Arsenic trioxide and bupivacaine combined medicine for inhibiting activity of tumor cells and application of arsenic trioxide and bupivacaine combined medicine
By combining arsenic trioxide with bupivacaine, the problems of insufficient dosage and high toxicity of arsenic trioxide in the treatment of liver cancer were solved, and significant inhibition of liver cancer cell proliferation and migration was achieved, with the effect of reducing toxicity and enhancing efficacy.
Patent Information
- Application Number
- CN202510931989.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-11-14
AI Technical Summary
Currently, arsenic trioxide is used in insufficient doses and has significant toxic side effects in the treatment of liver cancer, and there is a lack of safe and effective chemotherapy regimens.
Arsenic trioxide and bupivacaine were used in combination at concentrations of 2 μm and 200 μM, respectively, in a complete cell culture medium to prepare a drug for inhibiting liver cancer cells.
It significantly inhibits the proliferation and migration of liver cancer cells, reduces the dosage of arsenic trioxide, and minimizes toxic side effects, showing promising prospects for clinical translation.
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Figure CN120944820A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to a drug for inhibiting tumor cell activity by combining arsenic trioxide and bupivacaine and its application. Background Technology
[0002] Liver cancer is one of the most common malignant tumors worldwide, with consistently high incidence and mortality rates. Surgical resection is the preferred treatment for liver cancer; however, most patients are diagnosed at an advanced stage and are therefore ineligible for surgery. Thus, the use of chemotherapy drugs is particularly important. However, safe and effective chemotherapy regimens remain lacking in clinical practice.
[0003] Arsenic trioxide (ATO) is the main component of arsenic trioxide. In recent years, its broad anti-tumor effects have gradually attracted attention, and it has begun to be applied to other solid tumors. Among these, ATO has been found to have a significant inhibitory effect on liver cancer. However, ATO itself is highly cytotoxic, and the drug concentration reaching solid tumors is far lower than the therapeutic dose. Therefore, its effective dose in liver cancer is much higher than in hematologic malignancies, and the toxic side effects are correspondingly more severe. This is the main reason limiting the clinical application of ATO in liver cancer treatment. Therefore, how to reduce the dosage and toxic side effects of ATO is of significant research value. Summary of the Invention
[0004] The purpose of this invention is to address the issues of high dosage and significant toxic side effects in the treatment of liver cancer with arsenic trioxide, and to provide a novel synergistic anti-liver cancer regimen: the combination of arsenic trioxide and bupivacaine. This is also the first time that the combination of arsenic trioxide and bupivacaine has been shown to have a significant synergistic inhibitory effect on liver cancer cells, representing a potential therapeutic agent for liver cancer and providing a new strategy for its clinical treatment.
[0005] To achieve the above objectives, the technical means adopted by the present invention are as follows:
[0006] A drug for treating liver cancer by combining arsenic trioxide and bupivacaine, the drug comprising arsenic trioxide and bupivacaine, wherein the concentration of arsenic trioxide is 2 μM and the concentration of bupivacaine is 200 μM.
[0007] Furthermore, the solvent is a complete cell culture medium.
[0008] Furthermore, the complete cell culture medium consists of 89 vol% DMEM high glucose medium (Gibco, C11995500BT), 10 vol% fetal bovine serum (Gibco, c04001), and 1 vol% penicillin-streptomycin solution (Beyotime, C0222).
[0009] Furthermore, in the penicillin-streptomycin solution (100X), the content of penicillin is 10 kU / ml and the content of streptomycin is 10 mg / ml; this solution is prepared with 0.9% sodium chloride; the recommended working concentration of penicillin in cell culture medium is 100 U / ml and the working concentration of streptomycin is 0.1 mg / ml, that is, it can be used by dilution of 100 times.
[0010] A method for preparing the above-mentioned drug, wherein arsenic trioxide and bupivacaine are both diluted to the target concentration using a complete cell culture medium.
[0011] The application of one of the above-mentioned drugs that inhibit the activity of tumor cells in the preparation of drugs for treating cancer.
[0012] Furthermore, the cancer in question is liver cancer.
[0013] The advantages of this invention over the prior art are as follows:
[0014] (1) This invention is the first to propose the combined use of arsenic trioxide and bupivacaine to inhibit the activity of liver cancer cells. The two drugs have a strong synergistic effect in inhibiting liver cancer cells, which can achieve "reduced toxicity and enhanced efficacy" in the application of arsenic trioxide in the treatment of liver cancer. In addition, both arsenic trioxide and bupivacaine are drugs currently in clinical use and have good safety. This drug combination regimen has good prospects for clinical translation.
[0015] (2) The results of this study show that the combination of arsenic trioxide and bupivacaine can significantly inhibit the proliferation and migration of liver cancer cells and has a synergistic anti-liver cancer effect. The combination of small doses of arsenic trioxide and bupivacaine, which are ineffective when used alone, can significantly inhibit the proliferation and migration of liver cancer cells. The dosage of arsenic trioxide can be reduced, thereby reducing its toxic side effects.
[0016] (3) This invention proposes for the first time a drug combination based on the combined use of arsenic trioxide and bupivacaine to inhibit the activity of tumor cells, which has significant in vitro anti-liver cancer effect. Attached Figure Description
[0017] Figure 1 The effects of arsenic trioxide and bupivacaine alone on the survival of the human hepatocellular carcinoma cell line HepG2; (A) the inhibitory effect of arsenic trioxide alone on the survival of HepG2 cells; (B) the effect of arsenic trioxide alone on the IC50 of HepG2 cells. 50 (C) Inhibitory effect of bupivacaine alone on the viability of HepG2 cells; (D) Inhibitory effect of bupivacaine alone on the IC50 of HepG2 cells. 50 .
[0018] Figure 2 Heatmap of synergistic scores for drug combination use calculated using SynergyFinder software;
[0019] Figure 3 The inhibitory effect of 2 μM arsenic trioxide combined with different concentrations of bupivacaine on the survival of HepG2 cells;
[0020] Figure 4 The inhibitory effect of 200 μM bupivacaine combined with different concentrations of arsenic trioxide on the survival of HepG2 cells;
[0021] Figure 5 The effects of arsenic trioxide and bupivacaine alone or in combination on the proliferation of the human hepatocellular carcinoma cell line HepG2; (A) representative EdU images of HepG2 cells; (B) statistical data on the percentage of EdU-positive cells; (C) representative images of plate colony formation assay.
[0022] Figure 6 The effects of arsenic trioxide and bupivacaine alone or in combination on the migration ability of the human hepatocellular carcinoma cell line HepG2; (A) Representative scratch images of HepG2 cells; (B) Statistical graph of cell migration rate. Detailed Implementation
[0023] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.
[0024] This invention involved culturing the HepG2 human hepatocellular carcinoma cell line in vitro and treating it with gradient doses of arsenic trioxide and bupivacaine for 48 h. The viability of hepatocellular carcinoma cells in different treatment groups was detected using CCK8 assay. Combinations of arsenic trioxide and bupivacaine at various concentrations were designed to evaluate their synergistic effect and screen for effective combined doses. The effects of each drug treatment group on the proliferation of hepatocellular carcinoma cells were detected using EdU staining and colony formation assays; the effects on the migration of hepatocellular carcinoma cells were detected using a cell scratch assay. The results showed that 2 μM arsenic trioxide and 200 μM bupivacaine effectively inhibited the viability, proliferation, and migration of hepatocellular carcinoma cells. In conclusion, the combination of arsenic trioxide and bupivacaine exhibits significant in vitro anti-hepatocellular carcinoma effects. Further research into its anti-hepatocellular carcinoma effects and molecular mechanisms is of significant research importance.
[0025] Example 1:
[0026] Arsenic trioxide and bupivacaine, alone or in combination, can inhibit the growth of liver cancer cells to varying degrees.
[0027] 1 Experimental Methods
[0028] 1.1 Experimental group design:
[0029] Human hepatocellular carcinoma cell line HepG2 was cultured overnight at 37°C in a cell culture incubator containing 5% CO2. Cells were divided into four groups: a control group treated with arsenic trioxide at varying concentrations; a control group treated with bupivacaine at varying concentrations; a control group treated with 2 μM arsenic trioxide and different concentrations of bupivacaine; and a control group treated with 200 μM bupivacaine and different concentrations of arsenic trioxide. Both arsenic trioxide and bupivacaine were diluted to the appropriate concentrations using complete cell culture medium (DMEM high-glucose medium (Gibco, C11995500BT) + 10% fetal bovine serum (Gibco, c04001) + 1% penicillin-streptomycin solution (Beyotime, c0222) and treated with the appropriate concentrations for 48 h.
[0030] 1.2 CCK8 assay for the survival activity of liver cancer cells
[0031] In the human hepatocellular carcinoma cell line HepG2, after treatment with different concentrations of arsenic trioxide or bupivacaine for 48 h, CCK8 reagent was added to HepG2 cells, and the OD value at a wavelength of 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader to calculate cell viability.
[0032] 1.3 Data Processing
[0033] Experimental results are expressed as standard deviation ± standard error. Cell viability was calculated for each group based on OD values, and the IC50 of the drug was also calculated. 50 The graph was created using Graphpad Prism 8.0.
[0034] 2 Observation Results
[0035] 2.1 Arsenic trioxide or bupivacaine alone can inhibit the growth of HepG2 cells and have a high synergistic effect.
[0036] CCK8 results are as follows Figure 1 AD showed that, compared with the control group, the survival of HepG2 cells decreased in a concentration-dependent manner after treatment with arsenic trioxide or bupivacaine.
[0037] 2.2 The combined use of arsenic trioxide and bupivacaine can inhibit the growth of HepG2 cells.
[0038] Figure 2 This is a heatmap showing the synergistic inhibitory score of the combination of arsenic trioxide and bupivacaine on HepG2 cells. The results show that this drug combination has a strong synergistic inhibitory score on HepG2 cells. Figure 3 , 4The results showed that neither 2 μM arsenic trioxide nor 200 μM bupivacaine alone significantly inhibited the viability of HepG2 cells, while their combined use significantly reduced the viability of HepG2 cells. Therefore, a combination of 2 μM arsenic trioxide and 200 μM bupivacaine was chosen for subsequent experiments.
[0039] The above results indicate that both arsenic trioxide and bupivacaine, when used alone, can inhibit the viability of HepG2 cells in a concentration-dependent manner, and the combined use of the two has a strong synergistic effect. Low doses of arsenic trioxide, which are ineffective when used alone, can significantly inhibit the viability of HepG2 cells when used in combination with bupivacaine.
[0040] Example 2:
[0041] The combined use of arsenic trioxide and bupivacaine can inhibit the proliferation of liver cancer cells.
[0042] 1 Experimental Methods
[0043] 1.1 Experimental group design:
[0044] The experiment was divided into four groups: control group, 2 μM arsenic trioxide group, 200 μM bupivacaine group, and a combination of both. After HepG2 cells were treated with the drugs for 48 h, EdU staining was performed to observe cell proliferation under a fluorescence microscope; plate colony formation experiment was performed to observe cell colony formation.
[0045] 1.2 EdU staining assay to detect the proliferation ability of liver cancer cells
[0046] After HepG2 cells were treated with the drug for 48 hours, the culture medium was discarded, and the cells were washed three times with PBS. The cells were then fixed with 4% formaldehyde at 37°C for 15 minutes. Finally, the cells were stained according to the kit instructions, observed and photographed under a fluorescence microscope, and the number of EdU-positive cells was counted.
[0047] 1.3 Plate colony formation assay to detect the proliferative capacity of hepatocellular carcinoma cells
[0048] Prepare HepG2 single-cell suspensions and add them to 6-well plates at a density of 500 cells / well. Incubate overnight in a cell culture incubator at 37°C with 5% CO2. The next day, treat with different groups of cells for 48 hours, then change the culture medium every 3 days and continue culturing until clones are formed. Wash cells 1-2 times with PBS buffer, fix cells with 4% paraformaldehyde for 10 min, wash twice with distilled water, stain with crystal violet for 10 min, wash several times with distilled water, air dry and photograph.
[0049] 1.4 Data Processing
[0050] The results of this invention are expressed as standard deviation ± standard error. Graphs were plotted using Graphpad Prism 8.0, and the correlation between groups was measured using one-way ANOVA for pairwise comparison tests.
[0051] 2. Observation Results
[0052] 2.1 The combined use of arsenic trioxide and bupivacaine can inhibit the proliferation of liver cancer cells.
[0053] EdU staining results are as follows Figure 5 As shown in A and B, compared with the control group, there was no significant difference in the EdU-positive cell rate of HepG2 cells after treatment with 2 μM arsenic trioxide or 200 μM bupivacaine for 48 h. The combined use of the two significantly reduced the EdU-positive cell rate.
[0054] Results of plate colony formation experiment as follows Figure 5 As shown in Figure C, compared with the control group, there was no significant difference in the number of colonies formed in HepG2 cells after treatment with 2 μM arsenic trioxide or 200 μM bupivacaine for 48 h, but the number of colonies formed was significantly reduced after the two were used in combination.
[0055] The above results indicate that the combined use of arsenic trioxide and bupivacaine can inhibit the proliferation of liver cancer cells.
[0056] Example 3:
[0057] The combined use of arsenic trioxide and bupivacaine can inhibit the migration of liver cancer cells.
[0058] 1 Experimental Methods
[0059] 1.1 Experimental group design:
[0060] The experiment was divided into four groups: control group, 2 μM arsenic trioxide group, 200 μM bupivacaine group, and a combination of both. After HepG2 cells were treated with the drugs, cell migration was observed and photographed under a microscope at 0 h and 48 h.
[0061] 1.2 Scratch assay to detect the migration ability of liver cancer cells
[0062] Using a 200 μl pipette tip, scratches were made on the surface of HepG2 cells, ensuring that the scratch width was as consistent as possible. Cell migration on both sides was observed and photographed under a microscope at 0 h and 48 h after drug application.
[0063] 1.3 Data Processing
[0064] The results of this invention are expressed as standard deviation ± standard error. Graphs were plotted using Graphpad Prism 8.0, and the correlation between groups was measured using one-way ANOVA for pairwise comparison tests.
[0065] 2. Observation Results
[0066] 2.1 The combined use of arsenic trioxide and bupivacaine can inhibit the migration of liver cancer cells.
[0067] The results of the scratch test are as follows Figure 6 As shown in A and B, compared with the control group, there was no significant difference in cell migration rate over time after treatment with 2 μM arsenic trioxide or 200 μM bupivacaine, but the cell migration rate was significantly reduced after the two were used in combination.
[0068] The above results indicate that the combined use of arsenic trioxide and bupivacaine can inhibit the migration of liver cancer cells.
Claims
1. A drug for inhibiting tumor cell activity when arsenic trioxide is used in combination with bupivacaine, characterized in that: The drug comprises arsenic trioxide and bupivacaine, with arsenic trioxide at a concentration of 2 μM and bupivacaine at a concentration of 200 μM.
2. The drug for inhibiting tumor cell activity by combining arsenic trioxide and bupivacaine according to claim 1, characterized in that: The solvent is complete cell culture medium.
3. The drug for inhibiting tumor cell activity by combining arsenic trioxide and bupivacaine according to claim 1, characterized in that: The complete cell culture medium consists of 89 vol% DMEM high glucose medium, 10 vol% fetal bovine serum and 1 vol% penicillin-streptomycin solution.
4. The drug for inhibiting tumor cell activity by combining arsenic trioxide and bupivacaine according to claim 1, characterized in that: The working concentration of penicillin in cell culture medium is 100 U / ml, and the working concentration of streptomycin is 0.1 mg / ml, which means it can be used after a 100-fold dilution.
5. A method for preparing a drug for inhibiting tumor cell viability as described in any one of claims 1 to 4, characterized in that: The method is as follows: both arsenic trioxide and bupivacaine are diluted to the target concentration using complete cell culture medium.
6. The use of the drug for inhibiting tumor cell activity as described in any one of claims 1 to 4 in the preparation of a drug for treating cancer.
7. The application according to claim 6, characterized in that: The cancer in question is liver cancer.