Use of an RNA helicase dhx33 inhibitor in the preparation of a medicament for treating endometrial cancer

Abstract

This invention belongs to the field of biomedicine and discloses the application of RNA helicase DHX33 inhibitors in the preparation of drugs for the treatment or adjuvant treatment of endometrial cancer. This invention establishes the important role of DHX33 protein in the development of endometrial cancer, and the provided DHX33 inhibitor inhibits DHX33 helicase activity, exhibiting significant inhibitory activity against endometrial cancer cells, thus possessing significant pharmaceutical development value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to RNA helicase DHX33 inhibitors and their application in the preparation of drugs for the treatment of endometrial cancer. Background Technology

[0002] Endometrial cancer is a malignant tumor located in the epithelial cells of the uterine lining. It is one of the three major malignant tumors of the female reproductive system, second only to cervical cancer in incidence.

[0003] Endometrial cancer commonly occurs in postmenopausal women aged 50-60. The most common type is adenocarcinoma. Other unhealthy habits and genetic factors can also contribute to its development. Clinically, endometrial cancer is classified into type I and type II. Type I is hormone-dependent and has a better prognosis, while type II is non-hormone-dependent and has a poorer prognosis.

[0004] In recent years, the survival rate of endometrial cancer has reached as high as 95% due to its early detection and treatment. Endometrial cancer can be diagnosed at different stages based on its various clinical manifestations. Early-stage symptoms mainly include irregular vaginal bleeding; late-stage symptoms are more numerous, such as abdominal pain and distension, urinary frequency, and hematuria. In addition to these, endometrial cancer can be diagnosed and treated using other methods such as ultrasound, fractional curettage, hysteroscopy, MRI, cytology, and pathology. The most common treatment is surgery, often involving double resection. Patients diagnosed in the early stages often undergo minimally invasive treatment. Other treatment methods include radiotherapy, chemotherapy, and hormone therapy. However, drug treatment has limitations, necessitating the development of new drugs and treatment methods. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide an RNA helicase DHX33 inhibitor and its use in the preparation of a medicament or pharmaceutical composition for the treatment or adjuvant treatment of endometrial cancer.

[0006] To achieve the objectives of this invention, in a first aspect, the present invention provides an RNA helicase DHX33 inhibitor for the treatment or adjuvant treatment of endometrial cancer. In this invention, the RNA helicase DHX33 inhibitor (i.e., a DHX33 protein inhibitor) is selected from at least one of compound A or a pharmaceutically acceptable salt thereof or a prodrug:

[0007]

[0008] This invention discloses for the first time that DHX33 protein can be used as a therapeutic target for endometrial cancer. Therefore, in a second aspect, this invention provides the application of the above-mentioned RNA helicase DHX33 as a novel therapeutic target for endometrial cancer.

[0009] The reference sequence number of the DHX33 gene on NCBI is: NM_020162.4.

[0010] In a third aspect, the present invention provides the application of the above-mentioned RNA helicase DHX33 inhibitor in inhibiting the glutamine metabolic pathway in endometrial cancer cells.

[0011] In a fourth aspect, the present invention provides a targeted drug for the treatment or adjuvant treatment of endometrial cancer, wherein the target of the drug is RNA helicase DHX33, and the targeted drug can inhibit the activity of DHX33 helicase, thereby affecting some genes of the glutamine pathway regulated by DHX33 protein. The active ingredient of the targeted drug is compound A.

[0012] In a fifth aspect, the present invention provides the application of the aforementioned RNA helicase DHX33 inhibitor in inhibiting genes involved in regulating the glutamine pathway in endometrial cancer cells. In a specific embodiment of the present invention, the genes involved in regulating the glutamine pathway are SCL1A5, SCL2A1, SCL12A2, SCL10A5, SCL7A5, SCL3A2, PAST, GLUC, PYCR, GOT1, GPT2, or ASNA.

[0013] In a sixth aspect, the present invention provides the use of the above-mentioned RNA helicase DHX33 inhibitor in the treatment or adjuvant treatment of endometrial cancer.

[0014] In a seventh aspect, the present invention provides the use of the above-mentioned RNA helicase DHX33 inhibitor in the preparation of a medicament or pharmaceutical composition for the treatment or adjuvant treatment of endometrial cancer.

[0015] In an embodiment of the present invention, endometrial cancer is characterized by positive expression of the DHX33 protein.

[0016] In embodiments of the present invention, the frequency or dosage of the RNA helicase DHX33 inhibitor intake can be determined by a physician based on individual physical condition, age, sex, weight, and other factors. In specific embodiments, the intake frequency can range from once to three times per day. In embodiments of the present invention, the intake dose of the RNA helicase DHX33 inhibitor needs to ensure an effective drug exposure of 4000-7500 ng·h / mL per day. In specific embodiments, in mice, the oral dose of the RNA helicase DHX33 inhibitor can be 25 mg-300 mg / kg once, and the intravenous dose can be 2.5 mg-25 mg / kg per injection. In a specific implementation plan, in mice, the oral dose of the RNA helicase DHX33 inhibitor can be, for example, 35 mg-290 mg / kg, 45 mg-280 mg / kg, 55 mg-270 mg / kg, 65 mg-260 mg / kg, 75 mg-250 mg / kg, 85 mg-240 mg / kg, 95 mg-230 mg / kg, 105 mg-220 mg / kg, 115 mg-210 mg / kg, 125 mg-200 mg / kg, 135 mg-190 mg / kg, 145 mg-180 mg / kg, or 155 mg-170 mg / kg per dose. When administered intravenously in mice, the single injection dose of the RNA helicase DHX33 inhibitor can be, for example, 3.0 mg-24.5 mg / kg, 3.5 mg-24 mg / kg, 4.0 mg-23.5 mg / kg, 4.5 mg-23 mg / kg, 5.0 mg-22.5 mg / kg, 5.5 mg-22 mg / kg, 6.0 mg-21.5 mg / kg, 6.5 mg-21 mg / kg, 7.0 mg-20.5 mg / kg, 7.5 mg- 20mg / kg, 8.0mg-19.5mg / kg, 8.5mg-19mg / kg, 9.0mg-18.5mg / kg, 9.5mg-18mg / kg, 10.0mg-17.5mg / kg, 10.5mg- 17.0mg / kg, 11.0mg-16.5mg / kg, 11.5mg-16mg / kg, 12.0mg-15.5mg / kg, 12.5mg-15.0mg / kg or 13.0mg-14.5mg / kg.

[0017] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0018] This invention establishes the important role of DHX33 protein in the development and progression of endometrial cancer. The provided DHX33 inhibitor inhibits the activity of DHX33 helicase, thereby inducing cancer cell death. This DHX33 inhibitor can significantly inhibit the growth of endometrial cancer cells in vitro, thus achieving the goal of treating endometrial cancer and therefore has significant pharmaceutical development value. Attached Figure Description

[0019] Figure 1 In a preferred embodiment of the present invention, the expression level of DHX33 protein in representative human endometrial cancer tissues was significantly higher than that in normal endometrial tissues.

[0020] Figure 2 This is a comparison of the results of protein immunoblotting analysis of the content of DHX33 in endometrial cancer cells Hec-1-A and AN3CA and normal endometrial epithelial cells Heec cells in a preferred embodiment of the present invention.

[0021] Figure 3 The half-inhibitory concentration (IC50) of normal human skin fibroblasts (HSF) cells treated with DHX33 inhibitor compound A in a preferred embodiment of the present invention is analyzed, wherein the half-inhibitory concentration of compound A is >10 μm.

[0022] Figure 4 In a preferred embodiment of the present invention, the half-inhibitory concentration (IC50) of normal endometrial cells (Heec cells) treated with DHX33 inhibitor compound A is... 50 Analysis showed that the half-inhibitory concentration (IC50) of compound A was... 50 The value is 4.4487 μM.

[0023] Figure 5 The half-maximal inhibitory concentration (IC50) of Hec-1-A cells treated with DHX33 inhibitor compound A in a preferred embodiment of the present invention is shown in the figure. 50 Analysis showed that the half-inhibitory concentration (IC50) of compound A was... 50 The value is 22.7 nM.

[0024] Figure 6 The half-maximal inhibitory concentration (IC50) of AN3CA cells treated with DHX33 inhibitor compound A in a preferred embodiment of the present invention is shown in the figure. 50 Analysis showed that the half-inhibitory concentration (IC50) of compound A was... 50 The value is 54.1 nM.

[0025] Figure 7 The results of the analysis of the clonal growth of Hec-1-A cells treated with DHX33 inhibitor compound A in a preferred embodiment of the present invention are shown.

[0026] Figure 8 The analysis results of the independent growth of Hec-1-A soft agar treated with DHX33 inhibitor compound A in a preferred embodiment of the present invention are as follows:

[0027] Figure 9 This is the analysis result of AN3CA cell clone growth after treatment with DHX33 inhibitor compound A in a preferred embodiment of the present invention.

[0028] Figure 10 The results of the AN3CA soft agar experiment—independent suspension growth—after treatment with DHX33 inhibitor compound A in a preferred embodiment of the present invention are analyzed.

[0029] Figure 11 This invention provides a preferred embodiment of the changes in transcriptional levels of different genes in the glutamine pathway of Hec-1-A endometrial cancer cells treated with different doses of DHX33 inhibitor compound A for 6 hours.

[0030] Figure 12 for Figure 11 Analysis of the expression level of the target protein in cells treated with compound A.

[0031] Figure 13 This invention relates to a preferred embodiment of the transcriptional changes of different genes in the glutamine pathway in Hec-1-A endometrial cancer cells treated with #2 virus knockout DHX33. Detailed Implementation

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0033] 1. Cell Culture

[0034] Human endometrial cancer cell lines Hec-1-A and AN3CA, and human normal endometrial cell line Heec, were all purchased from Beyotime Biotechnology Co., Ltd. (Shanghai). Hec-1-A and AN3CA were cultured in McCoy's 5A medium and Minimum Eagle's Medium, respectively, while the normal endometrial cell line Heec was cultured in RPMI-1640 medium. McCoy's 5A medium, Minimum Eagle's Medium, and RPMI-1640 medium all contained 10% fetal bovine serum (FBS), 2 mM L-glutamine, non-essential amino acids, streptomycin, and penicillin. Culture conditions were a 37°C CO2 incubator with 60-70% humidity. Human skin fibroblast HSF was purchased from Shanghai Boson Biotechnology Co., Ltd., and cultured in Dulbecco's Modified Eagle Medium (high glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, non-essential amino acids, streptomycin, and penicillin.

[0035] 2. Real-time quantitative PCR

[0036] To analyze the molecular mechanism by which DHX33 protein promotes the growth of endometrial cancer cells, quantitative PCR (SYBR Green Supermix (Bio-Rad)) was used to analyze the expression changes of important genes in endometrial cancer cells. Cells were seeded at appropriate densities into 6-well plates. The next day, different concentrations of 0 nM, 20 nM, and 40 nM DHX33 inhibitors were added to the culture medium in the 6-well plates. The cells were treated with the inhibitors for 6 hours, after which the cells were harvested and RNA was extracted. Quantitative PCR analysis was then performed on the RNA samples. The target genes to be analyzed were: SCL1A5, SCL2A1, SCL12A2, SCL10A5, SCL7A5, SCL3A2, PAST, GLUC, PYCR, GOT1, GPT2, or ASNA. Primers were designed using the online "realtime PCR tool" of IDT (http: / / sg.idtdna.com / site) and purchased from BGI (Shenzhen) Co., Ltd.

[0037] The primer sequences for the genes involved in regulating the glutamine pathway in human cells are as follows (all primers are from 5'-3'):

[0038] Table 1. Primer sequences involved in the cellular glutamine pathway in human cells.

[0039]

[0040]

[0041] 3. Half-inhibitory concentration (IC50) 50 ) Measurement

[0042] Endometrial cancer cell lines Hec-1-A and AN3CA, as well as normal endometrial epithelial cell line Heec, were used at a concentration of 1×10⁻⁶. 4 100 μL / well of cells were seeded onto a 96-well plate and allowed to adhere completely. Compound A of this invention was then added to the corresponding cell culture medium at concentrations of 19 nM, 39 nM, 78 nM, 156 nM, 312 nM, 625 nM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM, and mixed thoroughly using a multichannel pipette. After 72 hours of incubation with compound A and cells, 1 ml of CCK8 reagent (Shanghai Yisheng Biotechnology Co., Ltd.) was mixed with 1 ml of PBS and added to the cell culture medium of each 96-well plate according to standard procedures. After incubation for 1 hour, the plates were read using an ELISA reader (OD). 450nm) The experiment was repeated three times, and inhibition curves of compound A at different concentrations were plotted (e.g., Figure 3 , 4 As shown in Figures 5 and 6, among which Figure 3 Data representing HSF cells, Figure 4 Data representing Heec cells. Figure 5 Data representing Hec-1-A cells, Figure 6 (Data representing AN3CA cells) Calculate the half-inhibitory concentration (IC50) of compound A. 50 The vertical axis of the curve in the figure represents the cell viability index, and the horizontal axis represents the logarithmic value of the concentration of compound A (μM).

[0043] 4. Immunohistochemical analysis

[0044] The tissue microarray was purchased from Shanghai Xinchao Biotechnology Co., Ltd., containing a total of 102 tissue samples, including 95 samples of endometrial cancer cells, 5 samples of normal physiological tissue, and 2 samples of undifferentiated endometrial cancer cells. Before use, the tissues were baked at 60°C for half an hour. The tissues were dewaxed in dewaxing solution and rehydrated in a series of solutions with gradually decreasing ethanol concentrations. Antigens were presented in a sterilizer using Tris buffer (pH 9.0). The tissues were then incubated in a methanol solution containing 1% H2O2 to inactivate endogenous peroxidase. After blocking with 5% FBS-PBS for 30 min at room temperature, the tissues were incubated overnight at 4°C with the primary antibody (anti-DHX33). The DAKO immunohistochemistry kit (DAKO Biotechnology, Denmark) was then used according to the manufacturer's instructions. The antibody used was anti-DHX33 (purchased from Santa Cruz Biotechnology, Inc.).

[0045] 5. Protein immunoblotting analysis (Western blot)

[0046] After cell harvesting, cells are resuspended in protein lysis buffer, lysed by sonication, and then centrifuged to obtain the supernatant. Protein concentration is calculated and quantified, and the loading amount is calculated based on the concentration. SDS-PAGE is then used for separation. Before electrophoresis, the transfer procedure must be prepared. After sample transfer, the membrane is blocked with blocking buffer for one hour, then incubated overnight at low temperature with primary antibody. The next day, the membrane is washed three times with TBST solution, then coated with secondary antibody. After the secondary antibody is applied, the membrane is washed three more times with TBST before developing the results. The above experimental steps are the standard operating procedure for Western blot.

[0047] 6. Soft agar test

[0048] 1.0×10 4 Each cell was mixed with 4.0 mL of DMEM medium containing 0.3% agar and 10% FBS and added to basal agar (4.0 mL of solidified DMEM medium containing 0.6% agar and 10% FBS). The plates were incubated at 37°C, and checked every 3 days, with 2.0 mL of DMEM medium containing 0.3% agar and 10% FBS added weekly. Colony growth was observed, and cell counts were performed after 2-3 weeks.

[0049] 7. Clonal growth of cells (Foci)

[0050] 2.0×10 3 Each cell was cultured in 10.0 mL of the corresponding cell culture medium (100 mm cell culture dish) in a CO2 incubator at 37°C, with the corresponding cell culture medium replaced weekly with fresh medium. The growth of cell clones was observed. After 2-3 weeks, when the cell clones had grown to a sufficient size, they were stained with Geimsa stain and photographed for statistical analysis.

[0051] 8. Data Statistical Analysis

[0052] Data are expressed as mean + SD. Statistical significance was determined using the Student's t-test. A p-value < 0.05 was indicated by *; a p-value < 0.01 was indicated by **; and a p-value < 0.001 was indicated by ***.

[0053] Example 1. High-efficiency expression of DHX33 protein in various endometrial cancer tissues

[0054] In this embodiment, immunohistochemistry was used to analyze the expression of DHX33 protein in human endometrial cancer tissue.

[0055] Microarrays of paraffin-embedded tissue sections of human endometrial cancer obtained from Shanghai ChipSuper Biotechnology included 102 different types of human endometrial cancer tissue, with 5 cases of normal endometrial tissue serving as controls and 2 cases of undifferentiated endometrial cancer tissue. The paraffin-embedded tissue microarrays were first incubated in a 60°C oven for 30 min, then rapidly deparaffinized in xylene, and gradually hydrated in a series of solutions with progressively decreasing ethanol concentrations (100%, 95%, 70%, 50%, and 25%) (gently shaken for 5 min each time, repeating the treatment once for each concentration of ethanol solution), and finally further hydrated in distilled water for 10 min. Antigens were then presented in a steam oven with 50 mM Tris hydrochloride buffer (pH 9.0) for 40 min, followed by cooling to room temperature. The tissues were then incubated in a methanol solution containing 1% H₂O₂ to inactivate endogenous peroxidase. After blocking with 10% FBS at room temperature for 1 h, the tissues were incubated with primary antibody overnight at 4°C. Then, a standard protocol was performed using the DAKO kit (DAKO, Denmark) as recommended by the manufacturer. The antibody used was anti-DHX33 (Santa Cruz Biotechnology, Inc.). Experimental results ( Figure 1 The dark, circular areas shown in the image indicate that DHX33 protein is highly expressed in various human endometrial cancer tissues, particularly in the cell nucleus. Table 2 below provides data for the aforementioned 102 tissues. This data shows that 40 pathological tissues exhibited high expression of DHX33 protein, accounting for nearly 40% of the total pathological samples. Analysis of the pathological sections revealed lower DHX33 expression in adjacent normal tissue areas and normal endometrial tissue.

[0056] Table 2: Pathological information of human malignant endometrial carcinoma tissues and immunohistochemical analysis data of DHX33 protein in five normal tissues.

[0057]

[0058]

[0059]

[0060] Note: A DHX33 staining score of 4 or higher is considered positive, and a score of 3 or lower is considered negative.

[0061] Example 2. DHX33 inhibitors can effectively inhibit the growth and proliferation of endometrial cancer cells.

[0062] In addition to analyzing DHX33 protein expression at the tissue level, cellular-level analysis was also conducted. Heec cells, an immortalized normal human endometrial cell line, were selected as a control, and the expression of DHX33 protein in two cancer cell lines, AN3CA and Hec-1-A, was analyzed. The results are as follows: Figure 2 As shown, the expression of DHX33 protein in cancer cells is significantly higher than in normal cells. This result validates the previous histological analysis at the cellular level.

[0063] Example 3. DHX33 inhibitors can specifically inhibit the growth and proliferation of DHX33-overexpressing endometrial cancer cells, but are insensitive to normal cells.

[0064] To analyze the therapeutic value of the DHX33 inhibitor compound A for endometrial cancer, we performed a drug sensitivity analysis. We treated human endometrial cancer cells with the DHX33 inhibitor and analyzed its inhibitory effect on these cells. Normal cells have low levels of DHX33 protein; therefore, we first analyzed the sensitivity of normal cells to the inhibitor using HSF and Heec cells. The results are as follows: Figure 3 and Figure 4 As shown, both normal cell lines exhibited low sensitivity and relatively high half-maximal inhibitory concentrations (WMCs). Compound A showed almost no significant inhibitory effect on HSF, with a WMC of 4.4487 μM in Heec, indicating that in normal cells with low DHX33 content, the inhibitory effect of compound A is negligible or weak. In contrast, the results of compound inhibition assays conducted in two representative endometrial cancer cell lines, Hec-1-A and AN3CA, are as follows: Figure 5 , Figure 6 As shown, the DHX33 inhibitor, compound A, exhibits nanomolar-level inhibitory activity against endometrial cancer cells, with inhibition curves indicating a decrease in cell count exceeding 50%. These experimental data demonstrate that the DHX33 inhibitor is specific in killing cancer cells compared to normal cells.

[0065] Example 4. DHX33 inhibitors can effectively inhibit the clonal growth and suspension-free growth of endometrial cancer cells.

[0066] To further verify that the DHX33 inhibitor can effectively inhibit the growth and proliferation of endometrial cancer cells, we selected Hec-1-A and AN3CA cells for further analysis. After treatment with compound A (20 nM), we found that compound A significantly inhibited the growth of endometrial cancer cells. Figure 7 , Figure 9 ; Figure 7 The cells were Hec-1-A. Figure 9 The cells are AN3CA.

[0067] In addition to the analysis of these two-dimensional cell culture systems, we also analyzed the inhibitory effect of DHX33 inhibitor compound A on endometrial cancer cells in a three-dimensional cell culture system. This experiment was conducted in a soft agar system. As mentioned earlier, it was observed that DHX33 inhibitor compound A significantly inhibited the growth of these two endometrial cancer cell lines. Suspension-independent growth is a key characteristic of cancer cells. Under treatment with compound A (20 nM), we found that Hec-1-A and AN3CA cell lines almost completely lost their ability to grow independently in suspension on soft agar, and could not form aggregates or clones. Figure 8 , Figure 10 The results of the above experiments show that compound A has a significant inhibitory effect on the growth of endometrial cancer cells.

[0068] In conclusion, DHX33 inhibitor compound A can inhibit the growth of endometrial cancer.

[0069] Example 5. DHX33 inhibitors can regulate genes related to the glutamine metabolic pathway.

[0070] Glutamine is an important metabolic fuel, helping rapidly proliferating cells meet their increasing demands for ATP, biosynthetic precursors, and reducing agents. Glutamine enters cells via the amino acid transporter ASCT2 / SLC1A5 and is converted to glutamate in mitochondria through deamination catalyzed by glutaminase (GLS), thus participating in the tricarboxylic acid cycle to provide energy for the cell. To analyze whether the DHX33 inhibitor compound A can regulate the expression of genes involved in the glutamine metabolic pathway, we performed real-time quantitative PCR analysis on cells treated with DHX33 inhibitor (compound A). Hec-1-A cells were treated with different doses of DHX33 inhibitor (compound A) for 6 h, and RNA was collected using these as templates for real-time quantitative PCR. The primer sequences are as described above. Figure 11 As shown, in Hec-1-A cells where DHX33 was inhibited, we found that the gene expression of several enzymes involved in glutamine metabolism, such as SCL1A5, SCL2A1, SCL12A2, SCL10A5, SCL7A5, SCL3A2, PAST, GLUC, PYCR, GOT1, GPT2, or ASNA, was downregulated. With increasing dosage of compound A, some genes showed significant downregulation (e.g., ...). Figure 11 (As shown). Analysis of the DHX33 protein content in cancer cells treated with the inhibitor revealed no change (e.g., ...). Figure 12(As shown). We silencing the DHX33 gene by infecting cells with lentivirus, and then performed real-time quantitative PCR analysis on the DHX33 knockout cells. The results showed that some genes still had significant changes (e.g., Figure 13 Examples include GOT1, GPT2, and SCL3A2, indicating that DHX33 inhibitor compound A has a potential regulatory effect on the glutamine pathway in Hec-1-A endometrial cancer cells.

[0071] The above experiments show that DHX33 inhibitors can significantly inhibit the growth of endometrial cancer cells, but have no significant inhibitory effect on normal cells. The mechanism by which it inhibits the growth of cancer cells is likely through downregulating the expression of glutamine metabolism genes.

Claims

1. The use of an RNA helicase DHX33 inhibitor in the preparation of a medicament or pharmaceutical composition for the treatment or adjuvant treatment of endometrial adenocarcinoma, characterized in that, The inhibitor is selected from compound A or its pharmaceutically acceptable salt. Compound A.

Images