CN109316482B - Application of clozapine in preparation of tumor treatment drug and application of clozapine serving as autophagy inhibitor and pharmaceutical composition - Google Patents

Abstract

The application discloses an application of clozapine in preparing a tumor treatment drug and an autophagy inhibitor, and also discloses a pharmaceutical composition. The application provides that clozapine can inhibit autophagy activity and can be used as an autophagy inhibitor. Clozapine can be used for preparing medicaments for treating tumors: clozapine has strong lethal capacity on various tumor cell lines including pancreatic cancer and breast cancer under the action of inhibiting autophagy activity and having no obvious toxicity on various normal tissue cells; clozapine has obvious inhibition effect on the growth of pancreatic cancer masses transplanted on mice; clozapine also has a significant inhibitory effect on tumor cell metastasis. Solves the technical problems that the tumor treatment medicament in the prior art kills tumor cells, kills cells of normal tissues and can not effectively control tumor growth and tumor cell metastasis.

Description

Application of clozapine in preparation of tumor treatment drug and application of clozapine serving as autophagy inhibitor and pharmaceutical composition

Technical Field

The application relates to the field of medicines, in particular to application of clozapine in preparation of a tumor treatment medicine and an autophagy inhibitor, and also relates to a pharmaceutical composition for treating tumors.

Background

Medically, cancer (cancer) refers to a malignant tumor that originates in epithelial tissue, and is the most common type of malignant tumor. According to the latest live report of the world health organization, tumors, i.e., cancers, are the second leading cause of death in the world (cardiovascular diseases are the first leading cause of death), and nearly one sixth of deaths are caused by cancers on a global basis. The highest mortality cancer types in the world are in turn: lung cancer, liver cancer, colorectal cancer, gastric cancer and breast cancer. The morbidity and mortality is not only sex related, but also in developed and less developed areas there are significant differences. Cancer is now the leading cause of death in our country.

Cancer cells are the root cause of tumor development and are the result of normal cellular variation. Cancer cells are different from normal cells, have three characteristics of infinite growth, transformation and metastasis, and can be infinitely proliferated and damage normal cell tissues. In addition to uncontrolled division, cancer cells locally invade surrounding normal tissues and even migrate to other parts of the body via the systemic circulation or lymphatic system, and are therefore difficult to destroy. And the cancer cells have strong capability of resisting death and are difficult to kill, so that the tumors are difficult to cure.

At present, the treatment of malignant tumor is mainly performed by means of operation, radiotherapy, chemical medicine treatment, Chinese medicine treatment, immunotherapy and the like. The curative effect of the tumor treatment medicines is seriously influenced in the aspects of toxicity, drug resistance and the like of the tumor treatment medicines, because most of the tumor treatment medicines kill tumor cells and cells of normal tissues at the same time, particularly bone marrow hematopoietic cells and gastrointestinal tract cells with vigorous proliferation, the immunologic function of a patient is easily reduced after the tumor treatment medicines are taken for a long time, even the gastrointestinal tract reaction of the patient is difficult to endure, so the treatment is forcibly interrupted, and the treatment fails. Therefore, it is an urgent technical problem to find a tumor therapeutic drug with good curative effect, no harm or less harm to human body, and effective control of tumor growth and tumor cell metastasis.

In order to provide increased energy demand for tumor cells, autophagy signals are activated to continuously degrade self-proteins to meet the malignant proliferation demand of tumor cells, so that the basal autophagy level of many types of tumor cells is significantly higher than that of normal cells. Therefore, the autophagy inhibition level in some kinds of tumor cells can inhibit the proliferation of the tumor cells, and a new idea is provided for the research and development of tumor treatment drugs.

Clozapine, also known as 8-chloro-11 (4-methyl-1-piperazinyl) -5H-dibenzo [ B, E][1,4]Diaza derivatives

It has good therapeutic effect on positive or negative symptoms of schizophrenia. Clozapine also acts as a non-specific agonist of TRPM7 and may potently activate the activity of TRPM 7. TRPM7 is a nonselective cation channel having both ion channel characteristics and protein kinase activity. Recent studies have found that TRPM7 activity regulates the release of zinc ion flux from this zinc reservoir, triggering a significant increase in the cytosolic zinc ion levels.

Other applications of clozapine are not reported at present, and particularly clozapine is not used in the field of tumor treatment and has not been found to have an autophagy inhibiting effect.

Disclosure of Invention

A large number of experiments prove that clozapine which has a better curative effect on positive or negative symptoms of schizophrenia in clinical application at present can block autophagy flow of cells and can be used as an autophagy inhibitor, and the clozapine has a killing effect on tumor cells and can inhibit growth and metastasis of the tumor cells based on the autophagy inhibition effect of the clozapine.

According to a first aspect of the application, there is provided the use of clozapine for the preparation of a medicament for the treatment of tumours.

Further, the tumors include pancreatic cancer and breast cancer.

Furthermore, the tumor treatment drug is a drug for inhibiting tumor growth.

Furthermore, the tumor treatment drug is a drug with a killing effect on tumor cells.

Furthermore, the tumor treatment drug is a drug for inhibiting tumor cell metastasis.

Furthermore, the clozapine is used for killing tumor cells when preparing the tumor treatment medicine.

Further, the clozapine is used for inhibiting the growth of tumors when being used for preparing the tumor treatment medicine.

Further, the clozapine is used for inhibiting the metastasis of tumor cells when preparing a tumor treatment medicament.

Furthermore, the clozapine is used for killing tumor cells and inhibiting the growth of tumors when being used for preparing the tumor treatment medicine.

Furthermore, the clozapine is used for killing tumor cells and inhibiting the metastasis of the tumor cells when preparing the tumor treatment medicine.

Further, the clozapine is used for inhibiting the growth of tumors and inhibiting the metastasis of tumor cells when preparing the tumor treatment medicine.

Furthermore, the clozapine is used for killing tumor cells, inhibiting the growth of tumors and inhibiting the metastasis of the tumor cells when preparing the tumor treatment medicine.

According to a second aspect of the application, there is provided the use of clozapine as an inhibitor of autophagy.

Further, clozapine is used as an autophagy inhibitor for killing tumor cells, inhibiting the growth of tumor cells or inhibiting the metastasis of tumor cells.

Further, the concentration of the clozapine is 10-40 mu M.

According to a third aspect of the application, there is provided a pharmaceutical composition for the treatment of tumors, the active ingredient being clozapine.

Further, the pharmaceutical composition is prepared into any one of oral liquid, granules, tablets, hard capsules, soft capsules, dripping pills, injections, nano preparations or targeted preparations.

Clozapine has strong lethality to various tumor cell lines including pancreatic cancer and breast cancer cell lines by inhibiting autophagy activity without obvious toxicity to various normal tissue cells, has obvious inhibition effect on the growth of pancreatic cancer masses transplanted on mice, and can also obviously inhibit the transfer capacity of the tumor cells. Solves the technical problems that the traditional technology can not effectively control the growth of the tumor and the metastasis of the tumor cells because the anti-cancer drug and the cancer treatment mode kill the tumor cells and kill the cells of normal tissues at the same time. Provides a new application of clozapine in the preparation of drugs for inhibiting autophagy and treating tumors, thereby expanding the application range of clozapine and having great practical significance.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, serve to provide a further understanding of the application and to enable other features, objects, and advantages of the application to be more apparent. The drawings and their description illustrate the embodiments of the invention and do not limit it. In the drawings:

FIG. 1 shows the staining results of the zinc ion probe in example 1 of the present application;

FIG. 2 shows the results of the Western blotting method in example 2 of the present application;

FIG. 3 is a histogram of the cell viability of the human pancreatic cancer cells Patu 8988t of example 3 of the present application;

FIG. 4 is a histogram of the cell activity of HPDE6c7 in the ductal epithelial cells of normal pancreas in example 3 of the present application;

FIG. 5 is a histogram of the cell activity of human breast cancer cell MCF7 in example 4 of the present application;

FIG. 6 is a histogram of the cell activity of normal mammary duct epithelial MCF 10A in example 4 of the present application;

FIG. 7 is a graph showing the growth of pancreatic cancer mass in mice after gavage treatment in example 5 of the present application;

FIG. 8 is a graph showing the growth of pancreatic cancer tumor mass in mice after tumor injection in example 6 of the present application;

FIG. 9 shows the results of crystal violet staining of cells metastasized with the human pancreatic cancer cell Patu 8988t of example 7 of the present application;

FIG. 10 is a graph showing the statistics of the migration rate and infiltration rate of human pancreatic cancer cells Patu 8988t in example 7 of the present application;

FIG. 11 shows the results of crystal violet staining of cells in which human breast cancer cell MCF7 is metastasized in example 8 of the present application;

FIG. 12 shows the statistics of the migration rate and infiltration rate of MCF7 in human breast cancer cells of example 8.

Detailed Description

In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that the term "comprises" and any variations thereof in the description and claims of this application and the above-described drawings are intended to cover non-exclusive inclusions, such as, for example, integers comprising a list of elements which are not necessarily expressly listed but may include other elements not expressly listed or inherent to such integers.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.

It should be noted that the specific operations not specifically recited in the examples of the present application are applicable to the routine operations or routine experimentation in the art, and it should be understood that those skilled in the art can reasonably recognize the operations according to the prior art.

Unless otherwise defined, all terms used herein should be interpreted as being open-ended or closed-ended based on the general knowledge in the art, and should be interpreted as being open-ended or closed-ended based on the general knowledge in the art.

Autophagy: autophagy is a process of phagocytosing self cytoplasmic proteins or organelles and encapsulating them into vesicles, fusing with lysosomes to form autophagosomes, degrading the encapsulated contents, thereby fulfilling the metabolic needs of the cell itself and the renewal of certain organelles.

The zinc ion probe FluoZin-3-AM staining method comprises the following steps: FluoZin-3-AM is a fluorescent probe for detecting zinc ions in cells, the fluorescent intensity of the probe is obviously increased after the probe is combined with the zinc ions in the cells, the cells can be incubated and dyed by the probe, and the fluorescent intensity is observed by using a fluorescence microscope and is used for reflecting the concentration of the zinc ions in the cells.

Western-blot detection method: western blotting is an experimental method frequently used in molecular biology, biochemistry and immunogenetics, and is an analytical method capable of performing qualitative and semi-quantitative analysis on proteins, which is to stain a cell or biological tissue sample treated by gel electrophoresis with a specific antibody, and obtain information on the expression of a specific protein in the analyzed cell or tissue by analyzing the location and depth of staining.

An EP pipe: a miniature centrifugal tube is a small centrifugal tube, is matched with a miniature centrifugal machine for use, and is used for separating a micro reagent.

ddH 2O: the abbreviation of double distilled water is water obtained by redistilling water after primary distillation.

BCA reaction working solution: BCA is an abbreviation for bicinchoninic acid protein, and BCA is a widely used protein quantification method in which the BCA reaction working solution used is a mixture of a reagent A (BCA alkaline solution) and a reagent B (copper sulfate solution) at a ratio of 50: 1.

loadingbuffer: the Western-blot assay uses loading buffer to indicate the progress of electrophoresis and to allow the sample to sink into the wells without floating.

Acr-Bis: acrylamide-methylene bisacrylamide solution.

Tris-HCl buffer: tris-hydroxymethyl aminomethane-hydrochloric acid buffer.

SDS buffer: sodium dodecyl sulfate buffer.

TEMED: tetramethyl ethylenediamine, used for preparing SDS-PAGE glue.

Marker: protein labeling, prestained or non-prestained proteins of various molecular weights, used for labeling the size and tracing of the protein in electrophoresis.

PVDF film: polyvinylidene fluoride (pvdf) is a solid support commonly used in western blotting.

TBST: contains Tris-HCl, NaCl and Tween20, and is one kind of buffering liquid commonly used in Western blotting.

HeLa cells: heila cells are artificially cultured cells with unlimited proliferation capacity, and are widely used in tumor research, biological experiments or cell culture in the medical field.

Trypan blue assay: the method is a rapid, simple and convenient method for detecting the cell survival rate by using a method that trypan blue can only stain dead cells into blue and living cells can not be stained.

Nude mouse tumorigenesis experiment: namely, injecting tumor cells into nude mice subcutaneously to observe the formation and development of tumors and the effect of antitumor drugs.

Transwell experiment: the Transwell chamber is placed in a culture plate, an upper chamber is called in the chamber, a lower chamber is called in the culture plate, an upper layer of culture solution is contained in the upper chamber, a lower layer of culture solution is contained in the lower chamber, the upper layer of culture solution and the lower layer of culture solution are separated by a polycarbonate membrane, cells are planted in the upper chamber, and due to the fact that the polycarbonate membrane is permeable, components in the lower layer of culture solution can affect the cells in the upper chamber, and therefore the influence of the components in the lower layer of culture solution on cell growth, movement and the like can be researched. The polycarbonate membranes with different pore diameters and different treatments are applied, and the research on various aspects such as cell migration, cell infiltration and the like can be carried out.

The Matrigel is polymerized to form a three-dimensional matrix with biological activity at room temperature, simulates the structure, composition, physical characteristics and functions of a cell basement membrane in vivo, is beneficial to culture and differentiation of cells in vitro, and can be used for research on cell morphology, biochemical function, migration, infection, gene expression and the like.

DMEM culture medium is one kind of culture medium containing amino acids and glucose.

BSA: bovine serum albumin, also known as the fifth component, is an albumin in bovine serum and contains 583 amino acid residues, has a molecular weight of 66.430kDa and an isoelectric point of 4.7.

OD 600: refers to the absorbance of a solution at a wavelength of 600nm to reflect the cell density in the solution.

The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.

Example 1: detection of clozapine in regulation of zinc ion level in cytoplasm

First, experiment grouping

Control group: DMSO treatment

Experimental group 1: 10 μ M clozapine treatment

Experimental group 2: treatment with 10. mu.M clozapine in combination with 1. mu.M FTY720

Experimental group 3: treatment with 10. mu.M clozapine in combination with 20. mu.M NS8593

Second, Experimental methods

The zinc ion probe FluoZin-3-AM staining method is adopted to compare the fluorescence intensity of Hela cell plasm after treatment of each group of drugs, and the specific method is as follows:

treating Hela cells with each group of drugs; washing the cells treated by the medicine with PBS for three times; the washed cells are incubated and stained for 30 minutes by using a zinc ion probe FluoZin-3-AM (2 mu M); the stained cells were photographed using a fluorescence microscope.

Third, experimental results

FIG. 1 is an image under a fluorescence microscope of a control group and 3 experimental groups after being stained with a zinc ion probe FluoZin-3-AM, which is sequentially from left to right: control group, experimental group 1, experimental group 2, and experimental group 3. Wherein the relevant content in figure 1 is translated or interpreted as follows: clozapine stands for Clozapine, FTY720 for one TRPM7 non-specific inhibitor and NS8593 for another TRPM7 non-specific inhibitor.

FIG. 1 shows a significant increase in fluorescence intensity in Hela cell paste after 10 μ M clozapine treatment compared to the control group; compared with the experimental group 1, the fluorescence intensity of Hela cytoplasm treated by 10 mu M clozapine and 1 mu M FTY720 is obviously reduced; the fluorescence intensity in the Hela cell paste was also significantly reduced after treatment with 10. mu.M clozapine in combination with 20. mu.M NS8593, compared to experimental group 1.

Fourth, conclusion

The use of clozapine as a non-specific agonist of TRPM7 can significantly increase the level of zinc ions in the cytosol, i.e. clozapine can modulate the level of zinc ions in the cytosol by activating the activity of TRPM7, and the effect of clozapine on the increase in zinc ion level can be counteracted by the overlay of a TRPM7 non-specific inhibitor (FTY720 or NS 8593).

The effect of clozapine on autophagy activity was verified by the following example 2, based on the demonstration in example 1 that clozapine can significantly elevate the level of zinc ions in the cytosol, which was found to be able to modulate autophagy flow in earlier studies.

Example 2: detection of inhibition of autophagy activity by clozapine

First, experiment grouping

Blank control group: normal medium + 10% fetal bovine serum

Experimental group 1: 20 μ M clozapine treatment

Experimental group 2: treatment with 20 μ M clozapine in combination with 5 μ M Bafilomycin A1

Experimental group 3: 5 μ M Bafilomycin A1 treatment

Experimental group 4: 50 μ M Rapamycin treatment

Second, Experimental methods

And (2) carrying out subsequent treatment and detection on each group of cells by adopting a Western-blot detection method (a protein immunoblotting method), wherein the specific method comprises the following steps:

A. protein extraction and concentration determination

(1) Cell: placing six-hole plate for culturing cells on ice, sucking off culture medium, adding 100 μ l cell lysate per hole, standing on ice for 3-5min, scraping off cells with cell scraper, collecting cell lysate in 1.5ml centrifuge tube, shaking on a vortex mixer for 20s, and standing on ice for 30 min.

(3) The standing EP tube was placed in a low-temperature centrifuge and centrifuged at 4 ℃ (15000 rpm. times.18 min).

(4) Gently pipette the supernatant into another well-labeled EP tube to obtain a total protein solution.

(5) And (3) detecting the protein concentration: mu.l of the protein solution was diluted 5-fold by adding 20. mu. lddH2O to each sample, and 10. mu.l was put in a 96-well plate and 2 duplicate wells were made for each sample.

(6) Protein standards A-H were added to the first column of the 96-well plate from high to low concentrations.

(7) Preparing a BCA reaction working solution (A: B ═ 50: 1) under the condition of keeping out of the sun, mixing uniformly, adding 100 mu l of the working solution into each hole, mixing uniformly by gentle shaking, and incubating in an incubator at 37 ℃ for 30min by wrapping the working solution with tinfoil in the condition of keeping out of the sun.

(8) And (3) detecting the Optical Density (OD) value of each hole and the concentration of the protein sample under the wavelength of 562nm by using a microplate reader.

(9) Packaging unused protein, and storing at-80 deg.C.

B. Denaturation of proteins

And calculating the volume of the added protein liquid according to the required protein amount and the experimental sample loading times. Taking the example of electrophoresis performed with 30 μ g total protein per well requiring 4 loading times to detect different assay indices, the volume of protein solution required (μ l) was calculated (30 μ g protein/protein concentration) × 5, then ddH2O was supplemented to 15 μ l × 5 to 75 μ l, 3 μ l × 5 to 15 μ l of 6 × loadingbuffer (6 × loadingbuffer: β -mercaptoethanol ═ 9:1) was added, the mouth was sealed, boiled in boiling water for 5min, and stored at 4 ℃.

C. SDS-PAGE gel electrophoresis

(1) Preparation of 10% polyacrylamide separation gel (2 gel portions): taking a special bottle for preparing the glue, adding the following reagents respectively, and carefully and uniformly mixing: ddH2O4 ml; 30% Acr-Bis3.3ml; 1.5M Tris-HCl buffer (pH8.8)2.5 ml; 0.1ml of 10% SDS buffer; 0.1ml of 10% Ammonium Persulfate (AP) and 0.004ml of TEMED0.004ml are mixed evenly, 1ml of separating glue is sucked by a gun and injected into a glass plate interlayer of an electrophoresis tank, about 2/3 small glass plate height is added without air bubbles, about 1ml of isopropanol is slowly added to seal a pressing line, and polymerization is carried out at room temperature for about 30 min.

(2) Preparation of 5% polyacrylamide concentrated gum (2 gum portions): taking a special bottle for preparing the glue, adding the following reagents respectively, and carefully and uniformly mixing: ddH2O2.7ml; 30% Acr-Bis0.67ml; 1.0M Tris-HCl buffer (pH6.8)0.5 ml; 0.04ml of 10% SDS buffer; 0.04ml of 10% ammonium persulfate; TEMED0.004ml and mix well.

(3) Pouring off the isopropanol on the top of the gel, sucking off the residual liquid with filter paper, injecting the concentrated gel on the top of the interlayer, inserting into a comb, and polymerizing at room temperature for 30 min.

(4) After the gel is solidified, the gel plate is fixed in an upper buffer chamber of the electrophoresis apparatus, the electrophoresis solution is added, the comb is pulled out, 18 mul of sample solution is sucked by a microsyringe and added into a sample adding hole, Marker is added into the first sample hole, 1 × loadingbuffer is added into the last sample hole, and the sample is placed into the electrophoresis apparatus poured with 1 × Tris-glycine electrophoresis buffer solution.

(5) And (3) switching on a power supply, wherein the voltage of the concentrated gel is 90V, the electrophoresis is carried out for about 30min, the voltage of the separation gel is 120V, and the voltage and the time for separating the gel are adjusted according to the molecular weight of the target protein in principle after the electrophoresis is carried out until the bromophenol blue reaches the bottom of the separation gel.

(6) And (3) turning off the power supply, taking down the gel, and cutting the gel containing the target protein according to a Marker for further film conversion.

D. Rotary film (Whole wet method)

(1) And (3) shearing a PVDF membrane with a proper size according to the size of the sheared target glue, marking a shearing angle on the PVDF membrane, soaking in methanol for 1-3min, and transferring into a1 Xmembrane transfer buffer solution.

(2) And (3) installing a membrane rotating device according to the sequence of negative electrode side-porous filter cotton-thick filter paper-glue-PVDF membrane-thick filter paper-porous filter cotton-positive electrode side, then placing the membrane rotating device into a Bio-RAD (Bio-Rad electrophoresis) electrophoresis apparatus, pouring 1 multiplied by membrane rotating liquid, carrying out ice-bath membrane rotating, and determining the membrane rotating time according to the molecular weight of target protein, wherein the membrane rotating time is generally determined by using a constant current of 350mA, and the membrane rotating time is transferred for 2 hours or 400mA and the membrane rotating time is transferred for 1.5 hours.

(3) After the film is turned, a corner of the PVDF film is cut off to mark the front and back sides of the film, the Marker is arranged on the left side, the increasing sequence is from bottom to top, the corner is arranged on the upper left, and the PVDF film is rinsed for 3 times by 10min by using 1 XTSST solution.

E. Immune response

(1) And (3) putting the rinsed transfer printing film into 5% skimmed milk powder sealing liquid, sealing for 2h at room temperature, and slowly shaking on a shaking table.

(2) The primary antibody was diluted to the appropriate concentration with 5% nonfat dry milk blocking solution, covered with the appropriate amount of antibody on the membrane, and incubated overnight at 4 ℃.

(3) Wash 3 times with 1 × TBST × 15min, shake on a shaker.

(4) Diluting the secondary antibody to a proper concentration by using 5% skimmed milk powder sealing solution, covering a proper amount of antibody on a membrane, and incubating for 1-2h at room temperature in a wet box.

(5) Wash 3 times with 1 × TBST × 15min, shake on a shaker.

(6) The two reagents A and B in ECL were mixed in equal volume, added to the membrane uniformly (protected from light), and photographed with a chemiluminescence gel imaging system.

Third, experimental results

FIG. 2 shows the results of the transformation of two forms of autophagy marker LC3 and the level of P62 protein in Hela cells detected by Western blotting, blank control, and experimental groups 1 to 4, 4 hours after the drug addition. Wherein the relevant content in fig. 2 is translated or interpreted as follows: LC3 represents tubulin-related protein 1 light chain 3, Normalized LC3II represents Normalized LC3II values, GAPDH represents GAPDH reference protein, P62 represents autophagy-selective substrate P62 protein, Normalized P62 represents Normalized values of P62 expression levels, CTL represents blank control, Clozapine represents Clozapine, Baf-A1 represents bafilomycin A1, and Rapamycin represents Rapamycin.

As shown in figure 2, the conversion of LC3II increased significantly after clozapine dosing for four hours, indicating that autophagy levels were regulated when the level of conversion of LC3I to LC3II increased, revealing that clozapine has a rapid regulatory effect on autophagy. Furthermore, when clozapine was administered in combination with Bafilomycin A1 (Baf-A1: a lysosomal inhibitor), the conversion of LC3II was not further increased, suggesting that clozapine inhibits autophagy activity. Rapamycin (Rapamycin) treated group (panel 4) had the effect of reducing the level of P62 protein, in contrast clozapine treated group (panel 1) increased the level of P62 protein, further verifying that clozapine inhibits autophagy activity.

Fourth, conclusion

Clozapine can inhibit the autophagy activity of Hela cells, and can be used as an autophagy inhibitor.

In order to provide increased energy demand for tumor cells, autophagy signals are activated to continuously degrade self-proteins to meet the malignant proliferation demand of tumor cells, so that the basal autophagy level of many types of tumor cells is significantly higher than that of normal cells. Therefore, on the basis that clozapine can inhibit autophagy in example 2, the application of clozapine in killing tumor cells, inhibiting tumor growth and inhibiting tumor cell metastasis is verified by the following examples 3 to 8.

Example 3: testing of lethality of clozapine to human pancreatic cancer cells Patu 8988t

First, experiment grouping

Blank control group: DMSO treatment

Experimental group 1: 10 μ M clozapine treatment

Experimental group 2: 20 μ M clozapine treatment

Experimental group 3: 40 μ M clozapine treatment

Second, Experimental methods

The cell is detected by adopting a trypan blue experiment method, specifically, after being digested by pancreatin, a blank control group (CTL) cell and an added medicine group (experiment groups 1, 2 and 3) cell are mixed with trypan blue reagent with a fixed volume, 10 mu L of mixed solution is sucked by a liquid transfer machine and added into a white blood cell counter, the white blood cell counter is placed under a microscope for counting, and the measurement is carried out after 24 hours, 48 hours and 72 hours of medicine addition are carried out respectively. Cells stained with trypan blue were counted as dead cells, whereas viable cells. The final ratio of viable cells to total cells per group was counted as cell viability.

Third, experimental results

FIG. 3 shows the difference in cell activity of human pancreatic cancer cells Patu 8988t at different concentrations of Clozapine (10. mu.M, 20. mu.M, 40. mu.M) for different durations (24h, 48h, 72h) of treatment. Wherein the relevant content in fig. 3 is translated or interpreted as follows: clozapine stands for Clozapine, CTL on the abscissa for blank control and Cell Viability on the ordinate for Cell activity.

FIG. 4 shows the difference in cell activity of HPDE6c7 in normal pancreatic ductal epithelial cells under treatment with Clozapine (10. mu.M, 20. mu.M, 40. mu.M) at different concentrations for different durations (24h, 48h, 72 h). Wherein the relevant content in fig. 4 is translated or interpreted as follows: clozapine stands for Clozapine, CTL on the abscissa for blank control and Cell Viability on the ordinate for Cell activity.

As shown in figure 3, clozapine significantly reduced cell activity and was in a time and dose dependent trend in pancreatic cancer Patu 8988t cells.

As shown in FIG. 4, the difference in cell activity between the drug-added group and the control group was small in the HPDE6c7 cell line of normal pancreatic ductal epithelial cell.

Fourth, conclusion

Clozapine is lethal to pancreatic cancer cells, Patu 8988t, but is less lethal to normal pancreatic ductal epithelial cells, HPDE6c 7.

Example 4: testing of the lethality of clozapine to human breast cancer cells MCF7

First, experiment grouping

Blank control group: DMSO treatment

Experimental group 1: 10 μ M clozapine treatment

Experimental group 2: 20 μ M clozapine treatment

Experimental group 3: 40 μ M clozapine treatment

Second, Experimental methods

Detecting cells by adopting a trypan blue experimental method, specifically, digesting blank control group (CTL) cells and cells of an adding medicine group (experimental groups 1, 2 and 3) by using pancreatin, uniformly mixing the cells with a trypan blue reagent with a fixed volume, sucking 10 mu L of uniformly mixed liquid by using a liquid transfer machine, adding the uniformly mixed liquid into a white blood cell counter, placing the white blood cell counter under a microscope for counting, and measuring after adding medicine for 48 hours. Cells stained with trypan blue were counted as dead cells, whereas viable cells. The final ratio of viable cells to total cells per group was counted as cell viability.

Third, experimental results

FIG. 5 shows the difference in cell activity of human breast cancer cells MCF7 after 48 hours of treatment with different concentrations of Clozapine (10. mu.M, 20. mu.M, 40. mu.M). Wherein the relevant content in fig. 5 is translated or interpreted as follows: clozapine stands for Clozapine, CTL on the abscissa for blank control and Cell Viability on the ordinate for Cell activity.

FIG. 6 shows the difference in cell activity of normal mammary ductal epithelial cells MCF 10A after 48 hours of treatment with different concentrations of Clozapine (10. mu.M, 20. mu.M, 40. mu.M). Wherein the relevant content in fig. 6 is translated or interpreted as follows: clozapine stands for Clozapine, CTL on the abscissa for blank control and Cell Viability on the ordinate for Cell activity.

As shown in fig. 5, clozapine significantly reduced cell activity in breast cancer MCF7 cells and was dose dependent.

As shown in fig. 6, in normal breast ductal epithelial cells MCF 10A, there was no significant difference in cell activity between the drug-added group and the control group.

Fourth, conclusion

Clozapine is lethal to MCF7, a breast cancer cell, but not normal breast ductal epithelial MCF 10A.

Example 5: experiment for inhibiting growth of mouse pancreatic cancer tumor by clozapine gavage treatment

First, experiment grouping

Control group: PBS gavage treatment (7 parallel groups)

Experimental groups: 1.2mg/ml intragastric clozapine (7 parallel groups)

Second, Experimental methods

A nude mouse subcutaneous tumor formation experiment:

1. when the Patu 8988t cells reach about 80-90% density, the fresh culture medium is replaced the night before the cells are collected.

2. Cells were trypsinized and washed twice with pre-cooled PBS in order to remove serum from the cells.

3. Blowing and beating the cell sediment by PBS or serum-free medium to a proper concentration, wherein the cell quantity inoculated to the subcutaneous tumor is (1-5) multiplied by 10⁶One cell/branch, the inoculation volume is 0.1ml, so that the concentration of the cell suspension is 1-5X 10⁷Individual cells/ml.

4. After the cells are digested, the cells should be inoculated under the skin of the nude mice as soon as possible, and the inoculation is generally completed within half an hour as possible, and the cell suspension is placed on ice in the process to reduce the metabolism of the cells.

5. The selected nude mice are 5-8 weeks old, and have a body weight of about 18-20g, and the planting parts are selected from areas rich in blood supply, such as the middle and rear parts of armpits and the middle and upper parts of groin.

6. Before inoculation, the cell suspension is blown off fully by a gun, so that cell agglomeration is prevented, and the cell survival rate is reduced.

7. During inoculation, the needle head is inserted a little bit deep into the subcutaneous needle, about 1cm deep, and the overflow of the cell suspension from the needle eye after injection is reduced.

Method for injecting tumor-in-tumor drug and measuring tumor volume of B mouse

After the mice had tumors of more than one centimeter, the mice were gazed daily with PBS or 1.2mg/ml clozapine, and the relevant parameters of the tumors were measured with a vernier caliper and the tumor volume was calculated as long diameter by short diameter/2.

Third, experimental results

FIG. 7 shows the growth curves of pancreatic cancer tumor mass in mice in PBS control group and Clozapine (1.2mg/ml) gavage group. Wherein the relevant content in fig. 7 is translated or interpreted as follows: PBS for phosphate buffered saline, Clozapine for Clozapine, Days after intragastric administration on the abscissa, Relative Tumor Volume on the ordinate.

As shown in figure 7, the tumor volume increase was significantly reduced in the clozapine gavage mice compared to the PBS gavage mice tumors (P <0.05, P < 0.01).

Fourth, conclusion

Clozapine is capable of inhibiting the growth of pancreatic cancer tumors in mice.

Example 6: experiment for inhibiting growth of mouse pancreatic cancer tumor by clozapine tumor injection

First, experiment grouping

Control group: PBS injection (10 parallel groups)

Experimental group 1: injection of 40. mu.M clozapine (10 parallel groups)

Experimental group 2: 40 μ M clozapine, 10 μ M FTY720 Combined injection (10 parallel groups)

Experimental group 3: 10 μ M FTY720 injection (10 parallel groups)

Second, Experimental methods

A nude mouse subcutaneous tumor formation experiment:

1. when the Patu 8988t cells reach about 80-90% density, the fresh culture medium is replaced the night before the cells are collected.

2. Cells were trypsinized and washed twice with pre-cooled PBS in order to remove serum from the cells.

3. Blowing and beating the cell sediment by PBS or serum-free medium to a proper concentration, wherein the cell quantity inoculated to the subcutaneous tumor is (1-5) multiplied by 10⁶One cell/branch, the inoculation volume is 0.1ml, so that the concentration of the cell suspension is 1-5X 10⁷Individual cells/ml.

4. After the cells are digested, the cells should be inoculated under the skin of the nude mice as soon as possible, and the inoculation is generally completed within half an hour as possible, and the cell suspension is placed on ice in the process to reduce the metabolism of the cells.

5. The selected nude mice are 5-8 weeks old, and have a body weight of about 18-20g, and the planting parts are selected from areas rich in blood supply, such as the middle and rear parts of armpits and the middle and upper parts of groin.

6. Before inoculation, the cell suspension is blown off fully by a gun, so that cell agglomeration is prevented, and the cell survival rate is reduced.

7. During inoculation, the needle head is inserted a little bit deep into the subcutaneous needle, about 1cm deep, and the overflow of the cell suspension from the needle eye after injection is reduced.

Method for injecting tumor-in-tumor drug and measuring tumor volume of B mouse

After the mice had tumors of more than one centimeter, the mice were gazed daily with PBS or 1.2mg/ml clozapine, and the relevant parameters of the tumors were measured with a vernier caliper and the tumor volume was calculated as long diameter by short diameter/2.

Third, experimental results

FIG. 8 shows the growth curves of pancreatic cancer tumor mass of mice in PBS control group, Clozapine injection group, Clozapine and FTY720 injection group, and FTY720 injection group. Wherein the relevant content in fig. 8 is translated or interpreted as follows: PBS for phosphate buffered saline, Clozapine for Clozapine, FTY720 for a non-specific inhibitor of TRPM7, Days post injection in the abscissa, Relative Tumor Volume in the abscissa.

As shown in fig. 8, the tumor volume growth of the clozapine tumor body-injected group (experimental group 1) mice was significantly slowed compared to the PBS group mice tumors, and the growth curve of experimental group 3 showed that the slowing of tumor mass growth after clozapine-dried prediction could be offset by the combined injection of FTY720 ([ P ] 0.05, [ P ] 0.01, [ P ] 0.001).

Fourth, conclusion

Clozapine is capable of inhibiting the growth of pancreatic cancer tumors in mice.

Example 7: experiment for inhibiting human pancreatic cancer cell Patu 8988t metastasis by clozapine

First, experiment grouping

Control group: DMSO treatment

Experimental group 1: 20 μ M clozapine treatment

Experimental group 2: combined treatment with 20. mu.M clozapine and 1. mu.M FTY720

Second, Experimental methods

Preparing a material A:

microscope for photography, Transwell cell (pore size 8 μm), cell culture plate 24-well plate for Transwell migration experiment (compatible with Transwell cell), Matrigel from BD, serum-free DMEM medium, (1% fetal bovine serum) DMEM medium, DMEM complete medium, sterile PBS, cotton swab, pancreatin (0.25% pancreatin), 4% paraformaldehyde stationary liquid or methanol, crystal violet dye (0.1% (g/ml) PBS crystal violet)

Step B and Process

1. Matrix glue paving: (required for cell infiltration experiments, migration experiments without this step)

With Matrigel 1 from BD: 8 dilution, coating the upper face of the bottom membrane of the Transwell cell, and standing at 37 ℃ for 30min to polymerize Matrigel into a gel. The base membrane is hydrated prior to use.

2. Preparation of cell suspensions

Firstly, before preparing cell suspension, the cells can be deprived of serum and starved for 12 to 24 hours, and the influence of the serum is further removed.

② digesting the cells, centrifuging after terminating digestion and discarding the culture solution, (washing 1-2 times with PBS), resuspending with serum-free DMEM medium containing BSA, and adjusting the cell density to 5 × 10⁵/ml。

3. Seeding cells

First, 100. mu.l of the cell suspension was taken and added to a Transwell chamber.

② 600 mul DMEM complete medium is added into the lower chamber of the 24-well plate.

③ culturing the cells: and culturing for 24h conventionally.

4. Statistics of results

First, the Transwell chamber was removed, the culture medium in the well was discarded, washed 2 times with calcium-free PBS, fixed with methanol for 30 minutes, and the chamber was appropriately air-dried.

② 0.1 percent crystal violet staining solution is used for staining for 20min, the upper layer of non-migrated cells are lightly wiped off by a cotton swab and washed 3 times by PBS.

Measuring the OD600 value of the eluted cell sap and counting.

And fourthly, calculating the migration rate and the infiltration rate of the cells (the OD600 value of the experimental group/the OD600 value of the control group).

Third, experimental results

FIG. 9 shows the results of crystal violet staining of human pancreatic cancer cells Patu 8988t that migrated or infiltrated into the lower layer of the migration chamber after treatment with Clozapine, Clozapine and FTY 720. Wherein the relevant content in fig. 9 is translated or interpreted as follows: control represents Control, Clozapine represents Clozapine, FTY720 represents a non-specific inhibitor of TRPM7, Migration represents Invasion represents Migration, and Invasion represents infiltration.

FIG. 10 shows the results of the migration (left panel) or infiltration (right panel) of human pancreatic cancer cells Patu 8988t into the lower layer of the migration chamber after treatment with Clozapine, and FTY 720. Wherein the relevant content in fig. 10 is translated or interpreted as follows: control represents Control, Clozapine represents Clozapine, FTY720 represents a non-specific inhibitor of TRPM7, Migration Rate represents mobility, and Invasion Rate represents infiltration.

As shown in fig. 9, pancreatic cancer cells migrating or infiltrating into the lower chamber layer after clozapine treatment were significantly reduced compared to the control group, and the comparison of experimental group 1 and experimental group 2 shows that the reduction effect by clozapine treatment disappears by superimposing FTY 720.

As shown in fig. 10, pancreatic cancer cell migration rate or infiltration rate of clozapine-treated pancreatic cancer cells was significantly reduced compared to the control group, and the comparison of experimental group 1 and experimental group 2 shows that the effect of reduction of migration rate or infiltration rate by clozapine treatment disappears due to the addition of FTY 720.

Fourth, conclusion

Clozapine has the ability to inhibit metastasis of pancreatic cancer cells Patu 8988 t.

Example 8: experiment for inhibiting MCF7 metastasis of human breast cancer cells by clozapine

First, experiment grouping

Control group: DMSO treatment

Experimental group 1: 20 μ M clozapine treatment

Experimental group 2: combined treatment with 20. mu.M clozapine and 1. mu.M FTY720

Second, Experimental methods

Preparing a material A:

microscope for photography, Transwell cell (pore size 8 μm), cell culture plate 24-well plate for Transwell migration experiment (compatible with Transwell cell), Matrigel from BD, serum-free DMEM medium, (1% fetal bovine serum) DMEM medium, DMEM complete medium, sterile PBS, cotton swab, pancreatin (0.25% pancreatin), 4% paraformaldehyde stationary liquid or methanol, crystal violet dye (0.1% (g/ml) PBS crystal violet)

Step B and Process

1. Matrix glue paving: (required for cell infiltration experiments, migration experiments without this step)

With Matrigel 1 from BD: 8 dilution, coating the upper face of the bottom membrane of the Transwell cell, and standing at 37 ℃ for 30min to polymerize Matrigel into a gel. The base membrane is hydrated prior to use.

2. Preparation of cell suspensions

Firstly, before preparing cell suspension, the cells can be deprived of serum and starved for 12 to 24 hours, and the influence of the serum is further removed.

② digesting the cells, centrifuging after terminating digestion and discarding the culture solution, (washing 1-2 times with PBS), resuspending with serum-free DMEM medium containing BSA, and adjusting the cell density to 5 × 10⁵/ml。

3. Seeding cells

First, 100. mu.l of the cell suspension was taken and added to a Transwell chamber.

② 600 mul DMEM complete medium is added into the lower chamber of the 24-well plate.

③ culturing the cells: and culturing for 24h conventionally.

4. Statistics of results

First, the Transwell chamber was removed, the culture medium in the well was discarded, washed 2 times with calcium-free PBS, fixed with methanol for 30 minutes, and the chamber was appropriately air-dried.

② 0.1 percent crystal violet staining solution is used for staining for 20min, the upper layer of non-migrated cells are lightly wiped off by a cotton swab and washed 3 times by PBS.

Measuring the OD600 value of the eluted cell sap and counting.

And fourthly, calculating the migration rate and the infiltration rate of the cells (the OD600 value of the experimental group/the OD600 value of the control group).

Third, experimental results

FIG. 11 shows the results of crystal violet staining of MCF7 cells from human breast cancer cells treated with Clozapine, Clozapine and FTY720, or of cells infiltrating the lower layer of the migration chamber. Wherein the relevant content in fig. 11 is translated or interpreted as follows: control represents Control, Clozapine represents Clozapine, FTY720 represents a non-specific inhibitor of TRPM7, Migration represents Invasion represents Migration, and Invasion represents infiltration.

Figure 12 shows the results of the migration or infiltration rates (left) of MCF7 into the lower layer of the migration chamber or the infiltration rate (right) of human breast cancer cells after treatment with Clozapine, Clozapine and FTY 720. Wherein the relevant content in fig. 12 is translated or interpreted as follows: control represents Control, Clozapine represents Clozapine, FTY720 represents a non-specific inhibitor of TRPM7, Migration Rate represents mobility, and Invasion Rate represents infiltration.

As shown in fig. 11, the migration or infiltration of clozapine-treated breast cancer cells into the lower chamber layer was significantly reduced compared to the control group, and the comparison of experimental group 1 and experimental group 2 shows that the reduction effect by clozapine treatment is lost due to the addition of FTY 720.

As shown in fig. 12, the mobility or infiltration rate of the breast cancer cells treated with clozapine was significantly reduced compared to the control group, and the comparison of the experimental group 1 and the experimental group 2 shows that the effect of the reduction in mobility or infiltration rate by clozapine treatment disappears by superimposing FTY 720.

Fourth, conclusion

Clozapine has the ability to inhibit the metastasis of MCF7 in breast cancer cells.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (5)

1. The application of clozapine in preparing a medicine for treating tumors is characterized in that the tumors are pancreatic cancer;

clozapine is used as an autophagy inhibitor for killing tumor cells, inhibiting the growth of tumors or inhibiting the metastasis of tumor cells.

2. The use of clozapine according to claim 1 for the preparation of a medicament for the treatment of tumours, wherein the clozapine is used for killing tumour cells.

3. The use of clozapine according to claim 1 for the preparation of a medicament for the treatment of a tumour, wherein the clozapine is used to inhibit the growth of a tumour.

4. The use of clozapine according to claim 1 for the preparation of a medicament for the treatment of tumours, for inhibiting the metastasis of tumour cells.

5. The use of clozapine in the preparation of a medicament for the treatment of a tumor according to claim 1, wherein the concentration of clozapine is 10-40 μ M when clozapine is used as an autophagy inhibitor.

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