Pharmaceutical composition for treating pancreatic cancer and application thereof

Abstract

The invention discloses a novel application of a composition of oleic acid, linoleic acid and Brusol or gemcitabine. According to the invention, oleic acid and linoleic acid are combined with different anti-tumor drugs, a pharmaceutical composition with a synergistic effect is screened out, and test results show that when oleic acid and linoleic acid in a specific proportion are combined with Brusol or gemcitabine, unexpected synergistic anti-pancreatic cancer activity can be obtained. The composition is expected to be developed into a new antitumor drug, can reduce the dosage of the antitumor drug, reduce drug resistance, reduce adverse reactions and improve the life quality of patients, and has important application prospects.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to an anti-tumor composition comprising oleic acid and linoleic acid and application thereof. Background Art

[0002] Liver cancer is a common malignant tumor of the digestive tract. Commonly used clinical treatment drugs include 5-uracil (5-FU), which is a fluoride of pyrimidine and an antimetabolite antitumor drug. It can inhibit thymidylate synthase, block the conversion of deoxypyrimidine nucleotides into thymidine nucleosides, and interfere with DNA synthesis. It also has a certain inhibitory effect on RNA synthesis. It has a certain therapeutic effect on colon cancer, liver cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, etc., but 5-uracil also has certain adverse reactions such as bone marrow suppression and gastrointestinal tract.

[0003] Capecitabine is an anti-metabolite fluoropyrimidine deoxynucleoside aminobamate drug that can be converted into 5-FU in the body. Capecitabine is widely used in the single-drug or combined clinical treatment of cancers such as breast cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, cervical cancer, pancreatic cancer, etc. However, capecitabine also has adverse reactions such as nausea and vomiting, diarrhea, gastrointestinal irritation and bleeding, and bone marrow suppression.

[0004] Pancreatic cancer is a common malignant tumor of the digestive tract. Due to its hidden symptoms, short course, strong invasiveness, rapid progression, and lack of sensitive diagnostic indicators for early diagnosis and treatment, most patients are in the late stage when they seek medical treatment. In recent years, the incidence of pancreatic cancer in China has increased rapidly, and pancreatic cancer has become the sixth largest cancer plaguing the Chinese people. At this stage, surgery is the only possible means of curing pancreatic cancer, but due to the difficulty in early diagnosis, only a small number of patients have the opportunity to receive surgical treatment, so drugs are still the main means of clinical treatment of pancreatic cancer.

[0005] Gemcitabine is one of the first-line chemotherapy drugs for the clinical treatment of pancreatic cancer. However, the median survival of patients taking gemcitabine is less than 6 months, and only 12% of patients have symptom relief. At the same time, clinically, patients often develop drug resistance and serious adverse reactions after long-term use of the drug. Therefore, there is an urgent need to develop safer and more effective new drugs and treatment methods.

[0006] Natural products have always been the focus of drug research and development. Quassin is a characteristic component of the traditional Chinese medicine Brucea javanica. Among them, Brusatol is an active compound against pancreatic cancer.

[0007] The use of combination therapy to treat tumors is one of the current research hotspots, but the combination of different drugs is a complex process. Different mechanisms of treating diseases will result in different effects such as antagonism or synergy. Summary of the Invention

[0008] Object of the Invention: To solve the above problems, through a large number of experimental screenings, the present invention combines oleic acid and linoleic acid with different anti-tumor drugs to screen out a drug composition with synergistic enhancement. Through a large number of experiments, the present invention discovers that a specific ratio of oleic acid and linoleic acid combined with Brusatol and gemcitabine can achieve unexpected synergistic anti-pancreatic cancer activity. The composition of the present invention can reduce the dosage of anti-tumor drugs, reduce adverse reactions, and improve the quality of life of patients.

[0009] Technical Solution: To achieve the above object, the technical solution adopted by the present invention is as follows: A pharmaceutical composition for treating cancer, which comprises oleic acid, linoleic acid and Brusatol or gemcitabine.

[0010] As a preferred embodiment, in the above-mentioned composition, the mass ratio of oleic acid and linoleic acid to Brusatol is 10-16:1, and the molar ratio of oleic acid and linoleic acid is 3:1-4:1.

[0011] Particularly preferably, the mass ratio of oleic acid and linoleic acid to Brusatol is 16:1, and the molar ratio of oleic acid and linoleic acid is 3:1 or 4:1.

[0012] The present invention discovers through a large number of experiments that a specific ratio of oleic acid and linoleic acid combined with Brusatol and gemcitabine has synergistic anti-pancreatic cancer activity.

[0013] The present invention can prepare the composition into a pharmaceutical preparation with a pharmaceutically acceptable carrier. The pharmaceutical preparations include common clinical dosage forms such as tablets, pills, capsules, granules, injections, etc. Brief Description of the Drawings

[0014] Figure 1 Shows that the optimal ratio of the combination of oleic acid and linoleic acid for synergistic anti-pancreatic cancer is 4:1. (A) Effects of different ratios of oleic acid and linoleic acid on the proliferation of pancreatic cancer MIA PaCa-2 cells; (B) ZIP, Bliss and HSA synergistic scores, inhibitory proliferation effects of 4O1L on pancreatic cancer MIA PaCa-2 cells for 24 h; (C) CI values of 4O1L and 3O1L on pancreatic cancer MIA PaCa-2 cells for 24 h. 3O1L: A combination of oleic acid and linoleic acid with a molar concentration ratio of 3:1. 4O1L: A combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1.

[0015] Figure 2It shows that the combination of oleic acid and linoleic acid has a significant synergistic inhibitory effect on pancreatic cancer growth with the anti-pancreatic cancer active compound (Brusatol). Among them, (A) tumor pictures; (B) tumor volume; (C) tumor weight; (D) schematic diagrams of HE (scale bar 50 µm), Ki67 immunohistochemistry (scale bar 50 µm), and TUNEL (scale bar 20 µm) in pancreatic cancer tumor tissues; (E) quantitative statistics of Ki67-positive cells in pancreatic cancer tumor tissues; (F) quantitative statistics of TUNEL-positive cells in pancreatic cancer tumor tissues; (G-K) changes in the protein ratios of cleaved Caspase 8 / Caspase 8 (H), cleaved PARP / PARP (I), cleaved Caspase 9 / Caspase 9 (J), and cleaved Caspase 3 / Caspase 3 (K) in pancreatic cancer tumor tissues after co-administration of oleic acid, linoleic acid, and Brusatol. Compared with the blank control, * P <0.05, ** P <0.01, *** P <0.001; compared with 3O1L + Brusatol, ■ P <0.05, ■■ P <0.01, ■■■ P <0.001; compared with 4O1L + Brusatol, ◆ P <0.05, ◆◆ P <0.01, ◆◆◆ P <0.001. 3O1L: The combination of oleic acid and linoleic acid with a molar concentration ratio of 3:1. 4O1L: The combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1.

[0016] Figure 3It shows that the combination of oleic acid and linoleic acid has a significant synergistic inhibitory effect on pancreatic cancer cells with the anti-pancreatic cancer active compound (Brusatol). Among them, (A) the effects of different doses of the combination of oleic acid and linoleic acid, Brusatol, and the combination of oleic acid and linoleic acid with Brusatol on the proliferation of pancreatic cancer MIA PaCa-2 cells for 24 h; (B) the CI value of the combination of oleic acid and linoleic acid with Brusatol on pancreatic cancer MIA PaCa-2 cells for 24 h; (C-D) the effects of the combination of oleic acid and linoleic acid with Brusatol on the apoptosis rate of pancreatic cancer MIA PaCa-2 cells; (E-I) the changes in the protein ratios of cleaved PARP / PARP (F), cleaved Caspase 3 / Caspase 3 (G), cleaved Caspase 9 / Caspase 9 (H), and cleaved Caspase 8 / Caspase 8 (I) after administration of the combination of oleic acid and linoleic acid with Brusatol. Compared with the blank control, * P <0.05, ** P <0.01, *** P <0.001; compared with 4O1L+Brusatol, + P <0.05, ++ P <0.01, +++ P <0.001. 4O1L: The combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1. Detailed implementation mode

[0017] The present invention will be further explained below in conjunction with the embodiments. It should be understood that the following embodiments are only used to explain the present invention, rather than limiting the protection scope of the present invention.

[0018] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For those reagents or instruments not specifying the manufacturer, they can all be purchased through regular channels.

[0019] Example 1 Anti-pancreatic cancer experiment of the combination of oleic acid and linoleic acid 1 Materials and methods 1.1 Experimental materials DMEM medium, fetal bovine serum, and horse serum were all purchased from Gibico; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Beyotime.

[0020] 1.2 Cell lines and cell culture The human pancreatic cancer cell line MIA PaCa-2 was passaged and cultured in our laboratory. It was inoculated into DMEM medium containing 10% fetal bovine serum, 2.5% horse serum, and 100 U / mL penicillin-streptomycin, and cultured in an incubator at 37°C, 5% CO 2 ₂, with saturated humidity, and passaged every 2 - 3 days.

[0021] 1.3 Detection of the effect of drugs on the proliferation of pancreatic cancer cells by MTT method Collect MIA PaCa-2 cells in the logarithmic growth phase, adjust the cell suspension concentration, and inoculate them into a 96-well plate, 100 μL per well, so that the density of the cells to be tested is adjusted to 3000 - 6000 cells / well, and the edge wells are filled with sterile PBS. Incubate in an incubator at 37°C, 5% CO 2 ₂, with saturated humidity. After the cells adhered, 100 μL of fresh culture medium was added to the control group, and 100 μL of culture medium containing different concentrations of drugs was added to the experimental groups respectively. The drug concentrations are listed in Table 1 below. After culturing for 24 h under the same conditions, 20 μL of MTT was added to each well, and the culture was continued for 4 h. Then, the culture medium was carefully aspirated, 150 μL of dimethyl sulfoxide was added to each well, and the mixture was shaken in the dark for 5 - 15 min. The absorbance (OD) value at 570 nm was measured with an enzyme-linked immunosorbent assay reader, and the cell survival rate and inhibition rate were calculated. Cell survival rate (%) = OD 药物 / OD 空白 ×100%. Inhibition rate = 1 - OD 药物 / OD 空白 .

[0022] Table 1 Concentrations of combined oleic acid and linoleic acid at different ratios acting on pancreatic cancer cells for 24 h

[0023] 1.3 Application of data model SynergyFinder 3.0 software was used to estimate the expected drug combination response based on three reference models: Bliss, ZIP, and HAS. This software allows interactive analysis and visualization of multi-drug combination analysis data. Positive and negative synergy score values can be observed in the synergy plot dose region, representing synergy (red) and antagonism (green) respectively. The model will automatically select the best synergy region, within which the synergy ability is the strongest within the selected concentration range.

[0024] 1.4 Calculation of the combination index (CI) value to evaluate the anti-pancreatic cancer activity of combined drugs The inhibition rates of cells at different drug doses were obtained through MTT experiments. The inhibition rates and corresponding drug doses were input into CompuSyn software to calculate the CI value. CI value < 1 indicates synergy, CI value = 1 indicates addition, and CI value > 1 indicates antagonism.

[0025] 2 Experimental Results As Figure 1 shown in A, cell proliferation experiments were carried out by mixing different doses of oleic acid and linoleic acid, and the inhibition rate was calculated. According to the inhibition rate, three data models were selected for synergy scoring (red: synergy, white: additivity, green: antagonism). The darker the red or green color, the stronger the synergy or antagonism. The model will automatically select the optimal synergy region, within which the synergy ability is the strongest within the selected concentration range. The data analysis results are as Figure 1 shown in B. When the ratio of oleic acid to linoleic acid is 4:1, the synergistic anti-pancreatic cancer effect is the strongest. Based on the CI value, the anti-pancreatic cancer synergy of the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 was compared with that of the combination of oleic acid and linoleic acid with a molar concentration ratio of 3:1. The results are as Figure 1 shown in C. The CI values of the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 are all less than those of the combination of oleic acid and linoleic acid with a molar concentration ratio of 3:1. The above results reveal that 4:1 is the optimal ratio for the combination of oleic acid and linoleic acid to synergistically anti-pancreatic cancer.

[0026] Example 2 Anti-pancreatic Cancer Experiment of the Combination of Oleic Acid and Linoleic Acid and the Anti-pancreatic Cancer Active Compound (Brusatol) 1 Materials and Methods 1.1 Experimental Materials DMEM medium, fetal bovine serum and horse serum were all purchased from Gibico; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell apoptosis detection kit were purchased from Beyotime. BALB / c nude mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. (Animal Certificate: B202310240002), male, 6-week-old, SPF grade, and raised in the Experimental Animal Center of Nanjing University of Chinese Medicine.

[0027] Cell Lines and Cell Culture Human pancreatic cancer cell line MIA PaCa-2 was passaged and cultured in our laboratory, inoculated in DMEM medium containing 10% fetal bovine serum, 2.5% horse serum, and 100 U / mL penicillin-streptomycin, and cultured in an incubator at 37°C, 5% CO 2 , saturated humidity, and passaged every 2 - 3 days.

[0028] MTT Method for Detecting the Effect of Drugs on the Proliferation of Pancreatic Cancer Cells Collect MIA PaCa-2 cells in the logarithmic growth phase, adjust the cell suspension concentration, inoculate in a 96-well plate, 100 μL per well, and adjust the density of the cells to be tested to 3000 - 6000 cells / well. The edge wells were filled with sterile PBS. At 37°C, 5% CO 2, cultured in an incubator with saturated humidity. After the cells adhered to the wall, 100 μL of fresh culture medium was added to the control group, and 100 μL of culture medium containing different concentrations of drugs was added to the experimental groups respectively. The drug concentrations are listed in Table 2. After culturing for 24 h under the same conditions, 20 μL of MTT was added to each well and cultured for another 4 h. Then, the culture medium was carefully aspirated, 150 μL of dimethyl sulfoxide was added to each well, and it was shaken in the dark for 5 - 15 min. The absorbance (OD) value at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate and inhibition rate were calculated. Cell survival rate (%) = OD 药物 / OD 空白 × 100%. Inhibition rate = 1 - OD 药物 / OD 空白 .

[0029] Table 2 Concentrations of the combination of Brusatol, oleic acid and linoleic acid, and the effects of oleic acid, linoleic acid and Brusatol on pancreatic cancer cells for 24 h

[0030] 1.4 Calculation of the combination index (CI) value to evaluate the anti - pancreatic cancer activity of combined drugs The inhibition rates of cells at different drug dosages were obtained through the MTT experiment. The inhibition rates and the corresponding drug dosages were input into CompuSyn software to calculate the CI value. A CI value < 1 indicates synergy, a CI value = 1 indicates addition, and a CI value > 1 indicates antagonism.

[0031] 1.5 Animal experiments (1) Establishment of a subcutaneous xenograft model of pancreatic cancer BALB / c nude mice were placed in a laminar flow rack with purified air in a sterile laboratory at a certain humidity and temperature. After 1 week of adaptive growth of the nude mice, the experiment was carried out to establish a subcutaneous pancreatic cancer xenograft model in nude mice. Animal experiments strictly complied with the experimental animal management and protection regulations of Nanjing University of Chinese Medicine. Logarithmic growth phase MIA PaCa - 2 cells were collected, resuspended with an appropriate amount of PBS, and 4×10 6 cells were injected subcutaneously under the right axilla of each nude mouse. When the tumor diameter reached 4 - 6 mm, the model was successfully established.

[0032] (2) Administration to animals After successful modeling, BALB / c nude mice were randomly divided into a blank control group, a Brusatol group, an oleic acid and linoleic acid group, and an oleic acid and linoleic acid combined with Brusatol group, with 6 mice in each group. The administration method was intraperitoneal injection, once a day for 28 consecutive days. The blank control group was given 0.9% sodium chloride injection, the Brusatol group was given 2 mg / kg Brusatol, the oleic acid and linoleic acid combination group was given 32 mg / kg, and the oleic acid and linoleic acid combination and Brusatol group was given different dose ratios of 10:1, 12.5:1, and 16:1 of the oleic acid and linoleic acid combination and Brusatol.

[0033] (3)Animal Observation and Detection After modeling, the tumor size was monitored daily to evaluate the anti-cancer effect of the drug. The tumor size was calculated as 0.52 × r 1 × r 2 × r 3 Every day, the body weight of the nude mice was measured to evaluate the safety of the drug. Three days after the last administration, the tumor tissue and related organs were dissected, weighed, and the tumor weight was counted. The tumor inhibition rate and organ index were calculated. Tumor inhibition rate = (1 - tumor weight of the administration group / tumor weight of the control group) × 100%; Organ index = organ weight (mg) / body weight (g). After rinsing the tumor tissue and related organs with 0.9% sodium chloride injection, they were quickly placed in liquid nitrogen or dry ice and stored in a -80 °C refrigerator.

[0034] 1.6 Western Blot Cells and tumor tissues were collected and lysed with lysis buffer. The protein concentration of the samples was measured using a DeNovix DS-11 FX + spectrophotometer. Equal amounts of protein (50 μg) were loaded onto 10% or 12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (PVDF). After blocking in 5% bovine serum albumin (BSA) for 1 - 2 hours, the membrane was incubated with the corresponding primary antibody overnight at 4 °C. After incubation with the secondary antibody, it was placed in an imaging system for exposure. Image analysis was performed using ImageJ 4.0 software.

[0035] 1.7 Paraffin Embedding Sections, HE Staining, Immunohistochemistry (IHC) Staining, and Tunel Detection of Tumor Tissues Tumor tissues were fixed with 4% paraformaldehyde and embedded in paraffin (4 μm). Then, hematoxylin and eosin staining was used to analyze histological changes.

[0036] Meanwhile, IHC staining was performed on tumor sections. The tumor sections were incubated with sodium citrate (pH 6.0) at 100 °C, and then dewaxed, hydrated, and antigen repaired in sequence. In 3% H 2 O2 After incubation in 5% normal donkey serum and 0.1% Triton X-100 to remove non-specific binding, the tumor sections were incubated with the primary antibody including anti-KI-67 overnight at 4 °C. Subsequently, the sections were incubated with the secondary antibody for 1 hour at room temperature. Finally, the signal of the antigen-antibody complex was detected using DAB. Panoramic photographs were taken using a microscope, and 5 fields of view were selected from each panoramic photograph for image analysis using Image J 4.0 software.

[0037] TUNEL assay was performed using a one-step Tunel apoptosis detection kit to calculate apoptosis in the prepared tumor sections. Apoptotic nuclei were quantified using Tunel staining (green fluorescence), and total nuclei were quantified using DAPI staining (blue fluorescence). The stained samples were observed under a fluorescence microscope. Cells with green fluorescence were defined as apoptotic cells. The apoptosis index was calculated as the ratio of the number of Tunel-positive cells to the total number of cells. Panoramic photographs were taken using a microscope, and 5 fields of view were selected from each panoramic photograph for image analysis using Image J 4.0 software.

[0038] 1.8 Detection of apoptosis rate by flow cytometry MIA PaCa-2 cells in the logarithmic growth phase were seeded in 6-well plates at 4×10 5 cells per well and divided into a blank control group, a single staining group, and a drug administration group. The drug administration group was further divided into Brusatol (25 nM), oleic acid and linoleic acid combination (312.5 µM), and oleic acid and linoleic acid combination (312.5 µM) with Brusatol (25 nM) groups, and treated for 24 h. Subsequently, according to the instructions of the apoptosis detection kit, the cells were harvested and incubated with Annexin V-FITC and PI for 10 - 20 minutes. The apoptosis ratio of the cells was determined by flow cytometry.

[0039] 1.9 Statistical analysis The experimental data were expressed as mean ± standard error (X̄±SEM). Graphpad 10.0 software was used for data processing. One-way analysis of variance (One-Way ANOVA) was used to compare the differences between multiple groups, and post hoc tests were performed. P <0.05 indicates that the difference is statistically significant.

[0040] 2 Experimental results 2.1 The combination of oleic acid and linoleic acid synergistically inhibits the growth of pancreatic cancer with Brusatol Compared with the single drug administration group, the inhibitory effect on tumor growth was significant when the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 or 3:1 was combined with Brusatol ( Figure 2(A-C). Among them, the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 and Brusatol at a mass ratio of 16:1 has the best synergistic inhibitory effect on tumors. The HE staining results show that it significantly reduces the cancer cell density in pancreatic cancer tumor tissues. The IHC experimental results further show that it significantly downregulates the expression of the proliferation marker KI-67 in pancreatic cancer tumor tissues ( Figure 2 (E). In pancreatic cancer tumor tissues treated with the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 and Brusatol at a mass ratio of 16:1, the percentage of TUNEL-positive cells is significantly increased ( Figure 2 (F). Western blot was used to detect the expression of apoptosis-related proteins in pancreatic cancer tumor tissues. The results show that the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 and Brusatol increases the protein expression levels of cleaved PARP, Caspase 3, Caspase 9, and Caspase 8 ( Figure 2 (G-K). It is revealed that the combination of oleic acid and linoleic acid synergistically inhibits the growth of pancreatic cancer with Brusatol, especially the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 and Brusatol has the best synergistic inhibitory effect on tumors.

[0041] 2.2 The combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 synergistically induces apoptosis of pancreatic cancer MIAPaCa-2 cells with Brusatol The combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 and Brusatol is a class of drug combinations with synergistic anti-pancreatic cancer effects ( Figure 3 (A-B). Flow cytometry analysis results show that after treatment with the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 and Brusatol, the apoptosis rate of pancreatic cancer MIA PaCa-2 cells is significantly increased ( Figure 3 (C-D). Western blot was used to detect the expression of apoptosis-related proteins in pancreatic cancer MIA PaCa-2 cells. The results show that compared with single drug use, the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 and Brusatol significantly increases the protein expression levels of cleaved PARP, Caspase 3, Caspase 9, and Caspase 8 ( Figure 3 (E-I). It is revealed that the combination of oleic acid and linoleic acid with a molar concentration ratio of 4:1 synergistically induces apoptosis of pancreatic cancer MIA PaCa-2 cells with Brusatol.

[0042] Example 3 Anti-pancreatic cancer experiment of the combination of oleic acid and linoleic acid and the first-line clinical drug for pancreatic cancer (gemcitabine) 1 Materials and methods 1.1 Experimental materials DMEM medium, fetal bovine serum, and horse serum were all purchased from Gibico; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Beyotime. 1.2 Cell lines and cell culture The human pancreatic cancer cell line MIA PaCa-2 was subcultured in our laboratory and inoculated into DMEM medium containing 10% fetal bovine serum, 2.5% horse serum, and 100 U / mL penicillin-streptomycin, and cultured in an incubator at 37 °C, 5% CO 2 2, and saturated humidity, with passage once every 2-3 days.

[0043] 1.3 Detection of the effect of drugs on the proliferation of pancreatic cancer cells by MTT method Collect MIA PaCa-2 cells in the logarithmic growth phase, adjust the cell suspension concentration, inoculate into a 96-well plate, 100 μL per well, and adjust the density of the cells to be tested to 3000-6000 cells / well. The edge wells were filled with sterile PBS. Incubate in an incubator at 37 °C, 5% CO 2 2, and saturated humidity. After the cells adhered, 100 μL of fresh culture medium was added to the control group, and 100 μL of culture medium containing different concentrations of drugs was added to the experimental groups respectively. The drug concentrations are listed in Table 3 below. After culturing for 24 h under the same conditions, 20 μL of MTT was added to each well, and the culture was continued for 4 h. Carefully aspirate the culture medium, add 150 μL of dimethyl sulfoxide to each well, shake in the dark for 5-15 min, and measure the absorbance (OD) value at 570 nm with an enzyme-linked immunosorbent assay reader to calculate the cell survival rate and inhibition rate. Cell survival rate (%) = OD 药物 / OD 空白 × 100%. Inhibition rate = 1 - OD 药物 / OD 空白 .

[0044] Table 3 Concentrations of gemcitabine, oleic acid and linoleic acid combination, and oleic acid and linoleic acid combined with gemcitabine acting on pancreatic cancer cells for 24 h

[0045] 1.4 Data model application SynergyFinder 3.0 software was used to estimate the expected drug combination response based on three reference models: Bliss, ZIP, and HAS, and calculate the score value. This software allows interactive analysis and visualization of multi-drug combination analysis data. When the score value < -10, it indicates antagonism; between -10 and 10, it indicates additivity; > 10 indicates synergy.

[0046] 2 Experimental results The scoring values of the combination of oleic acid and linoleic acid and gemcitabine in the treatment of pancreatic cancer MIA PaCa-2 cells are listed in Table 4 below, and the overall performance shows a synergistic effect. The results reveal that the combination of oleic acid and linoleic acid synergistically inhibits the proliferation of pancreatic cancer cells with gemcitabine, a first-line clinical drug for pancreatic cancer.

[0047] Table 4 Anti-pancreatic cancer effect of the combination of oleic acid and linoleic acid and gemcitabine 。

[0048] Example 4 Anti-hepatocellular carcinoma experiment of the combination of oleic acid and linoleic acid and a clinical drug for the treatment of liver cancer (5-fluorouracil) 1 Materials and methods 1.1 Experimental materials DMEM medium, fetal bovine serum and horse serum were all purchased from Gibico; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Beyotime. 1.2 Cell lines and cell culture Human hepatocellular carcinoma cell line HuH-7 was passaged and cultured in our laboratory, inoculated into DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin, and cultured in an incubator at 37°C, 5% CO 2 , saturated humidity, and passaged every 2 - 3 days.

[0049] 1.3 Detection of the effect of drugs on the proliferation of hepatocellular carcinoma cells by MTT method Collect HuH-7 cells in the logarithmic growth phase, adjust the cell suspension concentration, inoculate into a 96-well plate, 100 μL per well, so that the density of the cells to be tested is adjusted to 3000 - 6000 cells / well, and the edge wells are filled with sterile PBS. Cultured in an incubator at 37°C, 5% CO 2 , saturated humidity. After the cells adhered, 100 μL of fresh culture medium was added to the control group, and 100 μL of culture medium containing different concentrations of drugs was added to the experimental groups respectively. The drug concentrations are listed in Table 5. After culturing for 24 h under the same conditions, 20 μL of MTT was added to each well, and the culture was continued for 4 h. Carefully aspirate the culture medium, add 150 μL of dimethyl sulfoxide to each well, shake in the dark for 5 - 15 min, and measure the absorbance (OD) value at 570 nm with an enzyme-linked immunosorbent assay reader, and calculate the cell survival rate and inhibition rate. Cell survival rate (%) = OD 药物 / OD 空白 ×100%. Inhibition rate = 1 - OD 药物 / OD 空白 。

[0050] Table 5 Concentrations of 5-fluorouracil, the combination of oleic acid and linoleic acid, and the combination of oleic acid and linoleic acid and 5-fluorouracil acting on hepatocellular carcinoma cells for 24 h

[0051] 1.4 Application of Data Model Using SynergyFinder 3.0 software, the expected drug combination responses were estimated based on three reference models, Bliss, ZIP, and HAS, and the scoring values were calculated. This software allows for interactive analysis and visualization of multi-drug combination analysis data. When the scoring value < -10, it indicates antagonism; when it is between -10 and 10, it indicates additivity; when it > 10, it indicates synergy.

[0052] 2 Experimental Results The scoring values of the combination of oleic acid and linoleic acid combined with 5-fluorouracil acting on liver cancer HuH-7 cells are listed in the following table, showing an overall antagonistic effect. The results reveal that the combination of oleic acid and linoleic acid combined with the clinical drug for liver cancer treatment (5-fluorouracil) shows antagonism in liver cancer.

[0053] Table 6 Antitumor effect of the combination of oleic acid and linoleic acid combined with 5-fluorouracil on liver cancer

[0054] Example 5 Experiment on the combination of oleic acid and linoleic acid combined with the clinical drug for lung cancer treatment (capecitabine) against lung cancer 1 Materials and Methods 1.1 Experimental Materials DMEM medium, fetal bovine serum, and horse serum were all purchased from Gibico; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Beyotime. 1.2 Cell Lines and Cell Culture Human lung cancer cells NCI-H1299 and A549 were passaged and cultured in our laboratory, inoculated into DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin, and cultured in an incubator at 37°C, 5% CO 2 , with saturated humidity, and passaged every 2 - 3 days.

[0055] Detection of the effect of drugs on the proliferation of lung cancer cells by MTT method Collect NCI-H1299 and A549 cells in the logarithmic growth phase, adjust the cell suspension concentration, inoculate into 96-well plates, 100 μL per well, so that the density of the cells to be tested is adjusted to 3000 - 6000 cells / well, and the edge wells are filled with sterile PBS. Cultured in an incubator at 37°C, 5% CO 2 , with saturated humidity. After the cells adhered, 100 μL of fresh culture medium was added to the control group, and 100 μL of culture medium containing different concentrations of drugs was added to the experimental groups respectively. The drug concentrations are listed in Table 7. After culturing for 24 h under the same conditions, 20 μL of MTT was added to each well, and the culture was continued for 4 h. Then, the culture medium was carefully aspirated, 150 μL of dimethyl sulfoxide was added to each well, and shaken in the dark for 5 - 15 min. The absorbance (OD) value at 570 nm was measured with an enzyme-linked immunosorbent assay reader, and the cell survival rate and inhibition rate were calculated. Cell survival rate (%) = OD药物 / OD 空白 × 100%. Inhibition rate = 1 - OD 药物 / OD 空白 .

[0056] Table 7 Concentrations of the combination of capecitabine, oleic acid and linoleic acid, and the effects of oleic acid and linoleic acid combined with capecitabine on lung cancer cells for 24 h

[0057] 1.4 Application of data model Using SynergyFinder 3.0 software, the expected drug combination responses were estimated based on three reference models: Bliss, ZIP, and HAS, and the scoring values were calculated. This software allows for interactive analysis and visualization of multi-drug combination analysis data. When the scoring value < -10, it indicates antagonism; when it is between -10 and 10, it indicates additivity; when it > 10, it indicates synergy.

[0058] 2 Experimental results The scoring values of the combination of oleic acid and linoleic acid with capecitabine on lung cancer NCI-H1299 and A549 cells are listed in the following table, showing an overall antagonistic effect. The results reveal that the combination of oleic acid and linoleic acid combined with the clinical drug for lung cancer treatment (capecitabine) shows antagonism in lung cancer.

[0059] Table 8 Antitumor effect of the combination of oleic acid and linoleic acid with capecitabine against lung cancer

[0060] In the above screening experiments of the combination of oleic acid and linoleic acid with different antitumor drugs, the experimental results found that the combination of oleic acid and linoleic acid in a specific ratio combined with Brusatol or gemcitabine can achieve unexpected synergistic anti-pancreatic cancer activity. However, the combination of oleic acid and linoleic acid with capecitabine or liver cancer shows antagonistic effects.

Claims

1. A pharmaceutical composition for treating cancer, characterized in that: It includes oleic acid, linoleic acid, and Brusatol or gemcitabine.

2. The composition according to claim 1, characterized in that The mass ratio of oleic acid and linoleic acid to Brusatol or gemcitabine is 10-16:1, wherein the molar ratio of oleic acid to linoleic acid is 1:1-16:

1.

3. The composition according to claim 2, characterized in that The molar ratio of oleic acid to linoleic acid is 1:1, 1:2, 1:4, 1:8, 1:16, 2:1, 3:1, 4:1, 8:1, and 16:

1.

4. The composition according to claim 3, characterized in that The mass ratio of the oleic acid and linoleic acid composition to Brusatol or gemcitabine is 16:1, wherein the molar ratio of oleic acid to linoleic acid is 3:1 or 4:

1.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.

6. Use of the composition according to any one of claims 1 to 4 in the preparation of drugs against pancreatic cancer.

7. The use according to claim 6, characterized in that: The composition is prepared into a pharmaceutical preparation with a pharmaceutically acceptable carrier.

8. The use according to claim 7, characterized in that: The pharmaceutical preparations include tablets, pills, capsules, granules and injections.

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