CN114377003B - Application of octreotide bromide in resisting tumor - Google Patents

Abstract

The application relates to the technical field of medicines, and relates to a novel application of octreotide ammonium bromide (Otilonium Bromide) in tumor treatment. Specifically, the application discovers that the compound (octenium bromide) shown in the formula (I) has an enzyme activity inhibition effect on the deubiquitination hydrolase USP28 for the first time, and therefore, the application can down regulate the level of the USP28 substrate protein c-Myc in tumor cells, inhibit the proliferation and growth of the tumor cells, and is hopeful to be developed into an anti-tumor lead compound or drug targeting the deubiquitination hydrolase USP 28.

Description

Application of octreotide bromide in resisting tumor

Technical Field

The application relates to the technical field of medicines, in particular to application of octreotide ammonium bromide in resisting tumors.

Background

In the twentieth century, the living environment of human beings is continuously worsened, the pollution of the living environment is gradually increased, the contact between people and cancerogenic factors is more and more compact, the incidence rate of malignant tumors is gradually increased year by year, and the malignant tumors become the biggest enemy of human health beyond cardiovascular and cerebrovascular diseases. According to live reports of the world health organization, tumors, i.e. cancers, were the second leading cause of death in the world, and 1400 ten thousand people were diagnosed as cancer in 2012. It is estimated that by 2023, new cancer cases increase worldwide to 2200 ten thousand per year, and 2400 ten thousand per year by 2035, i.e., 20 years into the future, and cancer cases increase five to five. The report data shows that the new cancer cases and the death cases in China are located in the global crown. Thus, there is an urgent need in the art to develop effective antitumor agents.

Whereas deubiquitination hydrolase USP28 has important biological functions and is involved in the regulation of various tumor-associated signaling pathways. Targeted inhibition of USP28 would be an effective approach to inhibiting tumor cell proliferation.

The octreotide bromide is generally applied as spasmolytic in clinic and can relieve abdominal pain, flatulence and irritable bowel syndromeIs a recurrence of (2). The medicine is acetylcholine receptor inhibitor, and can inhibit intracellular acetylcholine-induced Ca ²⁺ A signal. However, the inhibition of the enzymatic activity of deubiquitinase USP28 and the inhibition of proliferation of tumor cells by octreotide have not been reported in the literature.

Disclosure of Invention

The application aims to provide a novel application of octreotide bromide in anti-tumor aspect. Specifically, the application discloses that the octreotide bromide can inhibit proliferation and growth of tumor cells by inhibiting the activity of USP28 and down regulating the level of USP28 substrate protein c-Myc in the tumor cells, thereby having effective anti-tumor effect.

In a first aspect of the application there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a formulation comprising said compound, for the preparation of

a) A medicament that inhibits USP28 enzymatic activity;

b) A drug that inhibits tumor cell proliferation;

c) A medicine for treating, relieving and preventing related diseases caused by tumors.

In another preferred embodiment, the tumor is a tumor associated with high expression of USP 28.

In another preferred embodiment, the tumor is selected from the group consisting of: rectal cancer, breast cancer, non-small cell lung cancer and glioma, or a combination thereof.

In another preferred embodiment, the formulation is oral or non-oral.

In another preferred embodiment, the formulation comprises: powder, granule, capsule, injection, tincture, oral liquid, tablet, buccal tablet, or dripping pill.

In another preferred embodiment, the formulation further comprises:

an anti-neoplastic agent selected from the group consisting of: capecitabine, irinotecan, oxaliplatin, trofloxuridine compound tablet, kang Naifei ni, regorafenib, pip Bai Xili or temozolomide.

In a second aspect of the present application, there is provided a pharmaceutical composition comprising:

i) A compound of formula I or a pharmaceutically acceptable salt thereof, or a prodrug thereof;

ii) other antineoplastic agents, which may include (but are not limited to) capecitabine, irinotecan, oxaliplatin, trafloxuridine compound tablet, bevacizumab, cetuximab, panitumumab, kang Nai non-drug

Nimoramide, regorafenib, pimozide Bai Xili, temozolomide, and alemtuzumab;

iii) A pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is an injection, a tablet, a capsule, a pill, a suspension or an emulsion.

In another preferred embodiment, the pharmaceutical composition is in the form of an oral dosage form, a transdermal dosage form, an intravenous or intramuscular injection.

In a third aspect of the application there is provided the use of a pharmaceutical composition according to the second aspect, the medicament being for the preparation of:

a) A medicament that inhibits USP28 enzymatic activity;

b) A drug that inhibits tumor cell proliferation;

c) A medicine for treating, relieving and preventing related diseases caused by tumors.

In a fourth aspect of the present application, there is provided a method of inhibiting USP28 enzymatic activity comprising the step of contacting a sufficient amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the second aspect, with a cell, such that USP28 enzymatic activity is inhibited.

In another preferred embodiment, the cell is a USP 28-expressing cell.

In a fifth aspect of the application, there is provided a method of inhibiting proliferation of a tumour cell comprising the steps of:

1) Administering to a subject in need thereof a pharmaceutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as claimed in claim 5;

2) Inhibit the activity of USP28 enzyme of tumor cells, thereby inhibiting proliferation of tumor cells.

In another preferred embodiment, the method is non-diagnostic, non-therapeutic.

In another preferred embodiment, the method is in vitro.

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a graph showing that octreotide bromide inhibits the deubiquitination activity of USP 28.

Figure 2, octreotide bromide inhibits proliferation and growth of a549, MCF7, U87, ls174T and HCT116 cells.

Figure 3. Octreotide bromide reduces the stability of c-Myc proteins in HCT116 and Ls174T cells.

Detailed Description

The inventors of the present application have conducted extensive and intensive studies and, for the first time, have unexpectedly found that octreotide bromide has an effect of inhibiting tumor proliferation, on the basis of which the present application has been completed.

Specifically, the research of the application shows that the octreotide bromide can lower the level of the substrate protein c-Myc of USP28 by inhibiting the activity of deubiquitination hydrolase USP28, thereby achieving the effect of inhibiting tumor proliferation.

Terminology

Active ingredient

As used herein, the term "compounds of the present application" refers to compounds of formula (i). The term also includes compounds of formula (I) and their various pharmaceutically acceptable salts.

As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the present application with acids or bases that are suitable for use as medicaments. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of the present application with acids. Suitable salts forming acids include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and the like; acidic amino acids such as aspartic acid and glutamic acid. One preferred class of salts is the salts of the compounds of the present application with bases. Suitable bases for salt formation include, but are not limited to: inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, and organic bases such as ammonia water, triethylamine, and diethylamine. These salts can be prepared from the compounds of formula (I) by known salt-forming methods.

Pharmaceutical compositions and methods of administration

Because the compound has remarkable USP28 enzyme activity inhibition effect and can inhibit proliferation and growth of tumor cells, the compound and pharmaceutically acceptable inorganic or organic salts thereof and a pharmaceutical composition containing the compound as a main active ingredient can be used for treating tumors related to high expression of USP 28. The pharmaceutical compositions of the present application comprise a safe and effective amount of a compound of the present application or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of the compound of the application per dose, more preferably 5-100mg of the compound of the application per dose. Preferably, the "one dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present application without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifying agents (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The mode of administration of the compounds or pharmaceutical compositions of the present application is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the application may be administered alone or in combination with other pharmaceutically acceptable compounds.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present application is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 1 to 2000mg, preferably 5 to 100mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Deubiquitination hydrolase USP28

Deubiquitination hydrolase USP28 has important biological functions and is involved in the regulation of various tumor-associated signaling pathways. USP28 is the first deubiquitinase found to antagonize ubiquitin ligase FBW7 function ^[4] . Overexpression of USP28 in cells will antagonize the ubiquitination modification of c-Myc and Cyclin E1 by FBW7, knocking out USP28 in tumor cells, with a concomitant decrease in c-Myc protein levels, and thus USP28 promotes c-Myc mediated proliferation of tumor cells. In addition, the stability of other non-FBW 7 related proteins is regulated by USP 28. USP28 increases intracellular stability of LSD1, LSD1 regulates cellular pluripotency and differentiation by participating in the demethylation process of H3K4me1/2, LSD1 overexpression is associated with the formation of various tumors. USP28 allows LSD1 to escape from the fate of degradation by proteasomes by removing its ubiquitination modifications and thus increases the intracellular stability of LSD1 and confers pluripotency to breast cancer cells as stem cells ^[5] . In addition to modulating the stability of the above-described protooncoproteins, USP28 is also involved in the modulation of other tumor formation-related signaling pathway proteins. A new study showed that USP28 allows HIF-1 to escape from the degraded fate by antagonizing ubiquitination of HIF-1 by GSK-3 beta and FBW 7. Modulation of HIF-1 alpha by GSK-3 beta/FBW 7/USP28 affects proliferation, differentiation and apoptosis of cells ^[6] . USP28 is closely related to the development and progression of a variety of tumors, particularly colorectal cancer: the research shows that USP28 has the effects of maintaining intestinal homeostasis and promoting colorectal cancer formation. Analysis of cancer cell samples from colorectal cancer patients showed significant increases in intracellular levels of USP28, c-Myc, NICD1 and c-Jun compared to normal individual colon cells ^[7] . Furthermore, studies on bladder cancer, non-small cell lung cancer, glioma also showed that there was an up-regulation of USP28 expression in all of these cancer cells ^[8-10] . Thus, USP28 has become a potential new target for cancer treatment.

The main advantages of the application include:

(1) The application discloses that the octreotide ammonium bromide has the inhibition effect of deubiquitination hydrolase USP28 for the first time, and can obviously inhibit proliferation and growth of A549, MCF7, U87, ls174T and HCT116 cells. Octreotide bromide provides a compound structural backbone reference for drug development targeting deubiquitinase USP 28.

(2) The octreotide bromide is a drug on the market and has better anti-tumor drug patent medicine prospect.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1 test method and results for the inhibition of the enzymatic Activity of USP28 by octenium bromide

In the embodiment, the enzyme activity inhibition effect of the octreotide bromide on the USP28 is detected by adopting a Ub-AMC hydrolysis inhibition experiment, and the specific experimental method and steps are as follows:

the experiment is mainly divided into 2 systems, namely a control group and an experimental group. Control group: the total experimental reaction system was 200. Mu.l, with a final concentration of USP28 of 20nM, a final concentration of Ub-AMC of 250nM and a DMSO content of 2%. Experimental group: the total experimental reaction system was 200. Mu.l, the final concentration of USP28 was 20nM, the final concentration of Ub-AMC was 250nM, the final concentration of octreotide bromide was set to 1,2,5,10,20,50,100 and 200. Mu.M, and the DMSO content was 2%. Each group of experimental samples are sequentially added into a black micro-pore plate (96-pore plate), the excitation light of a SpectraMax M5 enzyme-labeled instrument is set to 380nm, the emission light is set to 460nm, the reaction temperature is set to 37 ℃, and the reaction time is set to 10min. The percent inhibition of the enzymatic activity of deubiquitinase USP28 by the different concentrations of octreotide bromide was calculated from the enzyme catalytic reaction rate values. Non-linear fitting of the octreotide bromide concentration to the percent inhibition of enzyme activity was performed using GraphPad Prism 5 software to calculate the inhibition of enzyme activity IC of octreotide bromide on USP28 ₅₀ Values.

The test results are shown in fig. 1: ortemozolomide significantly inhibits the enzymatic activity of USP28 to catalyze hydrolysis of Ub-AMC, IC thereof ₅₀ The value was 6.90±0.90 μm, n=3.

Example 2 method and results for testing the inhibition effect of octenium bromide on proliferation and growth of tumor cells

In the embodiment, the SRB method is adopted to determine the proliferation and growth inhibition effect of the octreotide bromide on tumor cells, and the specific experimental method and steps are as follows:

(1) A549, MCF7, U87, ls174T and HCT116 cells, which were well conditioned and in log phase of growth, were digested with pancreatin, respectively, and counted. Diluting with culture medium to a certain concentration, mixing, inoculating cells into 60 wells in the center of 96-well plate with a row gun, adding 180 μl of cell suspension into each well, and counting 5000-6000 cells per well. After cell plating was completed, 200. Mu.L of sterilized PBS was added to a round of wells surrounding the 96-well plate, and the cells were placed at 37℃with 5% CO ₂ Culturing overnight in an incubator;

(2) After overnight anchorage growth of cells in 96-well plates, corresponding concentrations of octreotide bromide were added per well, with 0.5% dmso as a control. In the experiments with a549 and HCT116 cells, the final concentration of octreotide bromide was set at 1,5,10,15,20,25,30,50 and 80 μm, with DMSO content of 0.5%. In MCF7 cell experiments, the final concentration of octreotide bromide was set at 1,3,6,10,15,20,25,30 and 60 μm, with DMSO content of 0.5%. In the U87 cell experiments, the final concentration of octreotide bromide was set at 0.5,1,3,6,10,15,30,60 and 100 μm, with a DMSO content of 0.5%. In Ls174T cell experiments, the final concentration of octreotide bromide was set at 2,5,10,15,20,25,30,60 and 100 μm, with a DMSO content of 0.5%. 3 duplicate wells were set for each concentration condition. The cells were placed at 37℃in 5% CO ₂ Culturing in an incubator for 72 hours;

(3) After 72h of cell culture, 50. Mu.L of 50% trichloroacetic acid (wt/vol) was added to each well. Cells were fixed at 4℃for about 1h. After the cell fixation is completed, pouring out the liquid in the 96-well plate, slowly flushing the inside of the hole with tap water, and placing the 96-well plate in an oven for drying;

(4) mu.L of SRB dye (4 mg/mL) was added to each well, stained for 15min, unbound dye was rinsed with 1% glacial acetic acid, and the 96-well plate was placed in an oven for drying;

(5) 150. Mu.L of 10mM Tris solution was added to each well, and the 96-well plate was placed on a shaker and slowly shaken to allow the SRB dye to be fully dissolved and mixed well. The OD was then read by placing the 96-well plate in a SpectraMax M5 microplate reader ₅₁₀ Absorbance values. Respectively calculating proliferation of different concentrations of octenium bromide to the 5 tumor cells according to the absorbance valuePercent inhibition. Nonlinear fitting is carried out on the concentration of the octreotide bromide and the proliferation inhibition percentage by using GraphPad Prism 5 software, and proliferation inhibition IC (integrated circuit) of the octreotide bromide on the 5 tumor cells is calculated ₅₀ Values.

The test results are shown in fig. 2: otemozolomide significantly inhibits proliferation and growth of A549, MCF7, U87, ls174T and HCT116 cells, and Otemozolomide proliferation inhibition IC of the above 5 tumor cells ₅₀ The values were 17.60.+ -. 0.26. Mu.M, 9.19.+ -. 0.84. Mu.M, 26.30.+ -. 1.33. Mu.M, 18.60.+ -. 0.42. Mu.M and 13.20.+ -. 0.49. Mu.M, respectively, and n=3.

Example 3 method and results for detecting Ortebromoammonium to decrease stability of the USP28 substrate protein c-Myc in HCT116 and Ls174T cells

In this example, the effect of octreotide bromide on the stability of c-Myc protein in HCT116 and Ls174T cells was examined by immunoblotting.

The antibody information used in this example is shown below:

Anti-USP28(AbCam,Ab126604,1:1000),Anti-c-Myc(AbCam,Ab32072,1:1000),Anti-GAPDH(Cell Signaling Technology,#2118,1:1000)。

all three primary antibodies were Rabbit-derived antibodies, and the secondary antibodies used in this example were purchased from Absin and used in a dilution ratio of 1:4000.

The specific experimental method comprises the following steps:

3.1 Effect of octenium bromide on c-Myc protein levels in tumor cells:

(1) Ls174T and HCT116 cells in good condition and in log phase of growth were digested with pancreatin, respectively, and counted. The cell suspension is evenly mixed and inoculated into a 6-hole plate, and the number of cells in each hole is 80-90 ten thousand. The cells were placed at 37℃in 5% CO ₂ Culturing in an incubator;

(2) After the cell density grew to about 90%, the medium was aspirated, the cells were washed with PBS buffer, then the medium containing octreotide bromide was added, the octreotide bromide final concentration was set to 10,30,50,80 and 100 μm, and simultaneously 0.5% dmso was set as control group, and the treatment was performed for 2 hours;

(3) After drug treatment, cells were removed from the incubator, washed twice with PBS buffer, placed on ice and added with an appropriate amount of RIPA (Radio Immunoprecipitation Assay) lysate, cell lysate was collected into a 1.5mL EP tube, 12000rpm,4 ℃, centrifuged for 10min, cell lysate supernatant was aspirated, the total protein concentration of cell lysate was determined with Bradford kit, and the total protein concentration of all samples was adjusted to unity with RIPA lysate, the corresponding volume of 5 x loading buffer was added, and then the samples were boiled at 100 ℃ for 10min. The prepared sample is used for carrying out the subsequent western blotting experiment.

3.2 Effect of octreotide bromide on ubiquitin proteasome degradation pathway of c-Myc protein in tumor cells:

to demonstrate that the down-regulation of c-Myc protein levels in tumor cells by octreoum bromide is achieved by promoting the ubiquitin proteasome degradation process of c-Myc, we used the proteasome inhibitor MG132 to treat tumor cells.

(1) Ls174T and HCT116 cells in good condition and in log phase of growth were digested with pancreatin, respectively, and counted. The cell suspension is evenly mixed and inoculated into a 6-hole plate, and the number of cells in each hole is 80-90 ten thousand. The cells were placed at 37℃in 5% CO ₂ Culturing in an incubator.

(2) After the cell density had grown to about 90%, the medium was aspirated, the cells were washed with PBS buffer, and then medium containing the different experimental system components was added separately. In the experiment system without MG132, the control group only contains 0.5% DMSO, the experiment group only contains the octreotide bromide, the final concentration of the octreotide bromide is set to 25 mu M in the HCT116 cell experiment, and the final concentration of the octreotide bromide is set to 40 mu M in the Ls174T cell experiment; in the experiment system containing MG132, the control group contained only 10. Mu.M MG132 (0.5% DMSO), the experiment group contained octreotide bromide and 10. Mu.M MG132 (0.5% DMSO), the octreotide bromide final concentration was set to 25. Mu.M in the HCT116 cell experiment, and the octreotide bromide final concentration was set to 40. Mu.M in the Ls174T cell experiment.

(3) After 3h of drug treatment, cells were removed from the incubator, washed twice with PBS buffer, placed on ice and added with an appropriate amount of RIPA lysate, cell lysate was collected into a 1.5mL EP tube, 12000rpm,4 ℃, centrifuged for 10min, cell lysate supernatant was aspirated, the total protein concentration of the cell lysate was determined with Bradford kit, and the total protein concentration of all samples was adjusted to unity with RIPA lysate, the corresponding volume of 5 x loading buffer was added, and then the samples were boiled at 100 ℃ for 10min. The prepared sample is used for carrying out the subsequent western blotting experiment.

3.3 Western blotting experiments:

(1)SDS-PAGE

after the prepared samples are centrifuged, the samples are added into each pore canal by a liquid-transfering gun, 2 Marker pore canals are arranged in each glue, 2 mu L of protein markers are added into each pore canal, and the residual volume is filled with a loading buffer solution. After sample addition is completed, an electrode cover is covered, a sample is concentrated into a line in laminated glue by using 60V lamination low voltage, then the sample is separated rapidly by using 120V separation voltage, and electrophoresis is finished when bromophenol blue moves to the bottom of the separation glue;

(2) Transfer film

When SDS-PAGE is carried out, the PVDF membrane can be immersed into methanol for activation, and after the activation is finished, the PVDF membrane is put into membrane transferring liquid for stabilization for 10min for subsequent experiment use. Assembling a transfer film sandwich: the black face of the black-and-white clip was placed on the bottom, and a sandwich clip was prepared in the order of sponge sheet-filter paper-glue-PVDF film-filter paper-sponge sheet from negative to positive. In order to avoid influencing experimental results, the whole sandwich clamp preparation process is carried out in the transfer film liquid, and bubbles need to be avoided. And (3) placing the assembly in a film transfer groove, adding film transfer liquid stored at low temperature and an ice box, and preventing the film transfer process from excessively high temperature and transferring film for 3h at constant current of 0.25A. After the film transfer is completed, the power supply is turned off, the PVDF film is taken out, the Marker band position is used as a protein molecular weight reference, a target protein band is cut, a mark is written, and the PVDF film is put into an incubation box;

(3) Antibody incubation and development

After washing the PVDF membrane containing the target protein band with TBST buffer, it was blocked in TBST buffer containing 5% skimmed milk for 1 hour. Pouring out milk, adding a proper amount of TBST buffer solution for cleaning for 3 times, and shaking and washing on a side swinging table for 10min each time. The primary antibody was diluted with primary antibody in the dilution ratio indicated in the specification. The cleaned PVDF membrane was added to the corresponding primary antibody and incubated overnight with shaking at 4 ℃. The primary antibody was blotted off, washed 3 times with an appropriate amount of TBST buffer, 10min each time, and the non-specifically bound antibody was removed. The secondary antibody was formulated with TBST buffer containing 5% skim milk. The corresponding secondary antibody was selected according to the species source of the primary antibody and incubated with PVDF membrane for 1.5h at room temperature. The secondary antibody was blotted off, washed 3 times with an appropriate amount of TBST buffer, 10min each time, and the non-specifically bound antibody was removed. And uniformly mixing the developing solution A and the developing solution B in equal proportion, slowly and uniformly dripping the developing solution A and the developing solution B on the PVDF film, uniformly and slowly shaking the carrier plate to uniformly distribute the developing solution on the strip, and taking a photo by using a gel imaging and photographing system to obtain an immunoblotting result photo.

3.4 experimental results

The experimental results are shown in fig. 3: octreotide bromide reduces the stability of c-Myc proteins in HCT116 and Ls174T cells. FIG. 3A shows that octreotide bromide down regulates intracellular USP28 substrate protein c-Myc levels: as the concentration of the added octreotide bromide gradually increases, the intracellular c-Myc protein level gradually decreases, and the degree of decrease exhibits a dependence on the octreotide bromide concentration. Fig. 3B shows that the down-regulation of c-Myc protein levels in HCT116 and Ls174T cells by octreotide bromide is dependent on ubiquitin proteasome degradation pathways, thereby indicating that: downregulation of c-Myc protein levels is caused by inhibition of USP28 activity by octreotide bromide. After addition of MG132 during cell culture, the c-Myc protein levels in cells that were not used and treated with octreotide bromide were comparable.

Experimental results show that octreotide bromide significantly down-regulates c-Myc protein levels in HCT116 and Ls174T cells, and that the down-regulation of c-Myc protein content by octreotide bromide is achieved by promoting its degradation process via the ubiquitin proteasome pathway. Therefore, the compound is hopeful to be developed into an anti-tumor lead compound or a drug targeting deubiquitinase USP 28.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (11)

1. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a formulation containing said compound, for the preparation of

a) A medicament for inhibiting proliferation of tumor cells, wherein the tumor is selected from the group consisting of: rectal cancer, breast cancer, non-small cell lung cancer, glioma, or a combination thereof,

2. the use according to claim 1, wherein the tumor cell is a tumor cell highly expressing USP28 enzyme.

3. The use of claim 1, wherein said tumor cells are selected from the group consisting of: non-small cell lung cancer cell a549, breast cancer cell MCF7, diffuse glioma cell U87, colorectal adenocarcinoma cell Ls174T, colon cancer HCT116.

4. The use according to claim 1, wherein the formulation is oral or non-oral.

5. The use according to claim 1, wherein said formulation comprises: powder, granule, capsule, injection, tincture, oral liquid, tablet, or dripping pill.

6. The use according to claim 5, wherein the tablet is a lozenge.

7. The use of claim 1, wherein the formulation further comprises:

an anti-neoplastic agent selected from the group consisting of: capecitabine, irinotecan, oxaliplatin, trofloxuridine compound tablet, kang Naifei ni, regorafenib, pip Bai Xili or temozolomide.

8. A pharmaceutical composition, comprising:

i) A compound of formula I or a pharmaceutically acceptable salt thereof;

ii) other antineoplastic agents selected from the group consisting of: capecitabine, irinotecan, oxaliplatin, trafloxuridine compound tablet, bevacizumab, cetuximab, panitumumab, kang Naifei ni, regorafenib, pip Bai Xili, temozolomide and alemtuzumab;

iii) A pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is an injection, a tablet, a capsule, a pill, a suspension, or an emulsion.

10. Use of the pharmaceutical composition of claim 8, wherein the medicament is for the preparation of:

a) A medicament for inhibiting proliferation of tumor cells, wherein the tumor is selected from the group consisting of: rectal cancer, breast cancer, non-small cell lung cancer, glioma, or a combination thereof.

11. The use of claim 10, wherein said tumor cells are selected from the group consisting of: non-small cell lung cancer cell a549, breast cancer cell MCF7, diffuse glioma cell U87, colorectal adenocarcinoma cell Ls174T, colon cancer HCT116.