Application of combination of Molephantin and platinum chemotherapeutic drugs in preparation of drugs for treating nasopharynx cancer

Abstract

The invention discloses an application of a combination of Molephantin and a platinum chemotherapeutic drug in preparation of a drug for treating nasopharynx cancer. The purpose of the invention is to show the pharmaceutical application of Molephantin in tumor growth resistance. In-vitro and in-vivo experiments are combined, and the result shows that the growth of nasopharyngeal carcinoma can be inhibited by combining the Molephantin with the cis-platinum and the oxaliplatin. The invention provides a new application of the combination of the Molephantin and the cis-platinum, and provides a new drug source for adjuvant therapy of cancers.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology; specifically, it relates to the application of Molephantinin combined with platinum-based chemotherapeutic drugs in the preparation of drugs for the treatment of nasopharyngeal carcinoma. Background Art

[0002] Molephantinin is a novel compound extracted from the Asteraceae plant Elephantopus mollis Kunth, belonging to macrolides and having a sesquiterpene structure. Previous studies have shown that various extracts isolated from Elephantopus mollis exhibit significant biological activities. Its ethanol extract has anti-inflammatory and analgesic effects; the steroid component obtained by fractionation of the acetone extract can significantly reduce the blood glucose level of diabetic rats and improve insulin secretion; the ethyl acetate extract shows cytotoxicity, anti-proliferation and pro-apoptosis effects on liver and lung cancer cell lines. In this study, among 17 compounds extracted from Elephantopus mollis, Molephantinin was found to exhibit significant anti-nasopharyngeal carcinoma activity, with the expectation of providing a new candidate drug for the treatment of nasopharyngeal carcinoma.

[0003] Cisplatin is a classic platinum-based anti-cancer drug. By forming cross-links with DNA, it inhibits the proliferation of tumor cells and induces apoptosis, and has a wide application in the treatment of various solid tumors. Cisplatin monotherapy or combined chemotherapy regimens show significant anti-tumor activity in the treatment of nasopharyngeal carcinoma. Especially for patients with recurrent or metastatic nasopharyngeal carcinoma, cisplatin is often used as a first-line chemotherapeutic drug. However, there are certain limitations in cisplatin monotherapy, such as high recurrence rate and drug resistance, which limit its further clinical application effect.

[0004] Nasopharyngeal carcinoma is a malignant tumor with regional characteristics. Approximately 80% of the cases globally are concentrated in southern China and Southeast Asia. Although the combination of radiotherapy and chemotherapy has improved the survival rate of patients to a certain extent in recent years, problems such as high recurrence rate, drug resistance and treatment-related side effects still restrict the further improvement of clinical efficacy. For recurrent or metastatic nasopharyngeal carcinoma, the existing treatment methods are still limited, and the quality of life and prognosis of patients still need to be further improved. Therefore, developing new treatment strategies, especially combination drug regimens, to overcome drug resistance, reduce toxicity and improve treatment effect has become an important direction in current nasopharyngeal carcinoma research. Summary of the Invention

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention provides the application of Molephantinin combined with platinum-based chemotherapeutic drugs in the preparation of drugs for the treatment of nasopharyngeal carcinoma, and the platinum-based chemotherapeutic drug is cisplatin or oxaliplatin.

[0007] Preferably, the dosage concentration of cisplatin is greater than or equal to 4 μM.

[0008] Preferably, the dosage concentration of cisplatin is 8 μM; the dosage concentration of Molephantinin is 10 μM.

[0009] Preferably, the nasopharyngeal carcinoma is caused by CNE-1 cells, S18 cells or 58-F cells.

[0010] Preferably, the dosage concentration of Molephantinin is 10 μM, and the dosage concentration of oxaliplatin is 7 μM.

[0011] The present invention also provides a drug for treating nasopharyngeal carcinoma, comprising: Molephantinin and platinum-based chemotherapeutic drugs; the platinum-based chemotherapeutic drug is cisplatin or oxaliplatin.

[0012] Preferably, the dosage concentration of cisplatin is greater than or equal to 4 μM.

[0013] Preferably, the dosage concentration of Molephantinin is 10 μM, and the dosage concentration of oxaliplatin is 7 μM.

[0014] The present invention has the following beneficial effects but is not limited to:

[0015] The purpose of the present invention is to demonstrate the application of Molephantinin in anti-tumor growth.

[0016] Combining in vitro and in vivo experiments, the results show that the combination of Molephantinin and cisplatin can inhibit the growth of nasopharyngeal carcinoma. The present invention provides a new application of the combination of Molephantinin and cisplatin or oxaliplatin, providing a new drug source for the adjuvant treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to clearly show the specific implementation manners of the present invention and certain detection techniques used in the experiments, the following will describe the implementation manners and the techniques used, mainly introduced in the form of drawings.

[0018] Figure 1 The Q-value result diagram of the combination of Molephantinin and three chemotherapeutic drugs;

[0019] Figure 2 The result diagram of the effect of the combination of Molephantinin and cisplatin on the viability of human nasopharyngeal carcinoma cells;

[0020] Figure 3 The result diagram of the effect of the combination of Molephantinin and cisplatin on the tumorigenic ability of nasopharyngeal carcinoma cells in nude mice;

[0021] Figure 4 、Results graph of the combined use of Molephantinin and cisplatin on the kidneys and livers of nude mice;

[0022] Figure 5 、Results graph of the change in the proliferation index Ki67 after the combined use of Molephantinin and cisplatin;

[0023] Figure 6 、Chemical structural formula of Molephantinin;

[0024] Figure 7 、Chemical structural formula of cisplatin.

[0025] Specific implementation manners

[0026] The specific implementation manners of the present invention are assisted and explained by examples. Except for the technologies used in the detection which do not impose any form of limitation on the present invention, some of the solutions in the described examples belong to a part of the embodiments of the invention. For the embodiments obtained by ordinary technical operators in the art without creative achievements, they all fall within the protection scope of the present invention.

[0027] Example 1

[0028] Materials

[0029] Elephantopus mollis Kunth (Elephantopus tomentosus L.) is a plant widely distributed in tropical countries such as Central and South America, Africa, and East Asia. Its traditional uses include treating cough, fever, pharyngitis, and diarrhea, and it has the effects of clearing heat and detoxifying and diuresis. This plant contains various active ingredients, such as terpenoids, polyphenols, flavonoids, and sterol compounds. [1] . Research shows that the ethanol extract of Elephantopus mollis has anti-inflammatory and analgesic effects, can reduce the edema of the hind paws of mice, and inhibit abdominal permeability. [2] ; A steroid component obtained by fractionating the acetone extract of it can effectively reduce the blood glucose level of streptozotocin-induced diabetic rats and restore insulin levels at the same time. [3] . In addition, the ethyl acetate extract of Elephantopus mollis shows significant anti-tumor activity in liver cancer, lung cancer, and colorectal cancer cells, including inhibiting cell proliferation, inducing apoptosis, and inhibiting tumor growth through the ROS-mediated caspase-dependent apoptosis mechanism. [4-6] .

[0030] In this study, we screened a novel compound, Molephantinin, from 17 compounds extracted from Elephantopus scaber, and found that it could effectively inhibit the proliferation of nasopharyngeal carcinoma cells. Molephantinin is a macrolide compound with the structural characteristics of sesquiterpenes, with the chemical formula C20H34O5, and its structural formula is as Figure 6 shown. Cisplatin, also known as cis-diamminedichloroplatinum(II), with the structural formula as Figure 7 shown, is a well-known chemotherapeutic drug. It is used to treat a variety of human cancers, including bladder cancer, head and neck cancer, lung cancer, ovarian cancer, and testicular cancer. Cisplatin has efficacy against various types of cancers, including carcinoma, germ cell tumors, lymphomas, and sarcomas. Its anticancer mechanism of action is closely related to its ability to crosslink with purine bases in DNA. Cisplatin causes DNA damage by interfering with the DNA repair mechanism and ultimately induces apoptosis of cancer cells [7,8] . However, the use of cisplatin also faces some challenges and adverse side effects. Drug resistance is a major problem. In addition, cisplatin can also cause a variety of adverse reactions, including severe kidney problems, allergic reactions, reduced immunity to infections, gastrointestinal disorders, bleeding, and hearing loss, especially more significant in young patients [9] . Therefore, the treatment method of combining cisplatin with other drugs has been highly considered to overcome drug resistance and reduce toxicity.

[0031] References

[0032] 1. Kuete, V., et al., Cytotoxicity of the methanol extracts of Elephantopus mollis, Kalanchoe crenata and 4 other Cameroonian medicinal plants towards human carcinoma cells. BMC Complement Altern Med, 2017. 17(1): p. 280.

[0033] 2. Yam, M.F., et al., Anti-inflammatory and analgesic effects of Elephantopus tomentosus ethanolic extract. J Acupunct Meridian Stud, 2009. 2(4): p. 280 - 7.

[0034] 3. Daisy, P., et al., A novel steroid from Elephantopus scaber L., an ethnomedicinal plant with antidiabetic activity. Phytomedicine, 2009. 16(2 - 3): p. 252 - 7.

[0035] 4. Ooi, K.L., et al., Cytotoxic and Apoptotic Effects of Ethyl Acetate Extract of Elephantopus mollis Kunth. in Human Liver Carcinoma HepG2 Cells Through Caspase - 3 Activation. Integr Cancer Ther, 2014. 13(3): p. Np1 - 9.

[0036] 5. Bich Ngoc, T.T., et al., Elephantopus mollis Kunth extracts induce antiproliferation and apoptosis in human lung cancer and myeloid leukemia cells. J Ethnopharmacol, 2020. 263: p. 113222.

[0037] 6. Chan, C.K., et al., Elephantopus scaber induces apoptosis through ROS - dependent mitochondrial signaling pathway in HCT116 human colorectal carcinoma cells. J Ethnopharmacol, 2015. 168: p. 291 - 304.

[0038] 7. Dasari, S., et al., Pharmacological Effects of Cisplatin Combination with Natural Products in Cancer Chemotherapy. Int J Mol Sci, 2022. 23(3).

[0039] 8.Dasari, S. and P.B. Tchounwou, Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol, 2014. 740: p. 364-78.

[0040] 9.Galluzzi, L., et al., Molecular mechanisms of cisplatin resistance. Oncogene, 2012. 31(15): p. 1869-83.

[0041] Example 2

[0042] Experimental method steps

[0043] In vitro experiment: Nasopharyngeal carcinoma cells were treated with different drug combinations, and then cell viability was detected by CCK-8 assay to study the effects of different combinations of Molephantinin and cisplatin on the proliferation of nasopharyngeal carcinoma cells.

[0044] CCK-8 cell proliferation detection experiment: Cells were digested with trypsin, and 2000 cells of cultured CNE-1, S18, and 58-F cells were seeded into each well of a 96-well plate, with three replicate wells, and cultured overnight after plating. Cells were treated with different combinations of Molephantinin and cisplatin, and blank control wells with only medium without cells were set up. After 24 h, the cells treated with drugs were taken out, and cell viability was tested by CCK-8 assay. CCK-8 was added to the cultured cells at a working concentration of 10%, and incubated at 37 °C for 1.5 h. The absorbance was detected at 450 nm using a microplate reader.

[0045] In vivo experiment: By constructing a subcutaneous tumorigenesis model in female nude mice (balb / c-nu / nu) and treating with different drug combinations, the inhibitory effects of different combinations of Molephantinin and cisplatin on nasopharyngeal carcinoma in vivo were studied.

[0046] Animal experiment: Twenty 6-week-old female nude mice (balb / c-nu / nu) were selected, with 5 in the control group and 15 in the experimental group. A subcutaneous tumorigenesis model was constructed: (1) 58-F nasopharyngeal carcinoma cells were resuspended in PBS buffer and mixed with Matrigel at a ratio of 1:1, and 1×10 658-F cells; (2) Anesthetize the nude mice before the experiment, and evaluate the anesthetic level through painless and painful stimuli to determine that the nude mice are in an anesthetized state; (3) Use a microsyringe with a 25G needle to resuspend the cells and inject them subcutaneously into the nude mice. (4) Drug treatment: Ten days after subcutaneous injection, Molephantinin begins to be administered by gavage, and cisplatin is administered by intraperitoneal injection. A total of 3 drug combinations are set up in this experiment, namely Molephantinin 10mg / kg, cisplatin 5mg / kg, and Molephantinin 10mg / kg plus cisplatin 5mg / kg, with 5 in each group, and the drugs are administered twice a week. Molephantinin is dissolved in corn oil, and cisplatin is dissolved in physiological saline. Each mouse is administered 100 μL each time. Measure the tumor size and the body weight of the nude mice each time the drug is administered. After 3 weeks, euthanize the nude mice and remove the tumors and blood for subsequent experiments.

[0047] Serum detection experiment: Collect about 1 mL of whole blood from the orbital venous plexus. After the blood stands for 30 minutes, centrifuge it at 3000 rpm for 15 minutes at 4 °C, separate the serum, aliquot it, and store it at -80 °C. Commercial kits are used for detection. The urea enzyme-glutamate dehydrogenase method is used for urea detection, and the method recommended by IFCC is used for ALT / AST detection. The working solution is prepared strictly according to the instructions, and a series of concentration standard products are set (Urea: 2.5 - 50 mmol / L; ALT / AST: 5 - 200 U / L) and quality control serum. Detection is carried out using an automatic biochemical analyzer (such as Hitachi 7080). The determination conditions for urea are double wavelengths of 340 / 405 nm and a reaction at 37 °C for 5 minutes; the determination conditions for ALT / AST are a wavelength of 340 nm and a reaction at 37 °C for 3 minutes. The instrument automatically generates a standard curve (R 2 > 0.99) and calculates the concentration to ensure that the within-batch coefficient of variation < 5%, and the recovery rate is 95 - 105%. Avoid hemolysis throughout the experiment. Complete the detection within 2 hours after serum separation or store it at -80 °C, and keep the operation in a low-temperature environment. Data analysis is expressed as mean ± SEM, and statistical processing is carried out using GraphPad Prism. The judgment criteria are that ALT > 80 U / L indicates liver injury, AST > 120 U / L indicates myocardial and liver injury, and Urea > 15 mmol / L indicates renal function abnormality.

[0048] IHC experiment: First, prepare paraffin sections with a thickness of 4-5 μm. The tumor tissue fixed with 4% paraformaldehyde for 48 hours is dehydrated with gradient ethanol (70%-100%), cleared with xylene, and then embedded in paraffin. Bake the sections at 60 °C for 1 hour. During the experiment, perform dewaxing and hydration treatments in sequence: dewax twice with xylene, rehydrate with gradient ethanol for 5 minutes at each level, and rinse 3 times with PBS. Antigen retrieval is carried out using sodium citrate buffer or EDTA, with high-pressure retrieval at 121 °C for 2 minutes or microwave medium-fire retrieval for 15 minutes. After natural cooling to room temperature, rinse 3 times with PBS. Incubate with 3% H2O2 at room temperature for 10 minutes to block the activity of endogenous peroxidase, and rinse 3 times with PBS. After blocking with 5% BSA or normal serum at room temperature for 30 minutes, add an appropriate concentration of primary antibody and incubate overnight at 4 °C. Rinse 3 times with PBS, add HRP-labeled secondary antibody and incubate at room temperature for 1 hour. Rinse 3 times with PBS, develop color with DAB chromogenic solution (control the color development time under the microscope), and terminate the reaction with tap water. Counterstain with hematoxylin for 1 minute, differentiate with hydrochloric acid alcohol, bluing with ammonia water, dehydrate with gradient ethanol, clear with xylene, and mount with neutral gum. Finally, observe and collect images under an optical microscope, and perform quantitative analysis on the staining results using image analysis software such as Image-Pro Plus.

[0049] Jin's formula: That is, Q = Ea + b / (Ea + Eb - Ea*Eb), which is a method for evaluating the effect of drug combinations. Among them, Ea represents the inhibition rate of drug A, and Eb represents the inhibition rate of drug B. According to the value of Q, the effects of drug combinations can be divided into the following three categories: Antagonistic effect: When 0 < Q ≤ 0.85, it means that the effect of the two drugs used in combination is worse than that of using only one of the drugs alone, that is, there is an antagonistic effect between the drugs, resulting in a weakened overall effect. Additive effect: When 0.85 < Q ≤ 1.15, it means that the effect of the two drugs used in combination is equivalent to the sum of the effects of using the two drugs alone, that is, there is no obvious synergistic or antagonistic effect between the drugs, and the overall effect is additive. Synergistic effect: When Q > 1.15, it means that the effect of the two drugs used in combination is significantly better than that of using only one of the drugs alone, that is, there is a synergistic effect between the drugs, resulting in an enhanced overall effect.

[0050] Example 3

[0051] Analysis of experimental results

[0052] (1) In vitro cell experiment: The combination of Molephantinin and cisplatin has a synergistic inhibitory effect in inhibiting the progression of human nasopharyngeal carcinoma. Figure 1 The combination index is shown to indicate the synergistic effect. Specifically, Figure 1As shown, in this study, the combined effects of Molephantinin (10 μM) and three commonly used clinical chemotherapeutic drugs (cisplatin (DDP), gemcitabine (Gem), and oxaliplatin (Oxa)) were evaluated in nasopharyngeal carcinoma cells CNE-1. Through concentration gradient experiments (1 - 9 μM), it was found that the combination of Molephantinin and cisplatin showed a significant synergistic effect: when the cisplatin concentration ≥ 4 μM, the Q values of all combined groups were > 1.15, and the synergistic effect was most significant in the group combining 10 μM Molephantinin and 8 μM cisplatin, with the combination index calculated by the Chou-Talalay method reaching 1.366. In contrast, the Q values of gemcitabine were < 1.15 at all tested concentrations; for oxaliplatin, the Q value was 1.157 only when the concentration was 7 μM and Molephantinin was 10 μM, and it dropped below 1.15 again when the oxaliplatin concentration was 8 - 9 μM. These results indicate that the combination of Molephantinin and cisplatin can synergistically inhibit the growth of nasopharyngeal carcinoma, suggesting that Molephantinin may increase the sensitivity of cancer cells to cisplatin and a specific concentration of oxaliplatin. The combination of Molephantinin and cisplatin can inhibit the proliferation of human nasopharyngeal carcinoma cells. Figure 2 Showing the results of the cell CCK-8 assay. The cell proliferation activity of human nasopharyngeal carcinoma cells was affected by the combination of Molephantinin and cisplatin, and their activity was significantly lower than that of single drug use after the combination.

[0053] (2) In vivo animal experiments: The combination of Molephantinin and cisplatin can inhibit the growth of xenograft tumors of human nasopharyngeal carcinoma cells in nude mice. Figure 3 Showing the results of the subcutaneous tumorigenesis experiment in nude mice. The size of the tumors indicates that the subcutaneous tumorigenesis in nude mice was significantly inhibited after the combination of Molephantinin and cisplatin. Figure 4 Showing that there were no abnormal changes in urea (Urea) and liver function indicators (ALT, AST) in the blood of nude mice after the combination of the two drugs. This indicates that the combination of Molephantinin and cisplatin has no obvious drug toxicity, and there are no abnormal changes in the heart, liver, spleen, lung, and kidney tissues of nude mice. Figure 5 Showing the changes in the proliferation index Ki67 after the combination of the two drugs. The expression of Ki67 in the combination group of the two drugs was significantly lower than that of single drug treatment, indicating that the combination of Molephantinin and cisplatin in vivo can better inhibit the growth of nasopharyngeal carcinoma tumors.

Claims

1. The use of Molephantinin in combination with platinum-based chemotherapeutic drugs in the preparation of a drug for treating nasopharyngeal carcinoma, wherein the platinum-based chemotherapeutic drug is cisplatin or oxaliplatin.

2. The application according to claim 1, characterized in that: The dosage concentration of cisplatin is greater than or equal to 4 μM.

3. The application according to claim 1, characterized in that: The dosage concentration of cisplatin is 8 μM; the dosage concentration of Molephantinin is 10 μM.

4. The application according to claim 1, characterized in that, The nasopharyngeal carcinoma is caused by CNE-1 cells, S18 cells or 58-F cells.

5. The application according to claim 1, wherein: The dosage concentration of Molephantinin is 10 μM, and the dosage concentration of oxaliplatin is 7 μM.

6. A drug for treating nasopharyngeal carcinoma, characterized in that It includes: Molephantinin, platinum-based chemotherapeutic drugs; the platinum-based chemotherapeutic drug is cisplatin or oxaliplatin.

7. The drug according to claim 6, characterized in that: The dosage concentration of cisplatin is greater than or equal to 4 μM.

8. The drug according to claim 6, characterized in that: The dosage concentration of Molephantinin is 10 μM, and the dosage concentration of oxaliplatin is 7 μM.

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