Application of small molecule drug targeting ARPC2 / 3 in endometrial cancer

Abstract

The invention relates to the field of biological medicine, in particular to application of a small molecule drug targeting ARPC2 / 3 to treatment of endometrial cancer, the small molecule drug is CK636, the small molecule drug provided by the invention is CK636, the volume of endometrial cancer tumor can be reduced, and a new thought is provided for developing a novel anti-endometrial cancer drug.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of a small molecule drug targeting ARPC2 / 3 in the treatment of endometrial cancer. Background Art

[0002] Endometrial cancer (EC) is one of the most common malignant tumors of the female reproductive tract. EC mainly occurs in postmenopausal women, and its occurrence is related to a variety of factors, including gene mutations, endocrine disorders, obesity, infertility, delayed menopause, etc. The defects of endometrial cancer are mainly reflected in the complexity of its pathogenesis and resistance to treatment. Since the pathogenesis of the disease involves abnormalities in multiple genes and signaling pathways, it is relatively difficult to treat. At the same time, some endometrial cancer patients are insensitive to or resistant to traditional treatments such as surgery, radiotherapy and chemotherapy, resulting in poor treatment effects.

[0003] At present, the drugs for treating endometrial cancer mainly include hormone drugs (such as progesterone, anti-estrogen preparations), chemotherapy drugs (such as paclitaxel, carboplatin) and targeted drugs (such as bevacizumab). However, these drugs have certain disadvantages. For example, hormone drugs may cause side effects, chemotherapy drugs are highly toxic, and existing targeted drugs may cause hypertension and hypoproteinemia.

[0004] Therefore, in order to reduce side effects, improve treatment effects, and provide patients with new treatment options, it is urgent to study the relationship between targeted therapy drugs and endometrial cancer. Summary of the invention

[0005] In order to develop a drug with strong targeting and few side effects, the present invention provides the use of CK636 in the preparation of a drug for treating endometrial cancer. The CK636 inhibitor provided by the present invention can reduce the volume of endometrial cancer tumors, providing a new idea for the development of new anti-endometrial cancer drugs.

[0006] The present invention provides an application of a small molecule drug targeting ARPC5 in the preparation of a drug for treating endometrial cancer. The small molecule drug includes CK636 or an analog of CK636. The structural formula of CK636 is as follows: .

[0007] The drug CK636 in the present invention is a small molecule drug that specifically targets the ARPC2 / 3 complex. It interferes with the stability of the complex by inhibiting its activity, resulting in the obstruction of cytoskeleton reorganization, thereby inhibiting the invasion of cancer cells. At the same time, the small molecule drug CK636 is small in size and can easily penetrate the cell membrane to enter the cell. It can target a variety of molecules such as protein kinases, receptors, enzymes, nucleic acids, etc. The synthesis process is mature and the cost is low, providing new ideas for the research and development of drugs for ARPC5 and the precise treatment of endometrial cancer.

[0008] Furthermore, the small molecule drug is a drug composed of the CK636 and a pharmaceutically acceptable carrier and / or excipient.

[0009] Furthermore, the acceptable carriers and / or excipients include diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants and / or disintegrants.

[0010] Furthermore, the diluent is PBS.

[0011] Furthermore, the small molecule drug is a drug composed of CK636 and PD-L1 antibody.

[0012] Furthermore, the small molecule drug reduces the volume of endometrial tumors.

[0013] The present invention also provides an application of a small molecule drug targeting ARPC5 in the preparation of an agent for inhibiting the growth of endometrial cancer cells, wherein the small molecule drug is the CK636.

[0014] Furthermore, the endometrial cancer cells include Ishikawa, HEC-1-B, RL95-2, HEC-1-A, MFE-280, MFE-296, SNG-M, HECCL-1, KLE, ECC-1, ECC-10, ECC-12, HEC-151, HEC-251 or SK-UT-1.

[0015] Furthermore, the endometrial cancer cells are Ishikawa.

[0016] Use of a reagent for detecting the expression level of ARPC5 in preparing a diagnostic product for endometrial cancer.

[0017] Furthermore, the diagnostic product comprises anti-arpc5 / p16 ARC antibody.

[0018] ARPC5 is an important subunit of the ARPC2 / 3 complex, and its stability is essential for the normal function of the ARPC2 / 3 complex. ARPC5 regulates the dynamic changes of the actin network, and abnormal expression or dysfunction of this protein has been found to be closely related to the poor prognosis of various cancers.

[0019] Small molecule compounds are small in size and can easily penetrate cell membranes and enter cells. They can target a variety of molecules such as protein kinases, receptors, enzymes, nucleic acids, etc. The synthesis process is mature and the cost is low. Therefore, the development of more small molecule drugs targeting new targets for cancer treatment can bring more accurate and efficient treatment plans to cancer patients.

[0020] CK636 is a small molecule inhibitor that specifically targets the ARPC2 / 3 complex. It interferes with the stability of the complex by inhibiting its activity, resulting in the obstruction of cytoskeleton reorganization, thereby inhibiting the invasion of cancer cells. Therefore, CK636 is expected to be used as a targeted drug for ARPC5 function. By detecting the expression level of ARPC5, patients who are sensitive to CK636 can be selected for personalized cancer treatment, which has important potential in clinical applications.

[0021] The present invention found that ARPC5 is highly expressed in endometrial cancer cells, and its expression level is significantly correlated with prognosis.

[0022] The present invention provides a treatment scheme for endometrial cancer based on ARPC5 regulation. By using the small molecule compound CK636 to affect the skeleton remodeling of endometrial cancer cells, activate the cGAS signaling pathway, and enhance tumor immune response, it can be used as an effective strategy for combined immunotherapy.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The drug CK636 provided by the present invention is a small molecule drug that specifically targets the ARPC2 / 3 complex. It interferes with the stability of the complex by inhibiting its activity, resulting in the obstruction of cytoskeleton reorganization, thereby inhibiting the invasion of cancer cells. At the same time, the small molecule drug CK636 is small in size and can easily penetrate the cell membrane to enter the cell. It can target a variety of molecules such as protein kinases, receptors, enzymes, nucleic acids, etc. The synthesis process is mature and the cost is low, providing new ideas for the research and development of drugs for ARPC5 and the precise treatment of endometrial cancer.

[0024] The small molecule drug CK636 provided by the present invention is used to reduce the volume of endometrial cancer tumors. The therapeutic effect of the combination with PD-L1 (inhibition rate 87.70%) is better than that of the small molecule drug CK636 alone (inhibition rate 61.48%), and is also better than that of PD-L1 alone (55.68%). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1The high expression of APRC5 in endometrial cancer and its correlation analysis; Figure A is Western blot detection of ARPC5 expression levels in normal endometrial tissue and endometrial cancer tissue; Figure B is TCGA database analysis of the correlation between ARPC5 expression level and survival rate of endometrial cancer patients.

[0027] Figure 2 Figure 2 shows the inhibitory effect of CK636 on the activity of ARPC2 / 3 complex and the cytoskeleton reorganization of endometrial cancer cells. Figure A shows the effect of CK636 on the activity of ARPC2 / 3 complex and the level of γ-H2AX protein, a DNA damage marker, in the endometrial cancer cell line ishikawa detected by Western blot. Figure B shows the effect of CK636 on the growth of the endometrial cancer cell line ishikawa. Figure C shows that the expression of γ-H2AX protein, a DNA damage marker, was increased in the CK636-treated group by immunofluorescence, and the scale bar of Figure C is 20 μm. Figure D shows that the dynamic imbalance of microtubules in the CK636-treated group led to abnormal division and increased cytoskeleton disorder of cancer cells by immunofluorescence, and the scale bar of Figure D is 20 μm.

[0028] Figure 3 CK636 activates the cGAS-STING pathway and induces an immune response; in the figure, Figure A is a fluorescent microscopy image of cytoplasmic DNA leakage in cells after CK636 treatment (DAPI labels DNA, showing an increase in cytoplasmic DNA), the cGAS signal is significantly enhanced and co-localized with the cytoplasmic DNA, indicating that cGAS is activated; Figure B is a qPCR showing that the expression levels of interferon β (IFN-β) and other inflammatory cytokines IL-6 and CXCL10 related to the cGAS-STING pathway are increased after CK636 treatment, which usually indicates that the cGAS-STING pathway is successfully activated.

[0029] Figure 4 The combined therapeutic effect of CK636 and immune checkpoint inhibitors. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0031] The CK636 used in the present invention was purchased from selleck with the item number: S7497.

[0032] Example 1 High expression of ARPC5 in endometrial cancer and its correlation analysis Tissue sample collection: Endometrial tissue samples were obtained from endometrial cancer patients and healthy controls from the Third Affiliated Hospital of Guangzhou Medical University, and the protein expression level of ARPC5 was detected by Western blot.

[0033] The results are as follows Figure 1 Results showed that ARPC5 was significantly upregulated in endometrial cancer tissues, suggesting that it may play an important role in the occurrence and development of cancer.

[0034] Survival analysis: In the TCGA database, combined with patient follow-up data, the relationship between ARPC5 expression level and survival rate was analyzed using the Kaplan-Meier survival curve. It was found that patients with high ARPC5 expression had a poor prognosis, suggesting that it can be used as a potential marker for poor prognosis.

[0035] Example 2 Inhibitory effect of CK636 on ARPC2 / 3 complex activity and cytoskeletal reorganization in endometrial cancer cells Human endometrial cancer cell line Ishikawa was cultured in MEM medium containing 15% FBS, 1% P / S, and 1% NEAA at 37°C with 5% CO2.

[0036] CK636 was dissolved in DMSO and prepared as a 10 mM stock solution for storage. It was diluted with PBS to a 2 μM working solution when used. The pH of the PBS used was 7.4, the manufacturer was Biosharp, the product number was Biosharp 02-023-1 ACS, and the name was BiosharpBL310A D-PBS buffer.

[0037] Western blot detection: Take the logarithmic phase growing ishikawa cells, add CK636 to the culture plate, the final concentration is 2μM, and DMSO is added as a blank control. The culture plate is placed in a 5% CO2, 37℃ constant temperature incubator for culture. After 48h, extract the total cell protein (extract the total cell protein: discard the culture supernatant, wash once with pre-cooled PBS, add Beyotime / Biyuntian RIPA cell lysis buffer on ice for 10min, and then use a cell scraper to repeatedly scrape the protein, centrifuge at 4℃, 12000rpm for 10min to collect the supernatant). Then use Pierce™ Rapid Gold BCA Protein AssayKit (Thermo Scientific, Waltham, MA, USA) to quantify the protein concentration. Separate 50μg of protein by 12% SDS-PAGE at a constant pressure of 120V for 90min and transfer to a 0.2μm Immun-Blot® PVDF membrane (Bio-Rad, Hercules, CA, USA). Then the membrane was blocked with TBST solution containing 5% skim milk at room temperature for 60 min. The membrane was probed with primary antibody (1:1000) at 4 °C overnight. The membrane was then washed 3 times with TBST for 10 min each time and incubated with secondary antibody (1:1000) at room temperature on a low-speed shaker for 60 min. Afterwards, protein expression was exposed using ECL ultrasensitive detection reagent (GE Healthcare, Buckinghamshire, UK), and images were captured using the ChemiDoc™ MP imaging system and Image Lab 5.1 software (Bio-Rad, Hercules, CA, USA). Among them, the primary antibody was anti-arpc5 / p16 ARC antibody, the catalog number was ab51243, and the manufacturer was abcam.

[0038] The experimental results are as follows Figure 2 As shown in A, CK636 significantly inhibited the activity of ARPC5 and simultaneously led to the accumulation of DNA damage γ-H2AX.

[0039] Cell proliferation inhibition test (Prestoblue method): Collect cells that have been treated with CK636 inhibitor for 48 hours, adjust the cell density to 10,000 cells / 100 μL with culture medium, inoculate the cell suspension in a 96-well plate, 100 μL per well, incubate at 5% CO2, 37°C for 7 hours, add 10 μL of prestoblue (10×) solution to each well after the cells adhere to the wall, continue to culture for 10 minutes, take the supernatant and measure the absorbance of each well at 560nm~590nm on an automatic microplate reader. Detect once every 48 hours.

[0040] The experimental results are as follows Figure 2As shown in B, treatment of the endometrial cancer cell line Ishikawa with 2 μM CK636 significantly inhibited cell growth (inhibition rate was 50.8%).

[0041] Immunofluorescence detection of the inhibitory effect of CK636 on the skeleton reorganization of endometrial cancer cells: Ishikawa cells that have been treated with CK636 inhibitor for 48 hours were collected, inoculated on coverslips, and placed in 24-well plates to ensure that the cell density was 60%. After the cells adhered to the wall, 4% paraformaldehyde was added to each well and incubated at room temperature for 30 minutes. The cells were gently washed 3 times with PBS for 5 minutes each time to remove the paraformaldehyde. 0.2% Triton X-100 was added to the cells, incubated at room temperature for 15 minutes, and after permeabilization, they were washed 3 times with PBS for 5 minutes each time. Goat serum was added and blocked at room temperature for 60 minutes. The primary antibody working solution (rabbit anti-γ-H2AX antibody, diluted 1:200, mouse anti-tubulin, diluted 1:500) was prepared. The primary antibody was added and incubated overnight at 4°C. The cells were washed 3 times with PBS for 5 minutes each time to remove the unbound primary antibody. Prepare fluorescently labeled secondary antibody working solution (anti-rabbit IgG labeled with Alexa Fluor 488, anti-mouse IgG labeled with Alexa Fluor 488). Add secondary antibody and incubate at room temperature in the dark for 1 hour. After incubation, wash cells with PBS 3 times, 5 minutes each time, to remove unbound secondary antibody. Add DAPI (diluted 1:500) to stain nuclei and incubate at room temperature in the dark for 10 minutes. Wash with PBS 3 times, 5 minutes each time. Remove the coverslip from the well, add anti-fluorescence quenching mounting medium, and invert it on the slide to mount the slide. Observe the expression and localization of the target protein under a confocal microscope.

[0042] The experimental results are as follows Figure 2 As shown in C, the imbalance of microtubule dynamics in the CK636-treated group led to abnormal division, increased cell skeletal disorder, and accumulation of DNA-damaged γ-H2AX.

[0043] Example 3 CK636 activates the cGAS-STING pathway and induces immune responses Immunofluorescence detection of the effect of CK636 on the localization of CGAS in endometrial cancer cells: Collect cells that have been treated with CK636 inhibitors for 48 hours, inoculate them on coverslips, and place them in 24-well plates to ensure that the cell density is moderate. After the cells adhere to the wall, add 4% paraformaldehyde to each well, incubate at room temperature for 30 minutes, and gently wash the cells 3 times with PBS for 5 minutes each time to remove the paraformaldehyde. Add 0.1% Triton X-100 to the cells, incubate at room temperature for 15 minutes, and after permeabilization, wash them 3 times with PBS for 5 minutes each time. Add goat serum and block at room temperature for 60 minutes. Prepare the primary antibody working solution (rabbit anti-CGAS antibody, diluted 1:100, mouse anti-laminA / C, diluted 1:500). Add the primary antibody and incubate overnight at 4°C. Wash the cells 3 times with PBS for 5 minutes each time to remove the unbound primary antibody. Prepare fluorescently labeled secondary antibody working solution (anti-rabbit IgG labeled with Alexa Fluor 488, anti-mouse IgG labeled with Alexa Fluor 594). Add secondary antibody and incubate at room temperature in the dark for 1 hour. After incubation, wash cells with PBS 3 times, 5 minutes each time, to remove unbound secondary antibody. Add DAPI (diluted 1:500) to stain nuclei and incubate at room temperature in the dark for 10 minutes. Wash with PBS 3 times, 5 minutes each time. Remove the coverslip from the well, add anti-fluorescence quenching mounting medium, and invert it on the slide to mount the slide. Observe the expression and localization of the target protein under a confocal microscope.

[0044] The experimental results are as follows Figure 3 As shown in A, after CK636 treatment, the fluorescence microscopy image of cytoplasmic DNA leakage in the cells (DAPI labeled DNA, showing an increase in cytoplasmic DNA), the cGAS signal was significantly enhanced and co-localized with LaminA / C, indicating that cGAS was activated.

[0045] Q-PCR detection of the expression level of cGAS-STING pathway-related factors: Collect cells that have been treated with CK636 inhibitor for 48 hours, and extract RNA using the TRIzol method. Add 1 mL of TRIzol reagent to each well to fully lyse the cells. Add 200 μL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 12,000×g for 15 minutes at 4°C, and collect the supernatant. Add an equal volume of isopropanol, mix gently, and centrifuge at 12,000×g for 10 minutes. Wash the RNA precipitate with 75% ethanol and remove the ethanol by centrifugation. Dissolve the RNA precipitate with RNase-free water. Use NanoDrop to detect RNA concentration and purity (260 / 280 ratio should be 1.8-2.0). Use a commercial reverse transcription kit to reverse transcribe 1 μg of total RNA into cDNA.

[0046] Reaction system: RNA: 1 μg; 5x reverse transcriptase: 4 μL; RNase-free water: to 20μL.

[0047] Reaction conditions: 42°C 30min→85°C 5min→4°C storage. The synthesized cDNA was diluted 10 times for subsequent qPCR reactions.

[0048] qPCR reaction system: Total reaction volume 10 μL: 5 μL SYBR Green qPCR Mix; 1 μL forward primer (2 μM); 1 μL reverse primer (2 μM); 1 μL diluted cDNA; 2 μL RNase-free water.

[0049] Reaction conditions: 95℃ 5min (pre-denaturation) - 95℃ 10s, 60℃ 30s (40 cycles). After obtaining the Ct values ​​of the target genes (such as IFN-β, IL-6, CXCL10) and the reference gene 18s, the relative expression of the target genes was calculated using the formula: 2^−ΔΔCt. The differences between the groups were compared using the t-test, with a significance level of p<0.05. The primers are shown in Table 1. The results are shown in Figure 3 B in.

[0050] Table 1 Primer sequences Example 4 Combination therapy of CK636 and immune checkpoint inhibitors Test animals: 6-week-old female C57BL / 6 mice with normal immunity, female, bred in the SPF-grade breeding environment room of our experimental animals, with the indoor temperature controlled at 23±2℃, free food and water intake.

[0051] Establishment of mouse transplant tumor model: Use endometrial cancer cells from murine-derived cell line to establish mouse subcutaneous transplant tumor model, and use Matrix gel to adjust the cell concentration to 1×10 6 / 100 μL. The cell suspension was injected subcutaneously into the nipple of the mouse under sterile conditions. The growth of the tumor was observed 2 days after inoculation. When the tumor volume reached about 20 mm³ (usually 7d~10d after inoculation), drug treatment was started. It was divided into 4 groups, 3 mice in each group, and treated with CK636 alone (1 mg / kg), PD-L1 antibody alone (5 mg / kg) and combined treatment (CK636 1 mg / kg, PD-L1 antibody 5 mg / kg), and the tumor growth was observed. The long diameter (L) and short diameter (W) of the tumor were measured every 5 days using an electronic vernier caliper, and the tumor volume was calculated according to the following formula, and the growth curve was drawn. Among them, the PD-L1 antibody is InVivoPlus anti-mouse PD-L1 (B7-H1), the brand is BioXcell, and the product number is BP0101-5MG.

[0052] Calculate tumor volume: V = 1 / 2 × L × W 2 .

[0053] The experimental results are as follows Figure 4 Results: The combined treatment group showed significant tumor inhibition effect, which was better than the single-drug group.

[0054] Tumor volume inhibition rate (%) = (tumor volume of control group - tumor volume of experimental group) / tumor volume of control group × 100%.

[0055] The tumor volume inhibition rate (%) of CK636 group = (35.60 / 13.71) / 35.60×100%.

[0056] The tumor volume inhibition rate (%) of the PD-L1 group = (35.60 / 15.78) / 35.60×100%.

[0057] The tumor volume inhibition rate (%) of the CK636 and PD-L1 combination group = (35.60 / 4.28) / 35.60×100%.

[0058] In summary, CK636 is a small molecule inhibitor targeting the ARPC2 / 3 complex, which can specifically block the activity of ARPC5. CK636 interferes with the dynamic reorganization of the skeleton in endometrial cancer cells, leading to the release of cytoplasmic DNA and activating the cGAS-STING signaling pathway. The activated cGAS-STING signaling pathway induces the secretion of IFN-β and other inflammatory factors, enhancing the activity of immune cells in the tumor microenvironment. CK636 enhances the immune system's ability to recognize and eliminate tumors by reshaping the tumor immune microenvironment.

[0059] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0060] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a small molecule drug targeting ARPC2 / 3 in the preparation of a drug for treating endometrial cancer, characterized in that: The small molecule drug includes CK636 or an analog of CK636, and the structural formula of CK636 is as follows: .

2. The use according to claim 1, characterized in that: The small molecule drug is a drug composed of the CK636 and a pharmaceutically acceptable carrier and / or excipient.

3. The use according to claim 2, characterized in that: The acceptable carriers and / or excipients include diluents, binders, surfactants, wetting agents, adsorbent carriers, lubricants and / or disintegrants.

4. The use according to claim 3, characterized in that: The diluent is PBS.

5. The use according to claim 1, characterized in that: The small molecule drug is a drug composed of CK636 and PD-L1 antibody.

6. The use according to claim 1, characterized in that: The small molecule drug reduces the volume of endometrial tumors.

7. Use of a small molecule drug targeting ARPC5 in the preparation of an agent for inhibiting the growth of endometrial cancer cells, characterized in that: The small molecule drug is CK636 as described in claim 1.

8. The use according to claim 7, characterized in that: The endometrial cancer cells are any one of Ishikawa, HEC-1-B, RL95-2, HEC-1-A, MFE-280, MFE-296, SNG-M, HECCL-1, KLE, ECC-1, ECC-10, ECC-12, HEC-151, HEC-251, and SK-UT-1.

9. Use of the reagent for detecting the expression of ARPC5 as claimed in claim 7 in the preparation of a diagnostic product for endometrial cancer.

10. The use according to claim 9, characterized in that: The diagnostic product includes anti-arpc5 / p16 ARC antibodies.

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