WO2023077711A1 - Screening method of medicament for preventing prostatic cancer and application of nitazoxanide in pharmacy - Google Patents

Abstract

A screening method of a medicament for preventing prostatic cancer, comprising: carrying out 3D cell culture on prostatic cancer cells to obtain a tumor spheroid; carrying out a cell invasion experiment on the tumor spheroid by using medicaments to be screened, and screening out a medicament corresponding to the tumor spheroid with the invasion area smaller than 50%; adding the screened medicament into the tumor spheroid for culture, and screening out seven medicaments with the invasion of less than 70%; and measuring IC50 of the medicament, and screening out the medicament with the IC50 value smaller than 28 μM, namely the medicament for preventing the prostatic cancer. An application of nitazoxanide in preparation of drugs for preventing prostatic diseases, prostatic cancer or prostatic cancer bone metastasis.

Description

Screening method of drug for preventing prostate cancer and application of nitazoxanide in pharmacy

priority information

This application claims the priority of Chinese patent applications with application numbers 202111305388.2 and 202111296810.2 filed on November 3, 2021, the entire contents of which are incorporated in this application by reference.

technical field

The application relates to the technical field of biomedicine, in particular to a method for screening a drug for preventing prostate cancer and the application of nitazoxanide in pharmaceuticals.

Background technique

Prostate cancer is the most common malignant tumor of the male urinary system, and its incidence rate ranks fourth in the world. It is estimated that in 2020, 1,414,259 patients will develop prostate cancer, and 375,304 cancer patients will die from prostate cancer. Compared with European and American countries, although the incidence rate of prostate cancer in my country is relatively low, in recent years, with the aging of my country's social population, the improvement of economic level and the change of people's eating habits, the incidence rate of prostate cancer in my country is increasing year by year, which seriously threatens our country. Health of middle-aged and older men. According to the analysis of the current situation and epidemic trend of prostate cancer in China in 2013, the incidence of prostate cancer in Chinese men was only 3.52/100,000 in 1998, but it reached 11.00/100,000 in 2008, with an average annual growth rate of 12.07 in 10 years %. Bone metastasis of prostate cancer is one of the common complications of prostate cancer and the main cause of morbidity and death in patients. In the initial stage of prostate cancer, locally grown tumors can be successfully treated by surgery or radiation therapy, but about 20 to 30% of patients relapse. Although relapsed patients are initially sensitive to androgen deprivation therapy or androgen receptor antagonists, they inevitably develop castration-resistant prostate cancer (CRPC). This stage is accompanied by or rapidly followed by secondary tumors, and 90% of metastatic castration-resistant prostate cancer (mCRPC) metastasizes to skeletal sites. Although denosumab or zoledronic acid is recommended for the prevention or delay of skeletal related events (SREs) in CRPC patients with bone metastases, cancer-specific and overall survival No obvious curative effect was seen. Currently, docetaxel and cabazitaxel are the only chemotherapeutic drugs that have a survival benefit for mCRPC patients, but virtually all mCRPC will eventually develop drug resistance, and the prognosis of patients with bone metastases treated with docetaxel is still poor.

Contents of the invention

The main purpose of this application is to propose a method for screening drugs for the prevention of prostate cancer and the application of nitazoxanide in pharmaceuticals, aiming at screening new drugs that can prevent prostate cancer and proposing a method for the treatment of prostate cancer New uses for prostate disease.

In order to achieve the above purpose, the present application proposes a screening method for a medicament for preventing prostate cancer, comprising the following steps:

S10, performing 3D cell culture on prostate cancer cells to obtain tumor spheroids;

S20. Prepare the drug to be screened into a solution with a concentration of 10 μM, use the solution to conduct a cell invasion experiment on the tumor spheroid, calculate the invasion area of the tumor spheroid, and screen out tumor spheroids with an invasion area of less than 50%. The medicament corresponding to the body, wherein, the medicament to be screened is selected from a compound library listed on the FDA and included in the Pharmacopoeia;

S30. Prepare the screened medicaments into a plurality of solutions with different concentration gradients of 0-10 μM, add them into tumor spheroids for culture, calculate the invasion area of the tumor spheroids, and screen out the invasion area of less than 70%. 7 potions of

S40. Measure the IC50 of at least some of the seven medicines, and screen out the medicines with IC50 values less than 28 μM, which are the medicines for preventing prostate cancer.

In one embodiment, step S10 includes:

S11. Using prostate cancer cell PC3 and linker K369Q to construct prostate cancer highly metastatic PC3-KQ cells;

S12, performing 3D cell culture on the prostate cancer highly metastatic PC3-KQ cells to obtain tumor spheroids.

In one embodiment, the agent for preventing prostate cancer includes furamectin, nifuratel, nitazoxanide and retapalene.

Furthermore, the present application also proposes an application of nitazoxanide in the preparation of a medicament for preventing prostate diseases.

Furthermore, the present application also proposes an application of nitazoxanide in the preparation of a drug for preventing prostate cancer.

In addition, the present application also proposes an application of nitazoxanide in the preparation of a drug for preventing bone metastasis of prostate cancer.

In the technical solution provided by this application, a method for screening drugs for the prevention of prostate cancer is proposed. First, a high-concentration drug is used for cell invasion experiments, so as to perform the first screening in the FDA listed and pharmacopoeia-listed compound libraries. After that, set Concentration gradient of medicines, screen out 7 medicines that can inhibit the invasion area at low concentrations, and finally screen out medicines with IC50 values less than 28 μM according to the IC50 value of the medicines. The medicine screening method proposed in this application is high-throughput screening 3D screening in , which can more realistically simulate the in vivo cancer microenvironment while maintaining high-throughput properties. An application of nitazoxanide in treating prostate diseases is proposed. Nitazoxanide is a broad-spectrum antiparasitic drug and a broad-spectrum antiviral drug, which is used in medicine to treat various helminths, protozoa and virus infections. This application proposes a new application of nitazoxanide, which can treat prostate diseases, such as bone metastasis of prostate cancer, and develops a new drug for the treatment of prostate diseases. In addition, the screened nitazoxanide is an FDA-approved drug, and its drug safety has been verified; nitazoxanide can be obtained in large quantities from plant and animal sources, and the acquisition cost is low.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only For some embodiments of the present application, those skilled in the art can also obtain other related drawings according to these drawings without creative effort.

Fig. 1 is the result of the cell migration experiment of prostate cancer highly metastatic PC3-KQ cells and low metastatic PC3-KR cells in the embodiment of the present application;

Fig. 2 is an image of PC3-KQ tumor spheroids with high metastasis of prostate cancer and PC3-KR tumor spheroids with low metastasis of prostate cancer cultured in 3D according to the embodiment of the present application;

Fig. 3 is the result figure of the first screening of the embodiment of the present application;

Fig. 4 is the three-dimensional result figure of the second screening of the embodiment of the present application;

Fig. 5 is the statistical result figure of the second screening of the embodiment of the present application;

Figure 6 is a diagram of the area of inhibition of cell spheroid invasion by 5 of the 7 compounds at different concentrations in the examples of the present application;

Fig. 7 is the design drawing of the model of NTZ prevention mouse prostate cancer bone metastasis of the embodiment of the present application;

Fig. 8 is a success test diagram of in vivo imaging test PC3-KQ-LUC construction;

Fig. 9 is an effect diagram of in vivo imaging detection of NTZ anti-prostate cancer bone metastasis;

Figure 10 is a bioluminescent analysis diagram of the embodiment of the present application;

Fig. 11 is the effect diagram of the Micro-CT detection NTZ anti-prostate cancer bone metastasis of the embodiment of the present application;

Figure 12 is a graph showing the effect of NTZ on the body weight of mice during the administration period of the embodiment of the present application;

Fig. 13 is a HE staining analysis of the effect of NTZ on cancer cell metastases in the embodiment of the present application;

Figure 14 is the bioluminescence diagram of the PC3-KQ cells of the embodiment of the present application injected into the tail artery of the mouse for 7 days;

Fig. 15 is an effect diagram of in vivo imaging NTZ (nitazoxanide) in the treatment of prostate cancer bone metastases after 35 days of administration;

Figure 16 is a bioluminescence comparison diagram of the lower limb bone tissue of the control group and the NTZ group of the embodiment of the present application;

Figure 17 is a comparison chart of bioluminescence of the control group and the NTZ group before and after the statistical analysis of the embodiment of the application;

Figure 18 is the body weight change curve of the control group and the NTZ group mice during the administration of the embodiment of the present application;

Figure 19 is a diagram of the protective effect of NTZ on the bone tissue of living mice analyzed by Micro-CT in the embodiment of the present application;

Figure 20 is a diagram of the protective effect of NTZ on mouse bone tissue analyzed by Micro-CT in the embodiment of the present application;

Figure 21 is a diagram of the protective effect of NTZ on mouse trabecular bone analyzed by Micro-CT in the embodiment of the present application;

Figure 22 is a diagram of the HE staining detection of the invasion of PC3-KQ cells to bone tissue and the anti-invasion effect of NTZ in the embodiment of the present application;

Figure 23 is a diagram of NTZ reducing bone tissue cancer cell metastasis area in the embodiment of the present application;

Fig. 24 is a graph showing the results of immunohistochemical detection of Ki67 expression in the control group and NTZ group according to the embodiment of the present application.

The realization, functional features and advantages of the present application will be further described in conjunction with the embodiments and with reference to the accompanying drawings.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

In addition, the meaning of "and/or" appearing in the whole text includes three parallel schemes, taking "A and/or B" as an example, including scheme A, scheme B, or schemes that both A and B satisfy. In addition, the technical solutions of various embodiments can be combined with each other, but it must be based on the realization of those skilled in the art. When the combination of technical solutions is contradictory or cannot be realized, it should be considered that the combination of technical solutions does not exist , nor within the scope of protection required by the present application. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of this application.

Although denosumab or zoledronic acid is recommended for the prevention or delay of skeletal related events (SRE) in CRPC patients with bone metastases, no clear benefit was seen in terms of cancer-specific and overall survival. Currently, docetaxel and cabazitaxel are the only chemotherapeutic drugs that have a survival benefit for mCRPC patients, but virtually all mCRPC will eventually develop drug resistance, and the prognosis of patients with bone metastases treated with docetaxel is still poor.

In view of this, the present application proposes a method for screening agents for preventing prostate cancer, aiming at screening new agents capable of preventing prostate diseases.

The present application proposes a screening method for a medicament for preventing prostate cancer, comprising the following steps:

S10, performing 3D cell culture on prostate cancer cells to obtain tumor spheroids;

3D cell culture is a culture technology that can provide cells with a microenvironment closer to the living conditions in vivo during the cell culture process. It ensures high-quality, high-density cell reproduction, and breaks through the limitations of traditional culture dishes, culture bottles or microwell plates, which are time-consuming and tedious, and the cell yield is small.

Specifically, step S10 includes:

S11. Using prostate cancer cell PC3 and linker K369Q to construct prostate cancer highly metastatic PC3-KQ cells;

S12, performing 3D cell culture on the prostate cancer highly metastatic PC3-KQ cells to obtain tumor spheroids.

Studies have shown that prostate cancer high-metastatic PC3-KQ cells constructed from prostate cancer cell PC3 and linker K369Q have significantly stronger cell migration ability than prostate cancer low-metastatic PC3-KR cells constructed from prostate cancer cell PC3 and linker K369R .

S20. Prepare the drug to be screened into a solution with a concentration of 10 μM, use the solution to conduct a cell invasion experiment on the tumor spheroid, calculate the invasion area of the tumor spheroid, and screen out tumor spheroids with an invasion area of less than 50%. The medicament corresponding to the body, wherein, the medicament to be screened is selected from a compound library listed on the FDA and included in the Pharmacopoeia;

This step is mainly to carry out the first screening in the compound library listed in the FDA and included in the Pharmacopoeia. In the first screening, the concentration of the drug is relatively high, which is 10 μM, and the drug that can inhibit the increase of the tumor spheroid invasion area is mainly screened. Calculated using Image J software.

S30. Prepare the screened medicaments into a plurality of solutions with different concentration gradients of 0-10 μM, add them into tumor spheroids for culture, calculate the invasion area of the tumor spheroids, and screen out the invasion area of less than 70%. 7 potions of

In this step, the second screening is carried out among the drugs obtained from the first screening, and the drugs that still have the effect of inhibiting the increase of the tumor spheroid invasion area at a concentration lower than 10 μM are selected, and multiple different concentration gradients of 0-10 μM are selected. In the examples of this application, the concentrations used are 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, and 0 μM. The final results show that when the concentration is 1 μM and the invasion area is less than 70%, 7 The three agents are Furagin, Retapamulin, Ronidazole, Nitazoxanide, Nifuratel, Bortezomib, and Mitomycin_C.

S40. Measure the IC50 of at least some of the seven medicines, and screen out the medicines with IC50 values less than 28 μM, which are the medicines for preventing prostate cancer.

IC50 (half maximal inhibitory concentration) refers to the half inhibitory concentration of the antagonist being measured. It indicates how much a drug or substance (inhibitor) is inhibiting some biological process (or some substance involved in the process, such as an enzyme, a cell receptor, or a microorganism). In terms of apoptosis, it can be understood that a certain concentration of a certain drug induces 50% of tumor cell apoptosis, and this concentration is called 50% inhibitory concentration, that is, the corresponding concentration when the ratio of apoptotic cells to all cells is equal to 50%. The IC50 value can be used to measure the ability of a drug to induce apoptosis, that is, the stronger the induction ability, the lower the value. Of course, it can also reversely explain the tolerance of a certain cell to the drug.

According to the above screening method, four kinds of drugs can be obtained, furamectin, nifuratel, nitazoxanide and retapalene, all of which can be used as drugs for preventing prostate cancer, wherein the IC50 value of nitazoxanide lowest.

In addition, in the embodiment of this method, it is necessary to comprehensively screen related literatures to finally obtain the drug Nitazoxanide (nitazoxanide) for preventing prostate cancer.

The screening method of the medicament for the prevention of prostate cancer proposed by this application first uses a high-concentration drug to perform a cell invasion experiment, so as to perform the first screening in the compound library listed in the FDA and the Pharmacopoeia, and then set the concentration gradient of the medicament to screen out Seven drugs that can inhibit the invasion area at a low concentration, and finally screened out the drug with the lowest IC50 value, nitazoxanide, according to the IC50 value of the drug. The drug screening method proposed in this application is a three-dimensional method in high-throughput screening Screening can more realistically simulate the microenvironment of cancer in vivo while maintaining high-throughput characteristics. In addition, the screened nitazoxanide is an FDA-approved drug, and its drug safety has been verified; nitazoxanide can be obtained from Plant and animal sources are available in large quantities and at low cost.

Furthermore, the present application also proposes an application of nitazoxanide in the preparation of a drug for preventing prostate diseases, aiming to propose a new application of nitazoxanide for treating prostate diseases.

Nitazoxanide has antiprotozoal, anti-intestinal parasite, antibacterial, antiviral and other drug effects, and the antiparasitic spectrum and antibacterial spectrum are wider than albendazole, mebendazole and metronidazole, in the embodiment of this application Among them, nitazoxanide was used to prepare a medicament for preventing prostate diseases, and a new application of nitazoxanide in the field of medicine was developed. Nitazoxanide, a broad-spectrum antiparasitic and broad-spectrum antiviral drug, is used in medicine to treat various helminth, protozoan and viral infections. It is indicated for the treatment of Cryptosporidium and Giardia lamblia infections in immunocompetent individuals and has been repurposed for the treatment of influenza. Nitazoxanide has been shown to have potential against bladder cancer, glioblastoma, ovarian cancer, and colorectal cancer in cancer research, but has never been reported in prostate cancer, especially in bone metastases. Therefore, this application explores and discovers the new medical application of nitazoxanide for the first time by establishing a preclinical model, which provides a basis and foundation for the discovery of drugs for the treatment of prostate cancer bone metastasis.

Furthermore, the present application also proposes an application of nitazoxanide in the preparation of a drug for preventing prostate cancer. In the examples of the present application, nitazoxanide was used to prepare a medicament for preventing prostate cancer, and a new application of nitazoxanide in the field of medicaments was developed.

Furthermore, the present application also proposes an application of nitazoxanide in the preparation of a drug for preventing bone metastasis of prostate cancer. In the examples of this application, nitazoxanide was used to prepare a drug for preventing bone metastasis of prostate cancer, and a new application of nitazoxanide in the field of medicine was developed.

The technical solutions of the present application will be described in further detail below in conjunction with specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present application and are not intended to limit the present application.

The cell culture of the embodiment of the present application is carried out according to the following steps:

1. Cell proliferation and passage

The human prostate cancer cell lines PC3-KQ (high metastasis) and PC3-KR (low metastasis) used in this application are cultivated in a 37°C constant temperature _CO incubator with a humidity of 95% at a _CO concentration of 5%. The culture medium is conventional 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody. Observe the color change of the medium in the culture bottle under the microscope every day, pay attention to whether there is turbidity, observe the state of the cells, including the shape, density and extension of the cells, and change the medium after the color of the medium becomes light. If cell contamination is found during the cell culture process, discard all cells immediately, thoroughly disinfect the cells, resuscitate the cells, and wait for the cells to be in good condition before they can be used for experiments.

①Observe the cells under a microscope every day until they grow to 80-90% saturation and prepare for cell passage;

② Disinfect the ultra-clean table and the items used in the experiment with ultraviolet radiation for 30 minutes;

③Remove the culture medium, add PBS (2mL/dish), and carefully rinse the culture medium and cells remaining in the culture bottle;

④Remove the PBS, add 1.5mL 0.25% trypsin containing EDTA to each bottle, and let it digest for 2-3 minutes;

⑤ Observe the changes of the cells under the microscope until the cells shrink and become round, the intercellular space increases, and some cells fall off, then it can be considered that the digestion is complete. , until the cells attached to the wall of the flask are completely detached;

⑥ Transfer the blown cells and trypsin medium mixture to a 15mL centrifuge tube, and centrifuge at 1200rpm for 3min;

⑦ Discard the supernatant in the centrifuge tube, pay attention to protect the white sediment at the bottom of the centrifuge tube as the centrifuged cells, to prevent discarding the cells, add 2mL of fresh cell culture medium to the centrifuge tube, and repeatedly pipette the Pasteur pipette 40 times to resuspend the cells , forming a single-cell suspension;

⑧ Pipette 1 mL of the cells resuspended into a single cell suspension into a new 10 cm ² cell culture dish, and add 9 mL of fresh medium to the culture bottle;

⑨Pipe 1mL of the resuspended cells respectively, add them to the above-mentioned prepared culture flasks, put the culture flasks flat and shake slightly from side to side, so that the cells evenly cover the culture dishes;

⑩ Observe the cells under the microscope, mark the cell name and passage time, and place them in the incubator again to continue culturing; after tidying up the ultra-clean bench, wipe the ultra-clean bench with 75% alcohol, turn off the alcohol lamp, and perform ultraviolet disinfection.

2. Cell count

①Wipe the cell counting plate and coverslip with 75% alcohol, shake it left and right on the flame of an alcohol lamp for several times to dry, then completely cover the cell counting plate on the counting plate, and lay it flat without sliding the coverslip;

② Add trypsin to digest the cells according to the cell passage method, transfer the collected cells to a 15mL sterile centrifuge tube and prepare a single-cell suspension;

③ Pipette 20 μL of single cell suspension into a 1.5 mL centrifuge tube and mix with 20 μL of trypan blue;

④ Counting by cytometer;

⑤Record the cell count results and calculate the cell density per plate.

Example 1 Screening of Agents for Prostate Cancer Prevention

1. Construction of PC3-KQ cells with high metastases and PC3-KR cells with low metastases in prostate cancer

1. Conversion

Take the competent Escherichia coli out of the refrigerator at -80°C, and put it on ice to melt; add the ligation products (K369R, K369Q) constructed in the laboratory into the competent Escherichia coli, and incubate on ice; after heat shock at 42°C for 90s, place Place on ice for 2 minutes; add 200 μL of LB medium and 1 μL of antibiotics to the competent cells in a super-clean bench, pipette evenly, and then spread on a plate containing antibiotics; after coating, culture in a constant temperature incubator at 37°C overnight.

2. Plasmid amplification

Use a 10 μL pipette tip to pick up the monoclonal colonies on the plate and put them into EP tubes. Shake the bacteria in a shaker at 200 rpm for 12 hours, and extract the plasmids according to the plasmid extraction kit.

3. Virus packaging

When the growth confluence of 293T cells is about 80%, carry out plasmid transfection, collect the virus at 48h and 72h respectively, and centrifuge at 1500rpm for 5min; divide the prepared virus into 1.5mL EP tubes, and store in a -80°C refrigerator for later use .

4. Virus infection

Spread prostate cancer cell PC3 into a 6-well plate at a confluence of 30%, culture in complete medium, 2 mL per well;

Culture in a carbon dioxide constant temperature incubator at 37°C; 16-28 hours after the initial infection, suck off the old medium, add 500 μL of fresh complete medium and 500 μL of viruses (K369R and K369Q respectively) to each well, and finally add 1 μL of cation adsorbent (polybrene ), shake it evenly, and put it into a 37°C carbon dioxide incubator for cultivation. After 6 hours, add 1mL of fresh complete medium to each well. Mock represents the blank control for screening stable clones without adding virus; after 16-24 hours, secondary infection , aspirate the old medium and virus, add 500 μL of fresh complete medium, 500 μL of virus and 1 μL of cation adsorbent to each well, and cultivate in a 37°C carbon dioxide incubator. After 6 h, 1 mL of fresh complete medium was added to each well. After 16-24 hours, the infection is complete.

5. Screening of transgenic polyclonal cell lines

16-24 hours after the infection of prostate cancer cell PC3, the old medium and virus were sucked off, the cells were digested, transferred to a 10cm culture dish with 6mL of complete medium in each dish, and the culture was continued; after 24 hours, when the cells adhered to the wall, Aspirate the old medium, and the selection pressure is PC3: 800 μg/mL hygromycin B (Hygromycin B); replace the medium containing the selection pressure every two days; after two weeks, when the cells in the Mock dish are all After the selective pressure killing, it can be considered that the negative cells in the treatment are also killed by the selective pressure, and the surviving cells are the screened positive cells; the screened cells are expanded and cultured, and a part of them is used to collect RNA and lyse The protein is used to detect whether the target gene is successfully expressed; a part is used for cryopreservation; a part is used for continued cultivation to complete follow-up experiments.

6. Cell migration experiment

The K369R and K369Q cells grown to the logarithmic phase were digested with 0.25% trypsin, centrifuged at 1200rmp for 3min, the supernatant was discarded, and washed once with PBS. Suspend cells in serum-free medium, count cells with trypan blue, and adjust to 2.5×105 cells/mL. Take out the Transwell chamber, first add 800 μL complete medium (containing 15% fetal calf serum) of tumor cell chemokines to the lower chamber, and then inoculate 200 μL (containing 5×10 ⁴ ) cells of the prepared cell suspension on the upper chamber (Be careful not to generate air bubbles), then move the device to 37° C., 5% CO ₂ cell culture incubation for 24 hours. After incubation, wash with PBS slightly, and wipe off the tumor cells that do not penetrate the membrane on the surface of the filter membrane with a cotton swab. The Transwell chamber was soaked in 600 μL of 4% paraformaldehyde for 10 minutes, air-dried and then stained with 500 μL of crystal violet for 5 minutes. After the Transwell chamber was rinsed with tap water, air-dried, the cell distribution on the lower surface of the membrane was observed under a microscope, and 5 fields of view were taken for each well. Finally, add 500 μL of 33% acetic acid to the 24-well plate, place the Transwell chamber in it, soak the membrane, shake for 10 minutes, fully dissolve, take out the Transwell chamber, and place the 24-well plate on a microplate reader to measure the OD value at 570nm, to indirect Response cell number.

The results of cell migration experiments are shown in Figure 1. It can be seen that when acetylated KLF5 (KQ) and deacetylated KLF5 (KR) were stably transferred into PC3 cells, the migration ability of PC3-KQ cells was significantly stronger than that of PC3-KR cells.

2. 3D cell spheroid invasion screening drug system

1. 3D Culture of Tumor Spheroids

Prostate cancer high-metastatic PC3-KQ cells and low-metastatic PC3-KR cells were washed with PBS, digested with trypsin, and the separation of cells was checked under a microscope. When the cells became round, the trypsin was neutralized with complete growth medium. The cells were pipetted down with a P1000 pipette, and the cell suspension was centrifuged at 1200 rpm for 3 minutes. Remove the supernatant and resuspend the cell pellet in 1 mL of complete growth medium using a P1000 pipette. Count the cells with trypan blue and dilute the cell suspension to obtain 0.5-2 x ¹⁰⁴ cells/mL (need to determine the optimal cell density for each cell line in order to obtain tumors with a diameter of 300-500 μm 4 days after cell inoculation sphere). Transfer the cell suspension to a sterile sample tank, and use an electric multichannel pipette to draw the cell suspension (80 μL/well) and distribute it into an ultra-low attachment (ULA) 384-well circular bottom plate. The ULA384-well plate was centrifuged at 320 g for 4 min, and then the 384-well plate was transferred to an incubator (37° C., 5% CO ₂ , 95% humidity) for cultivation. After 4 days, the formation of tumor spheroids was visually confirmed. Referring to Figure 2, it can be seen that in the 3D cell culture system, the invasion ability of PC3-KQ is significantly stronger than that of PC3-KR.

2. First screening

Thaw Matrigel on ice. Store pipettes and tips for P10, P200 and P1000 in a -20°C freezer. Place the ULA384-well plate containing the 4-day-aged cell spheroids on ice. Using a multichannel pipette, gently remove 40 µL/well of the growth medium from the spherical plate. For this step, tip the tip to the inner wall of the U-shaped bottom hole, avoiding contact with the bottom of the hole and the location of the spheroid to minimize disturbing the spheroid. Transfer the Matrigel to an ice-cold tube using an ice-cold tip. For the drug evaluation research, first use the medium containing 1% FBS to prepare the drug (the drug is selected from the FDA listing and the pharmacopoeia collection compound library), so that the drug concentration reaches 40 μM, and then add 20 μL/well Matrigel to the containing medium using an ice-cold tip. Drug medium (20 μL/well) and swirl gently to mix well to avoid the formation of air bubbles. Gently dispense 40 µL of medicated matrigel into the wells at the bottom of the U-shape (final drug concentration is 10 µM), aligning the tip to the inner wall of the well (this step is the most critical, as for optimal image analysis, the Keep the sphere in the center of the hole). Using a microscope, visually check that the spheroid is centered. If not, centrifuge for 3 min at 4°C. Then at 24h, 48h and 72h, observe the degree of invasion of the 3D tumor spheres and take pictures, use Image J software to calculate the area of cell invasion, and screen out the drugs corresponding to the tumor spheroids whose invasion area is less than 50%.

Figure 3 is the result of the first screening, please refer to Figure 3, 87 compounds were screened out of 1987 compounds in the first screening.

3. Second screening

After the first screening of the compounds, the second line of drug screening is carried out. Firstly, 500 PC3-KQ cells were plated in each well of ULA384-well plate. After growing for 4 days, 40 μL medium was removed from the 384-well plate, and different concentrations of drug-containing medium (10, 1, 0.1, 0.01, 0.001, 0 μM) were mixed with Matrigel was mixed, and then slowly added to the cell sphere along the hole wall. After incubation for 48 hours, the drugs inhibited the invasion area of the cell sphere at different concentrations, and 7 agents with an invasion area of less than 70% were screened out.

Figure 4 and Figure 5 are the three-dimensional results and statistical views of the second screening, respectively. In this screening, 7 compounds were screened from 87 compounds, namely Furagin, Retapamulin, Retapamulin, Ronidazole (ronidazole), Nitazoxanide (nitazoxanide), Nifuratel (nifuratel), Bortezomib (bortezomib), Mitomycin_C (mitomycin C).

Among the 7 compounds, Bortezomib (bortezomib) and Mitomycin_C (mitomycin C) have been disclosed in the literature to have clear anti-cancer and metastasis effects, and are also approved for anti-cancer in clinical practice, so they will not be considered. Figure 6 is a map of the area of cell sphere invasion inhibited by 5 of the 7 compounds at different concentrations. It can be seen that compared with the other four drugs, Nitazoxanide (nitazoxanide) inhibited the increase of the cell sphere invasion area. The effect is obvious, and the lowest inhibitory effect is obvious at 1μM.

3. Determination of IC50

6×10 ³ /well PC3-KQ cells were seeded in 96-well plate, 100 μL/well, placed in a 37°C, 5% CO ₂ cell incubator for 24 hours, and 100 μL of drug-containing medium was added after the cells adhered to the wall. The final concentration of the drug added to the cells was 0 μmol/L, 0.015 μmol/L, 0.045 μmol/L, 0.137 μmol/L, 0.41 μmol/L, 1.2 μmol/L, 3.7 μmol/L, 11.1 μmol/L, 33.3 μmol/L L, 100 μmol/L, set up the control group (adding the same amount of culture medium) and the blank zero-adjustment group at the same time, and place 6 replicate wells in each well, place them in a CO ₂ incubator and continue to cultivate for 48 hours, absorb the culture medium, and then add 100 μL of CCK8 working solution was incubated in an incubator for 1.5 h, and the plate was placed at 450 nm on a microplate reader to measure the OD value.

The blank group was used to set zero, and the experiment was repeated 3 times, and the cell survival rate was calculated, and IC50 was obtained, wherein, the cell survival rate=(OD value of the drug treatment group/OD value of the control group)×100%.

Please refer to Table 1 for the IC50 of the 5 compounds.

Table 1 IC50 of 5 compounds

the	IC50(μM)
Furagin	27.93
Nifurate	18.89
Nitazoxanide	5.518
Retapamulin	12.17
Ronidazole	>100

It can be seen that Nitazoxanide (nitazoxanide) has the lowest IC50 and the strongest induction ability.

Example 2 Establishment of Prostate Cancer Bone Metastasis Model

1. Lentivirus infection to construct luciferase cells

1. Explore the optimal concentration of puromycin (puromycin)

Before screening, it is necessary to explore the lowest concentration of puromycin that can kill empty cells: the cells can be plated on a 24-well plate with a density of 5×10 ⁴ cells per well, and the complete medium with different concentrations of puromycin can be replaced after 24 hours, and the concentration of puromycin can be set 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 mg/mL were treated for 48 hours, and the lowest concentration that could kill more than 90% of empty cells was selected for subsequent experiments.

2. Lentivirus infection of cells

Day1: When the cell confluence is 80-90%, collect the cells and spread them on a 6-well plate with 1.5× ¹⁰⁵ cells per well; generally, the cell confluence rate is between 30%-50% when the virus infection is performed on the second day room, no antibiotics were added when laying the planks;

Day2: Before infecting the cells, take out the virus from the -80 degree refrigerator and slowly thaw it on ice, absorb the original medium of the cells, and add 1/2 volume of fresh medium (the medium may not contain double antibodies and serum) ;

According to the MOI value (multiplicity of infection, multiplicity of infection), virus is added for infection;

Add virus volume per well (μL)=MOI×cell number/virus titer (TU/mL)×1000

For cells that need to add Polybrane, Polybrane can be added at the same time (1:1000)

4 hours after infection, make up to the full culture volume with serum-containing medium;

Day3: On the second day after infection (about 24 hours), the culture solution containing the virus was aspirated, replaced with fresh complete culture solution, and continued to cultivate for 24 hours;

Day4: Replace with fresh complete culture medium with an appropriate concentration of puromycin, and screen cell lines stably expressing luc; during screening, it is necessary to set up a control group of cells not infected with the virus, and add an equivalent amount of puromycin at a concentration of 2.0ug/mL;

Day6: Add puromycin for 48 hours, and observe the death of untransfected cells in the control group. If the cell death rate in the control group exceeds 90%, remove puromycin and replace with fresh medium for culture.

After puromycin selection, the cells can be properly proportioned and subcultured when the cells are congested, and then one-fifth of puromycin can be used to maintain the resistance.

2. Establishment of bone metastasis model of prostate cancer in mice

Preparation of cell suspension: Take PC3-KQ cells with a confluence of 80-90%, digest with trypsin, stop digestion with complete medium, centrifuge at 1200rpm for 3min, and discard the supernatant. Add a certain amount of PBS to prepare a cell suspension, mix well and count the cells, prepare cells at 1×10 ⁶ /mouse, dilute and make a cell suspension, store on ice until use.

Inject cells into the tail artery: anesthetize the mice with 2.5% Avertin (120-150 μL per 10 g injection). Disinfect the tail artery; quickly inject 100 μL of the prepared cell suspension (1×10 ⁶ ) from the one-third of the tail of the mouse with an insulin needle. The time required for injection into the artery is less than 3 seconds, rotate the needle, and Press the injection site with a dry cotton ball for about 1 minute to prevent bleeding.

The results are shown in Fig. 7, Fig. 8 and Fig. 9, wherein, Fig. 7 is a model design drawing of NTZ (nitazoxanide) of the embodiment of the present application to prevent bone metastasis of mouse prostate cancer, inoculate PC3- Immediately after the KQ cells were administered with nitazoxanide, the Vehicle group was given 1% methylcellulose solution, and the treatment group was given nitazoxanide (100 mg/kg/day) treatment, and then the mouse live imaging and body weight were performed every week. The dose was administered continuously for 5 weeks, and mouse live imaging and micro-CT were performed at the end of administration. Figure 8 is a graph showing the success of PC3-KQ-LUC construction by in vivo imaging. In order to build a bone metastasis model in nude mice, we first constructed PC3-KQ-Luc cells stably expressing luciferase. For the mouse bone metastasis prevention model, PC3-KQ Vehicle group and PC3-KQ nitazoxanide group were set up respectively. Figure 9 is the effect of in vivo imaging detection of NTZ in preventing bone metastasis of prostate cancer. In vivo imaging of mice was performed on the 35th day. It can be observed that the PC3-KQ Vehicle group has obvious bioluminescence, and the luminescent parts are mainly concentrated in the bone tissue of the lower limbs of the mice. It can be seen that the bone metastasis model was successfully established by tail artery injection.

Example 3 The preventive effect of nitazoxanide on bone metastasis of prostate cancer

1. In vivo imaging of small animals and microCT

In vivo imaging in mice: In vivo imaging was performed 7 days, 14 days, 21 days, 28 days, and 35 days after injection of PC3-KQ-LUC cells. The specific operation is as follows: 10 minutes before imaging, start the in vivo imaging system, turn on the oxygen switch and the gas anesthesia switch, and then intraperitoneally inject the luciferase substrate (D-luciferin, Sodium Salt) prepared before the experiment into each group of mice, D - The concentration of the fluorescein working solution is 15 mg/mL, and the injection volume for each mouse depends on the weight of the mouse: 150 mg/kg; 10 minutes after injection into the mouse, the mouse is anesthetized for imaging analysis.

In addition, after the injection of PC3-KQ-LUC cells, nitazoxanide was administered immediately, and the administration method was intragastric administration, once a day, 100 mg/kg per mouse each time, the solvent was 1% CMC, and the administration amount was 100uL per mouse.

A small animal in vivo imaging system was used to detect the effect of cancer cell metastasis, and the observation was performed once a week for a total of 6 weeks. After the experiment was over, the mice were sacrificed by dislocation of the cervical spine, and small animal microCT was performed to observe the PC3-KQ cell transfer and bone tissue destruction.

2. HE staining

(1) After the mice in different treatment groups were killed by cervical dislocation, the femur and tibia were separated, and the excess connective tissue and muscle tissue around them were cut off, fixed in 4% paraformaldehyde for 48 hours, and then decalcified with EDTA decalcification solution for 14-21 hours sky;

(2) After the bone tissue is decalcified, it is dehydrated, and the procedure is as follows:

70% ethanol 1h

80% ethanol 1h

90% ethanol 1h

95% ethanol 1h

Absolute ethanol Ⅰ 1h

Absolute ethanol Ⅱ 1h

Xylene Ⅰ 1h

Xylene Ⅱ 1h

Paraffin Ⅰ 1h

Paraffin II 1h

Paraffin III 1.5h

(3) Embedding: Turn on the embedding machine 2 hours in advance, preheat and melt the paraffin; soak the embedding cassette in liquid paraffin for later use, place the bottom mold of the stainless steel embedding box in the paraffin on the other side, take a stainless steel bottom mold and place it on the table Squeeze in 1/3 of the paraffin, then place the tissue block in the center; then place the stainless steel bottom mold with the tissue on the freezing table, wait for it to solidify and turn white, put it on the heating table again and squeeze out the paraffin to cover the tissue , put the floor of the embedding box on the stainless steel mold, then add liquid paraffin to exceed the height of the bottom plate, place it on a freezing table to cool, when the paraffin block is frozen enough to be gently pulled out from the mold, pull out the tissue wax block, Then, the excess paraffin wax around the wax block is repaired by the heating platform of the embedding machine.

(4) Slicing: The bone tissue wax blocks were buried in ice in advance, and then frozen overnight in a refrigerator at 4°C to facilitate slicing. Take out the wax block from the refrigerator, and use a microtome to cut the tissue wax block longitudinally, with a slice thickness of 4 μm. Spread the slices with warm water at 42°C to fully expand the cut tissues, and then fix them on cationic detachment-resistant glass slides.

(5) Baking slices: Put the tissue slices into an oven at 62°C to bake the slices for 2-3 hours.

(6) Dewaxing: the procedure is as follows:

Xylene Ⅰ 10min

Xylene Ⅱ 10min

Xylene III 10min

Xylene IV 10min

Absolute ethanol Ⅰ 10min

Absolute ethanol Ⅱ 10min

95% ethanol 10min

85% ethanol 10min

75% ethanol 10min

H2O 5min

(7) Staining: Hematoxylin staining for 1.5 min, rinsed with running water for 1 min until the staining tank was colorless, soaked in PBS; differentiated with 5% acetic acid for 3 s, differentiated non-specific tissue staining; rinsed once with running water, soaked in PBS, observed the staining effect under a microscope, and The core should be sky blue;

(8) Dye with eosin dye solution for 2 to 3 minutes; quickly wash with tap water 2 times;

(9) Gradient ethanol dehydration: slices were subjected to 75% ethanol for 20 seconds, 85% ethanol for 20 seconds, 95% ethanol for 20 seconds, and 100% ethanol for 10 minutes, and then observed the results of eosin staining under the microscope; then continued with 100% ethanol for 10 minutes, xylene for 10 minutes twice.

(10) Seal the slide: take the above-mentioned treated cover glass, drop neutral gum onto the tissue of the glass slide, take the cover glass and gradually cover it from one end to avoid air bubbles.

The results are shown in Fig. 9, Fig. 10, Fig. 11, Fig. 12 and Fig. 13, among which, Fig. 9 shows the effect of in vivo imaging detection of NTZ in preventing bone metastasis of prostate cancer. On the 35th day, the PC3-KQ Vehicle group showed significant bioluminescence. However, the bioluminescence of the NTZ group was weakened. Figure 10 is the bioluminescence analysis diagram of the embodiment of the present application. Through statistical analysis, the bioluminescence value of the NTZ group is significantly lower than that of the Vehicle, and there is a significant difference (P<0.05). Figure 11 is the effect diagram of Micro-CT detection of NTZ anti-prostate cancer bone metastasis in the embodiment of the present application. On the 35th day, micro-CT and mouse in vivo imaging were performed on the PC3-KQ Vehicle group and the PC3-KQ nitazoxanide group respectively. As shown in Figure 11, in the PC3-KQ Vehicle group, the osteoclast behavior of the distal femur and proximal tibia of nude mice could be observed, while no obvious osteoclast behavior was observed in the nitazoxanide administration group. Figure 12 is a diagram of the effect of NTZ on the body weight of mice during the administration period of the embodiment of the present application. During the administration cycle (0-35 days), we weighed the nude mice in each group, weighing once a week, As a result, it was found that the body weight of the mice in the control group and the administration group increased with age, and there was no downward trend, which indicated that nitazoxanide had no obvious toxicity to the mice. Fig. 13 is a HE staining analysis of the effect of NTZ on cancer cell metastases in the bone tissue of the embodiment of the present application. The Vehicle bone tissue has obvious cancer cell invasion, while the NTZ group rarely has cancer cell invasion. From the above results, it can be seen that the drug nitazoxanide was administered on the first day after the cells were injected into the tail artery, and continued for 35 days. On the 35th day, in vivo imaging and microCT found that nitazoxanide could significantly prevent prostate cancer cells Transferred to bone tissue, and there was no significant change in the body weight of the control group and the drug-administered group, and both increased over time. HE staining found that almost no cancer cells invaded the bone tissue in the nitazoxanide-administered group, while the cancer cells in the control group obviously invaded the bone tissue.

Example 4 In vivo imaging system to observe the effect of nitazoxanide on bone metastasis of prostate cancer cells

1. Construction of highly metastatic PC3-KQ cells in prostate cancer

1. Conversion

Take the competent Escherichia coli out of the refrigerator at -80°C and thaw on ice; add the ligation product (K369Q) constructed in the laboratory into the competent Escherichia coli and incubate on ice; place on ice after heat shock at 42°C for 90s 2min; Add 200 μL of LB medium and 1 μL of antibiotics to the competent state in the ultra-clean bench, pipette evenly, and then coat the plate containing antibiotics; after coating the plate, culture in a constant temperature incubator at 37°C overnight.

2. Plasmid amplification

Use a 10 μL pipette tip to pick up the monoclonal colonies on the plate and put them into EP tubes. Shake the bacteria in a shaker at 200 rpm for 12 hours, and extract the plasmids according to the plasmid extraction kit.

3. Virus packaging

When the growth confluence of 293T cells is about 80%, carry out plasmid transfection, collect the virus at 48h and 72h respectively, and centrifuge at 1500rpm for 5min; divide the prepared virus into 1.5mL EP tubes, and store in a -80°C refrigerator for later use .

4. Virus infection

Spread prostate cancer cell PC3 into a 6-well plate at a confluence of 30%, culture in complete medium, 2 mL per well;

Culture in a carbon dioxide constant temperature incubator at 37°C; 16-28 hours after the initial infection, suck off the old medium, add 500 μL of fresh complete medium and 500 μL of virus (K369Q) to each well, and finally add 1 μL of cation adsorbent (polybrene), shake evenly Then put them into a carbon dioxide incubator at 37°C for cultivation. After 6 hours, add 1 mL of fresh complete medium to each well. Mock represents the blank control for screening stable clones without adding virus; Add 500 μL of fresh complete medium, 500 μL of virus and 1 μL of cation adsorbent to each well, and culture in a carbon dioxide incubator at 37 °C. After 6 h, 1 mL of fresh complete medium was added to each well. After 16-24 hours, the infection is complete.

5. Screening of transgenic polyclonal cell lines

16-24 hours after infection of prostate cancer cell PC3, the old culture medium and virus were sucked off, the cells were digested, transferred to a 10cm culture dish with 6mL complete culture medium in each dish, and the culture was continued; after 24 hours, when the cells adhered to the wall, Aspirate the old medium, and the selection pressure is PC3: 800 μg/mL hygromycin B (Hygromycin B); replace the medium containing the selection pressure every two days; after two weeks, when the cells in the Mock dish are all After the selection pressure kills, it can be considered that the negative cells in the treatment are also killed by the selection pressure, and the cells that survive are the positive cells that are screened out; One part of KQ cells is used to collect RNA and lysed protein to test whether the target gene is successfully expressed; one part is used for cryopreservation and conservation; the other part is used for continued culture to complete follow-up experiments.

2. Lentivirus infection to construct luciferase cells

1. Explore the optimal concentration of puromycin (puromycin)

Before screening, it is necessary to explore the lowest concentration of puromycin that can kill empty cells: the cells can be plated on a 24-well plate with a density of 5×10 ⁴ cells per well, and the complete medium with different concentrations of puromycin can be replaced after 24 hours, and the concentration of puromycin can be set 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 mg/mL were treated for 48 hours, and the lowest concentration that could kill more than 90% of empty cells was selected for subsequent experiments.

2. Lentivirus infection of cells

Day1: When the cell confluence is 80-90%, collect the cells and spread them on a 6-well plate with 1.5× ¹⁰⁵ cells per well; generally, the cell confluence rate is between 30%-50% when the virus infection is performed on the second day room, no antibiotics were added when laying the planks;

Day2: Before infecting the cells, take out the virus from the -80°C refrigerator and slowly thaw it on ice, suck off the original medium of the cells, and add 1/2 volume of fresh medium (the medium may not contain double antibodies and serum) ;

According to the MOI value (multiplicity of infection, multiplicity of infection), virus is added for infection;

Add virus volume per well (μL)=MOI×cell number/virus titer (TU/mL)×1000

For cells that need to add Polybrene (polybrene), Polybrane (1:1000) can be added at the same time

4 hours after infection, make up to the full culture volume with serum-containing medium;

Day3: On the second day after infection (about 24 hours), the culture solution containing the virus was aspirated, replaced with fresh complete culture solution, and continued to cultivate for 24 hours;

Day4: Replace with fresh complete culture medium with an appropriate concentration of puromycin, and screen cell lines stably expressing luc; during screening, it is necessary to set up a control group of cells not infected with the virus, and add an equal amount of puromycin;

Day6: Add puromycin for 48 hours, and observe the death of untransfected cells in the control group. If the cell death rate in the control group exceeds 90%, remove puromycin and replace with fresh medium for culture. The concentration of puromycin is 2.0ug/mL.

After puromycin selection, the cells can be properly proportioned and subcultured when the cells are congested, and then one-fifth of puromycin can be used to maintain the resistance.

3. The effect of nitazoxanide on bone metastasis of prostate cancer

Preparation of cell suspension: Take PC3-KQ cells with a confluence of 80-90%, digest with trypsin, stop digestion with complete medium, centrifuge at 1200rpm for 3min, and discard the supernatant. Add PBS to prepare a cell suspension, mix well and count the cells, prepare cells at 1×10 ⁶ per mouse, dilute and make a cell suspension, store on ice until use.

Inject cells into the tail artery: anesthetize the mice with 2.5% Avertin (120-150 μL per 10 g injection). Disinfect the tail artery; quickly inject 100 μL of the prepared cell suspension (1×10 ⁶ ) from the third part of the tail of the mouse with an insulin needle, and the time for injection into the artery should be less than 3 seconds. Press the injection site with a dry cotton ball for about 1 minute to prevent bleeding.

Seven days after the tail artery was injected with cells, nitazoxanide was administered by intragastric administration, once a day, 100 mg/kg per mouse, the solvent was 1% CMC, and the dosage was 100 uL per mouse.

In vivo imaging in mice: In vivo imaging was performed 7 days, 14 days, 21 days, 28 days, and 35 days after cell injection. The specific operation is as follows: 10 minutes before imaging, start the in vivo imaging system, turn on the oxygen switch and the gas anesthesia switch, and then intraperitoneally inject the luciferase substrate (D-luciferin, Sodium Salt) prepared before the experiment into each group of mice, D - The concentration of the fluorescein working solution is 15 mg/mL, and the injection volume of each mouse depends on the weight of the mouse: 150 mg/kg; 10 minutes after injection into the mouse, the mouse is anesthetized for imaging analysis.

Using the constructed PC3-KQ cells with high metastasis of prostate cancer, the PC3-KQ-LUC cell line was constructed by transfecting fluorescein LUC, and then injected into mice through the tail artery. The results are shown in Figure 14, Figure 15 and Figure 16 , Figure 17 and Figure 18, wherein, Figure 14 is the bioluminescence diagram of the PC3-KQ cell tail artery of the embodiment of the present application injected into the mouse body for 7 days, as shown in the figure, PC3-KQ-Luc has been Transferred to bone tissue of mice, divided into Control group and NTZ group according to the bioluminescence value; Figure 15 is the effect of in vivo imaging of NTZ (nitazoxanide) in the treatment of prostate cancer bone metastasis after 35 administration of the embodiment of the present application, through in vivo imaging It was observed that the bioluminescence of the control group was significantly stronger than that of the NTZ group; Figure 16 is a comparison of the bioluminescence of the lower limb bone tissues of the control group and the NTZ group of the embodiment of the present application. At the end of the administration, the mice were dissected and the bone tissue was taken for in vivo imaging; Figure 17 is a comparison chart of the bioluminescence of the control group and the NTZ group before and after the statistical analysis of the embodiment of the present application. On the 7th day, there was no difference in the bioluminescence of the control group and the treatment group, but on the 35th day, the bioluminescence value of the NTZ group was obvious Smaller than the control group, with significant differences (P<0.01).

From the above results, it can be seen that on the 7th day in vivo imaging, it was observed that the cancer cells had transferred to the bone tissue of the mice, and they were randomly divided into groups according to the bioluminescence value. In vivo imaging at 35 days found that, compared with the control group, the bioluminescence value of the administration group was significantly lower, with a significant difference. And, as shown in FIG. 18 , the body weight of the mice in the control group and the administration group increased with time.

Example 5 MicroCT detection of the protective effect of nitazoxanide on bone tissue in mice

After the experiment in Example 4 was over, the mice were sacrificed by cervical dissection, and the bone tissue was dissected and fixed with paraformaldehyde for 48 hours. The bone tissue was then immersed in a centrifuge tube containing PBS, and then small animal microCT was performed to observe the transfer of PC3-KQ cells. and bone tissue destruction, and detection of cortical bone destruction, and analysis of trabecular density (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) and other indicators.

The results are shown in Figure 19, Figure 20 and Figure 21. Among them, Figure 19 is a micro-CT analysis of the embodiment of the present application to analyze the protective effect of NTZ on the bone tissue of living mice. At the end of administration, the PC3-KQ control group was treated with And PC3-KQ NTZ group underwent micro-CT. As shown in Figure 19, in the PC3-KQ control group, it can be observed that osteoclastogenesis occurs in the distal femur and proximal tibia of nude mice, while no obvious osteoclastogenesis was observed in the nitazoxanide-administered group; Figure 20 is Micro-CT analysis of the protective effect of NTZ on mouse bone tissue in the example of this application. At the end of administration, the mouse bone tissue was dissected and micro-CT was performed for 3D reconstruction. It can be seen from the bone tissue section that the control group The area of trabecular bone was reduced and the bone cortex was damaged, while the NTZ group remained relatively intact; Figure 21 is a micro-CT analysis of the protective effect of NTZ on mouse trabecular bone in the embodiment of the present application, and the trabecular bone was analyzed by micro-CT Density (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp), found that NTZ can increase bone Beam density, trabecular number and trabecular thickness, reduce trabecular separation.

The mice in the control group showed osteoclast phenotype, while the integrity of the bone tissue in the nitazoxanide group was relatively intact, and compared with the control group, nitazoxanide could increase the bone trabecular density (BV/TV), the number of trabecular bone (Tb.N), bone trabecular thickness (Tb.Th) and reduce bone trabecular separation (Tb.Sp), have the effect of protecting bone tissue from cancer cells.

Embodiment 6 HE staining detects the therapeutic effect of nitazoxanide on mouse bone tissue

1. HE staining

(1) After the mice in different treatment groups were killed by cervical dislocation, the femur and tibia were separated, and the excess connective tissue and muscle tissue around them were cut off, fixed in 4% paraformaldehyde for 48 hours, and then decalcified with EDTA decalcification solution for 14-21 hours. sky;

(2) After the bone tissue is decalcified, it is dehydrated, and the procedure is as follows:

70% ethanol 1h

80% ethanol 1h

90% ethanol 1h

95% ethanol 1h

Absolute ethanol Ⅰ 1h

Absolute ethanol Ⅱ 1h

Xylene Ⅰ 1h

Xylene Ⅱ 1h

Paraffin Ⅰ 1h

Paraffin II 1h

Paraffin III 1.5h

(3) Embedding: Turn on the embedding machine 2 hours in advance, preheat and melt the paraffin; soak the embedding cassette in liquid paraffin for later use, place the bottom mold of the stainless steel embedding cassette in the paraffin on the other side, take a stainless steel bottom film and place it on the table Squeeze in 1/3 of the paraffin, then place the tissue block in the center; then place the stainless steel bottom mold with the tissue on the freezing table, wait for it to solidify and turn white, put it on the heating table again and squeeze out the paraffin to cover the tissue , put the floor of the embedding box on the stainless steel mold, then add liquid paraffin to exceed the height of the bottom plate, place it on a freezing table to cool, when the paraffin block is frozen enough to be gently pulled out from the mold, pull out the tissue wax block, Then, the excess paraffin wax around the wax block is repaired by the heating platform of the embedding machine.

(4) Slicing: The bone tissue wax blocks were buried in ice in advance, and then frozen overnight in a refrigerator at 4°C to facilitate slicing. Take out the wax block from the refrigerator, and use a microtome to cut the tissue wax block longitudinally, with a slice thickness of 4 μm. Spread the slices with warm water at 42°C to fully unfold the cut tissues, and then fix them on cationic detachment-resistant glass slides.

(5) Baking slices: Put the tissue slices into an oven at 62°C to bake the slices for 2-3 hours.

(6) Dewaxing: the procedure is as follows:

Xylene Ⅰ 10min

Xylene Ⅱ 10min

Xylene III 10min

Xylene IV 10min

Absolute ethanol Ⅰ 10min

Absolute ethanol Ⅱ 10min

95% ethanol 10min

85% ethanol 10min

75% ethanol 10min

H ₂ O 5min

(7) Staining: Hematoxylin staining for 1.5 min, rinsed with running water for 1 min until the staining tank was colorless, soaked in PBS; differentiated with 5% acetic acid for 3 s, differentiated non-specific tissue staining; rinsed once with running water, soaked in PBS, observed the staining effect under a microscope, and The core should be sky blue;

(8) Dye with eosin dye solution for 2 to 3 minutes; quickly wash with tap water 2 times;

(9) Gradient ethanol dehydration: slices were subjected to 75% ethanol for 20 seconds, 85% ethanol for 20 seconds, 95% ethanol for 20 seconds, and 100% ethanol for 10 minutes, and then observed the results of eosin staining under the microscope; then continued with 100% ethanol for 10 minutes, xylene for 10 minutes twice.

(10) Seal the slide: take the above-mentioned treated cover glass, drop neutral gum onto the tissue of the glass slide, take the cover glass and gradually cover it from one end to avoid air bubbles.

The results are shown in Figure 22 and Figure 23, wherein, Figure 22 is a graph of the HE staining of the embodiment of the present application to detect the invasion of PC3-KQ cells on bone tissue and the anti-invasion effect of NTZ, and the cancer cells in the control group invaded the bone tissue significantly more than the NTZ group ; Figure 23 is the embodiment of the application analysis of the control group and NTZ group bone tissue cancer cell metastasis area map, by analyzing the proportion of cancer cells in the bone tissue, it is found that compared with the control group, the NTZ group cancer cell invasion area ratio is significantly less in the control group.

In the control group, a large number of cancer cells invaded into the proximal end of the bone tissue joints, but few cancer cells in the nitazoxanide group invaded into the bone tissue. After statistical analysis, the area of cancer cells in the administration group was significantly less than that of the control group.

Example 7 Immunohistochemical detection of nitazoxanide inhibiting the growth of prostate cancer cells in bone tissue

Immunohistochemistry

(1) Paraffin sections are dewaxed to water;

(2) Wash with PBS for 5min×3 times;

(3) Incubate with 3% H ₂ O ₂ (80% methanol) at room temperature for 10 minutes to eliminate the activity of endogenous peroxidase;

(4) Wash with PBS for 5 min×3 times;

(5) protease antigen retrieval method for antigen retrieval;

(6) Wash with PBS for 5 min×3 times;

(7) Block with 5-10% normal goat serum (diluted in PBS), incubate at room temperature for 10-20 minutes, shake off the serum, do not wash;

(8) Add 50 μL of I antibody dropwise, and let stand at room temperature for 1 hour or overnight at 4°C or at 37°C for h;

(9) Rewarm at 37°C for 45 minutes after staying overnight at 4°C;

(10) Wash with PBS for 5 min×3 times;

(11) Add 45-50 μL of II antibody dropwise, 37°C for 1 hour (0.05% tween-20 can be added to II antibody);

(12) Wash with PBS for 5 min×3 times;

(13) DAB color develops for 5-10 minutes, and the degree of staining is grasped under a microscope;

(14) Rinse with PBS or tap water for 10 minutes;

(15) counterstain with hematoxylin for 2 minutes, and differentiate with hydrochloric acid and alcohol;

(16) Rinse with tap water for 10-15 minutes;

(17) Dehydration, transparency, sealing, microscopic examination.

Figure 24 is the result of immunohistochemical detection of Ki67 expression in the control group and NTZ group in the embodiment of the present application. As shown in Figure 24, Ki67 is a sign of cancer cell proliferation, and the expression of Ki67 in bone tissue cancer cells is analyzed by immunohistochemistry It can be seen that Ki67 is significantly expressed in the control group, but its positive expression is rarely observed in the administration group.

To sum up, the method for screening drugs for the prevention of prostate cancer proposed by this application has screened out a new drug that can prevent prostate diseases, and the screened nitazoxanide can effectively prevent bone metastasis of prostate cancer. The application of nitazoxanide in pharmaceuticals can treat prostate diseases, such as prostate cancer bone metastasis, and the therapeutic effect is better, and it has opened up a new drug for the treatment of prostate diseases, and nitazoxanide is an FDA-approved drug, and its drug safety It has been verified; nitazoxanide can be obtained in large quantities from plant and animal sources, and the acquisition cost is low. It can replace existing drugs and is widely used in the prevention of prostate cancer.

The above are only preferred embodiments of the present application, and are not intended to limit the patent scope of the present application. For those skilled in the art, various modifications and changes may be made to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this application shall be included within the scope of patent protection of this application.

Claims (6)

1. A screening method for a medicament for preventing prostate cancer, comprising the following steps:

   S10, performing 3D cell culture on prostate cancer cells to obtain tumor spheroids;

   S20. Prepare the drug to be screened into a solution with a concentration of 10 μM, use the solution to conduct a cell invasion experiment on the tumor spheroid, calculate the invasion area of the tumor spheroid, and screen out tumor spheroids with an invasion area of less than 50%. The medicament corresponding to the body, wherein, the medicament to be screened is selected from a compound library listed on the FDA and included in the Pharmacopoeia;

   S30. Prepare the screened medicaments into a plurality of solutions with different concentration gradients of 0-10 μM, add them into tumor spheroids for culture, calculate the invasion area of the tumor spheroids, and screen out the invasion area of less than 70%. 7 potions of

   S40. Measure the IC50 of at least some of the seven medicines, and screen out the medicines with IC50 values less than 28 μM, which are the medicines for preventing prostate cancer.
2. The screening method of the medicament for preventing prostate cancer as claimed in claim 1, wherein, step S10 comprises:

   S11. Using prostate cancer cell PC3 and linker K369Q to construct prostate cancer highly metastatic PC3-KQ cells;

   S12, performing 3D cell culture on the prostate cancer highly metastatic PC3-KQ cells to obtain tumor spheroids.
3. The screening method of the medicament for preventing prostate cancer according to claim 1, wherein the medicament for preventing prostate cancer comprises furamectin, nifuratel, nitazoxanide and retapalene.
4. Application of nitazoxanide in the preparation of medicaments for preventing prostate diseases.
5. Application of nitazoxanide in the preparation of medicaments for preventing prostate cancer.
6. Application of nitazoxanide in the preparation of drugs for preventing bone metastasis of prostate cancer.

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