CEMIP target inhibitor and application thereof

Abstract

The invention provides a CEMIP target inhibitor and application thereof, and belongs to the technical field of biological medicine. Umbelliferone is used as a CEMIP target inhibitor, expression of CEMIP can be reduced by combining with CEMIP, growth, proliferation and migration of cancer cells are inhibited, death of the cancer cells is promoted, and a good cancer treatment effect is achieved. In addition, the umbelliferone is low in cytotoxicity and cannot generate adverse effects on normal cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a CEMIP target inhibitor and application thereof. Background Art

[0002] Cancer is a serious disease that is extremely harmful to physical health. Among cancers, lung cancer and gastric cancer have a wide range of incidence, and their incidence and mortality rates are increasing rapidly. They are the two most serious malignant tumors that pose the most serious threat to people's health and life, and bring serious economic losses to patients and their families.

[0003] At present, the treatments for lung cancer and gastric cancer mainly include surgery, chemotherapy, radiotherapy, etc. However, nearly half of the patients rely on a combination of multiple broad-spectrum cytotoxic drugs. Unfortunately, this type of combined therapy has low selectivity, short response time, low remission rate and other problems, leading to rapid tumor progression and treatment failure. Chemotherapy and radiotherapy not only attack cancer cells, but also kill growing new cells, which has a great impact on the patient's health, and surgical treatment is difficult to cure. Recent studies have shown that inhibitors targeting lung cancer and gastric cancer pathogenic genes (such as EGFR, ER2, ALK, MET, etc.) can achieve better therapeutic effects, but patients are often forced to stop taking the drug due to toxic side effects during treatment. Leading to cancer progression or recurrence. Therefore, finding a new cancer treatment drug with high safety is crucial for the treatment of cancer.

[0004] In recent years, studies have found that cell migration inducing hyaluronan binding protein (CEMIP) is a new type of cancer-driving protein encoded by the deafness gene KIAA1199. In normal human tissues, CEMIP is expressed most in the brain, followed by the lungs, placenta and testes. It is highly expressed in a variety of solid tumors including gastric cancer, colorectal cancer, and lung cancer, and is positively correlated with poor chemotherapy sensitivity and poor prognosis in patients. It is a key factor in promoting tumor growth, metastasis and drug resistance. Duong et al. found that in the endosomes of colorectal cancer cells, a large amount of CEMIP binds to and phosphorylates MEK1, continuously activating downstream ERK1 / 2, thereby inducing c-MYC amplification and enhanced transcriptional activity, inducing cell resistance to selumetinib.

[0005] Since the CEMIP peptide sequence is long, consisting of 1366 amino acids, and has a complex spatial configuration, there is still no report on its protein crystal form and spatial structure, and there are very few known functional domains. Specifically, the N-terminus (M1-T31) of CEMIP contains 30 signal peptide sequences that allow it to be transported from the ER to the Golgi apparatus. The loss of this sequence leads to disordered CEMIP transport between organelles, and intracellular CEMIP diffuses into the cytoplasm and cannot synthesize functionally complete proteins. Immediately following, there is an important G8 domain in the Q44-K166 segment, which is considered to be the main functional domain of CEMIP for extensively regulating cell motility. In addition, the R187-N277 and D1244-S1330 segments are two ILEI functional domains that regulate the epithelial-mesenchymal transition of cells. The existence of this structure explains how CEMIP promotes its invasion and metastasis process in various tumor cells by regulating EMT changes. CEMIP also has two non-homologous GG domains (location unknown) and four pbH1 domains (D572-G594, S595-G617, I719-N741 and G798-S819), whose specific functions have not yet been resolved and annotated. In addition, there is a B domain at the C-terminus of CEMIP, which can form a suitable binding site for chaperone binding immunoglobulin protein (BiP) recognition, allowing CEMIP to be retained in the ER. These findings explain the mechanism of action of CEMIP on some biological changes of tumor cells at the molecular level, and provide theoretical support for the discovery of CEMIP inhibitors targeting different domains.

[0006] Traditional Chinese herbal medicine is a cultural treasure of the Chinese nation. Guangxi is home to many ethnic minorities. Ethnic minorities represented by the Zhuang and Yao have adapted to local conditions and have long used the rich local medicinal resources in the region to treat a variety of diseases, forming a unique ethnic medicine system. Traditional Chinese medicine and ethnic medicines and their extracts have attracted more and more attention due to their wide range of targets, low toxicity, and few side effects. They have great potential in the development of multi-target treatment strategies. At present, anticancer drugs widely used in first-line clinical practice, such as taxanes, camptothecins, and podophyllotoxins, are all derived from the active ingredients of natural products. Discovering the active ingredients and effective monomers of characteristic traditional Chinese medicine and ethnic medicines and providing a theoretical basis for the prevention and treatment of cancer with traditional Chinese medicine is a scientific problem that needs to be solved urgently.

[0007] Umbelliferone is a type of coumarin compound, and coumarin is widely found in more than 100 plants, including Leguminosae, Orchidaceae, Rutaceae, Apiaceae, Solanaceae and Asteraceae. Because umbelliferone has multiple functions such as antibacterial, smooth muscle relaxation, anticoagulant and photosensitizer, it is used as an inhibitor of Staphylococcus aureus, Escherichia coli, etc., as well as coronary artery dilator, anticoagulant and psoralen, but its cancer treatment effect has not yet been discovered. Summary of the invention

[0008] The purpose of the present invention is to provide a CEMIP target inhibitor and its application, which can improve the cancer treatment effect and inhibit the growth, proliferation and migration of cancer cells.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a CEMIP target inhibitor, wherein the CEMIP target inhibitor is umbelliferone.

[0011] Preferably, the structure of umbelliferone is as shown in formula (I):

[0012]

[0013] The present invention also provides an application of a CEMIP target inhibitor in the preparation of an anticancer drug. The CEMIP target inhibitor is umbelliferone, and the anticancer drug is an anti-lung cancer or gastric cancer drug.

[0014] Preferably, the umbelliferone can inhibit the growth, proliferation and migration of cancer cells and promote the death of cancer cells.

[0015] Preferably, the anticancer drug may also be added with a pharmaceutically acceptable carrier.

[0016] Preferably, the dosage form of the anticancer drug is one of powder, solution, capsule, granule, tablet, emulsion and suspension.

[0017] The beneficial effects of the present invention compared with the prior art are:

[0018] The present invention uses umbelliferone as a CEMIP target inhibitor, which can reduce the expression of CEMIP by binding to CEMIP, inhibit the growth, proliferation and migration of cancer cells, promote cancer cell death, and has a good cancer treatment effect. In addition, umbelliferone has low cytotoxicity and will not have adverse effects on normal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 is the inhibition rate of PC9GR cells treated with different concentrations of umbelliferone in Example 1;

[0021] Figure 2 is the inhibition rate of H1975 cells treated with different concentrations of umbelliferone in Example 1;

[0022] Figure 3 is the inhibition rate of MKN-7 cells treated with different concentrations of umbelliferone in Example 1;

[0023] Figure 4 is the inhibition rate of BGC823 cells treated with different concentrations of umbelliferone in Example 1;

[0024] Figure 5 is the inhibition rate of BESA-2B cells treated with different concentrations of umbelliferone in Example 1;

[0025] Figure 6 is the inhibition rate of BESA-2B cells treated with different concentrations of gefitinib in Example 1;

[0026] Figure 7 is the inhibition rate of PC9GR cells treated with different concentrations of gefitinib in Example 1;

[0027] Figure 8 is the viability of PC9GR and H1975 cells after umbelliferone treatment in Example 1;

[0028] Fig. 9 is the migration ability of H1975 cells after umbelliferone treatment in Example 1;

[0029] Fig.10 The expression of CEMIP protein in PC9GR cells after umbelliferone treatment in Example 2;

[0030] Fig.11 The expression of FLAG-tagged protein in HEK293T cells 48 hours after transfection with the empty plasmid, the full-length CEMIP plasmid, and the CEMIP truncated plasmids of different lengths carrying the tag protein FLAG in Example 2 (indicating that the plasmid transfection was successful);

[0031] Fig.12 The expression of FLAG in the CEMIP full-length plasmid sample after umbelliferone treatment in Example 2;

[0032] Fig.13 The expression of the tag protein FLAG in the A segment truncation sample after umbelliferone treatment in Example 2;

[0033] Fig.14 The expression of the tag protein FLAG in the B segment truncation sample after umbelliferone treatment in Example 2;

[0034] Fig.15 The expression of the tag protein FLAG in the C segment truncation sample after umbelliferone treatment in Example 2;

[0035] Fig.16 This is the expression of the tag protein FLAG in the D segment truncation sample after umbelliferone treatment in Example 2. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] Example 1 of the present invention detected the effect of umbelliferone on cancer cells, and the specific method is as follows:

[0043] (1) Effects of umbelliferone on the survival of cancer cells

[0044] A. Trypsin digestion of logarithmically growing human normal lung bronchial epithelial cells BEAS-2B, human lung cancer cells PC9GR, H1975 cells, human gastric cancer cells MKN-7, BGC823 cells in good growth state, digestion was terminated by culture medium, centrifuged and resuspended, and diluted to prepare the corresponding cell suspension according to 3000 cells per well and 100 μL cell suspension per well. 4 The cells were inoculated at a density of 100 μL / mL in a 96-well cell culture plate and incubated in a cell culture incubator overnight. When the cell density reached 90%, culture medium containing different drug concentrations was added, 100 μL per well, and 6 replicate wells were set at the same concentration. After incubating the cells for 48 hours, the cell survival rate was tested. The results were as follows: Figure 1-5 shown.

[0045] The formula is: Cell survival rate (%) = [(OD value of drug-treated group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%. Drug selection index (SI) = IC of BEAS-B 50 (umbelliferone or gefitinib) / IC of PC9GR 50 (umbelliferone or gefitinib).

[0046] See also Figure 1-5 According to the calculation formula, the IC values ​​of umbelliferone on normal human lung bronchial epithelial cells BEAS-2B, human lung cancer cells PC9GR, H1975, human gastric cancer cells MKN-7, and BGC823 cells for 48 hours are 50 The values ​​were 291.43±32.81μg / mL, 207.93±38.69μg / mL, 94.26±3.06μg / mL, 131.32±4.64μg / mL, and 108.43±12.41μg / mL, respectively. It can be seen that umbelliferone treatment for 48h can significantly reduce the survival rate of lung cancer cells and gastric cancer cells.

[0047] Depend on Figure 6-7 It can be seen that the IC of gefitinib, a routine drug for lung cancer, on human normal lung bronchial epithelial cells BEAS-2B and human lung cancer cells PC9GR for 48 hours is 50 They were 6.16±0.26μM and 28.07±10.81μM respectively.

[0048] The selection index (SI) is the safe range for judging the effect of drugs. The selection index (SI) is the IC50 of normal cells / IC50 of tumor cells. The selection index is greater than 1.00, which is effective. The larger the index, the larger the safety range. According to the formula, the selection index of umbelliferone on PC9GR and H1975 cells is 1.41 and 3.09 respectively; while the selection index of gefitinib on PC9GR cells is 0.22, indicating that umbelliferone can effectively inhibit lung cancer cells with a large safety range.

[0049] B. Take PC9GR and H1975 cells in the logarithmic growth phase, inoculate them in a 6-well plate at 5×105 cells / well, and grow adherently for 24 hours. Different concentrations of (50μg / mL, 100μg / mL) were added to the umbelliferone-treated group, and an equal volume of culture medium was added to the blank control group, and culture was continued for 24 hours. Digest and collect the cells and supernatant of each group, centrifuge, wash, and resuspend the cells in 100μL binding buffer. Add 5μL AnnexinV-FITC and 5μL PI dye solution respectively, mix thoroughly, incubate at room temperature away from light for 15 minutes, and add 300μL loading buffer to each tube at the end of staining, mix thoroughly, pass through a 400-mesh nylon sieve, and use a microcapillary cell flow cytometry platform to detect cell activity. The results are as follows Figure 8 shown.

[0050] Depend on Figure 8 It can be seen that compared with the control group, the number of surviving cells in the umbelliferone-treated group was significantly reduced, and the number of dead cells was significantly increased, indicating that umbelliferone can significantly reduce the survival of PC9GR and H1975 cells and promote cell death.

[0051] (2) Effects of umbelliferone on cancer cell migration ability

[0052] 12 h before the experiment, the serum-free medium was replaced to starve H1975 cells, and the logarithmic phase human lung cancer cells H1975 cells with good growth status were digested with trypsin. The digestion was terminated with complete medium, and the cells were resuspended by centrifugation in serum-free medium to adjust the cell density to 5×10 5 Each well contains 5×10 4 Cells were inoculated in the upper chamber of the Transwell chamber, and 650 μL of 1640 complete medium (containing 20% ​​fetal bovine serum) containing 100 μg / mL umbelliferone or no drug was added to the lower chamber. The cells were placed in a cell culture incubator and cultured for 16 hours. At the end of the experiment, each group of cells was fixed with 4% paraformaldehyde at room temperature for 15 minutes, and then stained with 0.1% crystal violet for 10 minutes. The crystal violet stain was aspirated and the remaining cells in the upper chamber were wiped. The migration of cells in the lower chamber was observed under a microscope, and photos were taken and recorded. The results are shown in Fig. 9 shown.

[0053] Fig. 9The results showed that compared with the control group, the number of migrating cells in the umbelliferone-treated group was significantly reduced, indicating that umbelliferone can significantly inhibit the migration ability of H1975 cells.

[0054] Example 2

[0055] Example 2 of the present invention uses a cell thermal shift experiment to detect the binding of umbelliferone to CEMIP.

[0056] (1) Cellular Thermal Shift Assay (CETSA) is a widely used biophysical technique introduced in 2013 to study the binding of ligands to proteins in intact cells. This method is an effective and direct method to measure the arrival and binding of drug molecules to target proteins. The principle is that the target protein usually becomes structurally stable after binding to the drug molecule, resulting in a positive shift in the melting temperature (tm). The tighter the binding, the higher the tm value. When the target protein can bind to the drug, the protein will be less likely to denature and precipitate due to the increased thermal stability. The precipitated unbound protein is then removed by centrifugation. Finally, the protein in the supernatant is detected by Western Blot or other protein detection techniques to obtain the protein spectrum bound to the drug, thereby studying its drug-target interaction and the corresponding off-target proteins. CETSA can monitor the changes in the thermal stability of drugs and specific proteins in cells and tissues of different species. It is widely used to verify and quantify the target binding of drugs in cells and tissues of different species.

[0057] Human lung cancer PC9GR cells growing in logarithmic phase were digested with trypsin, digested with RIPM-1640 medium containing 10% fetal bovine serum, centrifuged and resuspended, and the density was adjusted to 1×10 6 / mL, take 1mL and inoculate on the bottom area of ​​10cm 2The cells were placed in a cell culture dish and placed in a constant temperature and humidity incubator (37°C, 5% CO2, 98% relative humidity) for 48 hours. When the cell confluence reached 90-95%, they were replaced with 10 mL of serum-free RIPM-1640 medium containing 500 μg / mL umbelliferone (umbelliferone treatment group) or 10 mL of RIPM-1640 medium containing an equal amount of solvent (solvent group) and continued to be cultured for 1 hour. The cells of the solvent group and umbelliferone treatment group were collected, and 600 μL of phosphate buffer solution (PBS) was added to resuspend the cells and averaged to 6 200 μL tube centrifuge tubes (100 μL / tube). The cells of each group were placed in a gradient PCR instrument, and the temperature of each column was set to 37°C, 40°C, and 45°C. After heating for 3 minutes, they were incubated at room temperature for 3 minutes, and then placed in liquid nitrogen for rapid freezing for 3 minutes. After the sample was frozen, it was taken out and placed at room temperature for thawing. The cells were repeatedly frozen and thawed 3 times to lyse the cells. Centrifuge at 4°C, 15000g for 50 minutes, take 60 μL of supernatant from each group and mix evenly with 15 μL of 5× loading buffer, and boil in a water bath for 10 minutes to complete sample preparation.

[0058] The samples of each group were separated by 7.5% SDS-PAGE gel electrophoresis for 2 hours, and the proteins on the gel were transferred to nitrocellulose membranes in an ice bath for 2 hours. The transferred nitrocellulose membranes were soaked in 5% skim milk and blocked with slow shaking at room temperature for 1 hour. The corresponding membrane strips were soaked with rabbit anti-human CEMIP polyclonal antibody diluted 800 times with PBST, mouse anti-human tag protein FLAG monoclonal antibody diluted 5000 times with PBST, or mouse anti-human monoclonal internal reference (β-actin) antibody diluted 20000 times with PBST, respectively, and incubated at 4°C overnight, rinsed with PBST 3 times, 5 minutes each time, and then incubated with horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse IgG antibody diluted 20000 times with PBST at room temperature for 1 hour, and rinsed with PBST 3 times, 5 minutes each time. The protein bands on the membrane were developed by chemiluminescence, and the expression of CEMIP protein in the solvent group and umbelliferone treatment group after treatment at different temperatures was semi-quantitatively analyzed according to the gray value of the protein bands. The results are shown in the figure. Fig.10 shown.

[0059] Fig.10 The results showed that as the temperature increased to 45°C, the expression of CEMIP protein in PC9GR cells after umbelliferone treatment was higher than that in the corresponding solvent treatment group. The CEMIP expression in the solvent group was weaker after treatment at this temperature, indicating that umbelliferone can bind to the CEMIP protein in the cell.

[0060] (2) Explore the specific binding region of umbelliferone on CEMIP protein

[0061] Take HEK293 cells in logarithmic growth phase in good condition and adjust the culture medium to 1×10 6 / mL, take 1mL of each and inoculate to a bottom area of ​​10cm 2 The cell culture dish was filled with complete medium to 10 mL and placed in a constant temperature and humidity incubator (37°C, 5% CO2, 98% relative humidity) for 24 hours. The full-length CEMIP plasmid (1-1361aa)-3xFLAG containing FLAg-tagged protein and the CEMIP truncated plasmids containing different lengths (A segment: 1-177aa; B segment: 1-30&178-572aa; C segment: 1-30&573-819aa; D segment: 1-30&820-1361aa)-3xFLAG and empty plasmid were transfected into the tool cells HEK293T using lipo2000 transfection reagent, and the culture was continued for 48 hours until the plasmid was integrated and expressed.

[0062] According to the method of (1), serum-free medium containing umbelliferone and solvent was added to treat each group of cells and samples were prepared. The subsequent electrophoresis and transfer methods were the same as described in (1). After blocking with 5% skim milk, the cells were incubated overnight at 4°C with a 5000-fold diluted mouse anti-human FLAG tag primary antibody. The subsequent secondary antibody and development treatment were the same as described in 2.1. The expression of CEMIP protein in the solvent group and the umbelliferone treatment group after treatment at different temperatures was semi-quantitatively analyzed based on the gray value of the protein band. The results are shown in Figure 11-16 shown.

[0063] The protein expression in HEK293T cells 48 hours after transfection with empty plasmid, full-length CEMIP plasmid and CEMIP truncated plasmids of different lengths. Figure 11-16 As shown in the figure, as the temperature rises to 45°C, after umbelliferone treatment, the expression of the tag protein FLAG in the full-length (WT) and segment B truncated samples is higher than that in the corresponding solvent treatment group, while the FLAG protein expression in the corresponding solvent group is weaker, indicating that umbelliferone can bind to CEMIP, and its binding region is between 1-30 and 178-572aa. The expression of the tag protein FLAG in segments A, C, and D is lower than that in the corresponding solvent treatment group, indicating that umbelliferone fails to bind to the above three segments of protein regions.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A CEMIP target inhibitor, characterized in that The CEMIP target inhibitor is umbelliferone.

2. The CEMIP target inhibitor according to claim 1, characterized in that The structure of the umbelliferone is shown in formula (I):

3. A use of the CEMIP target inhibitor according to claim 1 in the preparation of anticancer drugs, characterized in that The CEMIP target inhibitor is umbelliferone, and the anticancer drug is an anti-lung cancer or gastric cancer drug.

4. The use of the CEMIP target inhibitor in the preparation of anticancer drugs according to claim 3, characterized in that: The umbelliferone can inhibit the growth, proliferation and migration of cancer cells and promote the death of cancer cells.

5. The use of the CEMIP target inhibitor in the preparation of anticancer drugs according to claim 3, characterized in that: The anticancer drug may also be added with a pharmaceutically acceptable carrier.

6. The use of the CEMIP target inhibitor in the preparation of anticancer drugs according to claim 3, characterized in that: The dosage form of the anticancer drug is one of powder, solution, capsule, granule, tablet, emulsion and suspension.

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