Application of methionine adenosine transferase 2A inhibitor AG270 in bladder cancer treatment

Abstract

The invention discloses an application of a methionine adenosine transferase 2A inhibitor AG270 in treatment of bladder cancer. The application specifically comprises: 1) an application of methionine adenosine transferase 2A as a molecular target in early-stage screening of cis-platinum resistance of bladder cancer patients; 2) application of methionine adenosine transferase 2A as a molecular target and an inhibitor AG270 of the methionine adenosine transferase 2A in treatment of bladder cancer; and (3) the methionine adenosine transferase 2A inhibitor AG270 is used for increasing the sensitivity of the cis-platinum and reducing the drug resistance reaction to the cis-platinum. According to the invention, the condition that the protein expression quantity of methionine adenosine transferase 2A (MAT2A) in a bladder cancer patient is increased, which is found for the first time, is taken as a basis for applying the methionine adenosine transferase 2A inhibitor AG270 to detection or treatment of bladder cancer, so that a new thought and a new drug resource are provided for treatment of bladder cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and bladder cancer technology, and specifically relates to application of a methionine adenosyltransferase 2A inhibitor AG270 in the treatment of bladder cancer. Background Art

[0002] Bladder cancer is one of the most common malignant tumors in the urinary system, with the highest incidence rate. Surgical removal of the bladder (tumor) combined with postoperative platinum chemotherapy is currently an effective treatment method. However, with the emergence of postoperative recurrence and drug resistance, the treatment effect of bladder cancer has always been quite limited, or even lacking. Therefore, research on bladder cancer resistance and research on enhancing the sensitivity of platinum drugs is quite urgent.

[0003] The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) is the rate-limiting enzyme in the production of the universal methyl donor S-adenosylmethionine (SAM). MAT2A has been shown to play an important role in a variety of cancers. In 2021, an oral MAT2A inhibitor, methionine adenosyltransferase 2A inhibitor AG270, was discovered. It has been shown to inhibit mRNA splicing in tumor cells and mediate DNA damage in tumor cells, thereby inhibiting tumors. However, whether the use of AG270 can reverse cisplatin resistance in bladder cancer to increase cisplatin sensitivity has not been reported. Summary of the invention

[0004] In view of the deficiencies in the above-mentioned prior art, the present invention provides an application of a methionine adenosyltransferase 2A inhibitor AG270 in the treatment of bladder cancer, which can provide new ideas and drug resources for the treatment of bladder cancer.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A first aspect of the present invention provides a use of a methionine adenosyltransferase 2A inhibitor AG270 in the treatment of bladder cancer.

[0007] Optionally, the use of methionine adenosyltransferase 2A as a molecular target in the preliminary screening of cisplatin resistance in bladder cancer patients.

[0008] Optionally, the expression level of methionine adenosyltransferase 2A is increased in cisplatin-resistant bladder cancer cells relative to cisplatin-sensitive bladder cancer cells.

[0009] Optionally, use of methionine adenosyltransferase 2A as a molecular target and its inhibitor AG270 in the treatment of bladder cancer.

[0010] Alternatively, the proliferation, invasion and migration abilities of bladder cancer cells are reduced by using a methionine adenosyltransferase 2A-specific knockdown virus or by using the methionine adenosyltransferase 2A inhibitor AG270.

[0011] Optionally, the methionine adenosyltransferase 2A inhibitor AG270 is used to increase sensitivity to cisplatin and alleviate resistance to cisplatin.

[0012] Alternatively, the methionine adenosyltransferase 2A inhibitor AG270 sensitizes bladder cancer cells to cisplatin in vitro.

[0013] Optionally, the methionine adenosyltransferase 2A inhibitor AG270 is used in combination with cisplatin to increase the apoptosis rate and cisplatin sensitivity of bladder cancer cells.

[0014] Optionally, oral administration of the methionine adenosyltransferase 2A inhibitor AG270 is combined with intraperitoneal injection of cisplatin to increase the therapeutic efficiency of cisplatin against bladder cancer in vivo.

[0015] The present invention provides an application of a methionine adenosyltransferase 2A inhibitor AG270 in the treatment of bladder cancer. The inventors first discovered that the protein expression of methionine adenosyltransferase 2A (MAT2A) in bladder cancer patients is increased, so that the methionine adenosyltransferase 2A inhibitor AG270 can be used in the detection or treatment of bladder cancer, thereby providing new ideas and drug resources for the treatment of bladder cancer.

[0016] The second aspect of the present invention provides a kit, comprising an antibody for detecting the expression of methionine adenosyltransferase 2A according to the first aspect and various embodiments.

[0017] The present invention provides a kit, which utilizes the characteristic that the protein expression of methionine adenosyltransferase 2A (MAT2A) in bladder cancer patients is increased, and uses methionine adenosyltransferase 2A (MAT2A) as a drug target for detecting bladder cancer. An antibody that can detect the expression of the above-mentioned methionine adenosyltransferase 2A is selected in the kit, so that the kit can achieve the purpose of detecting bladder cancer.

[0018] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the written description and the accompanying drawings.

[0019] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The immunohistochemistry provided in the exemplary embodiment of the present invention shows the difference in the expression level of MAT2A in cisplatin-resistant bladder cancer patients (resistant patient group) and cisplatin-sensitive bladder cancer patients (sensitive patient group);

[0021] Figure 2 is the difference in cisplatin IC50 between the drug-resistant bladder cancer cell line (RES group) and the sensitive bladder cancer cell line (NC group) provided in the exemplary embodiment of the present invention;

[0022] Figure 3 The difference in MAT2A protein expression between the drug-resistant bladder cancer cell line (RES group) and the sensitive bladder cancer cell line (NC group) provided in the exemplary embodiment of the present invention; P<0.05;

[0023] Figure 4 The effect of knocking down MAT2A or using AG270 on cell proliferation ability provided by the exemplary embodiment of the present invention; P<0.05;

[0024] Figure 5 The effect of knocking down MAT2A or using AG270 on cell migration and invasion abilities provided by the exemplary embodiments of the present invention; P<0.05;

[0025] Figure 6 The effect of AG270 and / or CIS on the apoptosis rate of bladder cancer cells provided by the exemplary embodiment of the present invention; P<0.05;

[0026] Figure 7 This is the effect of AG270 and / or CIS on subcutaneous tumors in mice provided in an exemplary embodiment of the present invention; P<0.05. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] The experimental methods described in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. described in the following examples are all commercially available unless otherwise specified.

[0029] The first aspect of the present invention provides a use of a methionine adenosyltransferase 2A inhibitor AG270 in the treatment of bladder cancer.

[0030] The present invention conducts immunohistochemical experiments on bladder cancer tissues of cisplatin-sensitive bladder cancer patients and cisplatin-resistant bladder cancer patients, and for the first time finds that the protein expression of methionine adenosyltransferase 2A (MAT2A) in bladder cancer patients is increased. In addition, the inventor also uses cisplatin to induce bladder cancer cells, constructs a cisplatin-resistant cell model, and finds that the protein expression of MAT2A is increased in the cisplatin-resistant cell model. Knockdown of MAT2A in bladder cancer cells, or use of inhibitor AG270, can specifically reduce the proliferation, invasion and migration ability of bladder cancer cells. At the same time, in apoptosis experiments, the inhibitor AG270 combined with cisplatin significantly increases the apoptosis rate of bladder cancer cells and increases cisplatin sensitivity; in addition, in in vivo mouse experiments, oral AG270 combined with intraperitoneal injection of cisplatin also increases the inhibitory effect of cisplatin on subcutaneous tumor formation, suggesting that the inhibitor AG270 has a sensitizing effect on cisplatin; it can be seen that by using the methionine adenosyltransferase 2A inhibitor AG270 in the treatment of bladder cancer, it can provide new ideas and drug resources for solving the drug resistance problem of bladder cancer.

[0031] In some possible embodiments, methionine adenosyltransferase 2A is used as a molecular target in the early screening of cisplatin resistance in bladder cancer patients.

[0032] Since the inventors found that the use of MAT2A-specific knockdown virus or the use of MAT2A inhibitor AG270 can reduce the proliferation, invasion and migration ability of bladder cancer cells, it can be determined that methionine adenosyltransferase 2A can be used as a molecular target in the early screening of cisplatin resistance in bladder cancer patients, thereby providing new ideas for the early screening of cisplatin resistance in bladder cancer patients.

[0033] In some possible embodiments, the expression level of methionine adenosyltransferase 2A in cisplatin-resistant bladder cancer cells is increased compared to that in cisplatin-sensitive bladder cancer cells.

[0034] It should be noted that the cisplatin-sensitive bladder cancer tissue specimens were from 7 patients with cisplatin-sensitive bladder cancer (sensitive patient group) admitted to the First Affiliated Hospital of China Medical University from July 2017 to June 2023; all 7 patients underwent radical cystectomy and bladder tumors were removed. The cisplatin-resistant bladder cancer tissue specimens were from 7 cisplatin-resistant patients (resistant patient group) who underwent radical cystectomy during the same period, and bladder tumors were removed. This study was approved by the hospital ethics committee and the patients gave informed consent.

[0035] Here, using immunohistochemistry experiments, the tissues corresponding to the above-mentioned sensitive patient group and resistant patient group were sliced ​​and stained in turn, and then the protein expression levels of methionine adenosyltransferase 2A (MAT2A) in the bladder cancer cells corresponding to the sensitive patient group and the resistant patient group were detected.

[0036] See also Figure 1 , it can be analyzed that: compared with the bladder cancer tissue corresponding to the sensitive patient group, the protein expression level of MAT2A in the bladder cancer tissue corresponding to the resistant patient group is significantly increased. Therefore, it can be explained that MAT2A is highly expressed in bladder tumors of cisplatin-resistant patients.

[0037] In this example, a western blot experiment was used to verify that the expression of MAT2A in cisplatin-resistant bladder cancer cells was higher than that in sensitive bladder cancer cells. The verification method included the following steps:

[0038] Step 1: using DMEM (10% FBS, 1% penicillin-streptomycin) to culture bladder cancer epithelial cells UM-UC-3 from bladder cancer patients; wherein the culture conditions are: temperature 37°C, CO2 concentration 5%, when the density of bladder cancer epithelial cells UM-UC-3 is 80%, use 0.25% trypsin to digest for subsequent experiments;

[0039] Step 2: Take cells in the logarithmic phase and use 1×10 5 The cells were plated at a density of 10 cells / well in a 96-well plate. After overnight culture, the half inhibitory concentration (IC50) of cisplatin was detected using the CCK8 method. Cells in the logarithmic growth phase (confluence 80%-90%) were treated with drugs at a low initial concentration (1 / 10-1 / 5 of the IC50 of the parental cell line) and cultured in an incubator at 37°C and 5% CO2. When the cell density reached 50%, the culture medium was discarded, the cells were washed twice with PBS, and the medium without drugs was replaced for continued culture. When the cell growth density recovered to 80%-90% again, the above drug treatment was repeated 6-8 times. After the cells grew stably at this concentration, the drug concentration was increased in turn and treated in the same way until the cells could grow stably at the final drug concentration to obtain drug-resistant bladder cancer cell lines. The IC50 of the drug-resistant bladder cancer cell lines was detected using the CCK8 method, and the resistance index (resistance index, RI), RI = IC50 of drug-resistant bladder cancer cell line / IC50 of parental cell line, RI>5 means that the drug resistance of drug-resistant bladder cancer cell line meets the requirements of drug-resistant strain;

[0040] Step 3: Extract cell proteins from drug-resistant bladder cancer cell lines and sensitive bladder cancer cell lines, detect concentrations, and use Western blot to detect differences in protein expression levels of MAT2A;

[0041] Step 4: Statistical analysis One-way ANOVA was performed using Prism software, and independent sample t-test was used for comparison between the two groups. P < 0.05 was considered to be significantly different. The results were described using mean ± standard deviation.

[0042] The results showed that the IC50 of drug-resistant bladder cancer cell lines (RES group) was significantly higher than that of sensitive bladder cancer cell lines (NC group). Figure 2 Also, see Figure 3 , it can be analyzed that P<0.05, that is, the expression level of MAT2A in the resistant bladder cancer cell line (RES group) was significantly higher than that in the sensitive bladder cancer cell line (NC group).

[0043] In some possible embodiments, methionine adenosyltransferase 2A is used as a molecular target and its inhibitor AG270 in the treatment of bladder cancer.

[0044] Here, the inventors found through EDU experiments and transwell experiments that knocking down MAT2A in bladder cancer cells or using the MAT2A-specific inhibitor AG270 can specifically reduce the proliferation, invasion and migration ability of bladder cancer cells. Therefore, it can be determined that methionine adenosyltransferase 2A can be used as a molecular target and its inhibitor AG270 in the treatment of bladder cancer, thereby providing new ideas and drug resources for the treatment of bladder cancer.

[0045] In some possible embodiments, a methionine adenosyltransferase 2A-specific knockdown virus or a methionine adenosyltransferase 2A inhibitor AG270 is used to reduce the proliferation, invasion and migration capabilities of bladder cancer cells.

[0046] It should be noted that the steps of using a methionine adenosyltransferase 2A-specific knockdown virus or using the methionine adenosyltransferase 2A inhibitor AG270 to reduce the proliferation, invasion and migration ability of bladder cancer cells are as follows:

[0047] Step 1: Use lentivirus sh-MAT2A to infect sensitive bladder cancer cells UM-UC-3, and perform subsequent experiments after purine selection (sh-MAT2A group); or use methionine adenosyltransferase 2A inhibitor AG270 (5μM) added to the culture medium to culture sensitive bladder cancer cells UM-UC-3 (AG270 group); or use negative control virus and methionine adenosyltransferase 2A inhibitor AG270 solvent to treat sensitive bladder cancer cells UM-UC-3 (NC group). Here, lentivirus sequence: "5'-UUUAACAAGGGAUUUUGCCAC-3'", "5'-GGCAAAAUCCCUUGUUAAA GG-3'";

[0048] Step 2: Take cells in the logarithmic phase and use 1×10 4 The cells were plated at a density of 10 cells / well in a 24-well plate, and after overnight culture, the EDU method was used to detect the changes in the proportion of proliferative cells in the two treatments compared with sensitive bladder cancer cells;

[0049] Step 3: Take cells in the logarithmic phase and use the transwell migration assay to detect changes in the migration ability of the two treatments compared to sensitive bladder cancer cells. Add 40 μl of 20% matrigel matrix gel diluted with serum-free medium to the transwell chamber, and perform the transwell invasion assay after solidification to detect changes in the invasion ability of the two treatments compared to sensitive bladder cancer cells;

[0050] Step 4: The experiment was repeated at least three times. The statistical analysis was performed using Prism software for one-way analysis of variance. The inter-group comparison was performed using t-test. P < 0.05 was considered to be significantly different. The results were described using mean ± standard deviation.

[0051] The results showed that the use of MAT2A-specific knockdown virus or its inhibitor AG270 could reduce the proliferation, invasion and migration ability of sensitive bladder cancer cells (P<0.05, Figure 4-5 ).

[0052] In some possible embodiments, the methionine adenosyltransferase 2A inhibitor AG270 is used to increase the sensitivity of cisplatin and alleviate the resistance to cisplatin.

[0053] Here, the inventors found in the cell apoptosis experiment that the cell apoptosis rate was increased in the group of methionine adenosyltransferase 2A inhibitor AG270 combined with cisplatin compared with the use of either drug alone, proving that AG270 can increase the sensitivity of cisplatin and reduce the resistance to cisplatin.

[0054] In some possible embodiments, the methionine adenosyltransferase 2A inhibitor AG270 promotes the sensitivity of bladder cancer cells to cisplatin in vitro.

[0055] Here, the inventors found in the apoptosis experiment that the combined use of the methionine adenosyltransferase 2A inhibitor AG270 and cisplatin can significantly increase the apoptosis rate of bladder cancer cells and increase the sensitivity to cisplatin. Therefore, it can be determined that the methionine adenosyltransferase 2A inhibitor AG270 can promote the sensitivity of bladder cancer cells to cisplatin in vitro and can be used in the treatment of bladder cancer, thereby providing new ideas and drug resources for the treatment of bladder cancer.

[0056] In some possible embodiments, the methionine adenosyltransferase 2A inhibitor AG270 is used in combination with cisplatin to increase the apoptosis rate and cisplatin sensitivity of bladder cancer cells.

[0057] It should be noted that the method of combining methionine adenosyltransferase 2A inhibitor AG270 with cisplatin comprises the following steps:

[0058] Step 1: Take sensitive bladder cancer cells in the logarithmic phase and use 3×10 5 The cells were plated at a density of 10 cells / well in a 6-well plate and cultured overnight;

[0059] Step 2: Add cisplatin and / or methionine adenosyltransferase 2A inhibitor AG270 to the culture medium to make the final concentration of cisplatin 10 μM and the final concentration of methionine adenosyltransferase 2A inhibitor AG270 5 μM, replace the normal culture medium, and after 48 hours, divide the cells into NC group, AG270 group, CIS group and AG270+CIS group, and use Annexin V / PI double staining method to detect cell apoptosis by flow cytometry;

[0060] Step 3: The experiments were repeated at least three times. Prism software was used for statistical analysis using one-way analysis of variance. Student's t-test was used for comparison between groups. P < 0.05 was considered significant. The results were described using mean ± standard deviation.

[0061] The results showed that the apoptosis rate of cells in the group of methionine adenosyltransferase 2A inhibitor AG270 combined with cisplatin was higher than that in the group of cells treated with either drug alone, which proved that the methionine adenosyltransferase 2A inhibitor AG270 could increase the sensitivity of cisplatin and reduce the drug resistance to cisplatin (P<0.05, Figure 6 ).

[0062] In some possible embodiments, oral administration of the methionine adenosyltransferase 2A inhibitor AG270 is combined with intraperitoneal injection of cisplatin to increase the therapeutic efficiency of cisplatin for bladder cancer in vivo.

[0063] Here, in order to prove that the combined use of oral administration of methionine adenosyltransferase 2A inhibitor AG270 and intraperitoneal injection of cisplatin can increase the therapeutic efficiency of cisplatin for bladder cancer, the combined use of oral administration of methionine adenosyltransferase 2A inhibitor AG270 and intraperitoneal injection of cisplatin was applied to the treatment of subcutaneous tumors in mice, wherein the combined use of oral administration of methionine adenosyltransferase 2A inhibitor AG270 and intraperitoneal injection of cisplatin significantly increased the therapeutic efficiency of cisplatin for subcutaneous tumors in mice, including the following steps:

[0064] Step 1: Take sensitive bladder cancer cells UM-UC-3 in the logarithmic phase and use 1×10 6The amount of cells / each was injected subcutaneously into the upper chest side of six-week-old Balb / c-nude mice. After the second week, the mice were divided into groups according to DMSO gavage and PBS intraperitoneal injection (NC group), AG270 gavage and PBS intraperitoneal injection (AG270 group), DMSO gavage and cisplatin intraperitoneal injection (CIS group), AG270 gavage and cisplatin intraperitoneal injection (AG270+CIS group), and the medication was given according to the dosage of cisplatin 4 mg / kg intraperitoneal injection every other day and AG270 100 mg / kg daily gavage. Here, the feeding and operation of experimental animals have passed the experimental animal welfare and ethics review of China Medical University and strictly followed the "Regulations on the Management of Experimental Animals";

[0065] Step 2: After two weeks of treatment, the nude mice were killed and dissected, and the subcutaneous tumor tissue was taken, weighed, measured, and photographed;

[0066] Step 3: Statistical analysis One-way ANOVA was performed using Prism software, and independent sample t-test was used for comparison between the two groups. P < 0.05 was considered to be significantly different. The results were described using mean ± standard deviation.

[0067] The results showed that compared with the use of either drug alone, the combination of oral administration of the methionine adenosyltransferase 2A inhibitor AG270 and intraperitoneal injection of cisplatin reduced tumor volume and weight, proving that the methionine adenosyltransferase 2A inhibitor AG270 can increase the sensitivity of cisplatin in the treatment of bladder cancer and reduce the drug resistance to cisplatin (P<0.05, Figure 7 ), and then it can be determined that the methionine adenosyltransferase 2A inhibitor AG270 provides new ideas and drug resources for solving the drug resistance problem of bladder cancer.

[0068] A kit provided in an embodiment of the second aspect of the present invention comprises an antibody for detecting the expression of methionine adenosyltransferase 2A in the embodiment of the first aspect and various embodiments described above.

[0069] Here, the antibody in the kit can be proteintech: 55309-1-AP.

[0070] Since the protein expression level of methionine adenosyltransferase 2A (MAT2A) is increased in bladder cancer patients, methionine adenosyltransferase 2A (MAT2A) can be used as a drug target for detecting bladder cancer. Therefore, when the kit includes antibodies for detecting the expression of methionine adenosyltransferase 2A in the above-mentioned first aspect embodiment and various embodiments, it can detect bladder cancer.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of AG270, a methionine adenosyltransferase 2A inhibitor, in the treatment of bladder cancer.

2. The use according to claim 1, characterized in that: The application of methionine adenosyltransferase 2A as a molecular target in the early screening of cisplatin resistance in bladder cancer patients.

3. The use according to claim 2, characterized in that: The expression of methionine adenosyltransferase 2A was increased in cisplatin-resistant bladder cancer cells compared with cisplatin-sensitive bladder cancer cells.

4. The use according to claim 1, characterized in that: The invention relates to the application of methionine adenosyltransferase 2A as a molecular target and its inhibitor AG270 in the treatment of bladder cancer.

5. The use according to claim 4, characterized in that: The use of methionine adenosyltransferase 2A-specific knockdown virus or the methionine adenosyltransferase 2A inhibitor AG270 reduced the proliferation, invasion and migration ability of bladder cancer cells.

6. The use according to claim 1, characterized in that: The methionine adenosyltransferase 2A inhibitor AG270 is used to increase the sensitivity of cisplatin and alleviate the drug resistance reaction to cisplatin.

7. The use according to claim 6, characterized in that: The methionine adenosyltransferase 2A inhibitor AG270 promotes the sensitivity of bladder cancer cells to cisplatin in vitro.

8. The use according to claim 7, characterized in that: The methionine adenosyltransferase 2A inhibitor AG270 is used in combination with cisplatin to increase the apoptosis rate and cisplatin sensitivity of bladder cancer cells.

9. The use according to claim 6, characterized in that: The methionine adenosyltransferase 2A inhibitor AG270 is administered orally and is used in combination with cisplatin intraperitoneal injection to increase the therapeutic efficiency of cisplatin on bladder cancer in vivo.

10. A kit, characterized in that: The invention comprises an antibody for detecting the expression of methionine adenosyltransferase 2A according to any one of claims 1 to 9.

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