WO2021174995A1 - Use of jq-1 in preparation of medicine for treating pancreatic cancer and method for verifying inhibition of jq-1 on exosome secretion of pancreatic cancer - Google Patents

Abstract

Disclosed is a use of JQ-1 in preparation of a medicine for treating pancreatic cancer. Further disclosed is an anti-pancreatic cancer pharmaceutical composition, which contains the JQ-1. Further disclosed is a method for verifying the inhibition effect of the JQ-1 on exosome secretion of pancreatic cancer cells. The method comprises the following processes: extracting cell supernatant exosome by using a low-temperature ultracentrifugation method; using a transmission electron microscope to verify a cup-shaped structure wrapped by a bilayer lipid membrane of the exosome; performing NTA to measure exosome concentration; lysing the collected exosome, then quantifying the protein by using a BCA kit, and reflecting the amount of the exosome by using the measured amount of the protein; and collecting pancreatic cancer tissues of a nude mouse subcutaneous tumorigenic model constructed by a CFPAC-1 cell line, and testing the mRNA levels of CD63 and Rab27a by means of PCR.

Description

Application of JQ-1 in the preparation of pancreatic cancer therapeutic drugs and its verification method for inhibiting the secretion of pancreatic cancer exosomes Technical field

The invention belongs to the technical field of biomedicine, relates to the technical field of application of JQ-1 (triphenylmethane triisocyanate), and specifically relates to the application of JQ-1 (triphenylmethane triisocyanate) in the preparation of pancreatic cancer therapeutic drugs and its inhibition Validation method for secretion of exosomes in pancreatic cancer.

Background technique

The special tumor microenvironment plays a key role in the occurrence, development, metastasis and drug resistance of pancreatic cancer. Pancreatic cancer helps its progression by secreting exosomes to shape the microenvironment of itself and other organs. Compared with normal cells and other tumors, pancreatic cancer cells secrete more exosomes, and highly malignant pancreatic cancer cells can transfer their cancerous characteristics to low-grade cancer cells through exosomes to promote their proliferation, Migration and invasion to accelerate disease progression. The exosomes derived from pancreatic cancer can also be taken up by Kupffer cells in the liver, causing Kupffer cells to secrete TGFβ, which in turn promotes the production of fibronectin by hepatic stellate cells, creating a microenvironment suitable for tumor growth to promote liver metastasis. It can be seen that finding effective means to inhibit exosomal secretion is of great significance for the clinical treatment of pancreatic cancer.

At present, GW4869, a non-competitive inhibitor of neutral Smase (sphingomyelinase), is basically a recognized exosome inhibitor, which can inhibit the secretion of exosomes. And studies have found that in prostate cancer cells, farnesyl transferase inhibitor (Manumycin A) inhibits the production and secretion of exosomes by inhibiting the expression of Ras signaling pathway and hnRNP H1. At present, there is no product that treats the production of disease-related exosomes as a drug strategy. This may be due to the complex mechanisms of exosomes regulating diseases and the unclear specific targets. Therefore, how to find a drug that can effectively inhibit the production and secretion of exosomes has become a problem that needs to be solved at present.

Summary of the invention

The technical problem to be solved by the present invention is to provide an application of JQ-1 in the preparation of pancreatic cancer therapeutic drugs and a verification method for inhibiting the secretion of pancreatic cancer exosomes, so as to solve the problems raised in the background art.

In order to solve the above technical problem, the embodiment of the present invention provides an application of JQ-1 in the preparation of a pancreatic cancer therapeutic drug.

Further, the pancreatic cancer drug is used to inhibit the secretion of pancreatic cancer cell exosomes.

The embodiment of the present invention provides an anti-pancreatic cancer pharmaceutical composition, characterized in that the pharmaceutical composition contains at least JQ-1.

Further, the pharmaceutical composition also includes medical auxiliary components added according to the requirements of the pharmaceutical preparation and dosage form.

The embodiment of the present invention also provides a method for verifying the inhibitory effect of JQ-1 on the secretion of pancreatic cancer cells exosomes, which is characterized by including the following processes: (1) Establishing a pancreatic cancer cell group after JQ-1 treatment and a control In the pancreatic cancer cell group, the cell supernatant exosomes were extracted by low-temperature ultracentrifugation; (2) Transmission electron microscopy was used to verify the goblet structure of exosomes wrapped in a double lipid membrane; (3) NTA was used to detect the concentration of exosomes; 4) After lysing the collected exosomes, use the BCA kit to quantify the protein, and use the measured protein amount to reflect the amount of exosomes; (5) Use the untreated CFPAC-1 cell line to construct subcutaneous tumors in nude mice Model, collect pancreatic cancer tissues, use PCR to detect the mRNA levels of CD63 and Rab27a.

Further, the step (1) specifically includes the following process: the step (1) specifically includes the following process:

(1-1) After treatment with JQ-1, the pancreatic cancer cell group and the control pancreatic cancer cell group were to collect 80 mL of cell supernatant under the standard of the same number of cells;

(1-2) Centrifuge at 300×g for 15 min at 4°C, centrifuge at 2000×g for 30 min, and centrifuge at 16500×g for 30 min to remove precipitation;

(1-3) The obtained supernatant was filtered through a 0.22μm filter and then 150,000×g low-temperature ultracentrifugation for 120min. The precipitate was collected and resuspended in 100μL PBS, and stored in aliquots at -80°C.

Further, the step (2) specifically includes the following process:

(2-1) Take 10μL of collected exosomes and fix them with 2.5% glutaraldehyde for 2h;

(2-2) Wash the exosomes with PBS and resuspend them in 100μL PBS;

(2-3) Take 20μL and drop it on the copper plate, and then negatively stain with 3% phosphotungstic acid in water for 1min, and observe by transmission electron microscope.

Further, the step (3) specifically includes the following process:

(3-1) Aspirate 8μL of collected exosomes, add PBS and dilute to 4mL, the dilution factor is 500 times;

(3-2) After the two are evenly blown and blown, they are driven into the nanoparticle tracking analysis instrument to detect the concentration and particle size of exosomes.

Further, the step (4) specifically includes the following process:

(4-1) Dilute the protein standard solution to 0.5mg/mL with PBS;

(4-2) Prepare the BCA working fluid according to the number of samples, and the BCA working fluid is now ready for use;

(4-3) Add the 0.5 mg/mL protein standard dilution solution to the 96-well plate in the order of 0, 1, 2, 4, 8, 12, 16, 20 μL, and make up to 20 μL with PBS. The diluted standard The product concentration is 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL; add 1μL exosomal protein sample to each well, add PBS to make up to 20μL;

(4-4) Add 200μL of BCA working solution to each well, incubate at 37°C for 20-30min, measure the absorbance at 562nm with a microplate reader, and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

Further, the step (5) specifically includes the following process:

(5-1) Collect untreated CFPAC-1 cells, make a cell suspension with 100μL PBS, and inject it into the nude mouse subcutaneously to construct a nude mouse subcutaneous tumor model. The modeling time of the model is 20 days;

(5-2) The nude mouse models are divided into experimental group nude mice and control group nude mice, each group contains 3 nude mice, and the experimental group nude mice are injected intraperitoneally with 30mg/kg/d JQ-1 every day, first and third , 5, 6 days for injection;

(5-3) After 7 days, collect the subcutaneous tumors of pancreatic cancer tissues from nude mice, extract RNA and reverse transcribed into cDNA, and use PCR to detect the mRNA levels of exosomes surface markers CD63 and Rab27a to reflect the number of exosomes .

The beneficial effects of the above technical solutions of the present invention are as follows: In the present invention, JQ-1 has the effect of inhibiting the secretion of pancreatic cancer cells. In view of the key role of exosomes secretion in pancreatic tumors, JQ-1 is applied to the preparation of pancreatic cancer therapeutic drugs , Provide a new theoretical basis for clinical patients to find potential drug targets; validated by the verification method of JQ-1 inhibiting the secretion of pancreatic cancer exosomes, thus confirming that JQ-1 has the effect of inhibiting the secretion of pancreatic cancer cells, further verifying The reliability of JQ-1 used in the preparation of therapeutic drugs for pancreatic cancer.

Description of the drawings

Figure 1 is a diagram of the morphology and number of pancreatic cancer cell exosomes in the control group and JQ-1 treatment group observed by transmission electron microscope in the present invention;

Figure 2 is a graph of the concentration of exosomes in pancreatic cancer cells in the NTA detection control group and the JQ-1 treatment group of the present invention;

Figure 3 is a graph showing the concentration of exosomal protein in pancreatic cancer cells detected by the BCA method in the control group and the JQ-1 treatment group in the present invention;

Fig. 4 shows the mRNA levels of CD63 and Rab27a in pancreatic cancer tissues of the control group and JQ-1 treatment group after the subcutaneous tumor formation of nude mice CFPAC-1 cells is detected by PCR in the present invention;

Among them, Figure 4A is a statistical diagram of CD63 mRNA levels in pancreatic cancer tissues of the control group and JQ-1 treatment group detected by PCR; Figure 4B is the statistics of Rab27a mRNA levels in pancreatic cancer tissues of the control group and JQ-1 treatment group detected by PCR picture.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

An application of JQ-1 in the preparation of a therapeutic drug for pancreatic cancer. The pancreatic cancer drug is used to inhibit the secretion of pancreatic cancer cell exosomes.

The embodiment of the present invention provides a pharmaceutical composition for anti-pancreatic cancer, the pharmaceutical composition at least containing JQ-1. The pharmaceutical composition is applied to inhibit exosomes secreted by pancreatic cancer cells.

The embodiment of the present invention also provides a method for verifying the inhibitory effect of JQ-1 on pancreatic cancer cells secreting exosomes, including the following processes: (1) Establishing a pancreatic cancer cell group after JQ-1 treatment and a control pancreatic cancer cell group , The use of low-temperature ultracentrifugation to extract exosomes from the cell supernatant; (2) Transmission electron microscopy to verify the cup-shaped structure of the exosomes wrapped in a double lipid membrane; (3) NTA method to detect the concentration of exosomes; (4) After the collected exosomes were lysed, the protein was quantified with the BCA kit, and the measured protein amount was used to reflect the amount of exosomes; (5) The untreated CFPAC-1 cell line was used to construct a nude mouse subcutaneous tumor model and collect In pancreatic cancer tissues, the mRNA levels of CD63 and Rab27a were detected by PCR.

In a further embodiment of the present invention, a method for verifying the inhibitory effect of JQ-1 on the secretion of exosomes of pancreatic cancer cells specifically includes the following steps: extracting cell supernatant exosomes using a low-temperature ultracentrifugation method. The detailed method is as follows: After JQ-1 treatment, the pancreatic cancer cell group and the control pancreatic cancer cell group are treated with the same number of cells and 80 mL of the cell supernatant is collected, centrifuged at 300×g for 15 min at 4°C, and centrifuged at 2000×g for 30 min. After centrifugation at 16,500×g for 30 minutes, the precipitation was removed. The supernatant was filtered through a 0.22μm filter and then ultracentrifuged at 150,000×g for 120 minutes at low temperature. The precipitate was collected and resuspended in 100μL PBS and stored in aliquots at -80°C.

Transmission electron microscopy verified the cup-shaped structure of exosomes wrapped in a double lipid membrane. Take 10μL of the collected exosomes and fix them with 2.5% glutaraldehyde for 2h, wash the exosomes with PBS and resuspend them in 100μL PBS, take 20μL drop on a small copper plate, and stain with 3% phosphotungstic acid in water for 1min, observe by transmission electron microscope . Among them, the morphology and number of exosomes were observed by transmission electron microscope, as shown in Figure 1, where Ctrl is the morphology and number of exosomes observed by transmission electron microscope in the control pancreatic cancer cell group; JQ-1 is the pancreatic cancer cells after JQ-1 treatment Group transmission electron microscope observation of exosomes morphology and number map.

The NTA method detects the concentration of exosomes. Detailed method: draw 8μL of collected exosomes, add PBS to dilute to 4mL, the dilution factor is 500 times. After the two are blown evenly, they are driven into the nanoparticle tracking analysis instrument to detect the concentration and particle size of exosomes. As shown in Figure 2, Vehicle is the control pancreatic cancer cell group; JQ-1 is the pancreatic cancer cell group after JQ-1 treatment.

After the collected exosomes were lysed, the BCA kit was used to quantify the protein, and the measured amount of protein was used to reflect the amount of exosomes. The detailed method is: dilute the protein standard solution to 0.5mg/mL with PBS; prepare the BCA working solution (reagent A: reagent B=50:1) according to the number of samples, and the BCA working solution is now ready for use. Add 0.5 mg/mL protein standard dilutions in the order of 0, 1, 2, 4, 8, 12, 16, 20 μL to a 96-well plate, make up to 20 μL with PBS, and the diluted standard concentrations are respectively 0 , 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL. Add 1μL of exosomal protein sample to each well, and add PBS to make up to 20μL. Add 200μL of BCA working solution to each well and incubate at 37°C for 20-30min. The microplate reader measures the absorbance at 562nm. Calculate the protein concentration of the sample based on the standard curve and the sample volume used. The protein quantification of the isolated exosomes was carried out with the BCA kit. As shown in Figure 3, it is a statistical graph of the concentration of exosomal protein detected by the BCA method in the control pancreatic cancer cell group, where Vehicle is the exosomal protein concentration data of the control pancreatic cancer cell group; JQ-1 is after JQ-1 treatment Exosomal protein concentration data of pancreatic cancer cell group.

In order to verify that JQ-1 can inhibit the secretion of exosomes, a nude mouse experiment was carried out in the present invention, which specifically includes the following steps: S1, nude mouse model preparation: collect untreated CFPAC-1 cells and prepare them with 100μL PBS The cell suspension was injected subcutaneously into nude mice to construct a nude mouse subcutaneous tumor model, the model forming time was 20 days; S2, the nude mouse model was divided into experimental group nude mice and control group nude mice, each group had 3 nude mice , The experimental group of nude mice were injected with 30mg/kg/d JQ-1 daily, and injected on the 1, 3, 5, and 6 days; after S3, 7 days, the nude mice were collected subcutaneously tumor-forming pancreatic cancer tissues, and the RNA was extracted and reversed It was recorded as cDNA, and RT-PCR was performed to detect the mRNA levels of exosomal surface markers CD63 and Rab27a to reflect the number of exosomes. Figure 4A is a statistical graph of CD63 and Rab27a mRNA levels in pancreatic cancer tissues of nude mice in the control group and JQ-1 treatment group; Figure 4B is a statistical graph of Rab27a mRNA levels in pancreatic cancer tissues of nude mice in the control group and JQ-1 treatment group . Among them, Control is the mRNA levels of CD63 and Rab27a in pancreatic cancer tissues of nude mice in the control group; JQ-1 is the mRNA levels of CD63 and Rab27a in pancreatic cancer tissues of nude mice in the experimental group; the test results show that: The number of exosomes secreted by pancreatic cancer cells in the experimental group of nude mice, that is, the group injected with JQ-1 into the abdominal cavity, decreased significantly.

Among them, the reagents in the embodiment of the present invention are prepared as follows:

Complete medium: add 50 mL of fetal bovine serum (Gibco, USA), 5 mL of Penicillin Streptomycin solution (Gibco, USA) to 450 mL of DMEM high glucose medium (Hyclone), mix and store at 4°C.

Cell PBS (Sangon Corporation).

1×PBST buffer: Dissolve 0.24g KH2PO4, 0.2g KCl, 1.44g Na2HPO4·12H2O and 1mLTween-20 in deionized water, dilute to 1000mL, and store at room temperature.

Primary antibody: Rabbit anti-mouse CD63 monoclonal antibody (Abcam, UK).

Secondary antibody: HRP-labeled goat anti-rabbit IgG (Jackson ImmunoResearch).

JQ-1-a bromodomain and extraterminated domain (BET) inhibitor, has anti-proliferation and apoptosis activity against many cancers. JQ1 is a BET bromodomain inhibitor that acts on BRD4 and binds to all bromodomains of the BET family, but not to the bromodomains outside the BET family. The BET protein is usually overexpressed in various types of human cancers and is clinically associated with these cancers. The present invention provides the use of JQ-1 to significantly inhibit the secretion of pancreatic cancer cells exosomes after inhibiting the BET protein. In view of the key role of exosomes secretion in tumors, this feature makes JQ-1 possible in clinical tumor treatment The important value provides a new theoretical basis for clinical patients to find potential drug targets.

The above are the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims (10)

An application of JQ-1 in the preparation of a therapeutic drug for pancreatic cancer. The use of JQ-1 in the preparation of a pancreatic cancer therapeutic drug according to claim 1, wherein the pancreatic cancer drug is used to inhibit exosomal secretion of pancreatic cancer cells. An anti-pancreatic cancer pharmaceutical composition according to claim 1, wherein the pharmaceutical composition contains at least JQ-1. An anti-pancreatic cancer pharmaceutical composition according to claim 3, wherein the pharmaceutical composition further comprises medical auxiliary components added according to the requirements of the pharmaceutical preparation and dosage form. A method for verifying the inhibitory effect of JQ-1 on the secretion of exosomes of pancreatic cancer cells, which is characterized by including the following processes: (1) Establish a pancreatic cancer cell group after JQ-1 treatment and a control pancreatic cancer cell group, using Low-temperature ultracentrifugation was used to extract exosomes from the cell supernatant; (2) Transmission electron microscopy to verify the cup-shaped structure of the exosomes wrapped in a double lipid membrane; (3) NTA to detect the concentration of exosomes; (4) The collected exosomes After the lysis of the exosomes, the BCA kit was used to quantify the protein, and the measured protein amount was used to reflect the amount of exosomes; (5) The untreated CFPAC-1 cell line was used to construct a nude mouse subcutaneous tumor model, and the pancreatic cancer tissues were collected , PCR was used to detect the mRNA levels of CD63 and Rab27a. The method for verifying the inhibitory effect of JQ-1 on the secretion of exosomes of pancreatic cancer cells according to claim 5, wherein the step (1) specifically includes the following process: (1-1) After treatment with JQ-1, the pancreatic cancer cell group and the control pancreatic cancer cell group were to collect 80 mL of cell supernatant under the standard of the same number of cells; (1-2) Centrifuge at 300×g for 15 min at 4°C, centrifuge at 2000×g for 30 min, and centrifuge at 16500×g for 30 min to remove precipitation; (1-3) The obtained supernatant was filtered through a 0.22μm filter and then 150,000×g low-temperature ultracentrifugation for 120min. The precipitate was collected and resuspended in 100μL PBS, and stored in aliquots at -80°C. The method for verifying the inhibitory effect of JQ-1 on the secretion of exosomes of pancreatic cancer cells according to claim 5, wherein the step (2) specifically includes the following process: (2-1) Take 10μL of collected exosomes and fix them with 2.5% glutaraldehyde for 2h; (2-2) Wash the exosomes with PBS and resuspend them in 100μL PBS; (2-3) Take 20μL and drop it on the copper plate, and then negatively stain with 3% phosphotungstic acid in water for 1min, and observe by transmission electron microscope. The method for verifying the inhibitory effect of JQ-1 on the secretion of exosomes of pancreatic cancer cells according to claim 5, wherein the step (3) specifically includes the following process: (3-1) Aspirate 8μL of collected exosomes, add PBS and dilute to 4mL, the dilution factor is 500 times; (3-2) After the two are evenly blown and blown, they are driven into the nanoparticle tracking analysis instrument to detect the concentration and particle size of exosomes. The method for verifying the inhibitory effect of JQ-1 on the secretion of exosomes of pancreatic cancer cells according to claim 5, wherein the step (4) specifically includes the following process: (4-1) Dilute the protein standard solution to 0.5mg/mL with PBS; (4-2) Prepare the BCA working fluid according to the number of samples, and the BCA working fluid is now ready for use; (4-3) Add the 0.5 mg/mL protein standard dilution solution to the 96-well plate in the order of 0, 1, 2, 4, 8, 12, 16, 20 μL, and make up to 20 μL with PBS. The diluted standard The product concentration is 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL; add 1μL exosomal protein sample to each well, add PBS to make up to 20μL; (4-4) Add 200μL of BCA working solution to each well, incubate at 37°C for 20-30min, measure the absorbance at 562nm with a microplate reader, and calculate the protein concentration of the sample based on the standard curve and the sample volume used. The method for verifying the inhibitory effect of JQ-1 on the secretion of exosomes of pancreatic cancer cells according to claim 5, wherein said step (5) specifically includes the following process: (5-1) Collect untreated CFPAC-1 cells, make a cell suspension with 100μL PBS, and inject it into the nude mouse subcutaneously to construct a nude mouse subcutaneous tumor model. The modeling time of the model is 20 days; (5-2) The nude mouse models are divided into experimental group nude mice and control group nude mice, each group contains 3 nude mice, and the experimental group nude mice are injected intraperitoneally with 30mg/kg/d JQ-1 every day, first and third , 5, 6 days for injection; (5-3) After 7 days, collect the subcutaneous tumor-forming pancreatic cancer tissue of nude mice, extract RNA and reverse transcribed into cDNA. Use PCR to detect the mRNA levels of exosomal surface markers CD63 and Rab27a to reflect the number of exosomes .

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