Application of pentadecanoic acid in preparation of medicine for preventing or treating bladder cancer

Abstract

The invention discloses application of pentadecanoic acid in preparation of a medicine for preventing or treating bladder cancer, and belongs to the technical field of treatment and prevention of bladder cancer. The invention relates to application of pentadecanoic acid in preparation of a medicine for preventing or treating bladder cancer. Wherein the pentadecanoic acid is found to have excellent anti-tumor activity for the first time and particularly has a remarkable inhibiting effect on bladder cancer, and the research finds that the pentadecanoic acid can remarkably inhibit invasion, migration and multiplication capacity of bladder cancer cells and inhibit growth of BBN-induced mouse bladder cancer and has potential for preventing or treating bladder cancer; the invention provides a theoretical basis for researching the treatment strategy of the bladder cancer, and provides an entry point for preparing a new medicine for preventing or treating the bladder cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bladder cancer treatment and prevention, and particularly relates to application of pentadecanoic acid in preparation of a drug for preventing or treating bladder cancer. BACKGROUND

[0002] Bladder cancer is a malignant tumor originating from bladder epithelial cells and is one of common tumors of the urinary system. Bladder cancer is insidious in onset, and nearly half of patients with early bladder cancer have no clinical symptoms, and there are many non-specific symptoms such as frequent urination, interrupted urine column, repeated urinary tract infection, etc., which are not easy to diagnose. Current diagnostic methods such as cystoscopy and urine cytology can provide certain diagnostic basis, but still have certain invasiveness and limitations. Chemotherapy is an important treatment for advanced bladder cancer, but bladder cancer cells have low sensitivity to chemotherapeutic drugs, and chemotherapeutic drugs cause serious damage to normal cells, and patients often suffer from significant adverse reactions, affecting the treatment effect and the quality of life of patients. Therefore, it is of great clinical significance to develop new, low-toxicity, high-efficiency and natural anti-bladder cancer drugs.

[0003] Fatty acids, as important bioactive molecules, play an important role in various physiological processes in the body. In recent years, odd-chain fatty acids, especially pentadecanoic acid, have gradually become a popular molecule in cancer research. Pentadecanoic acid is a naturally occurring odd-chain saturated fatty acid that mainly exists in dairy products and meat foods of ruminants and is usually obtained through dietary intake, and the human body is difficult to synthesize it by itself. As a natural fatty acid, pentadecanoic acid is considered to be one of the beneficial components in the diet. Recent studies have found that dietary intake of pentadecanoic acid is associated with a lower risk of cardiovascular disease, liver disease, chronic inflammation and certain specific cancers. Moreover, higher levels of pentadecanoic acid in the circulatory system are also associated with a reduced risk of various diseases, including type 2 diabetes, cardiovascular disease, liver disease and certain cancers. In particular, in tumor research, pentadecanoic acid has a significant dose-dependent anti-proliferative effect on various cancer cells such as human breast cancer (MCF-7, MDA-MB-231), lung cancer (A549), pancreatic cancer (PANC-1) and liver cancer (HepG2) cells. However, there are few studies on the effect of pentadecanoic acid intake on bladder cancer, and existing data are insufficient to determine its relevance and specific mechanism. SUMMARY

[0004] In view of the deficiencies in the prior art, the purpose of the present application is to provide the application of pentadecanoic acid in the preparation of a drug for preventing or treating bladder cancer, which solves the problems in the prior art.

[0005] The purpose of the present application can be achieved by the following technical solutions.

[0006] The application of pentadecanoic acid in the preparation of a drug for treating bladder cancer.

[0007] Further, the treatment of bladder cancer comprises inhibiting invasion, migration and proliferation of bladder cancer cells.

[0008] Further, the formula of the pentadecanoic acid is:

[0009]

[0010] Use of pentadecanoic acid in preparation of a drug for preventing bladder cancer.

[0011] Further, the bladder cancer is induced by BBN.

[0012] A drug for treating or preventing bladder cancer, comprising an active ingredient: pentadecanoic acid.

[0013] Further, the drug further comprises a nanoparticle carrier for encapsulating pentadecanoic acid.

[0014] Further, the nanoparticle carrier is a liposome or a polymer-based nanoparticle.

[0015] Further, the polymer-based nanoparticle is PLGA, PCL or chitosan.

[0016] A method for constructing a mouse model of bladder cancer, comprising: administering 0.2ml of 20% ethanol solution containing 5mg of BBN to the mouse by gavage once every two days for 12 weeks.

[0017] Advantages of the present application:

[0018] The present application first discovers that pentadecanoic acid has excellent anti-tumor activity, especially has a significant inhibitory effect on bladder cancer. The present application finds that it can significantly inhibit the invasion, migration and proliferation of bladder cancer cells, and inhibit the growth of BBN-induced bladder cancer in mice. It has potential for preventing or treating bladder cancer. The present application provides a theoretical basis for studying the treatment strategy of bladder cancer and provides a breakthrough point for preparing a new drug for preventing or treating bladder cancer. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiment or prior art description will be briefly introduced below. Those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0020] Figure 1 is a scratch test diagram of the migration ability of EJ cells treated with pentadecanoic acid or untreated at different time points in Example 1 and a statistical analysis of the migration rate thereof;

[0021] Figure 2This is a Transwell experiment diagram of EJ cells treated with or untreated with pentadecanoic acid in Example 1, along with a statistical analysis of the number of invasive cells.

[0022] Figure 3 These are the results of CCK-8 proliferation experiments on EJ cells treated or untreated in Example 1 at 24 hours, 48 ​​hours, and 72 hours.

[0023] Figure 4 This is the weight change over time for different groups (BC model group, BC PEAL group, BC PEAH group and control group) in Example 2;

[0024] Figure 5 These are representative images of bladder tissue from Example 2 and representative images of bladder tissue stained with H&E. The scale bar is 100 μm.

[0025] Figure 6 The percentages of normal tissue, reactive atypia, dysplasia, CIS, early invasion, and invasion are shown in each group of Example 2 at the stages of bladder cancer development (left) and the stages of bladder cancer progression (right).

[0026] Figure 7 These are representative images of Ki-67 staining in Example 2, with a scale bar of 100 μm;

[0027] Figure 8 This represents the percentage of Ki-67 positive cells in each group of Example 2 during the bladder cancer development stage (left) and the bladder cancer progression stage (right). Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The raw material sources involved in the preparation examples and embodiments are as follows:

[0030] The human bladder cancer EJ-1 cells were provided by Zhongda Hospital affiliated with Southeast University;

[0031] Pentadecanoic acid was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., product code:

[0032] P0035 (CAS No.:1002-84-2);

[0033] Pentadecanoic acid was purchased from MedChemExpress (MCE), product code:

[0034] HY-W004283 / CS-W004283 (CAS NO.: 1002-84-2);

[0035] Sodium Carboxymethyl Cellulose (CMC-NA) was purchased from Sigma-Aldrich;

[0036] Tween 80 was purchased from Beyotiom;

[0037] N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was purchased from Aladdin.

[0038] Preparation Example 1

[0039] The preparation process of pentadecanoic acid solution is as follows: a certain amount of pentadecanoic acid (product code HY-W004283 / CS-W004283) purchased from MedChemExpress (MCE) is weighed and dissolved in dimethyl sulfoxide (DMSO). A magnetic stirrer or ultrasonic treatment is used to ensure complete dissolution.

[0040] Preparation Example 2

[0041] A certain amount of CMC-NA is added to a suitable amount of distilled water. Stir well until the CMC-NA is completely dissolved to form a uniform viscous solution, and prepare a 0.1% CMC-NA solution. A certain amount of Tween 80 is added to the CMC-NA solution according to the required proportion, and stirred uniformly to obtain a 0.1% CMC-NA + 5% Tween 80 mixed solution. Next, pentadecanoic acid (product code P0035) purchased from TCI is added to the mixed solution according to the dosage requirement, and a magnetic stirrer or ultrasonic treatment is used to stir until the pentadecanoic acid is completely dissolved or uniformly dispersed. This solution can be used for subsequent experiments.

[0042] Example 1

[0043] In this example, the evaluation of pentadecanoic acid in inhibiting the migration, invasion and proliferation of EJ bladder cancer cells is introduced.

[0044] EJ bladder cancer cells were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and placed in a 37°C, 5% CO2 incubator. (1) Cell resuscitation: The frozen cells were immediately placed in a 37°C water bath and shaken; the thawed cell suspension was transferred to a 15mL sterile centrifuge tube containing 2mL of fresh medium and centrifuged at 1000rpm for 5 minutes; the supernatant was discarded, and the cells were pelleted with an appropriate amount of medium and transferred to a culture flask for static culture. After the cells adhered to the culture flask, the medium was replaced with fresh medium. (2) Cell passage: When the cells are in the logarithmic growth phase (i.e., when the bottom of the culture flask is 80%-90% confluent), remove the old culture medium and gently wash twice with PBS to remove residual serum; add 0.2% trypsin-EDTA digestion solution and digest at 37°C for 3-5 minutes; observe the changes in cell morphology under a microscope, or gently tap the culture flask to suspend most of the cells, and immediately add serum-containing culture medium to stop the digestion; collect the cell suspension, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in fresh culture medium and pipette; then aliquot into new culture flasks for continued culture.

[0045] Transwell cell invasion assay was used to assess changes in the invasive ability of bladder cancer EJ cells under pentadecanoic acid treatment. Matrigel pre-coated Transwell chambers were diluted with 200 μL of serum-free medium, and 5 × 10⁶ cells were seeded in the upper chamber. 5 Two bladder cancer EJ cells were used. The experimental group was treated with the reagents from Preparation Example 1, while 0.1% DMSO served as the control group. 500 μL of complete culture medium containing 12% FBS was added to the lower chamber. The cells were incubated in a 5% CO2 incubator at 37°C for 48-72 hours. After incubation, the chamber was removed, and the cells were washed with PBS. Cells were fixed with 4% formaldehyde for 5 min and then stained with 0.1% crystal violet. The unmigrated cells in the upper layer were gently wiped away with a moistened cotton swab. Images were taken randomly in each field of view under a microscope, and the number of cells that migrated to the membrane was counted. Each group was performed in triplicate, and the average number of migrating cells in each field of view was calculated as an indicator of migration ability.

[0046] The cell scratch assay was used to evaluate the migration ability of bladder cancer EJ cells under pentadecanoic acid treatment. First, cells were seeded in 5cm plates and cultured for 48 hours until a uniform monolayer covered each well. The culture medium was discarded, and the cells were washed once with PBS. A straight line was drawn on the cell monolayer using a sterile pipette tip of the same size. Cells were washed with PBS to remove residual cells, and serum-free culture medium was added. The experimental group was treated with the reagents from Preparation Example 1, while 0.1% DMSO served as the control group. The cells were then incubated at 37°C in a 5% CO2 incubator. The scratch healing was observed at different time points (0 h, 24 h, 48 h, 72 h) using an inverted microscope, and the scratch width was photographed and measured. Migration rate = (initial scratch width - measured width at each time point) / initial width × 100%.

[0047] The CCK-8 assay was used to assess changes in the proliferative capacity of bladder cancer EJ cells under pentadecanoic acid treatment. Log-grown cells were collected and analyzed at a concentration of 5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of 100 μL / well in 96-well plates, with 6 replicates per group. The experimental group received the reagents from Preparation Example 1, while the control group received the same volume of 0.1% DMSO. Cells were incubated at 37°C in a 5% CO2 incubator. At 24, 48, and 72 hours, the culture medium was discarded, and 10 μL of CCK-8 reagent and 100 μL of culture medium were added, followed by incubation for another 3 hours. After incubation, the absorbance (OD value) of each well was measured at 450 nm using an ELISA reader. Cell proliferation curves were plotted based on the calculated OD values.

[0048] The results of the scratch assay showed that PEA significantly inhibited the migration ability of EJ cells, such as Figure 1 In Transwell assays, PEA treatment significantly inhibited the invasive ability of EJ cells, with a statistically significant difference compared to the control group. Figure 2 In the CCK-8 assay, cell proliferation was measured at 24, 48, and 72 hours. At all time points, the absorbance (OD 450) of the PEA-treated group at 450 nm was lower than that of the control group, indicating a significant reduction in cell proliferation. Figure 3 As shown in the figure. These results demonstrate that PEA effectively inhibits the migration, invasion, and proliferation of EJ bladder cancer cells, highlighting its potential benefit in bladder cancer development.

[0049] Example 2

[0050] This embodiment describes the evaluation of pentadecanoic acid's inhibition of bladder cancer tumor occurrence and progression;

[0051] 1) Grouping of mice

[0052] After one week of acclimatization, male C57BL / 6 mice were stratified according to their initial body weight and then randomly divided into two stages, namely, the onset stage and the progression stage, with four groups in each stage: control group, bladder cancer model group (BC model), low-dose pentadecanoic acid intervention group (BC PEAL), and high-dose pentadecanoic acid intervention group (BC PEAH). There were 10 mice in each group in each stage, for a total of 80 mice.

[0053] 2) Establishment of a mouse model of bladder cancer

[0054] The bladder cancer model was established using the N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) chemical induction method. The bladder cancer model group, the low-dose PEA intervention group, and the high-dose PEA intervention group were administered 0.2 ml of 20% ethanol solution containing 5 mg BBN by gavage every two days for 12 weeks.

[0055] 3) Animal intervention program

[0056] The pentadecanoic acid intervention group received it daily via gavage starting one week before BBN administration (as in Example 2). The blank control group and the bladder cancer model group received the same volume of solvent via gavage. The intervention continued until the experimental endpoint. The low-dose pentadecanoic acid intervention group received a concentration of 35 mg / kg of pentadecanoic acid reagent; the high-dose pentadecanoic acid intervention group received a concentration of 350 mg / kg of pentadecanoic acid reagent.

[0057] 4) Sample collection

[0058] Sample collection was conducted at two time points: 10 mice from each group were sacrificed at week 15 to observe the occurrence of bladder cancer; 10 mice from each group were sacrificed at week 23 to observe the progression of bladder cancer. Mice were anesthetized with 5% chloral hydrate via intraperitoneal injection. Whole blood was collected from the eyes in 2ml EP tubes, incubated at room temperature for 30 minutes, and then centrifuged at 4°C, 3000 rpm for 15 minutes. The supernatant serum was separated and stored at -80°C for later use. Dissection was performed, and tissues and organs from each group of mice, including bladder, testes, liver, kidneys, stomach, intestines, spleen, pancreas, lungs, and brain, were collected, washed with physiological saline, and weighed. After weighing, a portion of the bladder was placed in EP tubes containing neutral formalin for pathological examination and incubated at room temperature; the remaining portion was placed in sterile EP tubes, nitrogen-filtered along with other tissues, and then transferred to -80°C for later use. Dissect the cecum of mice, collect 100 mg (1-2 tablets) of fecal sample in sterile EP tubes, pass through liquid nitrogen, and then transfer to -80℃ for cryopreservation.

[0059] 5) Indicator Testing

[0060] Bladder cancer case slides were stained, including hematoxylin and eosin (H&E) staining and Ki-67 immunohistochemistry, to qualitatively assess the histological grade of bladder cancer and quantitatively assess cell proliferation activity.

[0061] (1) Changes in mouse weight gain:

[0062] Weight gain such as Figure 4 As shown, the mice all experienced a weight increase relative to their initial weight before gavage, with varying degrees of increase (the difference between the initial and final weight at the end of the experiment). During the disease stage, the average weight increase in the control group was 10.62 ± 1.62 g, in the bladder cancer model group it was 9.39 ± 2.64 g, in the low-dose pentadecanoic acid intervention group it was 6.32 ± 1.63 g, and in the high-dose pentadecanoic acid intervention group it was 4.67 ± 1.44 g. Statistical calculations indicated significant differences between the groups. During the progression stage, the average weight increase in the control group was 12.22 ± 1.54 g, in the bladder cancer model group it was 10.88 ± 2.76 g, in the low-dose pentadecanoic acid intervention group it was 9.27 ± 1.69 g, and in the high-dose pentadecanoic acid intervention group it was 7.26 ± 2.39 g. Statistical calculations also indicated significant differences between the groups.

[0063] (2) Changes in the pathological morphology of tumor tissue:

[0064] Samples were taken from mouse bladder tissue, and representative images are shown below. Figure 5 As shown in (a) above. Fresh bladder tumor tissue was observed and photographed under a microscope after paraffin embedding, sectioning, spreading and mounting, baking, dewaxing, and H&E staining. Evaluation was performed according to the bladder cancer staging criteria. The results showed that the tumor tissue in the model group mainly exhibited higher stages (such as CIS, early invasive, and invasive lesions), characterized by significant tumor cell atypia, loose cell arrangement, dense chromatin, and irregular nuclei, suggesting strong invasiveness and malignant characteristics. In contrast, in the pentadecanoic acid intervention group, the proportion of milder stages (such as normal tissue or reactive dysplasia) significantly increased, tumor cell atypia decreased, cells were more compactly arranged, chromatin was more uniformly distributed, and nuclei were relatively normal in morphology. Figure 5 As shown in (b) above. Statistical analysis showed that, compared with the model group, the pentadecanoic acid intervention group exhibited more lower stages (such as normal or reactive dysplasia), and this difference was statistically significant. Figure 6 As shown in the figure. These results indicate that pentadecanoic acid can effectively inhibit the occurrence and progression of bladder cancer, delay or reduce tumor invasiveness, and improve tumor staging.

[0065] (3) Detection of cell proliferation in tumor tissue:

[0066] After dewaxing the paraffin sections of the tumor, immunohistochemistry was used to detect the expression of Ki-67 in the tumor tissue. Ki-67 is a marker of cell proliferation. Under a microscope, Ki-67-positive cells showed a brownish-yellow staining, representing dividing cells. Figure 7 As shown in the figure. Statistical analysis showed that the proportion of Ki-67 positive cells in the control group was significantly higher than that in the pentadecanoic acid intervention group, suggesting that pentadecanoic acid can effectively inhibit the proliferation of bladder cancer cells, such as... Figure 8 As shown in the figure. These results indicate that pentadecanoic acid can inhibit the growth of bladder cancer by suppressing cell proliferation, thus providing a new potential strategy for the treatment of bladder cancer.

[0067] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0068] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. Application of pentadecanoic acid in the preparation of drugs for treating bladder cancer.

2. The application according to claim 1, characterized in that, The treatment for bladder cancer includes inhibiting the invasion, migration, and proliferation of bladder cancer cells.

3. The application according to claim 1, characterized in that, The structural formula of the pentadecanoic acid is:

4. Application of pentadecanoic acid in the preparation of drugs for the prevention of bladder cancer.

5. The application according to any one of claims 1-4, characterized in that, The bladder cancer was induced by BBN.

6. A drug for treating or preventing bladder cancer, characterized in that, Including the active ingredient: pentadecanoic acid.

7. The drug according to claim 6, characterized in that, The drug also includes a nanoparticle carrier for encapsulating pentadecanoic acid.

8. The medicament according to claim 7, characterized in that, The nanoparticle carrier is a liposome or polymer-based nanoparticle.

9. The medicament according to claim 8, characterized in that, The polymer-based nanoparticles are PLGA, PCL, or chitosan.

10. A method for constructing a mouse model of bladder cancer, characterized in that, include: Mice were administered 0.2 ml of a 20% ethanol solution containing 5 mg BBN by gavage every two days for 12 weeks.

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