Application of daphnetin in medicine for treating drug-resistant ovarian cancer

Abstract

The invention belongs to the field of medicine application, and discloses application of daphnetin in medicine for treating drug-resistant ovarian cancer, and the daphnetin can be applied in the medicine for treating the drug-resistant ovarian cancer. The drug-resistant ovarian cancer treatment drug comprises daphnetin and platinum drugs. The platinum medicines in the medicine for treating the drug-resistant ovarian cancer comprise clinical platinum medicines such as cis-platinum, carboplatin, oxaliplatin, nedaplatin or lobaplatin. The dose of daphnetin contained in the medicine for treating the drug-resistant ovarian cancer is 0.45 g once, and three times a day. The dosage of platinum in the drug-resistant ovarian cancer treatment drug is calculated according to a clinical detection result of a patient. The method has the beneficial effects that experimental data confirms that the treatment scheme of combining daphnetin with cis-platinum can effectively overcome platinum drug resistance, and a medication strategy with conversion value is provided for the medication for clinically treating recurrent drug-resistant ovarian cancer.

Description

Technical Field

[0001] The invention belongs to the field of drug application, and relates to the application of a drug in a drug for treating drug-resistant ovarian cancer, and particularly relates to the application of daphnetin in a drug for treating drug-resistant ovarian cancer. Background Art

[0002] Currently, ovarian cancer is the malignant tumor with the highest mortality rate in the female reproductive system. Its clinical treatment faces a double dilemma: more than 70% of patients have progressed to the advanced stage (FIGO stage III / IV) when diagnosed, and most advanced patients have a 5-year survival rate of less than 40% due to acquired chemotherapy resistance. Although combined chemotherapy based on platinum drugs (such as cisplatin) is still the standard first-line treatment for advanced ovarian cancer, the occurrence of acquired resistance causes recurrence and progression of advanced patients within 2 years, and the median survival of patients is sharply reduced from 3-5 years to 1-1.5 years.

[0003] The mechanism of acquired drug resistance in ovarian cancer involves multidimensional pathological processes, which are mainly manifested in the following three aspects of synergistic effects: (1) Abnormal drug metabolism and pharmacokinetic imbalance: ① Transmembrane transport imbalance: Cancer cells reduce the uptake of platinum drugs by downregulating copper transporter 1 (CTR1) and at the same time upregulate the activity of efflux pumps such as ATP7A / B to accelerate drug excretion, resulting in insufficient accumulation of intracellular platinum drugs; ② Activation of the detoxification system: The glutathione (GSH)-mediated platinum detoxification pathway is enhanced, significantly reducing the efficiency of the formation of active platinum-DNA adducts. (2) Cell death resistance mechanism: ① Apoptosis pathway inhibition: B-cell lymphoma-2 (Bcl-2) / Bcl-2-associated X protein (Bax) protein imbalance hinders the activation of mitochondrial apoptosis pathway, and the activation level of caspase-3 is significantly reduced; ② Protective autophagy activation: Abnormal regulation of the AMP-activated protein kinase (AMPK) signaling pathway induces enhanced autophagy flow and promotes cell survival by maintaining mitochondrial function. (3) Tumor microenvironment remodeling: ① Metabolic adaptive changes: Hypoxia-inducible factor-1α (HIF-1α)-driven enhanced glycolysis provides tumor cells with a hypoxic microenvironment survival advantage; ② Immune escape mechanism: Ferropioid resistance leads to the formation of an immunosuppressive microenvironment, helping resistant cells escape immune clearance. In summary, acquired resistance is a self-protection mechanism constructed by tumor cells through multi-level adaptive changes such as metabolic reprogramming, death resistance, and microenvironment adaptation.

[0004] In recent years, although poly adenosine diphosphate ribose polymerase (PARP) inhibitors (Olaparib, etc.) and anti-angiogenic drugs (bevacizumab, etc.) have shown their value as maintenance treatment in patients with specific molecular classifications (homologous recombination repair deficiency [HRD] / BRCA1 / 2 mutations), their clinical application still faces significant limitations: ① The applicable population is limited, covering only about 10%-15% of the HRD-positive population; ② The objective response rate (ORR) is only about 30%-35%, and the median progression-free survival (PFS) is less than 13 months; ③ More than 40%-70% of patients with BRCA mutations develop acquired resistance within 24 months. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of poor prognosis caused by drug resistance in patients with advanced ovarian cancer, and to provide an application of daphnetin in a drug for treating drug-resistant ovarian cancer, and a drug for treating drug-resistant ovarian cancer.

[0006] The daphnetin provided by the invention can be used in drugs for treating drug-resistant ovarian cancer.

[0007] Drugs used to treat resistant ovarian cancer include daphneline and platinum drugs.

[0008] Platinum drugs used in the treatment of drug-resistant ovarian cancer include cisplatin, carboplatin, oxaliplatin, nedaplatin, or lobaplatin, which are clinically used platinum drugs.

[0009] The dose of daphnetin contained in the drug for the treatment of drug-resistant ovarian cancer is 0.45 g at a time, 3 times a day.

[0010] The dose of platinum in the treatment of drug-resistant ovarian cancer is calculated based on the patient's clinical test results.

[0011] Daphnetin (Daph) is a natural dihydroxycoumarin compound extracted from plants of the Thymelaeaceae family. It has a broad-spectrum biological activity and shows potential therapeutic value in the fields of inflammation, tumors, neurodegenerative diseases, cardiovascular diseases, fibrotic diseases, diabetes, etc. The present invention has confirmed for the first time through in vitro experiments that when daphnetin (20 mg / L) is used in combination with cisplatin, it can significantly enhance the synergistic killing effect on drug-resistant ovarian cancer cells (SKOV3 / DDP, A2780 / DDP), reverse cisplatin resistance, and provide a new drug for the treatment of drug-resistant ovarian cancer.

[0012] Beneficial effects of the present invention:

[0013] The daphnetin provided by the present invention can be used in drugs for treating drug-resistant ovarian cancer. In vitro experiments have confirmed that the combination of daphnetin (20 mg / L) and cisplatin (6 mg / L or 8 mg / L) can reduce the IC of the drug-resistant cell line SKOV3 / DDP.50 The value was reduced by 3 times (p<0.001), and the cell clone formation was significantly inhibited. The in vivo subcutaneous tumor model showed that the tumor volume inhibition rate of the daphneline combined with cisplatin treatment group was 80%, while the tumor volume inhibition rate of the paclitaxel combined with cisplatin treatment group was about 70%. Experimental data confirmed that the daphneline combined with cisplatin treatment regimen can effectively overcome platinum resistance and provide a drug strategy with translational value for the clinical treatment of recurrent resistant ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the in vitro enhancement of the drug sensitivity of drug-resistant ovarian cancer by combining daphnetin with cisplatin as described in the present invention.

[0015] Among them: A: Cell survival rate of ovarian cancer A2780 and drug-resistant ovarian cancer A2780 / DDP cells monitored by CCK-8 experiment under gradient concentration of cisplatin.

[0016] B: Cell survival rates of ovarian cancer SKOV3 and drug-resistant ovarian cancer SKOV3 / DDP cells monitored by CCK-8 assay under treatment with gradient concentrations of cisplatin.

[0017] C: IC of cisplatin in the four cell types in A and B 50 value.

[0018] D and F: Daphnetin combined with cisplatin treatment of A2780 / DDP cells and SKOV3 / DDP cells, respectively, visually represent the percentage of growth inhibition of A2780 / DDP cells (D) and SKOV3 / DDP cells (F) under single or combined treatment conditions (left). Synergy Finder 3.0 tool was used to evaluate the synergistic effect of various combinations of Daph and DDP (right).

[0019] E and G: The clone formation experiments of A2780 / DDP cells and SKOV3 / DDP cells treated with daphnetin, cisplatin, and daphnetin combined with cisplatin, respectively, intuitively showing the proliferation of A2780 / DDP (E) and SKOV3 / DDP (G) cells under single or combined treatment conditions.

[0020] Figure 2 This is a schematic diagram of the killing effect of daphnetin combined with cisplatin on drug-resistant ovarian cancer in vivo as described in the present invention.

[0021] Among them: A: Schematic diagram of the in vivo experimental design of mice. 21 days after tumor cell inoculation, the mice were randomly divided into: combined treatment group: cisplatin 5mg / kg + daphnetin 30mg / kg, cisplatin 5mg / kg + paclitaxel 20mg / kg (twice a week, intraperitoneal injection); control group (Control), cisplatin monotherapy group (DDP), daphnetin monotherapy group (Daph), daphnetin combined with cisplatin group (Daph+DDP), paclitaxel combined with cisplatin group (PTX+DDP), 6 mice in each group, and the treatment was continued for 15 days.

[0022] The body weight (B), tumor volume (C), and tumor weight (E) of the mice were recorded.

[0023] D: Representative images of ex vivo tumors.

[0024] F: Representative IHC images of tumor tissues of mice in different groups.

[0025] G: IHC score.

[0026] The results are expressed as SEM ± mean. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns = not significant. DETAILED DESCRIPTION

[0027] See also Figure 1 to Figure 2 As shown:

[0028] The daphnetin provided by the invention can be used in drugs for treating drug-resistant ovarian cancer.

[0029] Drugs used to treat resistant ovarian cancer include daphneline and platinum drugs.

[0030] Platinum drugs used in the treatment of drug-resistant ovarian cancer include cisplatin, carboplatin, oxaliplatin, nedaplatin, or lobaplatin, which are clinically used platinum drugs.

[0031] The dose of daphnetin contained in the drug for the treatment of drug-resistant ovarian cancer is 0.45 g at a time, 3 times a day.

[0032] The dose of platinum in the treatment of drug-resistant ovarian cancer is calculated based on the patient's clinical test results.

[0033] Daphnetin (Daph) is a natural dihydroxycoumarin compound extracted from plants of the Thymelaeaceae family. It has a broad-spectrum biological activity and shows potential therapeutic value in the fields of inflammation, tumors, neurodegenerative diseases, cardiovascular diseases, fibrotic diseases, diabetes, etc. The present invention has confirmed for the first time through in vitro experiments that when daphnetin (20 mg / L) is used in combination with cisplatin, it can significantly enhance the synergistic killing effect on drug-resistant ovarian cancer cells (SKOV3 / DDP, A2780 / DDP), reverse cisplatin resistance, and provide a new drug for the treatment of drug-resistant ovarian cancer.

[0034] The specific experimental process is as follows:

[0035] 1. In vitro experimental verification:

[0036] (1) Experimental materials:

[0037] Ovarian cancer cell lines (A2780, SKOV3), drug-resistant ovarian cancer cell lines (A2780 / DDP, SKOV3 / DDP), daphnetin, cisplatin, paclitaxel, CCK-8 kit, 4% paraformaldehyde, 0.5% crystal violet staining solution, phosphate-balanced saline (PBS).

[0038] (2) CCK-8 method to detect cell viability:

[0039] ① Cell plating: Digest the cells in the logarithmic growth phase and adjust the density to 1-5×10 using a hemocytometer. 4 cells / mL; inoculation of 96-well plate: add 100μL cell suspension to each well (final density 1,000-5,000 cells / well); fill the edge wells with 200μL PBS to reduce evaporation effect; pre-culture in a 37℃, 5% CO2 incubator for 24 hours.

[0040] ② Drug treatment: dilute the drug gradient into 6 concentration gradients with complete culture medium (1:3 isotropic dilution); medium replacement treatment: aspirate the original culture medium and add 100 μL of drug-containing culture medium to each well; set up a blank group (culture medium only, no cells) and a negative control group (equal amount of DMSO, concentration ≤0.1%).

[0041] ③CCK-8 detection: After treatment, add 10 μL CCK-8 reagent to each well (protect from light); gently shake to mix (amplitude 5 mm, frequency 120 rpm, time 30 seconds); continue to culture for 1 hour; set the microplate reader to shake for 5 seconds and then read the OD450 value.

[0042] ④Data analysis: Cell survival rate (%) = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100; GraphPad Prism software was used for four-parameter logistic curve fitting (4PL model) Y = Bottom + (Top-Bottom) / (1+10^((LogEC50-X)*HillSlope)).

[0043] ⑤HSA (Highest Single Agent) model synergy score (SynergyScore) for detecting the synergistic effect of combined drug therapy:

[0044] • Calculate the inhibition rate: .

[0045] • Convert inhibition percentage to a 0-1 scale (0 = no inhibition, 1 = complete inhibition).

[0046] • Calculate the maximum effect of a single drug to determine:

[0047] Extract the maximum inhibition rate of each drug treated alone (MaxSingleEffect):

[0048]

[0049] • Calculate theoretical additive effect:

[0050] For each concentration combination (i, j):

[0051]

[0052] • Calculate the synergy score:

[0053] The difference between the actual joint effect and the theoretical relative effect:

[0054]

[0055] Global Collaborative Scoring:

[0056]

[0057] •Result interpretation criteria:

[0058]

[0059] (3) Clone formation assay to detect cell proliferation activity:

[0060] ① Cell plating: Take cells in logarithmic growth phase, digest them with trypsin, and resuspend them in complete culture medium. Adjust the density to 250 cells / mL (adjust according to the cell proliferation rate) with a hemocytometer. Add 2 mL of cell suspension to each well of a 6-well plate (finally 500 cells / well). Gently shake the culture plate using the cross method to evenly distribute the cells. Pre-culture at 37°C for 24 hours (to allow the cells to adhere).

[0061] ②Drug treatment: Aspirate and discard the original culture medium, and divide the cells into groups according to the experimental design: blank control group (containing only culture medium solvent), control group (containing an equal volume of DMSO, final concentration ≤0.1%) and experimental group (drug concentration - daphnetin 20 mg / L, cisplatin 6 or 8 mg / L). Add culture medium to each well to a final volume of 2 mL, and culture at 37°C for 10-14 days (adjusted according to the cell proliferation rate, and the clone formation was observed every other day).

[0062] ③Terminate culture and staining: aspirate the culture medium, rinse gently twice with PBS (to avoid breaking up the clones), add 1 mL of 4% paraformaldehyde fixative to each well, let stand at room temperature for 15 minutes, aspirate the fixative, rinse three times with PBS to remove residual fixative, add 0.5% crystal violet staining solution (1 mL / well), stain at room temperature in the dark for 30 minutes, rinse slowly with running water until the background is colorless, invert to dry, and take pictures.

[0063] ④Clone counting standard: Valid clones are cell clusters visible to the naked eye with a cell number of ≥50.

[0064] 2. In vivo experimental verification:

[0065] (1) Construction of subcutaneous transplanted tumor model:

[0066] ① Animal selection: Beijing Weitonglihua BALB / c nude mice (6-8 weeks old, female, body weight 18-22g), 6 mice in each group.

[0067] ② Prepare cells: SKOV3 / DDP, subculture to the logarithmic growth phase in RPMI-1640 medium containing 15% FBS, and confirm that the viable cell rate is >95% using trypan blue staining.

[0068] (2) Construction of subcutaneous transplanted tumor model:

[0069] The model was constructed using the cell suspension transplantation method.

[0070] ① Preparation of cell suspension: After trypsin digestion, centrifuge (1000 rpm × 5 min), wash twice with PBS, resuspend in Matrigel-PBS mixture (Matrigel: PBS = 1:1, operate on ice), and adjust the cell density to 1 × 10 7 cells / mL.

[0071] ② Subcutaneous inoculation: 1.5% isoflurane was used for anesthesia. The mice were fixed in the lateral position and the cell suspension was injected subcutaneously under the right axilla (avoiding blood vessels and nerves) for each mouse. 6 cells), use a 27G needle, insert the needle at 45° and slowly push the needle in to avoid leakage.

[0072] ③Postoperative management and monitoring:

[0073] •Postoperative care: Apply iodine to the incision to prevent infection.

[0074] •Tumor monitoring: Use a vernier caliper to measure the long diameter (a) and short diameter (b) twice a week, calculate and record the tumor tissue volume: V = a × b × b / 2 (unit: mm 3 ); when the tumor volume is >1500mm 3 Or humane endpoint treatment when ulceration / necrosis occurs.

[0075] •Weight monitoring: Mice are weighed weekly (g), and humane endpoint treatment is performed if body weight loss > 20%.

[0076] (3) Drug sensitivity test:

[0077] The killing effects of daphnetin alone, cisplatin alone, daphnetin + cisplatin, and paclitaxel + cisplatin on tumor proliferation were tested.

[0078] ① Grouping: Control group: normal saline (0.1mL / 10g, intraperitoneal injection), cisplatin single-drug group: 5mg / kg (twice a week, intraperitoneal injection), daphnetin single-drug group: 30mg / kg (once a day, oral gavage), combined treatment group: cisplatin 5mg / kg + daphnetin 30mg / kg, cisplatin 5mg / kg + paclitaxel 20mg / kg (twice a week, intraperitoneal injection).

[0079] ② Preparation: Daphneline was dissolved in 5% DMSO + 30% PEG400 + 65% saline (final concentration 2 mg / mL), cisplatin was diluted to 0.6 mg / mL with saline, and paclitaxel was diluted to 2.4 mg / mL with saline.

[0080] ③ Grouping: After cell suspension inoculation, the tumor volume was monitored daily until the average volume reached 100-150mm 3 The patients were randomly divided into groups after 21 days (approximately 21 days), and a stratified randomization method was used to ensure that there was no statistical difference in the initial tumor volume among the groups (p>0.05).

[0081] ④ Dosage plan: Treatment for 15 consecutive days, cisplatin and paclitaxel are intraperitoneally injected on Mondays and Thursdays (operate in dark areas), and daphnetin is orally administered by gavage from 9:00 to 11:00 a.m. daily. If body weight loss is greater than 15%, the cisplatin dose is reduced to 3 mg / kg. If diarrhea occurs, daphnetin is suspended for 1 day.

[0082] ⑤ Monitoring and termination of the experiment: Monitor the tumor volume and weight of mice according to the method in 2 (2) ③. When the tumor volume is greater than 1500mm 3 Or when the body weight decreased by >20%, the mice were subjected to CO2 asphyxiation (flow rate 1.5 L / min, duration 5 min).

[0083] ⑥ Sample collection: Tumor tissue was divided into two portions, which were quickly frozen at -80°C and fixed with 4% paraformaldehyde. Blood was collected in heparin anticoagulant tubes (plasma was separated at 3000rpm×15min).

[0084] ⑦Immunohistochemistry (IHC):

[0085] •Dewaxing and hydration: Bake the slides in a 60°C oven for 30 minutes, dewax by immersing in xylene I and II for 10 minutes each, and then in gradient ethanol (100% → 95% → 80%) for 5 minutes each → rinse with distilled water for 3 minutes.

[0086] •Antigen retrieval: Heat retrieval method, microwave sodium citrate buffer (10mM, pH6.0) until boiling, put in the slices and maintain boiling on medium heat for 5 minutes, naturally cool to room temperature (about 30 minutes), rinse 3 times with PBS (pH7.4) for 3 minutes, and digest with 0.1% trypsin (pH7.8) at 37℃ for 10 minutes.

[0087] • Block endogenous peroxidase: Incubate with 3% H2O2 (in methanol) at room temperature for 10 minutes → rinse with PBS 3 times for 3 minutes.

[0088] •Block non-specific binding: add 5% normal goat serum (homologous to the secondary antibody), block at room temperature for 30 minutes → pour off the serum (do not wash).

[0089] •Primary antibody incubation: Add diluted primary antibody (Ki-67, 1:200), incubate overnight at 4°C in a wet box, return to room temperature the next day → rinse with PBS 3 times × 5 minutes.

[0090] • Secondary antibody incubation: Add HRP-labeled secondary antibody (anti-rabbit IgG, 1:500), incubate at room temperature for 30 minutes → rinse with PBS 3 times × 5 minutes.

[0091] •DAB color development: prepare DAB working solution according to the instructions of the kit (prepare before use), add DAB dropwise, control the color development time under a microscope (usually 1-3 minutes, until the positive signal is clear and the background is not colored), and immediately rinse with running water to terminate the reaction.

[0092] •Restaining and differentiation: Hematoxylin staining for 1 minute → rinse with running water to turn blue → 1% hydrochloric acid alcohol differentiation for 2 seconds → rinse with running water for 10 minutes.

[0093] •Dehydration and sealing: gradient ethanol dehydration (80% → 95% → 100% ethanol, 1 minute each) → xylene clearing 2 times × 5 minutes → neutral gum sealing.

[0094] •Photography: Photos were taken using an Olympus IX73 microscope.

[0095] • Calculate IHC score:

[0096] Strength classification:

[0097] 0 points (negative): no staining.

[0098] 1 point (weak positive): light yellow.

[0099] 2 points (moderately positive): brown-yellow.

[0100] 3 points (strong positive): dark brown.

[0101] Area classification (including the numbers on the left, excluding the numbers on the right):

[0102] 1 point: Positive cells account for 0-25%.

[0103] 2 points: Positive cells account for 25-50%.

[0104] 3 points: Positive cells account for 50-75%.

[0105] 4 points: Positive cells account for 75-100%.

[0106] IHC score = intensity grading score × positive cell percentage score.

Claims

1. A use of daphnetoside in the treatment of drug-resistant ovarian cancer.

2. The use of daphnetin in the treatment of drug-resistant ovarian cancer according to claim 1, characterized in that: The drug for treating drug-resistant ovarian cancer includes daphnetin and platinum drugs.

3. The use of daphnetin in the treatment of drug-resistant ovarian cancer according to claim 2, characterized in that: The platinum drugs in the drug-resistant ovarian cancer treatment drug include cisplatin, carboplatin, oxaliplatin, nedaplatin or lobaplatin, which are clinically used platinum drugs.

4. The use of daphnetin in the treatment of drug-resistant ovarian cancer according to claim 1 or 2, characterized in that: The dosage of daphnetin contained in the drug for treating drug-resistant ovarian cancer is 0.45 g at a time, 3 times a day.

5. The use of daphnetin in the treatment of drug-resistant ovarian cancer according to claim 3, characterized in that: The dosage of platinum in the drug for treating drug-resistant ovarian cancer is calculated based on the clinical test results of the patient.

6. A drug for treating drug-resistant ovarian cancer, characterized in that: The drugs include daphneline and platinum drugs.

7. The drug for treating drug-resistant ovarian cancer according to claim 6, characterized in that: The platinum drugs include cisplatin, carboplatin, oxaliplatin, nedaplatin or lobaplatin, which are clinically used platinum drugs.

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