CN117942337B

CN117942337B - Application of ESI-09 in preparation of medicines for treating bladder cancer

Info

Publication number: CN117942337B
Application number: CN202410127192.6A
Authority: CN
Inventors: 李攀; 王志平
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2024-01-30
Filing date: 2024-01-30
Publication date: 2024-07-09
Anticipated expiration: 2044-01-30
Also published as: CN117942337A

Abstract

The invention discloses application of ESI-09 in preparing a medicament for treating bladder cancer. The invention provides a new pharmaceutical activity of ESI-09, which is used for inhibiting proliferation of human bladder cancer cells and enhancing cisplatin drug sensitivity, so as to prepare a drug for treating bladder cancer, and belongs to the field of medicines. Through researches, ESI-09 has good in-vitro anti-tumor effect on bladder cancer cells, and can obviously reduce the expression of RAD51AP1, thereby inhibiting the proliferation of bladder cancer cells and enhancing the drug sensitivity of cisplatin. The ESI-09 is shown to provide a new treatment scheme for bladder cancer, and has good application prospect.

Description

Application of ESI-09 in preparation of medicines for treating bladder cancer

Technical Field

The invention provides a novel pharmaceutical activity of ESI-09-reducing expression of RAD51AP1 protein, further inhibiting proliferation of bladder cancer cells and enhancing sensitivity of the cancer cells to cisplatin, which is used for preparing a medicament for treating bladder cancer, and belongs to the field of medicines.

Background

Bladder cancer in advanced patients, cisplatin-based chemotherapy regimens are the first-line treatment option for patients, but such drugs can cause serious adverse effects in clinical settings, such as intrinsic resistance, systemic toxicity, etc., in some patients, and because of age and underlying disease factors, about half of patients are unsuitable for cisplatin chemotherapy depending on the existing dose. In recent years, the advent of therapeutic modalities such as immunotherapy that alter the immune state of tumors provides effective neoadjuvant therapy for patients with bladder cancer, but 70% -80% of patients are ineffective against these therapies. Thus, based on new molecular mechanisms, improving the sensitivity of patients to drugs, new therapeutic regimens are proposed to be critical for the treatment of bladder cancer.

RAD 51-related protein 1 (RAD 51AP 1) is a structure-specific DNA-binding protein that interacts with the homologous recombinant key protein RAD51, mediating repair of DNA double strand break damage. Is localized in the nucleus and distributed in the chromosome, telomere and other areas, and can bind RNA, single-stranded DNA and double-stranded DNA. The RAD51AP1 expression is obviously up-regulated in samples such as breast cancer, colon cancer, hepatobiliary cell carcinoma, esophageal squamous cell carcinoma, ovarian cancer and the like. Research shows that RAD51AP1 expression can enhance proliferation and invasiveness of tumors, and can obviously inhibit proliferation capability of the tumors when the RAD51AP1 is absent, and application of the RAD51AP1 in bladder cancer has not been reported at present.

ESI-09 is a potent, selective EPAC inhibitor that is used to promote mitochondrial uncoupling and to cause a futile cycle of mitochondrial respiration, resulting in reduced ATP production. However, the expression of the homologous recombination repair protein RAD51AP1 in bladder cancer cells is inhibited, the proliferation of the cancer cells is inhibited, the sensitivity of the cancer cells to cisplatin is improved, and the synergistic anticancer effect is not yet applied.

Disclosure of Invention

The invention aims to provide a new application of ESI-09 in preparing a medicament for treating bladder cancer, which is used for preparing the medicament for treating bladder cancer and aims to solve the problem that a clinical part of patients are insensitive to cisplatin medicament treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of ESI-09 in preparing medicines for treating bladder cancer.

The use is achieved by one or more of the following:

(A) ESI-09 inhibits proliferation of bladder cancer cells by inhibiting RAD51AP 1;

(B) ESI-09 enhances the sensitivity of bladder cancer cells to cisplatin drugs by inhibiting RAD51AP 1.

The bladder cancer is bladder cancer UMUC-3 cells.

The medicament is prepared by adding pharmaceutically acceptable carriers and/or auxiliary materials into ESI-09 serving as an active ingredient. The preparation is any one of tablets, sprays, granules, capsules, oral liquid, injection and suspension.

The invention first discovers that ESI-09 can inhibit proliferation of human bladder cancer cells. It was further found that ESI-09 can reduce the expression of RAD51AP1 protein, and the reduction of RAD51AP1 has an obvious inhibition effect on bladder cancer cell proliferation and enhances the drug sensitivity to cisplatin. Finally, the invention discovers that ESI-09 and cisplatin combined use can synergistically inhibit the proliferation of bladder cancer cells, and provides a new treatment scheme for treating bladder cancer.

The invention has the beneficial effects that:

The invention provides application of ESI-09 in preparing medicines for treating bladder cancer, and researches prove that the ESI-09 has good in-vitro anti-tumor effect on bladder cancer cells UMUC-3, has obvious inhibition effect on proliferation of bladder cancer cells UMUC-3, and has enhanced inhibition effect along with the increase of medicine concentration. Importantly, ESI-09 reduces the expression of the homologous recombination repair protein RAD51AP1, enhances the drug sensitivity to cisplatin, and reduces the treatment resistance of tumor cells to chemotherapy. Provides a new way and means for preparing new medicines for treating bladder cancer and provides a new idea for treating bladder cancer in medical application.

Drawings

FIG. 1 is a structural formula of ESI-09;

FIG. 2 shows the morphology of ESI-09, cisplatin after acting on human bladder cancer UMUC-3 cells;

FIG. 3 is a graph showing that ESI-09 inhibits proliferation of human bladder cancer UMUC-3 cells in vitro;

FIG. 4 shows that ESI-09 reduces the expression of RAD51AP1 protein in human bladder cancer UMUC-3 cells;

FIG. 5A shows that the decrease of RAD51AP1 in human bladder cancer UMUC-3 cells can inhibit the proliferation of tumor cells and improve the drug sensitivity of cisplatin;

FIG. 6 shows that ESI-09 in combination with cisplatin inhibits proliferation of UMUC-3 cells of human bladder cancer.

Detailed Description

The specific source of ESI-09 is not particularly limited, and ESI-09 can be synthesized by a conventional chemical synthesis method in the field, has good preparation and stability, and can be obtained by purchasing through a commercial way.

The structural formula of ESI-09 of the invention is shown in figure 1. In the present invention, bladder cancer cells are preferably UMUC-3 cells. In the invention, when ESI-09 is adopted to prepare the medicine for treating bladder cancer, the medicine can be used as the only active ingredient of the medicine, and can also be combined with other active ingredients with the effect of treating bladder cancer for preparing medicines.

The invention has no special limitation on the types of pharmaceutically acceptable auxiliary materials, and can adopt the conventional pharmaceutically acceptable auxiliary materials in the field. In the present invention, the dosage form of the drug includes injection, tablet, capsule or granule, and the injection includes intravenous injection and subcutaneous injection. The medicament can be applied to clinical practice through various administration ways such as oral administration, intravenous injection, subcutaneous injection and the like, has great flexibility and convenience, and can generate lower drug resistance to bladder cancer cells by taking ESI-09 as a medicament active ingredient, and enhance the sensitivity of cisplatin medicaments of the bladder cancer cells, thereby reducing the occurrence of treatment failure and recurrence phenomenon.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

In the following examples, conventional methods are used unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 ESI-09 inhibits proliferation of human bladder cancer UMUC-3 cells in vitro

(1) Seed plate: taking bladder cancer cells in logarithmic growth phase, carrying out pancreatin digestion, fully suspending the culture medium, preparing cell suspension, counting, adjusting the cell density to 1X 10 ⁵/ml respectively, adding 100 mu L of tumor cell suspension into each well of a 96-well plate, and culturing in an incubator.

(2) And (3) intervention: after observing that the cell wall is good under the microscope after culturing for 8-12 hours, sucking out the culture medium of each hole, respectively adding ESI-09 with different concentrations and normal culture medium of a control group into the corresponding hole, and continuing culturing for 48 hours.

(3) Absorbance was measured: 10. Mu.L of CCK-8 solution was added to each well and incubated for 2 hours, and absorbance at 450nm was measured by a microplate reader.

(4) GRAPHPADPRISM8 the semi-inhibitory concentration (IC 50 value) was calculated for each group of cell lines.

FIG. 2 shows the morphology of ESI-09, cisplatin after acting on human bladder cancer UMUC-3 cells. It was found that the dividing phase cells of UMUC-3 cells were significantly reduced and the cell morphology was disturbed and gradually elongated when the treatment was performed for 24 hours with ESI-09 and cisplatin at 4. Mu.M concentrations, respectively. When ESI-09. Mu.M was combined with cisplatin 1. Mu.M on UMUC-3 cells, dividing phase cells were more significantly reduced and some cell membranes were incomplete and died to varying degrees.

FIG. 3 shows the effect of ESI-09 on inhibiting proliferation of human bladder cancer cells in vitro. CCK8 experiments prove that ESI-09 significantly inhibits proliferation of human bladder cancer UMUC-3 cells through a concentration-dependent mode, and the ESI-09 acts on UMUC-3 cells for 24 hours, so that the half inhibition concentration (IC 50 value) of the cells is 27.98 mu M.

Example 2 ESI-09 decreases expression of human bladder cancer UMUC-3 cell RAD51AP1 protein

(1) And (3) paving: taking bladder cancer cell line in logarithmic growth phase, performing pancreatin digestion, completely suspending the cell line in culture medium, preparing cell suspension, counting, and paving 1×10 ⁵ bladder cancer cells in each hole of six hole plates.

(2) And (3) intervention: the following day cells were observed for adherence, the original medium was discarded and cells were incubated with medium containing different concentrations of ESI-09 for 24 hours.

(3) Cell protein extraction:

① Cleaning: when the adherent cells in the 6-hole plate are fused to about 90%, discarding the old culture solution, cleaning the adherent cells for 2 times by using PBS precooled at 4 ℃ and sucking the PBS buffer solution as much as possible;

② Cracking: 150 μl of precooled RIPA lysis mix (containing PMSF and phosphatase inhibitor) was added to each well, the 6-well plate was lysed on ice horizontally for 20min, the lysates were collected into 1.5mLEP tubes with clean precooled cell scrapes, and cells were sonicated 2 times at 200w for 5 seconds each;

③ Separating: the supernatant was centrifuged at 12000rpm at 4℃for 15min and the protein concentration was determined by transferring the supernatant to a freshly precooled EP tube.

(4) BCA assay to determine the protein concentration of interest:

① Respectively adding 20 mu L of the ready-to-use BSA standard into a 96-well plate;

② Adding 1 mu L of protein sample to be detected into a sample hole of a 96-well plate, setting 2 multiple holes, and then supplementing 19 mu L of PBS buffer solution to ensure that the volume of each hole is 20 mu L;

③ According to the calculated dosage of the required chromogenic working solution, preparing the chromogenic working solution from the reagent A and the reagent B according to the volume ratio of 50:1, fully and uniformly mixing, adding 200 mu L of chromogenic working solution into each hole, covering a 96-well plate cover, incubating for 30min at 37 ℃, and cooling to room temperature;

④ Measuring A562 of each sample and BSA standard by using an enzyme-labeled instrument, drawing a standard curve by Excel, calculating the protein concentration of the sample, and adjusting the protein concentration of each sample to be similar level by using PBS buffer solution;

⑤ 1/5 volume of 5 Xloading buffer was added to the protein sample, denatured at 100℃for 5 minutes in a metal bath, and sub-packaged and stored at-80 ℃.

(5) Protein gel electrophoresis:

① And (3) glue preparation: A. after washing with distilled water, drying the glass plate by a blower, and vertically fixing the glass plate on a special clamping plate to prepare glue; B. taking a piece of gel with the size of 1.0mm as an example, taking a one-step PAGE gel to rapidly prepare a lower-layer gel solution and a lower-layer gel buffer solution in the kit, respectively mixing 2.5 mL; taking the upper layer glue solution and the color upper layer glue buffer solution, and uniformly mixing 750 mu L of each upper layer glue solution and each color upper layer glue buffer solution; C. adding 60 mu L of improved coagulant into the lower glue mixed solution, lightly mixing, and injecting the mixed solution into a glue-making glass plate, wherein the distance between the liquid surface and the upper edge of the short glass plate is 0.5cm longer than that of the comb teeth; D. adding 15 mu L of improved coagulant into the upper glue mixed solution, gently mixing, gently injecting the mixed solution into a glue-making glass plate without waiting for the solidification of the lower glue, and inserting comb teeth; after the gel is fixed, the comb teeth are pulled out for electrophoresis;

② Loading electrophoresis: the volume of the protein sample added into each hole is 10 mu L and 50 mu g, the voltage of the electrophoresis apparatus is selected to be 80V for electrophoresis after the power is turned on, and the blue sample can be seen to be aligned at the bottom of the concentrated gel after about 20 minutes, and the electrophoresis voltage is adjusted to be 120V. Electrophoresis was continued for about 1 hour.

(6) Transferring:

① And (3) cutting glue: cutting glue according to the molecular weight of the target protein and referring to a Marker, and cutting PVDF films with corresponding sizes according to the molecular weights of the glue and the target protein;

② Soaking in methanol for 15 seconds to activate the PVDF membrane, soaking in ultrapure water for 2 minutes, and finally balancing in electrotransport liquid for 5 minutes;

③ The black plate is sequentially laid with filter paper, glue, PVDF film and filter paper under the black plate, and the black plate is inserted into a film transfer groove after air bubbles are exhausted, and the target protein molecular weight is less than 70KD, and the current is 300mA, and the electric transfer is carried out for 20 minutes.

(7) Closing: PVDF membrane is clamped by forceps and put into a closed incubation box filled with 5% skimmed milk powder for sealing, and the sealing is carried out by slowly shaking on a decolorizing shaker at room temperature for 1 hour.

(8) Incubating primary antibodies: after blocking was completed, PVDF membranes were washed 3 times with TBST for 5 minutes each, and then strips were placed in the corresponding primary antibodies and incubated overnight at 4 ℃.

(9) Incubating a secondary antibody: the membranes were washed 3 times with TBST the next day for 5min, 10min and 10min, respectively. Then, the primary antibody is incubated with a secondary antibody corresponding to the primary antibody, and the primary antibody is placed in a shaking table at room temperature for incubation for 1h.

(10) Exposure: after the secondary antibody incubation is completed, the TBST is used for washing the membrane for 3 times, the washing time is 5min, 10min and 10min respectively, and a OdysseyCLX near infrared double-color fluorescence imaging system is opened for exposure.

FIG. 4 shows that ESI-09 reduces the expression of RAD51AP1 protein in human bladder cancer UMUC-3 cells. Fig. 4A western blot results demonstrate that: ESI-09 reduced expression of RAD51AP1 protein levels in a concentration-dependent manner. FIG. 4B shows, by quantification of Western blotting results, that the expression level of RAD51AP1 was reduced to 77.47%, 71.06%, 12.69%, 0.05% in the control group by acting on UMUC-3 cells at 100nM, 1. Mu.M, 10. Mu.M, 100. Mu.M, respectively, for 24 hours, as compared to the control group. This may indicate that ESI-09 significantly reduced the expression level of RAD51AP1 protein in human bladder cancer UMUC-3 cells.

Example 3 reduction of RAD51AP1 in human bladder cancer UMUC-3 cells inhibits proliferation of tumor cells and enhances sensitivity to cisplatin

(1) Plasmid preparation:

① pLV3 plasmid: carrying knockdown sequence 5'-GCTCTACCAGAGAGACTTAGA-3' (RAD 51AP1 knockdown sequence shRNA 2) or nonsense sequence (no target of any gene, control sequence), double resistance of ampicillin and puromycin;

② psPAX2 helper plasmid: the expression product of the plasmid carrying the genes of the virus gag, pol, rev and tat can penetrate cell membranes more easily through an adhesion mechanism;

③ pMD2.G membrane protein plasmid: pMD2.G encodes VSV-G, forming the envelope of the viral particle.

(2) Plasmid transformation:

① Conversion: taking out Stbl3, inserting into ice, naturally dissolving (separating tube), adding about 10ng plasmid, continuously ice-bathing for 30min, heat-shocking at 42deg.C for 90 s, rapidly inserting into ice, adding 1mLSOC liquid culture medium, and resuscitating at 37deg.C for 30min at 209 r/min;

② Coating: extracting 50 mu L of bacterial liquid coating resistant LB solid medium; the cells were incubated overnight at 37℃for about 16 hours, the transformation effect was evaluated based on the number of single colonies (control group was set at the time of transformation), and the single colonies were picked for subsequent experiments.

(3) Plasmid extraction: the bacteria with plasmids are inoculated into 5-100mL of resistant LB liquid medium (the volume of the medium is determined according to specific requirements), cultured for 14-20h at 37 ℃ and 209r/min overnight, and plasmids are extracted according to a plasmid extraction kit.

(4) 293T cell preparation: the 293T cells are recovered in a T25 cell bottle, the high-sugar DMED complete medium is used for culturing (10% of fetal calf serum), when the cell fusion degree is about 80%, 1/3 passages are carried out, after 3 times of continuous passages, the cell fusion degree is 60%, the state is good, the transfection plasmid is used for virus packaging, and 2mL of the pre-warmed high-sugar complete medium is replaced 30min before transfection.

(5) Plasmid transfection: plasmid concentration was measured and transfection was performed in a safety cabinet. The system is as follows: PEI transfection reagent 20. Mu.l, psPAX 22.3. Mu.g, pMD 2.G1.5. Mu.g, target vector 4. Mu.g. The process is as follows:

① Reagent preparation: calculating the total amount of plasmid psPAX, pMD2.G, PEI transfection reagent and DMEM culture medium according to the packaging type number of the lentiviral vector, mixing the total PEI transfection reagent and 1/2 total DMEM culture medium, and standing for 10min;

② Plasmid preparation: mixing the total psPAX, pMD2.G and 1/2 total DMEM culture medium, subpackaging according to the types, respectively adding target gene vectors, adding the prepared PEIDMEM mixed solution, gently mixing, standing for 15min, slowly dripping into 293T cell culture medium, and gently shaking and mixing;

③ Virus collection: after 12h, 5mL of high sugar DMED complete medium was replaced, the medium was collected 48h after transfection, filtered through a 0.45 μm filter, and immediately transduced or sub-packaged for storage at-80 ℃. The addition of 4mL of high sugar DMED complete medium to the flask was continued, and the second wave lentiviral vector was collected 72h after transfection.

(6) Construction of RAD51AP1 knock-down stable transformants in human bladder cancer UMUC-3 cells:

① Resuscitating: resuscitating UMUC3 cells of bladder cancer, performing conventional culture, determining that the cell state is good, digesting, counting, paving 12 pore plates with 30000 cells/hole, and shaking uniformly;

② Infection: the next day the cell morphology is good, the fusion degree is about 10%, the culture medium is removed, 0.5mL of slow virus vector supernatant, 0.3mL of fresh culture medium and 10 mug/10 mu Lpolybrene are added, the mixture is uniformly mixed, and the fresh culture medium is replaced after 12-24 hours;

③ Screening: resistance screening was performed 72h after transduction: puromycin 1 μg/mL, screening for 3-5 days; g418400 μg/mL screening for about 1 week;

④ Amplification: and removing screening conditions, performing conventional culture for 3-5 days to obtain good cell state, transferring stable transfer cells to a culture flask for culture and amplification, and freezing.

(7) Western blotting: the RAD51AP1 target protein knockdown effect in stable transfer cells is detected by an experiment: the procedure is as in examples 2, (4) - (10).

(8) Cell proliferation assay:

① Seed plate: taking bladder cancer cells RAD51AP1 knockdown stable transgenic cells in the logarithmic growth phase and nonsense knockdown stable transgenic cells, digesting by pancreatin, fully suspending the culture medium, preparing cell suspension, counting, adjusting the cell density to 1X 10 ⁴/ml, adding 100 mu L of tumor cell suspension into each well of a 96-well plate, and culturing in an incubator. Setting groups of 0, 2, 4 and 6 days, wherein 3 groups are empty again;

② Absorbance was measured: 10. Mu.L of CCK-8 solution was added to the groups at days 0, 2, 4, and 6 after plating for 2 hours, and absorbance at 450nm was measured by a microplate reader.

(9) IC50 value determination

① Seed plate: taking bladder cancer cells RAD51AP1 knockdown stable transgenic cells in the logarithmic growth phase and nonsense knockdown stable transgenic cells, digesting by pancreatin, fully suspending the culture medium to prepare cell suspension, counting, adjusting the cell density to 1X 10 ⁵/ml respectively, adding 100 mu L of tumor cell suspension into each well of a 96-well plate, and culturing in an incubator;

② And (3) intervention: after observing that the cell wall is good under the microscope after culturing for 8-12 hours, sucking out the culture medium of each hole, respectively adding cisplatin with different concentrations and normal culture medium of a control group into the corresponding hole, and continuing culturing for 48 hours;

③ Absorbance was measured: adding 10 mu L of CCK-8 solution into each hole for incubation for 2 hours, and measuring the absorbance value at 450nm by using a microplate reader;

④ GRAPHPADPRISM8 IC50 values were calculated for each group of cell lines.

FIG. 5 shows the relationship between the decrease in RAD51AP1 in human bladder cancer UMUC-3 cells and the drug sensitivity of cisplatin to tumor cell proliferation. FIG. 5A is a western blot image of RAD51AP1 knockdown in human bladder cancer UMUC-3 cells. It can be seen that: compared with the control group, the RAD51AP1-shRNA2 and shRNA3 obviously reduce the expression of the RAD51AP 1. FIG. 5B shows the result of cell proliferation after RAD51AP1 knockdown in human bladder cancer UMUC-3 cells. CCK8 experiments demonstrated that: compared to day 0, the proliferation rates of control cells were 2.05, 6.84, and 13.71 on days 2,4, and 6, respectively, while the proliferation rates of RAD51AP1 knockdown cell lines were 1.22, 2.97, and 9.91 on days 2,4, and 6, respectively (p < 0.05). This may indicate that the knockdown of RAD51AP1 significantly inhibited proliferation of UMUC-3 cells. FIG. 5C, D shows drug sensitivity of RAD51AP1 pre-and post-knockdown cells to cisplatin in human bladder cancer UMUC-3 cells. CCK8 experiments demonstrated that IC50 values of cells for cisplatin were reduced from 5.662 μm to 3.327 μm to 58.76% of control cells following RAD51AP1 knockdown. This may indicate that the knockdown of RAD51AP1 increases the sensitivity of UMUC-3 cells to cisplatin (reduced IC50 values).

Example 4 synergistic inhibition of proliferation of human bladder cancer UMUC-3 cells by ESI-09 in combination with cisplatin

(2) And (3) intervention: after observing that the cell wall is good under the microscope after culturing for 8-12 hours, the culture medium of each hole is sucked out, and the concentration of ESI-09 and cisplatin is calculated according to the mole ratio of 1:2, preparing, namely adding corresponding holes into the normal culture medium of the control group according to different concentration gradients, and continuously culturing for 48 hours.

(4) GRAPHPADPRISM8 IC50 values were calculated for each group of cell lines. The synergy index (CI) is calculated according to the following formula. Synergistic effects are denoted by CI <1, ci=1 for additive effects and CI >1 for antagonistic effects. D1 is the dose of drug a required to produce 50% cell death when used in combination; d2 is the dose required for drug B to produce 50% cell death when used in combination; IC50,1 is the dose required for drug a to produce 50% cell death when used alone; IC50,2 is the dose required for drug B to produce 50% cell death when used alone.

FIG. 6 shows that the combination of ESI-09 and cisplatin inhibits proliferation of UMUC-3 cells of human bladder cancer. ESI-09 and cisplatin were allowed to act on UMUC-3 cells at a molar concentration ratio of 2:1 for 24 hours, with a semi-inhibitory concentration of 3.990. Mu.M for cisplatin and 7.980. Mu.M for ESI-09. Compared with the semi-inhibitory concentration of 5.662 mu M and 27.98 mu M of the cisplatin and ESI-09 single drug on UMUC-3 cells, the combined drug significantly reduces the IC50 value of the two drugs, and CI <1 shows that the two drugs have synergistic effect.

The results are shown by the experiments and the graphs, and the results show that: compared with the control group, ESI-09 can reduce the expression level of RAD51AP1 protein, and the reduced expression of RAD51AP1 protein can inhibit the proliferation of bladder cancer cells and improve the sensitivity to cisplatin medicaments. This can indicate that ESI-09 can inhibit proliferation of cancer cells and improve drug sensitivity of cisplatin by reducing RAD51AP1 protein expression level, and shows that ESI-09 has good in vitro anti-tumor effect on bladder cancer cells UMUC-3.

The above description is only of the preferred embodiments of the present application and it is not intended to limit the scope of the present application, but various modifications and variations can be made by those skilled in the art. Variations, modifications, substitutions, integration and parameter changes may be made to these embodiments by conventional means or may be made to achieve the same functionality within the spirit and principles of the present application without departing from such principles and spirit of the application.

Claims

Application of ESI-09 as unique active ingredient in preparation of medicine for treating bladder cancer, wherein ESI-09 has structural formula of。
2. Use of ESI-09 as defined in claim 1 as sole active ingredient for the manufacture of a medicament for the treatment of bladder cancer, wherein: ESI-09 inhibits bladder cancer cell proliferation by reducing expression of RAD51AP1 protein.
3. Use of ESI-09 as defined in claim 1 as sole active ingredient for the manufacture of a medicament for the treatment of bladder cancer, wherein: ESI-09 enhances the sensitivity of bladder cancer cells to cisplatin by reducing the expression of RAD51AP1 protein.
4. Use of ESI-09 as defined in claim 1 as sole active ingredient for the manufacture of a medicament for the treatment of bladder cancer, wherein: the bladder cancer is bladder cancer UMUC-3 cells.
5. Use of ESI-09 as defined in claim 1 as sole active ingredient for the manufacture of a medicament for the treatment of bladder cancer, wherein: the medicament is prepared by adding pharmaceutically acceptable carriers and/or auxiliary materials into ESI-09 serving as an active ingredient.
6. Use of ESI-09 according to claim 5 as sole active ingredient for the preparation of a medicament for the treatment of bladder cancer, wherein: the preparation is any one of tablets, sprays, granules, capsules, oral liquid, injection and suspension.