A new use of pancreatic elastase

Abstract

The present invention belongs to the technical field of cancer chemotherapy, and specifically relates to a new use of pancreatic elastase. The present invention provides the use of pancreatic elastase for preparing a drug for treating cancer. Experiments have found that pancreatic elastase has a therapeutic effect on cancers such as non-small cell lung cancer, liver cancer, brain neuroblastoma or cervical cancer, and in particular has a therapeutic effect on liver cancer that is better than the positive drug cisplatin. Therefore, pancreatic elastase has the potential to be used as a cancer treatment drug and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of cancer chemotherapy, and in particular relates to a new use of pancreatic elastase. Background Art

[0002] Cancer generally refers to all malignant tumors. Cancer has biological characteristics such as abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness and metastasis. Its occurrence is a complex process with multiple factors and steps, which can be divided into three processes: carcinogenesis, cancer promotion and evolution. It is closely related to smoking, infection, occupational exposure, environmental pollution, unreasonable diet and genetic factors.

[0003] Nowadays, the incidence of cancer is increasing day by day, which seriously affects people's health and happiness. Therefore, the treatment of cancer has always been an important research topic. The treatment of cancer mainly includes surgical treatment, chemotherapy and radiotherapy. Among them, chemotherapy is the use of drugs that can kill cancer cells to treat cancer. Since the biggest difference between cancer cells and normal cells is the rapid cell division and growth, the working principle of anticancer drugs is usually to inhibit the growth of cancer cells by interfering with the mechanism of cell division, such as inhibiting DNA replication or preventing chromosome separation.

[0004] The disadvantage of chemotherapy is that most chemotherapeutic drugs have no specificity and will kill normal tissue cells that undergo cell division at the same time, thus often damaging healthy tissues that need to divide to maintain normal function. On the other hand, the combination of multiple drugs in chemotherapy can produce better therapeutic effects, which is called "comprehensive chemotherapy". Therefore, people also hope to develop as many chemotherapeutic drugs as possible so that there are more options in the chemotherapy of cancer. In short, due to the needs of the above-mentioned drug side effects and drug combination, the development of new cancer chemotherapeutic drugs remains an important goal in this field.

[0005] Pancreatic elastase (Uniprot: Q9UNI1) is a serine protease extracted from porcine pancreas. It is mainly used in the clinic to treat hyperlipidemia, atherosclerosis and fatty liver. At present, it has been reported that pancreatic elastase can be used for the diagnosis of pancreatic cancer (Zhang Ling, Gao Jun, Li Zhaoshen, et al. Detection of methylation status of elastase 3B (ELA3B) gene in pancreatic cancer and chronic pancreatitis [J]. Gastroenterology, 2007, 12(8):4). However, there is no report on whether pancreatic elastase can be used to treat cancer. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides a new use of pancreatic elastase, aiming to provide a new chemotherapeutic drug.

[0007] Use of pancreatic elastase (Uniprot: Q9UNI1) for preparing a drug for treating cancer.

[0008] Preferably, the drug is used to treat liver cancer.

[0009] Preferably, the medicament is used to treat hepatocellular carcinoma.

[0010] Preferably, the drug is used to treat non-small cell lung cancer.

[0011] Preferably, the drug is used to treat brain neuroblastoma.

[0012] Preferably, the drug is used to treat cervical cancer.

[0013] Preferably, the drug is a drug that promotes apoptosis of cancer cells.

[0014] The present invention has found through experiments that pancreatic elastase has an inhibitory effect on cancers such as non-small cell lung cancer, liver cancer, brain neuroblastoma or cervical cancer, and in particular has an inhibitory effect on HepG2 that is superior to that of the positive drug cisplatin. HepG2 is a cell line derived from the liver tissue of patients with hepatocellular carcinoma (HCC). Therefore, the above experiments have proved that pancreatic elastase has the potential as a therapeutic drug for cancer (especially hepatocellular carcinoma) and has a good application prospect.

[0015] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0016] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the drug dilution method in Example 1;

[0018] Figure 2 This is a schematic diagram of adding drugs to a 96-well plate in Example 1;

[0019] Figure 3 The results of the experiment on the inhibitory effect of pancreatic elastase on various cell lines in Example 1 are shown;

[0020] Figure 4 The results of the positive control drug cisplatin's inhibitory effect on each cell line in Example 1 are shown in Table 1.

[0021] Figure 5The apoptosis results of A549 cells in Example 1 with 35 μM elastase;

[0022] Figure 6 This is the apoptosis result of HepG2 cells in Example 1 induced by 35 μM elastase. DETAILED DESCRIPTION

[0023] The reagents and materials used in the following examples are all commercially available unless otherwise specified.

[0024] Experimental Example 1 Antitumor Effect of Pancreatic Elastase

[0025] This experiment investigated the inhibitory effect of pancreatic elastase on 8 tumor cell lines, and used the CCK-8 method to detect the effect of elastase on 4 normal cell lines to preliminarily evaluate its effect on normal cells.

[0026] 1. Experimental Methods

[0027] Determination of IC of elastase in 8 tumor cell lines 50 The drug treatment time was 24, 48, and 72 hours, with 8 concentrations, 3 replicates for each concentration, and the experiment was repeated twice.

[0028] Detailed dosage and specific layout plan are shown in ①②③.

[0029] ① The maximum final concentration of the drug to be tested is 100 μg / ml. 8 concentration gradients are set. The specific dilution ratios of the working solution concentrations are as follows:

[0030]

[0031] ② The highest concentration of the positive drug cisplatin is 50 μM, and 7 concentration gradients are set. The specific dilution ratios of the working solution concentration are as follows:

[0032]

[0033] like Figure 1 : Elastase and cisplatin drugs were prepared from the mother solution according to the dilution method in the figure, and the final concentration was prepared after dilution 10 times for detection.

[0034] ③ Schematic diagram of drug addition in 96-well plate Figure 2 As shown in the figure, the green circled part is the drug-added part of the experiment; the red font part is the test drug with different concentrations; the black font part represents the positive control drug cisplatin with different concentrations.

[0035] 1. Calculation method of main observation indicators

[0036] Tumor cell inhibition rate, %, the IC50 of the drug on tumor cells is calculated by the inhibition rate. The inhibition rate calculation formula is:

[0037] Inhibition rate (%) = [OD (0 drug addition) - OD (drug addition)] / [OD (0 drug addition) - OD (blank)] x 100%

[0038] OD (drug added): absorbance value of wells with cells, culture medium, CCK-8 solution and drug solution

[0039] OD(blank): absorbance value of wells with culture medium, CCK-8 solution, and no cells

[0040] OD (0 drug addition): absorbance value of the well with cells, culture medium, CCK-8 solution, and no drug solution

[0041] The IC50 value was calculated using Graph Pad-Prism data analysis software.

[0042] 2. Experimental Materials

[0043] 2.1 Experimental Materials

[0044]

[0045] Note: All cells were cultured at 37°C and 5% CO2

[0046] 2.2 Experimental reagents and consumables

[0047]

[0048]

[0049] 3. Experimental steps

[0050] 3.1 Cell culture

[0051] Resuscitate 12 kinds of cells with corresponding culture medium, maintain cell growth to the logarithmic phase, and collect cells by centrifugation at 1000rpm for 5 minutes. After centrifugation, remove the supernatant and add appropriate amount of culture medium to resuspend the cells.

[0052] 3.2 Inhibitory effects of the tested samples on 8 cell types

[0053] On the first day, cell plating: Take the vigorously growing cells to prepare a single cell suspension, and calculate the cell density through a hemocytometer. According to the cell inoculation amount (when plating a 96-well plate, generally 5×103 cells / well) and the total volume of each well, calculate the required volume of cell suspension and fresh culture medium. Then, according to the calculation results, prepare the cell suspension, mix it thoroughly, and use a 300μL spray gun to spread the cell suspension into a 96-well plate (the total volume of each well is 180μL). Gently shake the plate to evenly distribute the cells in the wells, and then place it in an incubator and culture it until the next day when the cells are fully attached to the wall.

[0054] The next day, drug treatment: After the cells adhere to the wall, according to the concentration of the drug mother solution and the working concentration to be detected, aspirate the corresponding volume of the mother solution into fresh culture medium, prepare a series of dilutions with a concentration gradient, mix thoroughly, add 20 μL per well to a 96-well plate, and then place the cells in an incubator and culture for 24, 48, and 72 hours, repeating twice.

[0055] Day 3-5, CCK-8 assay: Take out the 96-well plate and add 10 μl CCK-8 reagent to each well, and put the cells back into the incubator for 2 hours. After incubation, measure the OD value at a wavelength of 490 nm using a multifunctional microplate reader.

[0056] 3.3 Preparation of test and reference substances

[0057] The compound solution was prepared in a biological safety cabinet or clean bench.

[0058] Dosing concentration:

[0059] ◎The storage solution concentration of the drug to be tested is 10 mg / ml, the maximum final concentration of the working solution is 100 μg / ml, and 8 concentration gradients are set; the storage solution concentration of the positive control cisplatin is 10 mM, the maximum final concentration of the working solution is 50 μM, and 7 concentration gradients are set.

[0060] Note: Mix well before use to ensure the preparation is homogeneous.

[0061] ◎ Specific information on the preparation methods of drugs of different concentrations:

[0062] The preparation of the working solution of the test sample is as shown in ① above;

[0063] The preparation of the working solution of the reference substance cisplatin solution is shown in ② above.

[0064] Note: Gloves and masks should be worn when preparing medicines;

[0065] 3.4 Administration

[0066] When the confluence of cells reaches 60%-70% after plating, add drugs according to the above methods ①②③.

[0067] 3.5 Statistical analysis

[0068] GraphPad Prism software was used to draw S-shaped dose-inhibition rate curves using nonlinear regression models and calculate IC 50 value.

[0069] 2. Experimental Results

[0070] The statistical analysis results of CCK-8 data at 24h, 48h, and 72h after cells were treated with pancreatic elastase and positive control cisplatin are shown in the following table and Figure 2-4 As shown:

[0071]

[0072]

[0073] It can be seen that pancreatic elastase has a significant inhibitory effect on cell lines such as LN229 (neuroblastoma of the brain), HELA (cervical cancer), A549 (non-small cell lung cancer), CaCo2 (colorectal adenocarcinoma) and HepG2 (liver cancer). Especially for HepG2 (liver cancer), after 72 hours of drug addition, the IC50 was significantly lower than that of the control experimental group of the positive drug cisplatin, proving that pancreatic elastase has a very significant therapeutic effect on liver cancer.

[0074] In addition, the inhibitory effect of pancreatic elastase on normal cell lines has its own advantages and disadvantages compared with the positive drug cisplatin, but overall, the inhibitory effect of pancreatic elastase on normal cells is weaker, which indicates that pancreatic elastase has a certain selectivity for tumor cells.

[0075] The apoptosis results of A549 cells and HepG2 cells at 35 μM elastase are shown in Figure 5-6 As shown in the figure, it can be seen that elastase induces significant apoptosis in A549 and HepG2 cells, and the degree of apoptosis is similar to that of the control group. It can be inferred that the anti-tumor effect of elastase is achieved through the apoptosis pathway.

[0076] From the above experimental examples, we can see that pancreatic elastase has an inhibitory effect on cancers such as non-small cell lung cancer, liver cancer, brain neuroblastoma or cervical cancer, especially for liver cancer, it has an inhibitory effect better than the positive drug cisplatin. Therefore, pancreatic elastase has the potential to be used as a cancer treatment drug and has a good application prospect.

Claims

1. Use of pancreatic elastase for preparing a drug for treating cancer, characterized in that: The Uniprot number of the pancreatic elastase is Q9UNI1; the drug is used for treating liver cancer.

2. The use according to claim 1, characterized in that: The medicament is used for treating hepatocellular carcinoma.

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