft assay on the combined use of tannin and cisplatin on liver cancer tumor growth in vivo: (1) Preparation of experimental animals: All animal experiments were approved by the Ethics Committee of China Pharmaceutical University. Eighteen 5-week-old female BALB / c nude mice, weighing 16-18g, were selected. Each mouse was subcutaneously inoculated with 5×10 6 One Huh7 / CDDP cell.

[0042] (2) Grouping and administration: When the tumor grows to 100 mm 3 Mice were randomly divided into groups of 6. The model group received no treatment and served as a control to observe the natural growth of the tumor. The cisplatin group was treated with cisplatin (10 mg / kg) via tail vein injection. The combination therapy group was treated with gavage of tannin (15 mg / kg) and tail vein injection of cisplatin (10 mg / kg).

[0043] (3) Measurement of observation indicators: Tumor volume and weight are measured every 2-3 days. The tumor is calculated using the formula: 1 / 2 × length × width 2.

[0044] The test results are shown in the attached diagram in the instruction manual, and the conclusion is: Figure 1 CCK8 results showed that cell viability in the cisplatin group was lower than that in the control group ( p <0.05%, the cell survival rate in the group treated with tannin and cisplatin was significantly lower than that in the cisplatin group ( p <0.001); Figure 2 Cell scratch assay results showed that the cell invasion rate in the cisplatin group was lower than that in the control group within 24 hours. p <0.05%, the cell invasion rate in the group treated with tannin and cisplatin was significantly lower than that in the cisplatin group. p <0.001); Figure 3 Cell Transwell assays showed that the number of cells migrating in the cisplatin group was significantly lower than that in the control group. p <0.01, the number of cells migrating in the group treated with tannin and cisplatin was significantly lower than that in the cisplatin group ( p <0.01); Figure 4 Clonal formation results showed that the number of clones formed in the cisplatin group was lower than that in the control group ( p <0.05, the number of clones formed in the combination therapy group of tannin and cisplatin was significantly lower than that in the cisplatin group ( p <0.001); Figure 5 Subcutaneous tumor-bearing experiments showed that the combined use of tannin and cisplatin inhibited the proliferation of Huh7 / CDDP cells in vivo. These results indicate that the combined use of tannin and cisplatin can inhibit the proliferation of Huh7 / CDDP cells.

[0045] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process method article or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process method article or apparatus.

[0046] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a combination of mimosine and cisplatin in the preparation of a medicament for treating liver cancer, characterized in that: The application discloses a drug for inhibiting proliferation of liver cancer cells, which is prepared by combining acacetin with cisplatin. ​ 2.The use of a combination of gossypol and cisplatin in the preparation of a liver cancer treatment drug according to claim 1, characterized in that: The application discloses a drug for inhibiting migration and invasion of liver cancer cells, which is prepared by combining acacetin with cisplatin. 3.The use of a combination of gossypol and cisplatin in the preparation of a liver cancer treatment drug according to claim 1, characterized in that: The pathological type of the liver cancer treated by the application is hepatocellular carcinoma.

4. The application of the combination of acacetin and cisplatin in the preparation of a liver cancer treatment drug according to claim 1, wherein the ratio of acacetin to cisplatin is (2.0-2.3):

50.

5. The use of a combination of gossypol and cisplatin in the preparation of a medicament for treating liver cancer according to claim 4, characterized in that: The application of the combination of acacetin and cisplatin in the preparation of a drug for reducing the side effects of single cisplatin treatment of liver cancer.

6. The use of a combination of gossypol and cisplatin in the preparation of a medicament for treating liver cancer according to claim 4, characterized in that: The application of the combination of acacetin and cisplatin in the preparation of a drug for reducing the drug resistance of liver cancer to cisplatin.

7. The use of a combination of gossypol and cisplatin in the preparation of a medicament for treating liver cancer according to claim 1, characterized in that: The application discloses that the combination of acacetin and cisplatin has inhibiting effects on the survival, proliferation and migration of Huh7 / CDDP cells through in-vitro experiments; the Huh7 / CDDP cells are cells with cisplatin-resistant characteristics screened from cells treated by cisplatin. 8.The use of a combination of gossypol and cisplatin in the preparation of a liver cancer treatment drug according to claim 7, characterized in that: The in-vitro experiments include CCK8, clone formation, scratch test and Transwell. 9.The use of a combination of aurostilben and cisplatin in the preparation of a liver cancer treatment drug according to claim 1, characterized in that: The application discloses that the combination of acacetin and cisplatin has inhibiting effects on the growth of liver cancer tumor in vivo through in-vivo experiments by subcutaneously inoculating the cultured Huh7 / CDDP cells.

10. A medicament for treating liver cancer, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof. The combination of acacetin and cisplatin is included.

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